transcriptional regulation of meiosis and spore morphogenesis

252
Transcriptional Regulation of Meiosis and Spore Morphogenesis in Saccharomyces cerevisiae by Shelley Chu DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biochemistry in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA SAN FRANCISCO Approved: Committee in Charge Date University Librarian Degree Conferred. . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Transcriptional Regulation of Meiosis and Spore Morphogenesisin Saccharomyces cerevisiae

by

Shelley Chu

DISSERTATION

Submitted in partial satisfaction of the requirements for the degree of

DOCTOR OF PHILOSOPHY

in

Biochemistry

in the

GRADUATE DIVISION

of the

UNIVERSITY OF CALIFORNIA SAN FRANCISCO

Approved:

Committee in Charge

Date University Librarian

Degree Conferred. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DEDICATION

To my grandparents, Lena and David Miao,

and my parents, Vida and T.K. Chu

iii

ACKNOWLEDGEMENTS

I am indebted to Ira Herskowitz for giving me the opportunity to learn

science in such a productive atmosphere. During my first years in the lab, I

found his insightfulness and god-like ability to take a step back and see the

"big picture" very refreshing. This last year of graduate school I have also

become aware of his other talents -- a clear and concise writing style and a

simplified yet dynamic speaking style. I will always strive to reach Ira's

standards of clear thinking and presentation.

I must next thank my collaborators, Jon Mulholland, David Botstein,

Pat Brown, and especially Joe DeRisi. Working with this talented group was

one of the most exciting and enjoyable parts of graduate school. To me, Joe is

the ideal scientist: he has showed me how to do careful, cutting-edge science

while having a lot of fun at the same time. His patience and dedication -- Joe

is truly "doing it for honor"-- are inspirational. Working with all our

colleagues at Stanford has been a real privilege for me.

In addition, I would like to thank Arnie Levine, James Sherley, and

Cathy Finlay, for believing in me before there was a rationale reason to. I also

deeply appreciate the encouragement and useful advice I received from my

thesis committee members, Tris Parslow, Peter Walter, and especially

Andrew Murray. Discussions with Andrew and crashing the Murray lab

journal clubs have both contributed significantly to my growth as a scientist

and a person. I have also been fortunate enough during graduate school to

have met not one, but two, role models, Sue Biggins and Jais Lingappa, who

are both successful female scientists and much more. I also enjoyed

Ž2à.

**%****

~!|-º

2. – 2-->

iv.

interactions with John Watson, Pat O'Farrell, and members of their

respective labs. And I am grateful towards Sue Adams and Jana Toutolman

for coming to my rescue multiple times during the past five years.

There are so many people in the yeast and meiosis community that

have made graduate School an exciting time for me. It was a suggestion by

Nancy Klecker which changed the fate of my graduate career. In addition, I

am indebted to Liuzhong Xu, Andrew McKee, Scott Keeney, Yona Kassir,

Aaron Mitchell, Renee Reijo, Mary Clancy, Mike Eisen, Margaret Fuller, Bob

Malone, Christina Hull, Marion Shonn, and many, many others, for all their

help, generosity, and enthusiasm.

I also appreciate the contributions by members of the Herskowitz lab,

both past and present. I would especially like to acknowledge Flora Banuett

for her unfailing interest in my work, Anita Sil for her patience, and Mary

Maxon for teaching me almost everything I know about biochemistry and

volleyball. I thank Dora Alaya and Annie Poon for their reliable support, and

I will miss sharing "an office" and many laughs with Laura Baxter. I am

further indebted to the night crew of Flora Banuett, Seiko Ishida, Wei Wei,

and Sean O'Rourke for making the wee hours in lab more bearable. I also

appreciate the recent interest shown by Kirsten Benjamen and Linda Huang

in continuing aspects of my graduate work.

I thank Kelvin and Dan for their fathomless support always when I

needed it the most. And to my parents, I owe all my success. Their example

has given me the inspiration, curiosity, and drive essential for getting

through graduate school. And finally, I am grateful to my grandmother and

grandfather who by sharing their wisdom have helped me to keep life in

perspective. Their encouragement and love have been a great source of

comfort and strength. I especially thank my grandmother for filling these

past several years with beauty, laughter, music, and sunshine.

A version of Chapter Two originally appeared as Chu and Herskowitz

(1998), Molecular Cell 1: 685-696.

*à3º s

:º-:***

º:

vi

UNIVERSITY OF CALIFORNIA, SAN FRANCISCO

Stºkºlay - DAVIS • NYNE • Los ANGELE5 - Rlvºsipº - $AN bit GU - $AN PRANCISCU ■W

ucsf.edu

DEPARTMENT OF BROCHEMISTRY AND BIOPHYSICS (415) 476–4985, phfºomsaºneous (415) 502-5145, fax

July 29, 1998

Cell Press

1050 Massachusetts Avenue gºCambridge, MA 02138 ***fax: 617-661-7061 f\\-To whom it may concern,

I would like permission from to reprint the article Molecular Cell 1: 685–696,1998: "Gametogenesis in Yeast is Regulated by a Transcriptional Cascade Dependenton Ndt&O" (authored by myself and Ira Herskowitz) for my PhD thesis. Mydisseration will also be transfered to microfilm by University Microfilms, which also

requests permssion to supply single copies on demand. Please respond by fax to(415) 502-5145,

Thank you very much.

Sincerely,

----,*:

§4. (4–"-----

-: Shelley Chu& the artina, ms' . . .” --- - - - rtúatºtº copyright is rºu tº Geº Pºmººr serwise." ºsººn

*Oº■■ 0■ .NYaNAc Ø\xrl,

&\,{&vii

Transcriptional Regulation of Meiosis and Spore Morphogenesis

in Saccharomyces cerevisiae

Shelley Chu

ABSTRACT

Meiosis is a specialized form of cell division necessary for sexual

reproduction in eukaryotes. I was interested in understanding the molecular

basis for the difference between meiosis and mitosis in budding yeast. I began

by characterizing a gene, NDT80, expressed only during sporulation (the

combined processes of meiosis and spore formation) which is necessary for

progression from meitoic prophase into the first meiotic division (Xu et al.,

1995). I found that Ndt80 is a transcription factor responsible for activating

expression of many genes during sporulation, including most of the B-type

cyclins, the regulatory subunits of the Cdc28/cyclin complex. In addition,

Ndt80 activity appears to be under checkpoint control, being dependent on

successful completion of meiotic recombination. These and other

observations support the hypothesis that sporulation is regulated by a

transcriptional cascade. To pursue this line of study further, I embarked on a

comprehensive analysis of gene expression during sporulation at the whole

genome level in collaboration with Joe DeRisi and others at Stanford

University. Nearly one-tenth of the yeast genome is induced during

sporulation. Ndt80 is responsible for transcription of the largest of seven

classes of genes induced during sporulation. Using microarray analysis as an

unbiased genetic screen, we characterized three novel genes induced with

viii

different kinetics during sporulation and found each to be essential for

sporulation. We hope that this and additional studies based on the

sporulation expression data will bring us one step closer to understanding

how meiosis is a specialized form of the cell cycle.

rix

TABLE OF CONTENTS

CHAPTER ONE Introduction 1

CHAPTER TWO Gametogenesis in Yeast is Regulated by a 26Transcriptional Cascade Dependent on Ndt80

CHAPTER THREE The Transcriptional Program of Germ Cell 68Development in Budding Yeast

CHAPTER FOUR Conclusion 106

APPENDIX ONE Clb2 can Substitute for Clb1 in Meiosis 117

APPENDIX TWO Potential Regulators of NDT80. Ime4, 130Ime?, and Ids2

APPENDIX THREE A role for Clb5 in Meiosis 148

APPENDIX FOUR NDT80 and its regulation 173

REFERENCES 196

LIST OF TABLES

1-1.

1-2.

2-1.

3–1.

A1-1.

A1-2.

A1-3.

A1-4.

A2-1.

A3–1.

A3–2.

A3–3.

A4–1.

Putative Regulatory Elements of Classes of Sporulation Genes

Cyclin Expression in Meiosis

Mid Sporulation Elements (MSEs) and Ndt80-Regulated Genes

Table of Clusters Expressed during Sporulation

Oligonucleotides Used to Generate Plasmids

Yeast Strains for Appendix One

Clb2 Does Not Affect Progression through Sporulation

Clb2 Can Functionally Substitute for Clb1 during Sporulation

Yeast Strains for Appendix Two

Yeast Strains for Appendix Three

B-type Cyclins in Meiosis

Gal-Clb5 can Complement the Sporulation Defect of a Clb5Deficient Strain

Yeast Strains for Appendix Four

xi

LIST OF FIGURES

1-1.

1-2.

1–3.

1-4.

1–5.

1-6.

2-1.

2-2.

2-4.

2–5.

2-6.

3–1.

3–2.

3–3.

3–4.

Meiosis versus Mitosis

Chromosomal Behavior in Meiosis

Proposed Transcriptional Cascade

Cdc28 and Ndt80 are Required at PachyteneModels for Ndt80 and Cdc28 Function

Coordination of Transcription of Genes Involved in Meiotic

Division and Spermatid Differentiation in Drosophila

Transcription Pattern of Sporulation Genes in Wild-Type andNdt80-Deficient Cells

Ndt80 is Necessary for Transcriptional Induction of CLB1, CLB3,

CLB4, CLB5, and CLB6 in Meiosis

Ectopic Expression of Ndt80 Induces Middle Gene Expression in

Vegetative Cells

Regulation of NDT80 Transcription

Ndt80 Recognizes the SPS4 MSE

The Meiotic Recombination Checkpoint Gene RAD17 Controls

Transcription of the Ndt80-Regulated Gene CLB1

Ndt80 is a Central Regulator of the Sporulation Transcriptional

Cascade

Landmark Events of Sporulation

Cluster Analysis of Induced Sporulation Genes

Ultrastructural Landmarks of Sporulation

Mitochondrial Morphology during Late Stages of Sporulation

Sequence Alignment

xii

A1-1.

A1-2.

A2-1.

A2-2.

A2-3.

A2-4.

A2-5.

A3-1.

A3–2.

A3-3.

A3-4.

A3–5.

A3-6.

A4-1.

A4-2.

A4–3.

A4–4.

A4-5.

A4–6.

The Two Stages of Gametogenesis

Parallels between Sporulation and Spermatogenesis

Meiosis is a Specialized Form of Mitosis

Models for Clb1 and Clb2 Functions in Meiosis

CLB1 and CLB2 under Control of CLB1 5' and 3' Regulatory

Regions

Initiation of Sporulation

Ime4 is Necessary for Ndt80 and NDT80 Transcript Levels

Ndt80 Induces Transcription of IME2

Potential Regulators of NDT80: Ime4, Ime2, and Ids2

Models for Different Levels of NDT80 Expression

B-type Cyclins in Meiosis and Mitosis

Models for Clb5 Function during Meiotic Division

Clb5 is Required for Normal Progression through Premeiotic

DNA Synthesis

A Role for Clb5 in Sporulation

Clb5 Expression during Sporulation

Immunoprecipitation of Clb5-HA (in SK1 Background)

Modes of Regulation of Ndt80 Activity

Ndt80 Co-Localizes with DNA throughout Meiosis

Ndt80-HA is Detectable beyond Meiotic Prophase

GAL-NDT80-HA Inhibits Vegetative Growth

Ndt80 is a Target of the Meiotic Recombination CheckpointNdt80-HA is Detectable in dmc1 Strains

xiii

CHAPTER ONE

INTRODUCTION

Mºisindgametoge

A■ undamentalWºls. Most cells t

*s in the formatio

(mº■ t identical to th

*is undergo a ■ ohºld daughter cells

"Tipiental cell*ular level how n

Hº■ t l-l).

Meiosis is neces

ºnisms. Before atºsis DNA an

*sºns (see Figºutive meiotic n

*sº and me

*ional Cell r

Meiosis and gametogenesis

A fundamental process of life is the ability of a cell to divide to yield

two cells. Most cells undergo a form of cell division called mitosis, which

results in the formation of two daughter cells, each of which have a DNA

content identical to that of the original cell. However, certain diploid cells

can also undergo a form of cell division called meiosis, which yields four

haploid daughter cells, or gametes, each with half of the DNA content of the

original parental cell. The focus of my studies has been to understand at the

molecular level how meiosis is a specialized form of the mitotic cell cycle (see

Figure 1-1).

Meiosis is necessary for sexual reproduction in all eukaryotic

organisms. Before a cell can undergo meiotic nuclear division, it first

replicates its DNA and then pairs and recombines its homologous

chromosomes (see Figure 1-2). The cell then proceeds through two

consecutive meiotic nuclear divisions: meiosis I (segregation of homologous

chromosome) and meiosis II (separation of sister chromatids, as in mitosis).

Each functional cell resulting from a meiotic division is called a gamete. Two

compatible, haploid gametes can fuse to form a zygote in which the DNA

content is restored to the diploid level (see Figure 1-1).

Gametogenesis is the developmental process involving both meiotic

division and gamete morphogenesis. In the budding yeast, S. cerevisiae,

gametogenesis is also referred to as sporulation. Because the essential steps of

meiosis are conserved in all organisms, our characterization of sporulation in

yeast should provide a framework for understanding gametogenesis in

higher eukaryotes.

figu■ t 11 Mitosis v

MS Cells, such

& Tiego mitosis

& Whispect to be#ifted form of cel

Hºmele beta sp

Wºment of the p:

ºmision of two §3.

# devel |D). In c

"ip■ ºduces Cell

“Tºtas the parental

Figure 1-1. Mitosis versus Meiosis

Most cells, such as a human liver cell or a vegetatively growing yeast

cell, undergo mitosis which yields two cells identical to the original parent

cell, with respect to both amount and content of DNA (A). Meiosis is a

specialized form of cell division which produces four cells, or gametes (B).

Each gamete, be it a sperm or egg in humans or a spore in yeast, has half the

DNA content of the parent cell. Meiosis allows for sexual reproduction, since

upon fusion of two gametes, the DNA content of the zygote is restored to the

diploid level (D). In contrast, fusion of cells resulting from a mitotic division

would produce a cell with a tetraploid DNA content with twice as much DNA

content as the parental cell (C).

CELL DIVISION

A. MITOSIS B. MEIOSIS

G.) (W)WW

lQo S&DOhumans:liver cell sperm or eggyeast: vegetative cell spore

CELL FUSIONC. "G) (S)

Y ■

(V)humans:fetus

yeast: aggote

Figure 1-2. Chromosome Behavior in Meiosis

Following DNA synthesis, homologous chromosomes pair and

recombine during meiotic prophase. In meiosis I, each pair of homologous

chromosomes segregates apart. In meiosis II, sister chromatids separate.

(Meiosis I is also referred to as a reductional division since the number of

centromeres per incipient cell is reduced by half. In contrast, meiosis II and

mitosis known as equational divisions since sister chromatid separation

maintains the same number of centromeres per cell.) Pachytene is the

substage of meiotic prophase in which synaptonemal complex formation is

complete and SPBs have duplicated. Desynapsis, SPB separation, and meiotic

division have yet to occur.

DNAREPLICATION

HOMOLOG PARIING&

MEIOTIC RECOMBINATION

MEIOSIS I(reductional)

MEIOSIS II(equational)

Precedent for how meiosis might be a modified form of the mitotic cell cycle

comes from work in other organisms. Drosophila has two homologs of the

cdc25 phosphatase: string, which is expressed in somatic tissues, and twine,

which is expressed in germ cells (Alphey et al., 1992). One explanation for the

differential expression pattern is that twine may have functions (such as

substrate recognition) which are specifically required during spermatogenesis

and cannot be carried out by String.

Sporulation is proposed to be regulated by a transcriptional cascade

Most studies of sporulation have focussed on the regulation of

initiaton of sporulation (reviewed by Mitchell, 1994). Two inputs (mating

type and nutritional starvation) are required to initiate sporulation. In cells

expressing the a102 repressor, synthesis of Rmel, a repressor of meiosis, is

inhibited (Covitz et al., 1991). A diploid a■ o cell in late G, will enter the

sporulation differentiation pathway, instead of committing to another round

of cell division, if it is starved for both nitrogen and a fermentable carbon

source (see Figure 1-3).

The developmental programming of sporulation has been proposed to

be regulated by a transcriptional cascade based on the discovery of several sets

of genes that are expressed in a sequential, temporal manner during

sporulation (Kurtz and Lindquist, 1984; Percival-Smith and Segall, 1984;

Weir-Thompson and Dawes, 1984; Kaback and Feldberg, 1985; Nag et al., 1997).

The hypothesis of a transcriptional cascade implies that one transcription

factor activates expression of a second transcription factor, which, in turn,

activates expression of a subsequent set of genes.

Regulation of expression of the first set of sporulation genes -- the earlygenes -- has been well characterized. The absence of Rmel in a/O cells leads to

Figure 1-3. Proposed Transcriptional Cascade

Sporulation is thought to be regulated by a transcriptional cascade.

Under nutrient starvation, a diploid a ■ o cell alo.2 prevents Rmel from

inhibiting IME1 transcript accumulation (Kassir et al., 1988; Covitz et al.,

1991). Imel then activates expression of the early class of sporulation genes,

which are involved in homolog pairing and recombination. At least three

other sets of genes are expressed sequentially during meiosis, at times

approximately correlating with their function: the middle genes involved in

spore wall formation, the mid-late genes involved in spore coat maturation,

and the late genes involved in spore maturation.

starvation T->

a1-02 – RME1 – IME1 YEARLY GENESpairing & recombination

MIDDLE GENESspore wall formation

MID-LATE GENESspore coat maturation

LATE GENESspore maturation

º º

iº º&

Tetrad of4 spores

expression of the inducer of meiosis, IME1, encoding the major transcription

factor of the early genes (Kassir et al., 1988; Covitz et al., 1991). One of the

targets of Imel activation includes the sporulation-specific serine/threonine

kinase, Ime2 (Komianami et al., 1993). Once expressed, Ime2 can replace Imel

function for early gene expression, possibly by phosphorylating and thus

activating another transcription factor. Because Ime2 is not a transcription

factor (Komianami et al., 1993) and because both Imel and Ime2 are thought

to act directly only on the early genes and not a subsequent class of

sporulation genes, Imel and Ime2 do not constitute a transcriptional cascade.

In addition to the early gene, other sets of sporulation genes include

the middle, mid-late, and late genes, which are expressed during successive

developmental stages of sporulation (reviewed by Mitchell, 1994). Based on

the function of known genes in each set, there is a general correlation

between when a gene is expressed and its function. Early genes are involved

in aspects of homologous chromosome pairing and recombination (reviewed

by Roeder, 1997). Middle genes are involved in spore formation (Hepworth et

al., 1995; Ozsarac et al., 1997). Both mid-late and late genes function in spore

maturation (Briza et al., 1990; Law and Segall, 1988). The early gene set is the

largest classes, without about 17 known members (reviewed by Kupiec et al.,

1997). The middle class has about a dozen genes, and the mid-late and late

each only have a couple representative members.

How the later classes of sporulation genes are regulated remains to be

determined. In addition to Imel, other transcription factors have been

suggested to function during meiosis. These include Abfl (see below) and

Ids2, which is necessary for Ime2-dependent expression of middle but not

early genes (Gailus-Durner et al., 1996; Sia and Mitchell, 1995). However,

10

none of these factors have been shown to be directly responsible for activation

of the middle, mid-late, or late genes.

Regulatory sites for different classes of sporulation genes

Elements required for proper regulation of the early and middle classes

of genes have been defined (see Table 1-1). The best characterized is the URS1

element, the major regulatory site for early genes. The URS1 motif

(Tº/cGGCGG*/CT) is recognized by the Imel/Ume6 complex. Imel provides

a transcriptional activation domain, and Ume■ contains a DNA-binding

domain which binds the URS1 (REF). Umeå is necessary not only to tether

Imel to the URS1 site for sporulation-specific activation of the early genes

(Bowdish et al., 1995) but also to tether Sin3 and the histone deacetylase, Rpd3,

to prevent vegetative expression of the early genes (Kadosh & Struhl, 1997).In addition to the URS1 site, which functions as both a mitotic

repressor and an early meiotic activator site, two additional sites are often

found in the promoters of early genes. These include the TAC site and the

UASH site. Recent characterization has shown that the UASH site is required

for non-specific activation of early genes (Prinz et al., 1995). The Abfl protein

may be responsible for UASH -associated transcriptional activity, as the Abfl

binding site (CGTNNNNNº/,"/CGA'/c) overlaps with the UASH site in the

promoters of several early genes, including HOP1, RED1, ZIP1, and REC104

(Gailus-Durner et al., 1996). One model is that the URS1 site mediates

repression of transcription of early genes in vegetative cells but that in

meiotic cells, both the URS1 and UASH sites are necessary for full early gene

expression (Vershon et al., 1992). Abfl may also function at later stages of

sporulation since Abfl sites are found upstream of several middle genes:

SPR3, SMK1, SGA, and SPR1 (Gailus-Durner et al., 1996).

11

Table 1-1. Putative Regulatory Elements of Classes of Sporulation Genes

las Element Possible factor ExamplesEarly URS1 motif (Tº/cGGCGG"/CT) Imel HOP1, RED1

UASH Abfl HOP1TAC 2

Middle MSE motif (CGCAAA*/r) Ndt80 SPS1

Mid-Late NRE motif Tup1 DIT1, DIT2

Late 2 2 SPS100

12

A common regulatory site has also been found in the promoter region

of the set of middle genes (Hepworth et al., 1995; Ozsarac et al., 1997). The

MSE (middle sporulation element) is sufficient to confer sporulation-specific,

middle-gene expression to a heterologous promoter (Hepworth et al., 1995).

A putative MSE-binding activity was reported in vegetative cells, suggesting

that the mechanism for middle gene transcription might parallel that of the

early genes: conversion of a vegetative repression site to a meiosis-specificactivation site.

Multiple regulatory elements are required for proper regulation of the

divergently transcribed mid-late genes, DIT1 and DIT2. The most well

defined of these elements is a putative negative regulatory site (NRE). This

activity was found to be dependent on the Tup1-repressor (Friesen et al.,

1997). Consistent with the model that Tup1 mediates proper regulation of the

mid-late genes by delaying their expression until the proper time, vegetative

cells deficient in Tup1 have increased levels of DIT1 message (DeRisi et al.,

1997). An MSE-like motif was also identified in the upstream region of the

DIT genes, but its function remains to be further characterized (Friesen et al.,

1997).

Pachytene is a major regulatory point of gametogenesis

Two unique steps are essential to meiosis. First, in early prophase,

high levels of recombination occur between paired homologous

chromosomes. Second, in the first meiotic division, homologous

chromosomes segregate apart (see Figure 1-2). Poised between these two key

steps is the pachytene substage of prophase. A cell in pachytene is

cytologically defined to have fully paired synaptonemal complexes and

duplicated spindle pole bodies (SPBs), prior to desynapsis, SPB separation, and

13

meiotic division. Exit from pachytene is a key regulatory step to ensure a

successful meiosis. If a cell is allowed to progress through meiosis I before

completing recombination, for example, in a cell deficient for Spol1, the

gametes which form are inviable (Klapholz et al., 1985).

In yeast, exit from pachytene is also referred to as the transition from

prophase into metaphase of the first meiotic division (or "prophase-to

metaphase I transition". This transition is regulated by proteins known to

have checkpoint functions, such as Radl'Z, Rad24, and Mec1 (Lydall et al.,

1996). A defect resulting in the accumulation of recombination

intermediates, such as a mutation of the meiosis-specific RecA homolog,

Dmc1, causes a cell to arrest at pachytene. In the absence of Rad17, a dmc1

mutant will progress through a lethal meiosis. (It is worth noting that

although Spol1-deficient cells do not complete recombination, they do not

trigger a checkpoint arrest because these mutants do not accumulate

intermediates of recombination complexes.)

In spermatogenesis, pachytene is also a major regulatory step.

Mutations in any of several genes (including BAX, CREM, DAZLA, A-MYB,

MLH-1, and ATM) cause spermatocytes to arrest at pachytene (reviewed by

Sassone-Corsi, 1997; Clancy, 1998). The mechanism responsible for this

similar arrest point is not understood.

Genes required for progression through pachytene.

Several genes are required for exit from pachytene. These include

CDC28, CDC36, CDC39 and NDT80. (Shuster & Byers, 1984; Xu et al., 1995).

CDC28 encodes the active subunit of the cyclin/Cdk complex in yeast. CDC36

and CDC39 encode negative regulators of transcription in vegetative cells

(Collart & Struhl, 1994). NDT80 was originally identified in a screen for

14

mutants which do not form mature spores with a fluoresent outer coat (Xu et

al., 1995). Wild-type spores have a dityrosine crosslink in their outer coat

which allows them to fluoresce in short-wave ultraviolet light. NDT80

stands for non-dityrosine. (Xu et al., 1995). NDT80 is expressed only during

sporulation, and not during vegetative growth. Based on this similar arrest

phenotype of Cdc28 and Ndt80-deificient cells -- neither mutant progresses

through meiotic division or forms spores (see Figure 1-4), it was proposed

that Cdc28 and Ndt80 might functionally interact (Xu et al., 1995). The

prophase-to-metaphase I transition is parallel to the G./M transition of the

vegetative cell cycle. However, because it is so critical that nuclear division

(which is dependent on active Cdc28/cyclin) occurs only after meiotic

recombination is complete, a meiosis-specific activator may be imposed on

Cdc28/cyclin. Ndt80 might be such a regulator (see Figure 1–5; Xu et al., 1995).

Another possibility is that in order to drive the unique chromosomal

segregation events of meiosis I, relevant substrate(s) for Cdc28 may be

presented specifically in meiosis. Ndt80 might be such a substrate (Xu et al.,

1995).

Exit from pachytene is also the point of “commitment" to sporulation

(Esposito & Esposito, 1974; Horesh et al., 1979; reviewed by Esposito &

Klapholz, 1981). Prior to commitment, a cell will revert back to vegetative

growth if rich nutrient conditions are restored. After the commitment point,

cells will complete sporulation independent of nutrient status. Therefore it is

possible that the action of Cdc28 and Ndt80 may “commit" a cell to

sporulation.

º■ ciºsses 1sºassº tº sº

ºn tº-

*...*T)**

-****

~~~ºut ºf

15

Figure 1-4. Cdc28 and Ndt80 are Required at Pachytene

Mutations in either Cdc28 or Ndt80 cause cells to block at pachytene, a

substage at the end of meitoic prophase. By pachytene, meiotic recombination

is complete, the synaptonemal complex is fully formed, and the SPB has

duplicated but not separated. Neither cdc28 nor ndt&0 mutants progress

through meiotic division or form spores.

16

prophaseS

Figure 1-5. Models for Ndt80 and Cdc28 Function

NDT80 is expressed only in cells undergoing sporulation, not in

vegetatively growing cells. Based on the similar requirement for both Ndt80

and Cdc28 for progression thorugh meiotic prophase, it is possible that Ndt80

is required for a meiosis-specific activity of Cdc28. Model A: Ndt80 is a

meiosis-specific activator of Cdc28 function. Model B: Ndt80 is a meiosis

specific substrate of Cdc28 function (see Xu et al., 1995)

18

ModelA:Ndt80isa

meiosis-specificactivatorofCdc28

Ndt80

e

MEIOSIS

Cdc28/cyclin)—-GCdc28/cyclin)—-SpecIFIC

ACTIVEFUNCTIONS

inactiveModelB:Ndt80isa

meiosis-specificsubstrateofCdc28 Cdc28/cyclin ACTIVE

-QNdt80 Ndt80

inactive

MEIOSIS—-SppCIFIC

FUNCTIONS

ACTIVE

F-A \O

Cyclin expression during gametogenesis

Given the requirement for Cdc28 at prophase of meiosis and the role of

the B-type cyclins (Clbs), the regulatory partners of Cdc28 in promoting

mitosis, an obvious question is what is the role of the Clbs in meiosis. In S.

cerevisiae, Clb1-, Clb3-, and Clb4-associated H1 kinase activities increase just

prior to entrance into meiosis I; in contrast, Clb2 protein and activity are not

induced during meiosis (Grandin & Reed, 1993). Clb5 and Clb6 protein and

associated activities have not been examined, but it was noted by Epstein &

Cross (1995) that Clb5 is required for meiosis. Based on these analyses, Clb1

was concluded to be the major B-type cyclin (supplanting the role of Clb2 in

mitosis) (Grandin & Reed, 1993; Dahmann & Futcher, 1995). However, a

Clb1-deficient strain has no sporulation phenotype in the W303 background,

most likely due to functional redundancy (Dahmann & Futcher, 1995). Clb1

and Clb4 together promote meiosis II, as a clb1 clb4 double mutant sporulates

with reduced efficiency and forms dyads (asci containing two spores) most of

which have only undergone meiosis I (Dahmann & Futcher, 1995). A strain

deleted for all three cyclins -- Clb1, Clb3, and Clb4-- only sporulates with 5%

efficiency (Dahmann & Futcher, 1995).

Evidence also exists from other eukaryotes for a role for the B-type

cyclins at pachytene based on expression analysis (See Table 2). In fission

Yeast, cac13, which encodes a cyclin B homolog, is transcribed just prior to

rrneiosis I (Iino et al., 1995). In fruit flies, cyclin B is expressed just prior to

rrneiosis I in primary spermatocytes (Lin et al., 1996). Similarly, in mouse,

transcripts for both cyclin B2 and the germ cell-specific cyclin A1 increase** rarmatically at pachytene (Chapman annd Wolgemuth, 1993; Sweeney et al.,

**96). Genes necessary for cyclin transcription in meiosis have been

20

Table 1-2; Cyclin expression in meiosis

Organism

fission yeast

fruit fly

IIlC)11Se

Induction of RequiredmRNA prior Geneto Meiosis I Product

cdc13 (cyclin B) Mei 4

cyclin B Aly

cyclin A2 2

21

identified -- the meia gene in fission yeast, which encodes a forkhead protein;

and the aly gene in fruit flies. However, a direct role for either of these

proteins in activating cyclin expression hsa not bee established (White-Cooper

et al., 1998; Horie et al., 1998). It is of note that both Meiq and aly have been

implicated in transcription of genes in addition to the cyclins just prior to

meiosis. For example, the messages for both cdc25 and cam1 increase with

that of cdc13 in fission yeast (Iino et al., 1995). Similarly, aly is also necessary

for expression of genes involved in sperm morphogenesis, such as fºo (Lin et

al., 1996). Based on these and other observations, Lin et al. (1996) proposed

that transcription of genes involved in meiotic division and sperm

morphogenesis may occur in parallel (see Figure 1-6).

Gene expression during gametogenesis

A clue to understanding the molecular mechanisms that occur diruing

a developmental process is to identify which genes are expressed. Studies in

spermatogenesis suggest that germ cell transcripts correspond to genes with

Somatic functions and to genes uniquely expressed during gametogenesis.

This latter class of germ cell-specific transcripts may be isologous to somatic or

unique transcripts (reviewed by Eddy & O'Brien, 1998). Genes expressed

ciuring spermatogenesis include those encoding structural proteins, such as

rhuclear lamins, histones, and synaptonemal complex components. In

acidition, genes whose products are required for DNA repair and

recombination, transcription, RNA processing, cell division, cytoskeleton,

and energy metabolism are also induced (see review Eddy & O'Brien, 1998).To identify additional meiosis-specific transcripts which may be**hechanistically required for meiotic division, others are in the process of

22

Figure 1-6. Coordination of Transcription of Genes Involved in Meiotic

Division and Spermatid Differentiation in Drosophila

aly is required for expression of genes (such as cyclin B or twine)

involved in either meiotic division and in sperm morphogenesis (such as

fzo). Figure 1-6 is taken from White-Cooper et al. (1998).

23

Spermatid MeioticDifferentiation Division

fzo cyclin Btwine

24

constructing spermatocyte-specific libraries at different stages of meiosis to

identify additional transcripts (R. Reijo, personal communication).

With the completion of the S. cerevisiae genome sequence, it is now

possible to examine the expression of nearly every yeast gene under different

physiological conditions (Lashkari et al., 1997). For example, DeRisi et al.

(1997) followed the widespread changes in gene expression which occur

during the diauxic shift from fermentation to respiration. With this

technology, gene expression during any developmental program, including

sporulation, can be characterized and used to further our understanding of

the biological mechanisms behind each process.

25

CHAPTER TWO

GAMETOGENESIS IN YEAST

IS REGULATED BY A

TRANSCRIPTIONAL CASCADE

DEPENDENT ON Ndt80

A version of Chapter Two originally appeared as Chu and Herskowitz (1998),Molecular Cell 1: 685-696.

||■ º

26

Abstract

Gametogenesis requires the successful coordination of two key

processes, meiotic nuclear division and gamete morphogenesis. A central

regulatory step in progression through gametogenesis occurs at the pachytene

stage of meiotic prophase. We find that Ndt80 functions at pachytene of yeast

gametogenesis (sporulation) to activate transcription of a set of genes required

for both meiotic division (e.g. B-type cyclins) and gamete formation (e.g.

SPS1). Ectopic synthesis of Ndt80 in vegetative cells induces transcription of

these genes, and recombinant Ndt80 protein binds to a conserved sequence in

their upstream region. Transcription of NDT80 itself is dependent on Imel,

which activates expression of early sporulation genes. Transcription of the

Ndt80-regulated gene, CLB1, is mediated by the checkpoint gene, RAD17.

Thus, Ndt80 is a pivotal component of a transcriptional cascade programming

yeast gametogenesis and may also be a target of meiotic checkpoint control.

Introduction

Meiosis is the developmental pathway by which sexually reproducing

diploid organisms generate haploid germ cells. It can be regarded as a

specialized form of the mitotic cell cycle with two major differences. First,

following DNA synthesis, high levels of recombination occur between

homologous chromosomes during meiotic prophase. Second, after

recombination is complete, consecutive nuclear divisions occur without

intervening DNA replication. In the first, reductional division (meiosis I),

homologous chromosomes separate. In the second, equational division

(meiosis II), sister chromatid segregation occurs.

27

Progression from prophase into the first meiotic division is a critical

regulatory step in meiosis. It would be detrimental to the differentiating cell

to enter nuclear division before finishing recombination, for example, with

unrepaired double-strand breaks, or before completing preparation for the

meiotic divisions that follow. In both metazoan spermatogenesis and yeast

sporulation (the process of meiosis and spore formation), mutations in

several genes result in prophase arrest (reviewed by Sassone-Corsi, 1997;

Roeder et al., 1997). This arrest in yeast is triggered by defective meiotic

recombination complexes and is dependent on checkpoint genes, e.g. RAD17

(Lydall et al., 1996; Xu et al., 1997). In animal oogenesis, entry into the first

meiotic division is mediated by maturation promoting factor (MPF), which

was originally identified by its ability to induce development of amphibian

oocytes beyond their natural arrest point in prophase. Purified MPF consists

of a catalytic subunit, the p34** protein kinase, and its regulatory partner, aB-type cyclin (reviewed by Murray and Hunt, 1993).

In the budding yeast, Saccharomyces cerevisiae, both the cdc2

homologue, CDC28, and a meiosis-specific gene, NDT80, are required for

progression from prophase into meiosis I (Shuster and Byers, 1989; Xu et al.,

1995). Cells with mutations in either CDC28 or NDT80 undergo normal

levels of meiotic recombination but then arrest at the end of prophase, at a

stage called pachytene, with duplicated spindle pole bodies (SPBs) and fully

synapsed homologous chromosomes. Several possibilities exist for the

function of Ndt80. For example, it might activate the cyclin-Cdk complex or

be a meiosis-specific target of Cdc28 kinase activity.

Budding yeast has six B-type cyclins, Clb1-Clb6 (reviewed by Nasmyth,

1996). Clb1, Clb3, and Clb4 have been shown to have a role in meiotic

28

division, with Clb1 being the key meiotic cyclin. Depending on strain

background, mutations in CLB1 alone or in combination with either CLB3 or

especially CLB4 severely reduce meiotic division, as assayed by sporulation

efficiency (Grandin and Reed, 1993; Dahmann and Futcher, 1995). Epstein and

Cross (1994) note a requirement for Clb5 in meiosis. In contrast to its

predominant role in mitosis (Fitch et al., 1992), Clb2 protein is barely

detectable in meiosis (Grandin and Reed, 1993).

Sporulation has been proposed to be regulated by a transcriptional

cascade, based on the observed temporal expression pattern of four sets of

sporulation-specific genes -- early, middle, mid-late, and late genes (reviewed

by Mitchell, 1994). Transcription of the early genes, whose products are

involved in chromosome synapsis and recombination, is activated by Imel

(reviewed by Kupiec et al., 1997). The middle, mid-late, and late gene

products are thought to function in aspects of yeast gamete morphogenesis,

i.e. spore formation and maturation (see Mitchell, 1994). A DNA sequence

found upstream of several of the middle genes, termed the midsporulation

element (MSE), is both necessary and sufficient for proper meiosis-specific

expression of a heterologous reporter (Hepworth et al., 1995; Ozsarac et al.,

1997).

We show that Ndt80 is a transcription factor required for expression of

the middle sporulation genes, which include both genes involved in spore

morphogenesis and five of the six CLB genes. We furthermore show that

meiotic transcription of CLB1 is under control of the checkpoint gene RAD17

(Lydall et al., 1996), suggesting that Ndt80 function may be regulated by this

meiotic checkpoint. Our findings on Ndt80 demonstrate the existence of a

transcriptional cascade during gametogenesis in yeast and provide a

29

framework for understanding progression through pachytene in

spermatogenesis of higher eukaryotes.

Results

Ndt80 Activates Expression of Middle Sporulation Genes

We initially found that a fusion protein in which Ndt80 was joined to

the DNA-binding domain of LexA activated transcription from a reporter

containing upstream LexA operators (data not shown). This result and the

observation that ndt&0 mutants are unable to form spores (Xu et al., 1995)

raised the possibility that Ndt80 might activate expression of genes required

for spore morphogenesis, such as the middle sporulation genes (Garber and

Segall, 1986).

To determine whether Ndt80 is required for transcription of these

genes, we compared the expression pattern of two previously identified

middle genes, SMK1 and SPS1, in wild-type and Ndt80-deficient cells. SMK1

encodes a sporulation-specific MAP kinase (Krisak et al., 1994); SPS1 encodes a

sporulation-specific STE20 kinase homologue (Friesen et al., 1994). Both SPS1

and SMK1 transcripts were induced midway through sporulation in wild-type

cells (Figure 2-1A; Friesen et al., 1994; Krisak et al., 1994). In contrast, in ndt&0

cells, although there was still a basal level of SMK1 message, induction of

both SPS1 and SMK1 transcripts was strikingly absent (Figure 2-1A). This

finding indicates that Ndt80 is necessary for full induction of these middle

sporulation genes.

The absence of middle gene expression in Ndt80-deficient cells might

be due to an arrest of sporulation several steps prior to induction of middle

gene transcription, rather than a more direct requirement for Ndt80

30

Figure 2-1. Transcription Pattern of Sporulation Genes in Wild-Type andNdt80-Deficient Cells

(A) Ndt80 is Necessary for Transcriptional Induction of Middle Sporulation

Genes

Wild-type (YSC328) and ndt&0 mutant (YSC330) cells were transferred

to sporulation medium at t=0. RNA was harvested at hourly intervals and

used to make duplicate Northern blots, one probed with SPS1 and TCM1

(upper), the other with SMK1 and TCM1 (lower). TCM1 codes for the

ribosomal L3 protein and served as a loading control.

(B) Transcription of NDT80, CLB1, and SPC42 Relative to Representative

Early and Mid-Late Genes

RNA samples obtained from the same time course as Figure 2-1 used to

make duplicate Northern blots. The first blot was hybridized with probes for

DMC1 (an early sporulation gene), NDT80, CLB1, and TCM1 (upper; see

Figure 2-2). The second blot was probed with SPC.42, DIT1 and TCM1 (lower;

see Figure 2-2).

31

A +/4. ndt&0A/ndt&0A| | |

t = 0 3 4 5 6 78.59 0 3 4 5 6 78.5 9 h|

3.

SMK1 *

º

TCM1

+/4. ndt&0A/ndt&0A| | | |

t = 0 1 2 3 4 5 6 78.59 0 1 2 3 4 5 6 78.59 h|

º*…º.º.º.º.º.º.º.

NDT80&

CLB1 . º º # * * * º * *

tº º & --------sº

- - - Sºº -

---. -3.

º ºg º… º.º. ºf & > . 3.&… º. is

32

transcriptional activity. Because both NDT80 and the representative middle

gene, SPS4, are normally expressed only in sporulating cells (Xu et al., 1995;

Garber and Segall, 1986), we used vegetative cells as a heterologous system to

study the ability of Ndt80 to promote meiotic gene expression. Ndt80 protein

was ectopically expressed in vegetative cells under control of a galactose

inducible promoter. An HA-tagged Ndt80 protein, which complements the

sporulation defect of an nat30 mutant (data not shown), was used to assay

Ndt80 protein levels. Middle gene expression was assayed with wild-type

UASSPS4-lacz reporters which contain 15 or 29 basepairs of the SPS4 upstream

region and are induced in a/o cells during sporulation (Hepworth et al., 1995).

Cells producing Ndt80-HA induced ■ -galactosidase expression 60- and 690

fold from the 15 and 29 basepairs segment reporters, respectively, relative to

cells containing an empty pCAL vector. Expression of untagged Ndt80

protein caused similar levels of UAS8P84 induction (data not shown). In

contrast, Ndt80-HA did not induce expression from a UASCYC1 reporter (data

not shown). These observations show that ectopic synthesis of Ndt80 in

vegetative cells is sufficient to activate the UAS8P84.

Based on deletion analysis and alignment of the upstream regions of

known middle sporulation genes, Ozsarac et al. (1997) define the MSE

consensus sequence as gNCRCAAA(A/T), where g represents a non

conserved guanine nucleotide, N is any nucleotide, and R is a purine. To

determine whether the conserved nucleotides are important for Ndt80

dependent activation, mutational analysis was perfomed within the MSE of

the UASSPS4 reporter. We found that changes of the nine basepair consensus

drastically decreased Ndt80-dependent induction. Deleting three A

nucleotides from the A tract or changing GCCACAAAA to GCCAGTAAC,

33

TCATGTAAG, GCCACGCTG, or GCCTCAAAA all reduced induction, from

690-fold to 2- to 8-fold. These observations indicate that Ndt80-dependent

induction requires the conserved basepairs of the MSE.

B-type Cyclin Genes (CLB) Are Also Middle Genes

The above results indicate that Ndt80 activates expression of known

middle sporulation genes. Both smk1 and sps1 mutants proceed through

meiotic divisions normally and are defective only in spore formation

(Friesen et al., 1994; Krisak et al., 1994). In contrast, ndt&0 mutants arrest in

pachytene without undergoing nuclear division or spore formation (Xu et al.,

1995). Therefore, we reasoned that Ndt80 might also control transcription of

genes that regulate meiotic nuclear division.

Given the known role of the B-type cyclins in mitotic nuclear division,

we examined the transcription pattern of the CLB genes during meiosis. We

found that five of the B-type cyclin genes exhibited a similar expression

pattern. A striking increase in mRNA levels for CLB1, CLB3, CLB4, CLB5, and

CLB6 occurred between four and five hours (Figure 2-2). This rise in CLB

message took place one hour prior to the initiation of meiotic division at six

hours, as assayed by the presence of two DAPI-staining bodies, which are

indicative of cells in meiosis I. In contrast, CLB mRNA levels did not

increase in an isogenic nat30 strain (Figure 2-2). Although we cannot rule out

the possibility that Ndt80 activates transcription of only a subset of the CLBs

which in turn stimulate expression of other CLBs (Amon et al., 1993), the

simplest interpretation of our findings is that Ndt80 is necessary for meiotic

induction of CLB1, CLB3, CLB4, CLB5, and CLB6 RNA synthesis. Unlike the

other five B-type cyclins, only a slight increase in CLB2 message was observed

during meiosis. Consistent with their expression patterns, we identified

34

Figure 2-2. Ndt80 is Necessary for Transcriptional Induction of CLB1, CLB3,

CLB4, CLB5 and CLB6 During Meiosis

The same blots of Figure 2-1B were hybridized with probes for CLB1,

CLB2, CLB5, and TCM1 (upper), or CLB3, CLB4, CLB6, and TCM1 (lower).

Progression through meiotic division was monitored at each interval

by fixing and staining cells with the DNA-specific dye, DAPI. From t=0 to t-5

hours, 99% of the cells were mononucleate (single DAPI-staining body). By

t=6, 58% of the cells were mononucleate, 15% were binucleate (indicative of

cells which have completed meiosis I), and 27% were tetranucleate (indicative

of cells which have completed meiosis I and II). The percentages of mono-,

bi-, and tetranucleate for subsequent intervals were the following: t-7 (24%,

16%, 60%); t-8.5 (20%, 7%, 73%); t-9 (16%, 8%, 74%).

***.***

2.*

a sº

* 1:£.sº tº

35

+/-- ndt80A/ndt&0A|

3 4 5 6 78.5 9 h| | | | | | |

*** * * * * * *

º ºx * * * * *• *

-

* * * * * * **• * ~ * * * * ~ *

• * ******º • * **

CLB3

CLB4

CLB5

CLB6

TCM1

TCM1

36

MSE-like sequences upstream of the CLB1, CLB3, CLB4, CLB5 and CLB6 open

reading frames (Table 2-1; see Discussion).To characterize when the increase in CLB mRNA occurred relative to

other transcriptional events during sporulation, we examined the timing of

CLB1 expression relative to representative early, middle, and mid-late genes.

The transcript for the early gene DMC1 appeared within one hour after

transfer to sporulation medium (Figure 2-1B, Bishop et al., 1992). By five

hours there was a large increase in transcript levels not only for the

previously identified middle genes, SMK1 and SPS1 (Figure 2-1A; Friesen et

al., 1994; Krisak et al., 1994), but also for CLB1 and SPC42 (Figure 2-1B). SPC42

encodes a major component of the yeast spindle pole body (reviewed by

Marschall and Stearns, 1997), and its upstream region contains an MSE (see

Table 2-1). A representative mid-late gene, DIT1, was not expressed until

seven hours. Based on this temporal expression pattern, it is clear that the B

type cyclins, CLB1, CLB3, CLB4, CLB5, and CLB6, are also middle sporulation

genes.

Having observed that CLB5 and CLB6 transcript levels are induced

midway through meiosis, we wondered whether their gene products might

have a role in meiotic progression as has been shown for Clb1, Clb3, and Clb4

(Grandin and Reed, 1993; Dahmann and Futcher, 1995). We first observed

that clb5 cells sporulate at levels only 12% that of wild-type, with an equal

distribution of tetrads, dyads and monads. In contrast, isogenic wild-type cells

produced 96% tetrads, 3.8% dyads, and 0.2% monads. FACS analysis revealed

that Clb5-deficient cells were severely compromised in their ability to

undergo DNA synthesis (data not shown). The requirement for Clb5 in

***-º-º:

º

*****

sº a

º

37

Table 2-1. Mid Sporulation Elements (MSE) and Ndt80-Regulated Genes

A. q N C R C A A A A/TSMK1 – 69 × – – G T C A C A A A TSPR3 –289 --> G A C A C A A A. A

– 14 - - - G A C G C A A A. ASPS1 –370 – — » G A C A C A A A TSPS4 – 191 --> G C C A C A A A ASPC42 – 175 –– S tº A C A C A A A. ACLB1* –648 –-> C T C A C A A A. A

–579 --> G A C A C A A A. A– 99 – – a T C A C A A A. A

CLB3 -213 --> G G a A (C A A A A) 3 g A A A A C A A A. ACLB4 – 151 --> G A a A C A A A A A C A A A A A C A A T ***

CLB5 —233 × – – a A C G C A A A T *CLB6 –394 –-> a C C A C A A A. A *

–354 × – – G T C G C A A A. A gº

NDT8O –221 --> G A C A C A A A A sº

– 78 : - - G A C A C A A A. A *

B. C N C R C A A A A/T *SPR6 –273 --> t A C A C A A A. A *-SPR28 –211 <-- a C C A C A A A T -*

– 89 <-- G A C A C A A A. A º

SPS2 – 30 <-- G C C A C A A A. A

SPS1.8% -107 3 – – G T C A C A A A. A ºSPS19 + —191 – -> G T C A C A A A. A º

DIT1 * –565 & – – G T C G C A A A. A *–342 – — » C T C A C A A A T -

DIT2 * –555 × – – C T C A C A A A T º–332 --> G T C G C A A A. A º

CDC3 * -106 × -- a A C A C A A A. A º

– 96 :- — tº A C A C A A A. ACDC10 * – 133 --> a C C A C A A A TSGA1 –194 –-> G T C A C A A A TMPS1 –200 --> G A C A C A A A A

– 13 --> a T C tº C A A A. AGIP1 –353 --> a A C A C A A A A

–265 –– P G A C G C A A A A– 43 — — » tº T C G C A A A T

MSEs that fit the consensus, gNCRCAAAA/T (Ozsarac et al., 1997),where g is a non-conserved guanine nucleotide, N is any nucleotide, and R isa purine.

A. MSEs of known Ndt80-dependent middle sporulation genes

MSEs of middle sporulation genes whose expression is shown here to be

Ndt80-dependent by Northern analysis (Figures 2-1 and 2-2, and data not

38

shown), GFP reporter (Figure 2-4), or £-galactosidase reporter (text and datanot shown).

B. MSEs of other sporulation genes

MSEs of other genes with previously reported sporulation-specific expression

pattern and/or function (see Kupiec et al., 1997 and references within).

References for the characterization of expression during sporulation are

given.

x+ indicates that this gene and an adjacent gene are divergentlytranscribed.

--> indicates that the MSE and its corresponding open reading frame areon the same strand.

<-- indicates that the MSE and ORF are on opposite strands.

Numbers refer to the distance between an MSE and the presumed start of its

open reading frame. Only MSEs found between adjacent open reading frames

are listed. Non-consensus residues are in lower case. The following genes

have additional MSEs that differ from the consensus only at position 4 (R) of

the MSE: SMK1 (-353; -245), SPS1 (-646; -581; -92), SPS2 (-45), SPS4 (-66), CDC3

(-585), CDC10 (–212), CLB2 (-553), CLB4 (-62), CLB5 (-529), CLB6 (-543), and

NDT80 (-197).

39

premeiotic DNA synthesis precluded determining whether Clb5 also has a

later role in meiotic prophase. This earlier activity may be mediated by

residual Clb5 protein synthesized before meiosis or from the low level of

Ndt80-independent CLB5 (Figure 2-2). In seeking a possible role for Clb6 in

meiosis, we found that a Clb6-deficient strain had no sporulation phenotype

either singly or in the background of a clb1 clb3 clb4 strain, which has

previously been reported to have a low sporulation efficiency (Dahmann and

Futcher, 1995).

Ndt80 Can Induce Synthesis of CLB RNA in Vegetative Cells

We showed above that ectopic Ndt80 expression in vegetative cells is

sufficient to induce ■ º-galactosidase activity from the SPS4 UAS. A similar

analysis was done to determine whether Ndt80 can also activate CLB

transcription. RNA was harvested from asynchronous, exponentially

growing cells expressing Ndt80-HA under control of the galactose-inducible

promoter. As observed with the UAS8P84 reporter, we found that Ndt80

induced expression of the middle genes SPS1 (Figure 2-3C) and SMK1 (data

not shown) in vegetative cells. In addition, ectopic NDT80 expression also

induced transcription of the five B-type cyclin genes, CLB1 (Figure 2-3A, lanes

1-2), CLB3 and CLB4 (Figure 2-3B), and CLB5 and CLB6 (Figure 2-3C). In

contrast, CLB2 message remained unchanged (Figure 2-3A, lanes 5-6). There

was no difference in budding index between strains carrying p(SAL and pCAL

NDT80-HA during galactose induction (data not shown), indicating that

increases in cyclin messages are not due to accumulation of cells at a

particular stage of the cell cycle.

To determine if Ndt80-stimulated transcription is sufficient to account

for the observed increase in CLB message levels, we carried out the following

40

Figure 2-3. Ectopic Expression of Ndt80 Induces Middle Gene Expression in

Vegetative Cells

RNA was harvested from vegetative cells (YSC531, YSC552, and

YSC553) in the absence or presence of ectopic Ndt80-HA expression.

Northern blots were hybridized with probes for CLB1-CLB6.

(A) Comparison of CLB1 and CLB2 expression in strains containing pcAL

NDT80-HA grown with (lanes 2, 4 and 6) or without (lanes 1, 3 and 5)

galactose treatment. Asynchronous a/o (YSC531; lanes 1, 2, 5 and 6) or o

factor treated a haploid (YSC553; lanes 3 and 4) cells were used.

(B) Comparison of CLB3 and CLB4 expression in an asynchronous

population of cells from strains containing pCAL (YSC552) and pCAL

NDT80-HA (YSC553) grown in the presence of galactose. Similar induction

was observed when these blots were probed with CLB1, CLB4, and CLB6 (data

not shown).

(C) Comparison of CLB5, CLB6, and SPS1 expression in an asynchronous

population of cells from strain YSC531 processed as described in (A).

41

asyn- O'- asynchronous factor chronous

T- r– H– + — —H – + GAL

CLB1 * * * CLB2 w" -TCM1 ºenº Úº TCM1 tº ºp

1 2 3 4 5 6

H– + GALsº º

: : i C

*

y - x

CLB3 -º 3.

~ * CLB6 - *§§

---

SPS1 tº

TCM1 Oes TCM1 tº º11 2 2

42

analysis. Cells were treated with o-factor to promote arrest in G1, when CLB1

message is normally not detectable (Figure 2-3A, lane 3; Surana et al., 1991;

Ghiara et al., 1991). Ectopic Ndt80 expression was then induced by treatment

with galactose. We found that in the presence of Ndt80, G1 cells synthesized

CLB1 message (Figure 2-3A, lane 4). This result indicates that Ndt80 is a

positive regulator of CLB1 transcription and not simply an inhibitor of CLB1

message turnover. The ability of Ndt80 to induce SPS1 and CLB messages in

either asynchronous or G1 cells indicates that middle gene transcription is

activated by Ndt80.

Ndt80 is a Central Component of the Sporulation Transcription Cascade

The observation that Ndt80 activates middle gene expression led us to

hypothesize that it governs a step in the proposed sporulation transcription

cascade. Such a role for Ndt80 leads to several predictions. First, transcription

of NDT80 should be dependent on Imel, the transcription factor for early

sporulation genes (see Kupiec et al., 1997). Second, NDT80 should be

expressed before its targets, the middle genes. Third, in the absence of Ndt80,

not only the middle genes, but all subsequent classes should not be expressed.

To determine whether Imel is required for NDT80 transcription, we

compared NDT80 transcripts in wild-type and Imel-deficient sporulating cells

of the W303 background. NDT80 message was present by 11 hours in wild

type cells but not detectable in imel strains even after 15 hours (Figure 2-4A).

Ime1 has been shown to associate with the DNA-binding protein, Umeå, to

recognize URS1 sites upstream of early genes (see Kupiec et al., 1997). We

found that vegetative strains deficient in Umeå express measurable levels of

NDT80 message (data not shown), a characteristic of early gene transcripts

(reviewed by Kupiec et al., 1997). These observations indicate that NDT80

43

Figure 2-4. Regulation of NDT80 Transcription

(A) NDT80 Transcription is Dependent on Imel

Wild-type (YSC7) and imel mutant (YSC794) cells were transferred to

sporulation medium at t=0. RNA was harvested at the indicated intervals.

The Northern blot was probed with NDT80 and TCM1. TCM1 served as a

loading control.

(B) Ndt80 Can Activate Its Own Synthesis

Exponentially growing strains were induced with galactose and

examined by phase (panels i, iii, v, and vii) or immunfluorescence (panels ii,

iv, vi, and viii) microscopy. A functional GFP-tagged Ndt80 protein under

control of the NDT80 promoter (pNDT80-NDT80-GFP) was expressed in

strains carrying p(SAL-NDT80 (YSC921; panels i and ii) or pGAL (YSC918;

panels v and vi). A pcAL-NDT80-GFP construct was also expressed in either

background of pcAL-NDT80 (YSC922; panels iii and iv) and pCAL (ysc919;

panels vii and viii). Glucose-grown cells exhibited no immunofluorescence

(data not shown).

A. +/4 ime1A/ime1A| | | |

t = 0 3 5 7 9 11.5 13 15 0 3 5 7 911.5 13 15 hours| | | | | | | | | | | | | | | | in SPM

NDT80 - --> ~

TCM1 *****-*-we

B.

pNDT80-NDT80-GFP pGAL-NDT80-GFP

::

45

transcription is dependent on Imel, which, together with Umeå, presumably

acts on the URS1 site present in the NDT80 upstream region.

NDT80 message appears at approximately the same time (four hours in

sporulation medium in the SK1 background) as CLB1, SPS1 and SPC42

transcripts (Figure 2-1). Upon longer exposure of the same autoradiograms, a

low but detectable level of NDT80, but not SPS1, can also be seen even by

three hours (data not shown). NDT80 is not transcribed with the early genes

such as DMC1 (Xu et al., 1995; Figure 2-1B), yet middle gene transcription

depends on Ndt80 (Figures 1 and 2). Therefore we infer that NDT80 belongs

to a new class of "delayed-early" genes which are expressed after the early

genes but before the middle genes. It remains to be determined whether

other previously classified middle genes, for example, SMK1, are actually also

delayed-early genes. Although induction of SMK1 message is clearly Ndt80

dependent (Figure 2-1), it should be noted that SMK1 message is first

detectable before SPS1 message and that in the absence of Ndt80, a basal level

of SMK1, but not SPS1 transcript, remains (Figure 2-1A). Ndt80 is also

required for transcription of the mid-late gene, DIT1 (Figure 2-1B), which is

synthesized after the middle genes (Briza et al., 1990). These results strongly

support the hypothesis that Ndt80 governs at least one step of the sporulation

transcriptional cascade.

Our identification of MSE sequences within the NDT80 upstream

region (Table 2-1A) led us to examine whether Ndt80 can induce its own

synthesis. To test this possibility, we expressed a functional GFP-tagged Ndt80

protein under control of the NDT80 promoter (pNDT80-NDT80-GFP) in a

strain that produces NDT80 under galactose control (pCAL-NDT80). In the

presence of galactose, a fluorescent GFP signal was present in 60% of the cells

**** * ** * *

2. º* *

*:::::******

* 1:f.was 4 º'

ºsº |

*-*.*

****º sº*** ºr a

***** ****

*** * * *

46

containing both pCAL-NDT80 and pnDT80-NDT80-GFP (Figure 2-4B; panel

ii). In contrast, a GFP signal was not detectable in cells containing both pCAL

and pnDT80-NDT80-GFP (Figure 2-4B; panel vi). As a positive control for

galactose-inducibility, a pcAL-NDT80-GFP construct expressed in either the

pGAL or pGAL-NDT80 strains gave a galactose-dependent GFP signal in

approximately 56% of the cells (Figure 2-4B; panels iv and viii). Taken

together, these data show that Ndt80 induces its own synthesis, presumably at

the transcriptional level.Ndt80 Binds to the MSE

To determine whether Ndt80 contributes to a MSE DNA binding

activity, gel mobility shift assays were performed using as a probe the 29

basepair segment of the SPS4 promoter containing an MSE (Hepworth et al.,

1995). We first looked for MSE-binding activity in yeast extracts from strains

expressing HA-tagged Ndt80. Extracts from galactose-induced strains

containing p(SAL-NDT80-HA (Figure 2-5A, lane 2), but not those containing

pCAL (data now shown), could form a protein-DNA complex. The top band

could be disrupted by excess unlabeled wild-type competitor (Figure 2-5A, lane

3, 4, and 9). An additional faster migrating band could not be competed away

by excess oligonucleotide and was therefore not specific (Figure 2-5A, asterisk).

To determine whether the conserved basepairs of the MSE are

important for the Ndt80-dependent binding activity, we tested various

mutant MSEs and found they could not prevent complex formation when in

excess. The mutant competitors contained deletions of three of the four

conserved A nucleotides (lanes 5-6) or changes from GCCACAAAAAC to

GCCAGTAACAC (lanes 7-8), GCCACGCTGAC (lanes 10-11), or

TCATGTAAGAC (lanes 12-13). Taken together, these findings indicate that

º

º

i

47

Figure 2-5. Ndt80 Recognizes the SPS4 MSE

(A) Extracts From Cells Expressing Ndt80-HA Have a DNA-Binding Activity

Specific for the SPS4 MSE

Extracts made from a galactose-induced pCAL-NDT80-HA strain

(YSC562, lanes 2-15) were incubated with a [32P) labeled MSE probe. The probe

alone was run in lane 1. The reactions contained no competitor (lane 2), 29

basepair wild-type competitor (A; lanes 3, 4 and 9), or mutant competitors

GCCAAC (B; lanes 5-6), GCCAGTAACAC (C; lanes 7-8), GCCACGCTGAC (D;

lanes 10-11), or TCATGTAAGAC (E: lanes 12-13). A change from the

consensus is indicated by an underline. 10- or 100-fold excess competitor was

added to the reactions as indicated. Arrow indicates protein-DNA complex

specific to GAL-NDT80-HA extracts. Asterisk indicates non-specific complex.

(B) Mbp-Ndt80 Isolated from E.coli Binds the MSE

Gel mobility shift assays were performed using Mbp (lane 2) or Mbp

Ndt80 fusion protein (lanes 3-13) purified from E. coli and incubated with a

[32P)-labeled MSE probe. The probe alone was run in lane 1. The reactionsincluded no competitor (lanes 3 and 13), wild-type competitor (A; lanes 4-6),

or mutant competitor oligonucleotides GCCAGTAACAC (C; lanes 7-9) or

TCATGTAAGAC (E: lanes 10-13). A change from the consensus is indicated

by an underline. 10-, 100- or 200-fold excess competitor was added to thereactions as indicated.

-gººº

.:º

48

A.

coldcompetitor ;

probe

1 2 3 4 5 6 7 8 9 10 11 12 13

B. A C E| | | | | |

cold 24 x: X; X: ?: ;competitor — & 3 8 & 3 8 & 3 3 —

~ v-H CN r + º- CN r" rº-H CN

*- : *******

probe

º

1 2 3 4 5 6 7 8 9 10 11 12 13

49

extracts from vegetative yeast cells producing Ndt80-HA contain an activity

that specifically binds the MSE DNA sequence.

To address whether Ndt80 protein directly binds to the MSE, we

examined the ability of purified Ndt80 to interact with the MSE by the gel

shift assay. Full-length Ndt80 was produced in E. coli as a fusion protein with

maltose-binding protein (Mbp) and purified as described in Experimental

Procedures. A molar excess of unfused Mbp alone did not shift the labeled

MSE (Figure 2-5B, lane 2). In contrast, Mbp-Ndt80 demonstrated specific MSE

binding (Figure 2-5B, lanes 3 and 13), as determined by competition analysis

with unlabeled oligonucleotides (Figure 2-5B, lanes 4-12). The binding

activity could be disrupted by 200-fold excess competitor containing a wild

type MSE (Figure 2-5B, lanes 4-6) but not by excess competitor in which the

sequence was changed to either GCCAGTAACAC (lanes 7-9) or

TCATGTAAGAC (lanes 10-12). Thus the conserved basepairs of the MSE are

important for both in vivo Ndt80-dependent transcriptional activation and in

vitro Ndt80-dependent DNA binding. These observations indicate that direct

binding of Ndt80 to the MSE is necessary for activating transcription of the

middle sporulation genes.

The Meiotic Recombination Checkpoint Machinery Controls Transcription of

the Ndt80-Regulated Gene, CLB1

Defective meiotic recombination, for example, due to a mutation in

DMC1, the sporulation-specific RAD51 homologue, causes cells to arrest at

pachytene (Bishop et al., 1992). This arrest is dependent on both mitotic

checkpoint gene products, Radl?, Rad24 and Mec1 (Lydall et al., 1996), and

meiosis-specific checkpoint gene products, Red1 and Mek1 (Xu et al., 1997). In

contrast, pachytene arrest exhibited by ndt&0 mutants occurs without any

50

Figure 2-6. The Meiotic Recombination Checkpoint Gene, RAD17, Controls

Transcription of the Ndt80-Regulated Gene, CLB1, but not NDT80

radl? (YSC927), dmc1 (YSC907), and dmg 1 radl 7 (YSC928) strains were

transferred to sporulation medium at t=0. RNA was harvested at hourly

intervals. The Northern blot was probed with NDT80, CLB1 and TCM1.

51

■■

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*

-||||§4º5*&z_1&8&9sº8zi€,$Z$$$$ži

|

ZIppa/ZIppuLoup/LõuupLõuup/LõuupZIppa/ZIppa

08JLCIN|

0=}

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52

gross disruption in the recombination process and therefore does not appear

to be a consequence of the recombination checkpoint (Xu et al., 1995). Because

both the meiotic recomination checkpoint and Ndt80 deficiency cause arrest

at pachytene, we wondered whether the meiotic recombination checkpoint

machinery might somehow act by inhibiting Ndt80 expression or function.

To test whether the meiotic recombination checkpoint regulates

transcription of NDT80, we compared NDT80 mRNA expression in

synchronized, sporulating strains in which the meiotic checkpoint was either

activated due to the accumulation of recombination intermediates (dmc1) or

bypassed due to a second mutation in a checkpoint gene (dmc1 radl?).

Checkpoint arrest was assayed by DAPI staining of nuclei (97%mononucleate). We found that NDT80 was transcribed in both dmc1 and

dmc1 radl 7 strains (Figure 2-6). These observations indicate that NDT80

transcription is not under checkpoint control.

To test whether Ndt80 activity might be a target of the meiotic

checkpoint, meiotic transcription of CLB1, which we have shown to be Ndt80

dependent (Figures 2 and 3), was assayed. We found that although

checkpoint-activated dme1 strains expressed NDT80, they did not produce

CLB1 transcript (Figure 2-6). In contrast, radl? and dme1 radly strains

expressed both NDT80 and CLB1 (Figure 2-6). Similarly, we found that dmc1

red1 and dmg1 mek1 strains also transcribed both NDT80 and CLB1 (data not

shown). Taken together, these findings suggest that the meiotic

recombination checkpoint machinery inhibits Ndt80 function at a step

subsequent to NDT80 transcription. We have detected a post-translationalmodified form of Ndt80 (data not shown) which may be relevant to this

checkpoint control.

-

53

Discussion

We have shown that Ndt80 is a central component of a transcription

cascade governing gametogenesis in yeast. Ndt80 functions at pachtyene of

meiotic prophase to activate transcription of the middle sporulation genes,

which are required for both meiotic division and gamete morphogenesis. We

furthermore find that transcription of the representative middle gene, CLB1,

is controlled by the meiotic recombination checkpoint. Thus the step

governed by Ndt80 may be a target for meiotic checkpoint control.

Ndt80 Activates Transcription of the Middle Genes

The conclusion that Ndt80 recognizes the MSE to activate transcription

of the middle sporulation genes is based on the following observations. First,

Ndt80 is required for middle gene RNA synthesis (Figures 1 and 2). Second,

ectopic synthesis of Ndt80 in vegetative cells activates middle gene

transcription (Figure 2-3). Third, activation by Ndt80 is dependent on the

conserved basepairs of the MSE. Fourth, Ndt80 isolated from yeast or bacteria

recognizes the MSE sequence in vitro (Figure 2-5). Thus the consensus

basepairs of the MSE are required for both Ndt80-dependent transcriptional

activation in vivo and Ndt80-dependent DNA-binding activity in vitro.

Consistent with its role as a transcriptional activator, we have localized Ndt80

to the nucleus of cells undergoing both meiotic divisions (data not shown).

It has recently been reported that partial depletion of H2A-H2B histone

dimers also results in a prophase arrest without any gross defects in meiotic

recombination (Tsui et al., 1997). Ndt80 might therefore alter nucleosome

structure. It has also been observed that the presence of an adjacent 14

basepairs upstream of the SPS4 MSE increases MSE-dependent expression 10

fold during either sporulation (Hepworth et al., 1995) or vegetative growth in

54

the presence of Ndt80 (as we observed). Therefore, another factor may

recognize this sequence and enhance Ndt80 activity in vivo.

Ozsarac et al. (1997) identified MSE sequences upstream of several

sporulation genes. We extend the set of MSE-containing genes to include five

B-type cyclins, NDT80 itself, SPC.42, and several other genes implicated in

sporulation (Table 2-1). We do not yet know whether these MSE sequences

are functional. An MSE sequence is present directly upstream of the open

reading frame for CLB1 but not CLB2 (Table 2-1A). Thus there is a striking

correlation between the presence of an MSE and Ndt80-dependent

transcription (Figure 2-3). Although the sequence requirements for MSE

recognition remain to be determined, a perfect fit to the putative consensus is

not required. For example, CLB3 and CLB4 lack a consensus MSE but share a

variant MSE sequence followed by multiple CAAAA(A) repeats (Table 2-1A).

Ndt80 is a Central Regulator of the Sporulation Transcriptional Cascade

A transcriptional cascade has been postulated to govern orderly

progression through sporulation, with members of one set of genes triggering

transcription of the subsequent set (reviewed by Mitchell, 1994). Our findings

demonstrate the existence of a transcriptional cascade and show that Ndt80 is

a central component of it. Transcription of NDT80 is dependent on the early

sporulation gene activator, Imel. Ndt80 then activates expression of the

middle sporulation genes. Thus early genes involved in chromosome

synapsis and recombination (e.g. HOP1 and DMC1) are expressed before later

genes involved in meiotic nuclear division and spore formation (e.g. CLB1

and SPS1) (Figure 2-7). Ndt80 is also required, either directly or indirectly, for

later transcriptional events, such as expression of mid-late genes, e.g. DIT1

(Figure 2-1).

55

Figure 2-7. Ndt80 is a Central Regulator of the Sporulation TranscriptionalCascade

Both nutritional and mating type signals initiate entrance into

sporulation by activating the transcription factor Imel (reviewed by Malone,

1990). Imel together with its DNA-binding partner, Ume■ , turn on

expression of the early genes involved in chromosome synapsis and

recombination (see Kupiec et al., 1997). NDT80 transcription is dependent on

Imel and occurs after early gene expression. Ndt80 can activate its own

transcription and that of the middle sporulation genes. Transcription of the

middle genes, which function in both meiotic division, e.g. CLBs, and spore

formation, e.g. SPS1 (Friesen et al., 1994), is regulated by the meiotic

recombination checkpoint machinery. The activities responsible for

subsequent transcription of the mid-late and the late genes, whose products

are required for spore wall maturation (see Mitchell, 1994), remain to be

identified. The Ndt80-dependent, coordinate induction of genes involved in

nuclear division and gamete morphogenesis results in the production of an

ascus containing a tetrad of four haploid spores.

.**º

ºº

º

56

WEARLY GENES

meioticrecombinationcheckpoint

MIDDLE GENES

spore formation|nuclear division

W MEIOSIS IMID-LATE

GENES !W MEIOSIS II

LATE GENES

W

ÖTetrad of 4 spores

57

NDT80 itself appears to have two modes of transcription. In the first,

NDT80 RNA synthesis is activated by Imel. Why NDT80 transcription is

delayed relative to typical early genes is not known (Xu et al., 1995; Figure 2-1).

A second mode of NDT80 expression is stimulated by Ndt80 itself, which

presumably acts on an MSE in its upstream region to maintain its own

synthesis (Figure 2-4B). This proposed two-stage regulation of Ndt80

synthesis is analogous to that of lambda cI (Reichardt and Kaiser, 1971; Echols

and Green, 1971) and Drosophila Sxl (Bell et al., 1988) and may contribute tocommitment to meiotic division.

Activation of B-type Cyclins in Yeast and in Other Organisms May Stimulate

Entry into Meiosis I

The Ndt80-dependent induction of CLB1 and CLB3-CLB6 classifies

them as middle sporulation genes. We propose that this simultaneous burst

of five CLBs promotes the prophase to metaphase I transition, thus

explaining the similar pachytene arrest phenotypes of ndt&0 and cdc28

mutants. This transcriptional induction also provides a molecular

explanation for the previously reported increase of both protein and

associated H1 kinase activity for Clb1, Clb3, and Clb4, but not Clb2, midway

through meiosis (Grandin and Reed, 1993). Clb5- and Clb6-associated kinase

activities during meiosis have not been examined.

Induction of cyclin transcription before meiosis I has also been

observed in other organisms. In mouse spermatocytes, the message levels for

both cyclin B2 and the germ cell-specific cyclin A1 increase dramatically at

pachytene (Chapman and Wolgemuth, 1993; Sweeney et al., 1996). In fission

yeast, a large induction of cdc13 transcript, which encodes a cyclin B

homologue (Hagan et al., 1988), occurs one hour prior to meiosis I and is

:

58

dependent on the meiosis-specific transcription factor, Mei-4 (Iino et al., 1995).

Thus, induction of cyclin transcription by Ndt80 or its analogues in other

systems may be one means by which entry into meiotic division is regulated

in both yeast and male germ cell gametogenesis.

The simultaneous transcription of five CLBs at meiotic prophase is

strikingly different from their sequential expression during the vegetative cell

cycle (reviewed by Koch and Nasmyth, 1994) and might reflect a general

requirement for high Cdc28 kinase levels during meiosis. It is also possible, º

based on their known mitotic roles, that different cyclin-Cdc28 species have ■some specificity. For example, Ndt80-induced Clb1-Cdc28 activity may º

promote meiotic nuclear division, and Clb3- and Clb4-associated kinase !

activity at pachytene may ensure that SPB separation (Fitch et al., 1992; |

Richardson et al., 1992) in meiosis occurs only after DNA replication, s

recombination, and synapsis are complete. Clb5 may have a dual role in

meiosis -- an early function in premeiotic DNA synthesis (as in vegetative {

cells; reviewed by Stillman, 1996) and a later, Ndt80-induced activity in |

meiotic progression. Metazoan cyclin A, which is functionally analogous to

Clb5 and Clb6 in several respects (Pagano et al., 1992; Dahmann et al., 1995),

functions in the meiotic divisions of both mouse and Drosophila

spermatogenesis (Sweeney et al., 1996; Gönczy et al., 1994; Thomas et al., 1994).

Sporulation and Spermatogenesis are Analogous Meiotic DifferentiationProcesses

There are several parallels between sporulation and spermatogenesis.

Both are continuous processes in which meiotic division is coupled to gamete

morphogenesis (Lin et al., 1996) to produce four highly differentiated,

functional cells -- spores or sperm. As noted above, in both sporulation and

59

spermatogenesis, B-type cyclin transcription is induced at pachytene. In

addition, in both systems the progression from prophase into metaphase I

may be under checkpoint control. Control of transcription of cyclins and

other genes by Ndt80 in yeast, or its analogue in spermatogensis, may be

responsible for these parallels.

Coordinate induction of transcription of genes involved in meiotic

division and gamete formation may ensure that these two processes can begin

at approximately the same time. In yeast, spore morphogenesis appears to

initiate just prior to meiosis I, as indicated by a subtle enlargement of the

outer plaque of the SPBS (Moens and Rapport, 1971). In Drosophila, aly is

required for transcription not only of genes involved in meiotic division,

such as cyclin B (White-Cooper et al., 1997), but also of genes required for

spermatid differentiation, e.g. fuzzy onions (Hales and Fuller, 1997). Ndt80

and aly may play analogous roles in initiating meiotic division and gamete

morphogenesis.

A variety of defects in both human and mouse spermatogenesis and in

yeast gametogenesis interrupt the prophase to metaphase transition.

Spermatocytes of humans with testicular maturation arrest, a male infertility

syndrome, are blocked at pachytene (Soderstron and Suominen, 1980).

Mutations in several mouse genes, including Dazla, A-myb, Mlh1, and ATM,

trigger pachytene arrest in spermatogenesis (Ruggiu et al., 1997; Toscani et al.,

1997; reviewed in Sassone-Corsi, 1997). In yeast, mutations resulting in

defective interhomolog recombination intermediates also result in a

pachytene arrest which is under checkpoint control (Lydall et al., 1996; Xu et

al., 1997). Our finding that meiotic transcription of the Ndt80-regulated gene,

CLB1, is under control of the recombination checkpoint is consistent with the

ºº

60

hypothesis that Ndt80 is a target of the meiotic recombination checkpoint

machinery. Thus the prophase to metaphase transition in both yeast and

male germ cells may be controlled by the regulated transcription of the cyclin

component of MPF.

61

Materials and Methods

Media and Culture Conditions

Yeast were grown in complete (YPD) or synthetic minimal media (SD)

as described in Sherman et al. (1986). For YPA, 1% potassium acetate was

substituted for glucose of YPD. For SRaf, 0.2% raffinose was substituted for

glucose of SD. For galactose inductions, cells were grown in SRaf to early log

phase (OD600 0.3) and then with 1.7% galactose for the 3.5 h. Sporulation

medium (SPM) contained 0.3% potassium acetate and 0.02% raffinose and

synchronized as described by Cao et al. (1990).

£-galactosidase Assays

Ten ml exponentially growing cells in SD-ura or 10 ml cells in SPM

were processed for £-galactosidase assays as described by Miller (1972) Values

reported are an average of those from two independent transformants.

DAPI Staining and Immunofluorescence

500 pil cells in SPM were fixed with 70% ethanol then stained with 1

pg/ml 4,6-diamino-2-phenylindole (DAPI). Cells were visualized with an

Olympus BX60 microscope.

Gel Mobility Shift Assays

Yeast extracts were made according to Goutte and Johnson (1988). Gel

mobility shift analysis followed the protocol of Hepworth et al. (1995). The

sequence of the top strand of the probe was 5'GGATCGCCGATTGAC

62

GCGCGCCACAAAAACG3' with overhang nucleotides underlined. Each

DNA-binding reaction mixture contained 10 pul A' buffer, 0.65 mM

dithiothreitol, 11 mM MgCl2, 50 pm ZnSO4, 2 pig poly dI-dC, 0.005 pmol of

radioactively labeled probe, and competitor oligonucleotide as indicated.

Added last to each reaction was 4.4 pil crude extract (Figure 2-5A) or 1 ng

recombinant protein (Figure 2-5B).

Genetic and Molecular Biological Methods

Recombinant DNA manipulations were performed as described by

Sambrook et al. (1989). Yeast genetic methods were carried out as in Rose et

al. (1990). Yeast transformations were carried out by the lithium acetate

protocol of Ito et al. (1983).

NDT80-HA and NDT80-GFP construction. A BgllI fragment containing

the NDT80 ORF from pnkY1213 (Xu et al., 1995) was inserted into the BamhI

site of the integrating vector pKS306 (Sikorski and Hieter, 1989) to generate

pSC85. Site-directed mutagenesis of pSC85 was used to introduce a unique

BgllI site (pSC88) or Clal site (pSC89) directly preceding and in frame with the

NDT80 stop codon. A BamhI fragment containing two copies of an 11 amino

acid HA epitope (YPYDVPDYASL) was introduced at the BgllI site of pSC88 to

form pSC101. A triple ligation between a Cla■ -BamhI GFP-containing

fragment, a 2.2 kb Not-Clal NDT80 fragment of pSC89, and pSC88 digested

with BgllI and Not■ was carried out to construct pnDT80-NDT80-GFP (pSC94).

To construct pCAL-NDT80-GFP (pSC382), a Not-BamhI 1.9 kb fragment of

pSC131 which includes the GAL promoter was dropped into pSC94 digested

with Noti and BamhI. Both pSC101 and pSC94 were integrated at the NDT80

locus by digesting with BamhI prior to transformation (Rothstein, 1991).

pSC382 was integrated at the LIRA3 locus by digesting with Nsi■ .

:º :!º

º

:

63

pGAL-NDT80 construction. A unique Ndel site was introduced into

pSC85 at the ATG of NDT80 by site-directed mutagenesis to generate pSC87.

To construct pCAL-NDT80 (pSC131), a Not■ -Ndel fragment containing the

GAL1-10 promoter was exchanged with a Not■ -Ndel fragment of pSC87 which

contains the NDT80 promoter. To construct pCAL-NDT80-HA (pSC193), a

Not-BamhI fragment of pSC131 replaced a Not-BamhI fragment of pSC101.

To construct pCAL (pSC192), a Ndel-SmaI fragment spanning the NDT80

open reading frame was dropped out of pSC131. pSC131, pSC192, and pSC193

were integrated at the LIRA3 locus by digesting with Stu■ or at the NDT80

locus by digesting with BamhI. HIS3-marked versions of pcAL, pCAL

NDT80 and pCAL-NDT80-HA (pSC232, pSC233, pSC235, respectively) were

made by inserting the respective Not■ -Sal■ fragments of pSC131 and pSC192

193 into the Not■ -Sal■ sites of the integrating vector pKS303 (Sikorski and

Hieter, 1989). These plasmids were integrated at HIS3 after digesting withNheI.

MBP-NDT80 construction. To construct pSC86, a unique BgllI site was

introduced by site-directed mutagenesis into pSC85 directly downtream and

in frame with the ATG of NDT80. The BgllI-Sal■ fragment of pSC86 was

inserted directly downstream of maltose binding protein (Mbp) at the BamhI

Sall sites of pVAL-p2 (New England Biolabs) to create an in-frame fusion of

Mbp-Ndt80 (pSC220).

MSE-lacz construction. Reporter constructs were based on those of

Hepworth et al. (1995). Oligonucleotides contained overhangs of 5GATC at

both ends followed by 29 basepairs of mutant or wild-type SPS4 MSE sequence

(ATCGCCGATTGACGCGCGCCACAAAAACG) and Sal■ and Bambi■ sites

(AGATCTGTCGACC). The sequence of the complete wild-type MSE

64

oligonucleotide was as follows: 5'GATCATCGCCGATTGACGCGCGC

CACAAAAACGAGATCTGTCGACCTCGA3'. Overhang residues at the ends

are underlined. Internal residues which were subsequently mutated (see

Table 2-1 and Figure 2-6) are also underlined. These oligonucleotides were

cloned into the BamhI/XhoI sites of pDCA312SASS, a cycl-lacz reporter

plasmid which lacks the cycl UAS (constructed by Aaron Mitchell; from Jason

Moffitt and Brenda Andrews), and confirmed by sequence analysis (BRC

facility, UCSF).Purification of MBP-NDT80

pMAL-p2 (Mbp) and pSC220 (Mbp-Ndt80) were transformed into

protease-deficient E. coli strain NB42. Early log phase cells were induced at

37°C for 3 h in the presence of IPTG. Harvested cells were resuspended in 35

ml PBS, sonicated, and lysed with 1% Triton for 30 min at 4°C. Cell debris was

removed by a 20 min spin at 10 krpm. The supernatant was batch bound to 1

ml amylose resin slurry for 2 h before packing into a column and then elutedwith 10mM maltose.

Northern Blot Analysis

Northern analysis was performed as described by Cross and

Tinkelenberg (1991). RNA was harvested from exponentially growing or o

factor treated cells in vegetative medium or from synchronized, sporulating

cells. 20 pg/ml o-factor was added every 2 h throughout the experiment.

After 3.5 h of exposure to mating pheromone (99% shmooed or unbudded

cells), galactose was added to 1.7% for an additional 3.5 h (94% shmooed cells).

Each lane was loaded with 30 pg total RNA. All probes were random-prime

labeled using the Prime-It Kit (Stratagene). The templates for random

priming, except those for CLB2 and TCM1, were gel-purified PCR products

65

made with oligonucleotides corresponding to each open reading frame

(Research Genetics, Inc). The CLB2 probe was a 1.36 kb Spel-HindIII fragment

within the CLB2 open-reading frame from pmC213 (p.GAL-CLB2; Li and Cai,

1997), which was gel purified twice before use. The TCM1 probe was a 0.8 kb

HpaI-Sall fragment from pab309A (Schultz and Friesen, 1983). Blots were

stripped (Krisak et al., 1994) and re-exposed before subsequent hybridization.Yeast Strains

Yeast strains used in this study were the following. Those of the SK1 2.background (ho::LYS2 ura■ leu2::hisG) include YSC328/NKY611 (a/o wild- ~.type), YSC330/NKY2296 (a/o ndt&0A::LEu2/ndt&0A::LEu2), YSC907 (a/o tº

dmc1::ARG4/dmc1::ARG4 trpl;hisG/trp1::hisG argº-Bgl/arga-Bgl), YSC927 2.(a/o radl 7::hisG-URA3/rad 17::his G-URA3), YSC928 (a/o radl 7:::his G- 2.URA3/rad 17::hisG-LIRA3 dmc1::ARG4/dmc1::ARG4 trp 1::his G/trp1::his G -sº

arga-Bgl/arga-Bgl). Those of the W303 background (ade2-1 his3-11,15 leu2- |-3,112 trpl-1 ura■ -1 can1-100) include YSC7 (a/o wild-type), YSC531 (a/o 2.uRA3::pCAL-NDT80-HA), YSC553 (a URA3::pCAL-NDT80-HA), YSC561 (a/o -.

HIS3::pCAL), ySC562 (a/o HIS3::pCAL-NDT80-HA), YSC794 (a/o imelA:hisG

uRA3/ime1A::hisG-URA3), YSC918 (a/o HIS3::pGAL; pnDT80-NDT80-GFP),

YSC919 (a/o HIS3::pCAL; ura■ ::pGAL-NDT80-GFP); YSC921 (a/o HIS3::pGAL

NDT80; pnDT80-NDT80-GFP); ySC922 (a/o HIS3::pCAL-NDT80; ura3::pCAL

NDT80-GFP). All tagged Ndt80 constructs were integrated at the NDT80 locusunless otherwise noted.

66

Acknowledgements

We are indebted to Nancy Kleckner and members of her laboratory,

especially Liuzhong Xu, Andrew McKee, Scott Keeney, and Sean Burgess, for

their advice, generosity, and encouragement. We thank Minx Fuller, Renee

Reijo, Sue Biggins, Marion Shonn, Joe DeRisi, Christina Hull, Mark Winey,

Yona Kassir, Andrew Murray, and Pat O'Farrell for enlightening discussions;

and Yona Kassir and Jacqueline Segall for plasmids. We thank Liuzhong Xu,

Andrew Murray, Jacqueline Segall and her laboratory, Renee Reijo, and Flora -:

Banuett for comments on the manuscript. This work was supported by a * = .

research grant (AI18738) from the National Institutes of Health to I.H. and a ::

Medical Scientist Training Program grant from the NIH, supplemented by the 2Sussman Fund, the Markey Program in Biological Sciences, and the Herbert gº tº

W. Boyer Fund to S.C.

67

CHAPTER THREE

■ º a

THE TRANSCRIPTIONAL PROGRAM r:

OF GERM CELL DEVELOPMENT 2.IN BUDDING YEAST gº tº

gº - i.

* * ***

** a -

Contributing Authors:Shelley Chu”, Joe DeRisi"#, Jon Mulholland#,David Botstein #, Patrick O. Brown?, Ira Herskowitz

* The first two authors contributed equally to this work.

#Department of BiochemistryStanford University School of MedicineHoward Hughes Medical InstituteStanford, CA 94305-5428

68

Abstract

The program of germ cell development in Saccharomyces cerevisiae

involves a transcriptional cascade. We have carried out a comprehensive

analysis of gene expression during sporulation (the coordinated process of

meiosis and spore formation) using DNA microarrays. This analysis reveals

the temporal program of gene expression as it unfolds during sporulation and

suggests possible functions for a large number of previously unknown genes.

Eight classes of genes, four of which are novel, were defined based on their

expression profiles during sporulation. Several putative regulatory elements

were identified by searching for common motifs in the promoters of

regulated genes. The sporulation-specific transcription factor, Ndt80, appears

to activate transcription of more than 100 middle genes, as many of these

genes were induced by ectopic expression of Ndt80 and required Ndt80 for full

expression during sporulation. A connection between function and

expression profile was also observed, thus implicating novel genes in specific

steps of meiosis and spore formation. The functions of several novel genes

belonging to different temporal classes were tested genetically, and each was

found to be essential for sporulation. Several genes induced during

sporulation have homologs with temporally regulated induction during

spermatogenesis, suggesting that the yeast genes we identified are a rich

Source of new candidates for roles in gametogenesis in higher eukaryotes.

Introduction

All sexually reproducing organisms undergo the developmental

Pathway of gametogenesis to produce germ cells with haploid DNA content.Gametogenesis in yeast (sporulation) involves two overlapping processes,

69

meiosis and spore morphogenesis (see Figure 3-1). In meiosis, pairs of

replicated, homologous chromosomes align and undergo meiotic

recombination during prophase. Two consecutive nuclear divisions then

follow, in which, first, homologous chromosomes segregate apart (meiosis I)

and then sister chromatids separate (meiosis II). In spore morphogenesis, a

flattened membrane sac forms from each modified outer plaque of the

meiosis II spindle pole body (SPB). This sac grows and fuses to form a double

bilayer prospore membrane which encapsulates each of the four lobes of the

meiotic nucleus. The immature spores then mature by the deposition of

several distinct layers of spore-wall material within the lumen of the

prospore membranes (reviewed by Byers, 1981).

Progression through gametogenesis involves the regulated function of

gene products required for meiotic division and germ cell morphogenesis.

Sporulation in yeast is characterized by sequential transcription of at least four

sets of genes -- early, middle, mid-late, and late (reviewed by Mitchell, 1994).

For many genes, the timing of their expression reflects the timing of action of

their gene products. Several early genes are involved in pairing of

homologous chromosomes or recombination. Their expression is regulated

by the Umeå/Ime1 complex, which recognizes a conserved site (URS1) found

in their upstream region (see Kupiec et al., 1997). Products of the middle

genes are required for the concomitant events of meiotic nuclear divisions

and spore formation (Chu and Herskowitz, 1998). Ndt80 is a meiosis-specific

transcription factor that acts at the end of prophase to induce transcription of

the middle genes through recognition of the MSE (middle gene sporulationelement) motif found upstream of many of the middle genes (Hepworth etal., 1995; Xu et al., 1995; Ozsarac et al., 1997; Chu and Herskowitz, 1998). The

70

Figure 3-1. Landmark Events of Sporulation

The developmental program of sporulation involves both the

processes of chromosom distribution and spore formation. In meiosis, pairs

of replicated, homologous chromosomes align and undergo recombination

during prophase. Two consecutive nuclear divisions then follow, in which,

first, homologous chromosomes segregate apart (meiosis I), and then sister

chromatids separate (meiosis II) (reviewed by Kleckner, 1996; Roeder, 1997).

In spore morphogenesis, spindle pole bodies (SPBs) duplicate and separate to

form the meiosis I spindle. As the SPBs duplicate again for meiosis II, each of

their outer plaques becomes modified and nucleates the formation of a

flattened membrane sac. This sac grows and fuses to form a double-bilayer

prospore membrane which encapsulates each of the four lobes of the meiotic

nucleus. Deposition of distinct layers of spore-wall material within the

lumen of the prospore membrane leads to the formation of a spore (reviewed

by Byers, 1981). Sporulation of a cell yields a tetrad of four spores. The major

transcription factors for sporulation include Imel (reviewed by Kupiec et al.,

1997), which activates the early genes, and Ndt80 (Chu & Herskowitz, 1998),

which activates the middle genes. Representative genes for each of the classes

(early class I and II, early-mid, middle, mid-late, and late) are specified

(reviewed by Kupiec et al; also see Figure 3-2).

71

MAJORTRANMAIORTRANGENE

SPOREMEIOTIC --SCRIPTIONAL FORMATIONDIVISIONACTIVATORTCLUSTER (S)REPL.&y■ IME1EarlyI:

RECOMB.ZIP1,DMC1,FKH1,YOR177 WEarlyII:

©POL30,DBF4,SMC3,YMR144

|Early-Mid:

PDS1,CDC14,SPC42,YGR225

MEIOSISI

NDT80 W

||||

MEIOSISII

W

||||

s

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*

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s

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f

id

Middle: CLB4,CDC20,SPO20,YDR104 Mid-Late: CDC26,HCT1,DIT1,STE5 Late: SPS100,YKL050,YMR322º

*!

NS

mid-late class includes genes necessary for formation of the outer layer of the

spore wall (Briza et al., 1990), and the late genes are thought to have a role in

spore maturation (Law and Segall, 1988). The result of the sporulation

program is four haploid spores, each of which is capable of germinating and

fusing with a spore of the oppposite mating type, analogous to the fusion of

egg and sperm.

Genes necessary for sporulation have been identified by mutant hunts

using visual assays for spore formation (see Esposito et al., 1972; Briza et al.,

1990; and Xu et al., 1995, for examples) or functional assays for processes such

as recombination (reviewed by Esposito & Klapholz, 1981). In addition, genes

have been identified based on differential expression during sporulation, as

assayed by Northern analysis (reviewed by Kupiec et al., 1997) or by ■ º

galactosidase activity with random lacz insertions (Burns et al., 1994). As a

result, roughly 150 genes have been identified to pay a role in sporulation

(Kupiec et al., 1997).

To systematically explore the transcriptional program during

sporulation, we used DNA microarrays containing 97% of the known or

predicted genes of S. cerevisiae (DeRisi et al., 1997). Electron and light

microscopy were used to monitor morphological transformations occuring in

parallel with expression changes. Approximately 500 genes were found to be

induced during sporulation and whose products are therefore likely to have

roles in sporulation. The results provide a global view of the choreography of

the genetic program of meiosis and spore formation, and facilitate the

identification of additional proteins involved in the different steps of

gametogenesis in yeast and higher eukaryotes.

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73

Results

We used the microarray technique described by DeRisi et al. (1997) to

study the program of transcription during sporulation in a global manner. In

our first analysis, the relative mRNA concentrations for each gene were

measured from wild-type yeast cells at t=0,0.5, 2, 5, 7, 9, and 11.5 hours (h)

after transfer to sporulation medium. These times were chosen based on the

expression pattern of known canonical early, middle, mid-late, and late genes

(DMC1, SPS4, DIT1, and SPS100, respectively). To monitor progression of the

cell population through sporulation, we examined cells by both light and

electron microscopy for landmark morphological changes. Further analysis

of the middle gene set was done with two additional microarray experiments.

In particular, we examined the set of genes induced by ectopic expression of

Ndt80 in vegetative cells and the requirement of Ndt80 for transcription of

the temporally-defined middle sporulation genes. The program of gene

expression in wild-type yeast also provided a screen to identify genes

involved in aspects of sporulation and allowed grouping of subsets of genes

into putative functional categories.

Classes of Sporulation Genes

We organized the expression data into temporal classes based on

similarity of transcriptional profiles using a clustering algorithm (Eisen et al.,

1998). Among the resulting set of more than 1000 genes, approxmiately half

were induced during sporulation, while the other half were repressed.

Previous work based on a relatively small number of genes defines four

temporal classes of sporulation-specific genes (reviewed by Mitchell, 1994).

Our analysis confirms the existence of these four major classes, adds many

new members of sporulation-induced genes to each class, and identifies four

74

new classes (see Table 3-1). The early genes can be subdivided into two gene

sets, class I and class II. We define the set of early-mid genes as those induced

early which then receive a second boost midway through sporulation. The

middle genes constitute the largest set of induced genes, with more than 140

members, in contrast to the mid-late and late gene sets. More than 50 genes

implicated in basic metabolic processes are transiently induced during

sporulation. In addition, over six hundred genes are repressed throughout

sporulation. In summary, cluster analysis has revealed at least eight

temporally distinct classes of genes: early class I, early class II, early-mid,

middle, mid-late, late, metabolic, and repressed.

Early Class I Gene Cluster

The early class I cluster contains 89 genes. This group is characterized

by early induction, 0.5 h after transfer to sporulation medium, which is

generally maintained throughout the rest of the time course (see Figure 3-2).

Inspection of the upstream sequences belonging to this cluster revealed that

over one-third contained a URS1 consensus site (5' GGCGGCT3'). Many early

class I genes with previously studied functions are involved in aspects of

homolog pairing (e.g. HOP1) or recombination (e.g. DMC1), suggesting that

newly identified members of this class may have related roles.

Early Class II Gene Cluster

A second class of early genes, early class II, is distinguished by a slightly

delayed increase in mRNA levels relative to the early class I genes (see Figure

3-2). This lag may be due to a later onset of transcription or may reflect a

lower rate of transcription relative to class I genes. Many of the proteins

encoded by early class II genes are involved in aspects of DNA replication (e.g.POL30,

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75

Table 3-1.

Eight clusters of genes are induced or repressed during sporulation.

The approximate number of genes in each cluster, timing of their expression,

general function, potential regulatory sites, and percentage of genes in each

cluster with such a site are given.

Table of Clusters Expressed during Sporulation

/**

, ■ º

.

OnsetofPotential(%ofgenes

#ofExpressionRegulatoryincluster

ClusterGenes>1.5-foldGeneralFunctionsSitewithsite)SPORULATION-INDUCED

EarlyClassI890.5h

homologpairing,URS1(36%)

recombination

EarlyClassII272hDNAreplication,SCBandMCB(33%)? Early-Mid

850.5–2handchromosomebehavior,2

5–7hspindle,SPBdynamicsMSE(50%)?

Middle1465hmeioticdivision,MSE(75%)

S;sporemorphogenesisactivation?:CCWKYGCTTT

repression(invegetativecells)?:

GGGWDWTGSS

Mid-Late777h

meioticdivision,MSE(42%)?

sporemorphogenesis2

Late7

9-11.5hsporematuration2

Metabolic530.5–2h

metabolic2

(transient)

SPORULATION-REPRESSED Repressed6540.5or2h"ribosomalandothers2

*:**:*******:::*:*:

UU)■LDNAV

Lº*,7.--

º -º,*º!.Yc*-º–-***-?--~*–“T!,

chromosomebehavior

RFA2) or chromatid cohesion (e.g. SMC3, PDS5). Such proteins may be less

abundant than components of the synaptonemal complex (early class I

proteins).

Most early class II genes are likely to be under different transcriptional

control than class I genes. Only three of the early class II genes contain a

consensus URS1 site in their promoter region. We searched the upstream

regions of the remaining 24 genes for common motifs and found that 33%

have one or more SCB or MCB sites. These sites are targets of cell-cycle

regulatory factors, SBF and MBF, and may serve the same function in

sporulating cells, as well (see Leem et al., 1998) or be recognized by a novel

transcription factor.

Early-Mid Gene Cluster

The early-mid cluster consists of 85 genes whose expression is first

induced within 0.5 to 2 h upon transfer to sporulation medium, similar to the

early genes. The early-mid genes are then further induced by 5 or 7 h, similar

to the middle and mid-late genes, respectively. Several early-mid genes are

involved in aspects of spindle and SPB dynamics (e.g. SPC42) or chromatid

behavior (e.g. PDS1).

How the expression of the early-mid genes is regulated is not known.

Their early induction is unlikely to be directly mediated by Imel/Ume6, since

only four of the 85 genes contain a classic URS1 site in their promoters. With

respect to the second boost in expression midway through sporulation, 50% of

the early-mid genes contain one or more MSE sites (of the consensus

CRCAAAA/T) in their promoter regions. Ndt80 may be responsible for the

induction of this subset of genes.

1/.

ºi.

78

Middle Gene Cluster

The middle gene cluster of 146 genes constitutes the largest set, most of

which are strongly induced by 5 h after transfer to sporulation medium (see

Figure 3-2). Many of the known middle genes are involved in aspects of

meiotic division (e.g.CLB6) and spore morphogenesis (e.g. SPO20). 75% of the

middle genes have one or more MSE sites in their upstream regions which

are presumable recognized by Ndt80 to activate their transcription. The

expression of some of the 37 middle genes which lack a perfect MSE may still

be dependent on Ndt80, either directly or indirectly. For example, Ndt80 may

recognize a divergent MSE or regulate a transcription factor which directly

activates this subset of genes. Some middle genes also appear to have Ndt80

independent expression. Analysis of the middle genes lacking perfect MSE

sites indicated that 16 (43%) share a potential activation site

(5'CCNNYGCTTT3') in their upstream regions. (The genome-wide frequency

of this site is only 1.4%.) This subset includes both CLB3 and CLB4, which

previously have been shown by Northern analysis to have some level of

Ndt80-independent expression (Chu & Herskowitz, 1998).Mid-Late Gene Cluster

The mid-late gene cluster contains 77 genes which are induced strongly

by 7 h in sporulation medium. Many of these genes are also involved in

aspects of both meiotic division (e.g. CDC26) and spore formation (e.g. DIT1).

Nearly half have one or more MSE sites in their promoter region. A negative

regulatory site may delay utilization of the MSE relative to middle genes (seeFriesen et al., 1997).

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79

Figure 3-2. Cluster Analysis of Induced Sporulation Genes

The approximately 500 genes induced during sporulation were grouped

based on the similarity of their expression patterns with use of a clustering

algorithm (Eisen et al., submitted). Data from the wild-type sporulation time

course were used for cluster analysis. Seven clusters are specified: metabolic

(M), early class I (E-I), early class II (E-II), early-mid (E-M), middle (MID), mid

late (M-L), and late (L). Each bar represents and individual measurement, and

each row represents data for an individual gene. Time progresses on the

horizontal axis, with the first column representing t = 0 h, and each

subsequent column corresponding to t = 0.5, 2, 5, 7, 9, and 11.5 h in

sporulation medium. The data for the ectopic expression of Ndt80 in

vegetative cells is shown in the eighth column (Gal-Ndt80). The presence of

a consensus sequence, as elucidated by motif analysis (see Meme analysis,

Materials and Methods), for the MSE and URS1 sites are shown in blue and

yellow, respectively. The degree to which the sequence matches theconsensus is indicated by the brightness of the bar.

80

-

O /2 2 5 7 9 11

#

I

g

Hours

|

Gal-Ndl30MSE

URs.1

|

7.

Late Gene Cluster

The late gene cluster exhibits induction by 9 or 11.5 h and contains only

seven members, including the prototype, SPS100, necessary for spore wall

maturation (Law and Segall, 1988). Consistent with the model of a

transcriptional cascade, none of these genes are expressed during sporulationin the absence of Ndt80.

Metabolic Gene Cluster

A cluster of 53 genes exhibits mostly transient expression during

sporulation. While the majority (84%) of these genes have known functions

in basic metabolic processes, especially with respect to nitrogen starvation,

two are involved in cell wall biosynthesis, and five are novel. Due to their

roles in metabolism, it is likely that members of this cluster are regulated by

nutrient conditions. In addition, the promoters of ten of these genes contain

a URS1 motif, for which the binding of Umeå is well-characterized in the

Cases of ACS1, INO1, and CAR1 (Kratzer and Schuller, 1997; Jackson and

Lopes, 1996; Duboi and Messenguy, 1997). The differences in expression

between these ten transiently-induced genes and the more stably-inducedearly class I genes -- both of which have URS1 sites -- is striking and suggeststhe use of an another regulatory element, in addition to the URS1 site.Repressed Gene Cluster

Over 600 genes are repressed during sporulation. These fall into three

§eneral subsets. The first includes many genes encoding ribosomal proteins

which are repressed within 0.5 h of transfer to sporulation medium, but by11.5 h have returned to their original t-0 levels. The second and third subsetsare repressed by 0.5 and 2 h, respectively, and remain low throughoutSPOrulation.

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Regulation of middle genes by Ndt80As noted above, Ndt80 is required for transcriptional induction of

several middle genes (SPS1, SPC.42, CLB3, and CLB6 among others) as assayed

by Northern analysis (Chu & Herskowitz, 1998). The cluster analysis

described here reveals a set of 146 middle genes which are also induced by 5 h

upon transfer to sporulation medium. To characterize the relationship

between middle gene expression and Ndt80, we examined the consequences

of expressing Ndt80 ectopically in vegetative cells and of eliminating Ndt80

during sporulation.

Prior work showed that ectopic expression of Ndt80 in vegetative cells

induces transcription of several representative middle genes, such as SPS1, as

assayed by Northern analysis (Chu & Herskowitz, 1998). We found that

under these conditions, more than 200 genes are induced -3-fold, as assayed

by microarray analysis. Of these candidate Ndt80 targets, over one-third weremiddle genes (see Figure 3-2). (In contrast, fewer than 7% belonged to each ofthe early, early-mid, mid-late, or late gene clusters.) Of all the members of themiddle gene cluster, 70% are induced 22-fold in vegetative cells expressingNdt8O. There is thus a positive correlation between genes induced by ectopicSynthesis of Ndt80 and those belonging to the middle gene cluster. Some

exceptions to this correlation may be noteworthy. For example, YLR368 and

SPA2 are both strongly induced during sporulation (>5-fold) but not inVegetative cells expressing Ndt80 (<2-fold). These genes contain presumptive

Ndt80 binding sites in their promoters but may require an additionalSPOrulation-specific transcription factor for induction or may be also regulated

by a vegetative repressor. Of the 44 middle genes which are not induced 22fold by ectopic Ndt80 synthesis in vegetative cells, 30% of them, including

83

YLR368 and SPA2, share a potential repressor site

[5'GGGNNNTG(C/G)(C/G)3] in their upstream regions. (The genome-wide

frequency of this site is only 2.2%).

Approximately one-third of all the genes in the yeast genome, and also

one-third of all early genes, have one or more MSE sites in their promoter

regions (see Figure 3-2). In contrast, approximately 75% of all middle genes

and 60% of all genes induced by ectopic Ndt80 expression (by >3-fold) contain

at least one MSE site in their upstream regions. Therefore there is also a

positive correlation between the frequency of an MSE site with both middle

gene expression and inducton by Ndt80.

To characterize further the dependence of middle gene expression on

Ndt&O, we analyzed expression of these genes at 6 h in sporulation medium

in a strain lacking Ndt80. We found that the average level of Ndt80

independent expression was 33% that of a wild-type strain at a correspondingtime point, indicating that full expression of middle genes requires Ndt80.For several middle genes, however, there is clearly also a substantial level ofNdt&0-independent expression. For example, although both SPS2 and SPR28contain MSE sites in their promoter regions and are strongly induced in

Vegetative cells synthesizing Ndt80, their levels of expression in ndt&0 cells

under sporulation conditions approximate their wild-type levels for acorresponding time point. Such genes may be activated by both Ndt80 andanother factor.

Cytological Changes during SporulationIn parallel with the gene expression time course, cytological changes of

meiotic division and spore morphogenesis were followed using fluorescent,light, and electron microscopy (see Figure 3-3 and Figure 3-4). Progression

º: -

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114

■ is ºssº

has

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84

Figure 3-3. Ultrastructural Landmarks of Sporulation.

A) Premeiotic cells. An example of several premeiotic cells (t = 0 h) showing

numerous lightly stained mitochondria (n = nucleus). B and C) Cells at the

beginning of meiosis I (t = 2h). In B, dense body (DB) and synaptonemal

complexes (SC) are located near the nuclear envelope (NE); in C, side-by-side,

duplicated SPBs (arrows) are observed with associated nuclear spindle. D, E

and F) Formation of prospore membrane in meoisis II (t = 7 h). D shows

distinctive meiosis II SPBs. The prospore membrane is associated with the

enlarged outer plaque and has an electron-dense bulb at its leading edge (long

arrows). Short arrow denotes nuclear envelope in which SPB is embedded. E

shows a prospore membrane leading edge, which is bulb-shaped and studded

with a distinct protein coat. F shows a cell completing prospore formation; a

lobe of the nucleus (N) is being "pinched' off, and mitochondria (M) are being

Sequested into the forming spore; arrows denote the bulbous leading edges of

the prospore membrane. G, H, and I) Spore maturation. G (t = 7 h) is an

example of a prospore, showing aggregation and accumulation of lipid

droplets (L) around the double bilayer prospore membrane (arrow). H) Tetradformation. H (t=9 h) is an example of a tetrad (partially shown) ofimmature spores; spore walls (arrow) have begun to form, and lipid dropletsare rrhostly absent. I) Mature spore. I (t = 11.5 h) is an example of a mature

SPOre within an ascus; the spore wall is thick and has a darkly staining outer

layer (arrow). Vacuole (V). Bars: A, 1 pum; B and F, 0.25 pum; C and D, 0.125

Plm; E, 0.05 pum; G, H, and I, 0.5 p.m.

85

-- - -

·|×·■ ae|-

■ |-|-

86

through meiosis was monitored in the light microscope by DAPI staining ofcell nuclei. The first indication of meiotic division was seen at 5 h, at which

point two DAPI-staining bodies (representative of meiosis I) were detected in2% of the cells. The second meiotic division appeared to be well underway by7 h, at which time 14% of cells were binucleate, and 28% were tetranucleate,

indicative of cells undergoing meiosis II. At 9 h, 7% and 42%, and at 11.5 h,

15% and 50%, bi- and tetranucleate cells were observed, respectively. The

final sporulation efficiency after more than 24 h was 67%.

Upon initial transfer to sporulation medium (t=0), cells appeared

morphologically similar to vegetative cells as assayed by electron microscopy

(see Figure 3-3A). However, in contrast to vegetatively growing cells in rich

medium, cells contained many mitochondria located all along their periphery

(NMiyakawa et al., 1984).

By 2 h, a prominent feature within the nucleus was a large, dark body

(see Figure 3-3B), which may correspond to the "dense body" postulated to be

Part of the incipient synaptonemal complex (Moens and Rapport, 1971b).

Fiber-like structures that may be synaptonemal complexes (Engels and Croes,1968) were observed in about 20% of the nuclei. These structures were

Observed only at this time point. Also seen only at 2 h were duplicated but

unseparated SPBs which form the meiosis I spindle (see Figure 3-3C).

:: ****

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87

Figure 3-4. Mitochondrial Morphology during Late Stages of Sporulation

A) Several mitochondrial profiles are shown; darkly stained, parallel

structures are shown in the lower right mitochondrion and 'tooth-like'

structures in the left-most mitochondrion. B) A longitudinal section through

a mitochondrion. Two parallel structures, one of which is directly associated

along its entire length with the mitochondrial inner membrane are evident

C and D) In cross-section, the parallel structures are seen as two rows of

multiple 'teeth" which are generally located to one side of the mitochondrion

and occasionally around the entire inner membrane (D). Bars: A and B, 0.2* Geº**.

!!mrm; C and D, 0.1 mm.º

t

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89

By 5 h, only a few cells showed signs of nuclear division and prosporemembrane formation. Vacuoles were no longer observed, but most cellscontained six to eight lipid droplets, which may help to form the prosporemembrane.

By 7 h, many of the cells had spindles and SPBs with a large outer

plaque (see Figure 3-3D). This modified outer plaque marks the second

meiotic division and serves to nucleate prospore membrane formation

(Moens and Rapport, 1971a; Byers, 1981). The leading lip of the prospore

membrane was observed to be bulb-shaped and studded with electron dense

material (see Figure 3-3E-F). Additionally, lipid droplets were observed

aggregated around the prospore membrane (see Figure 3-3G).

At 9 h, growth of the prospore membrane and spore wall were seen to

continue, and the number of cytoplasmic lipid droplets decreased

correspondingly (see Figure 3-3H). By 11.5 h, mature spores with a dark outer

wall became readily apparent (see Figure 3-3I). At both 9 and 11.5 h, the

mitochondrial morphology in the ascal and spore cytoplasm was drastically

altered, with novel tooth-like structures associated with the inner

mitochondrial membrane (see Figure 3-4). This morphological change may

reflect an alteration in the energy-producing requirements necessary for

germination.

Microarray analysis as a genetic screening method

Specific induction of a gene during sporulation presumably reflects a

need for its function in an aspect of meiotic division or spore morphogenesis.

We have characterized three novel genes, each belonging to a different

temporal expression classes: YPRO07, an early gene; YGR225, an early-mid

*

90

gene; and YDR104, a middle gene. In contrast to their strong inductionduring sporulation, their expression is not regulated in a cell cycle-dependent

manner in vegetative cells (P. Spellman, personal communication).

YPR007, an early class I gene, is predicted to encode a protein with

secluence similarities to Scol of S. cerevisiae (Michaelis et al., 1997; see Figure

3-5A) and Rec8 of S. pombe (Molnar et al., 1995). YPRO07 may hold sister

chromatids together specifically during meiosis. Deletion of YPRO07 does not

inn pair vegetative growth, but homozygous YPRO07 cells do not undergo

meitoic division as assayed by DAPI staining and are unable to sporulate. We

designate the gene SPO69, for sporulation microarray.

YGR225 belongs to the early-mid cluster. It has homology to the family

of FIZZY/CDC20 genes of D. melanogaster, S. cerevisiae, and other organisms,which are essential for proteolysis events during mitosis (reviewed byTownsley and Ruderman, 1998; see Figure 3-5B), suggesting that it might be ameiosis-specific activator of APC/cyclosome-mediated proteolysis. We have

fourd that YGR225, now designated SPO70, is not essential for vegetative

&rovth but is required for sporulation. DAPI-staining of SpoZ0-deficient cells*hcler sporulation conditions is abnormal. It is of note that cluster analysisPlaces YGR225 adjacent to APC4, which encodes a known component of the

Proteolysis machinery (reviewed by Glotzer, 1995). (A precise deletion of onlythe YGR225 open reading frame prevents sporulation. However, the adjacent

*Ovel open reading frame, YGR226, is only 27 bp away and is also expressed as

an early-mid gene. Therefore regulatory elements for YGR226 may also be

affected in the YGR225 deletion mutant.)

YDR104 is a novel middle gene with an MSE in its promoter region. It

is predicted to encode a potential transmembrane protein. YDR104-deficient

=_*

=3

* R.

i

91

Figure 3-5. Sequence Alignment

A. Spo69 (YPR007) and ScC1. Spo69 and the sister-chromatid cohesion

protein, ScC1, share homology at both their N- and C-termini (37).B. SpoZ0 (YGR225) and Fzy/Fzr family. SpoZ0 shares homology with the first

two of seven distinctive WD repeats of the FZY/FZR family of ubiquitin

conjugating specificty factors (39). Comparison is shown for YGR225, Cdh1

and Cdc20 of S. cerevisiae, fizzy-related proteins of Drosphila and Xenopus, D

Fzr and X-Fzr, respectively, and Cdc20 of C. elegans. Identical residues are red;

cornserved residues are purple. 2******

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/* {

92

YPROO7 39SCC1 28

95

84

153140

202

200

261

257

3.19

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617

SNKSN-STGNISSSTVKK--KDIVNISIPKTCDEIQNFENDF-SLRYISNLLYGVTICYNSNMSNIPRGSVIQTHIAESAKEIAKAS---GCDD-ESGDNEYITLRTSGELLCGIVRVYS

KKTEYVLNDLNHLLVQLQKNDVYAFKAKNKSTR-INGLNSNNSIIGNKNNNYTEECVFKQATFLLTDIKDTLTKISM----LFKTSQKMTSTVNRLNTVTRVHQLMLEDAVTEREVLV

- - - - -FDDDPLYDITKVPALEFLNTTLQDNVSFIEEAKSIRR----QDYI-NELSNSNR

TPGLEFLDDTTIPVGLMAQENSMERKVQGAAPWDTSLEVGRRFSPDEDFEHNNLSSMNLD

FELH-GDMTNSDAQSNLGSNVRNSFPLDEIPVDVDFNLDLDDIVSHQGTPLGSHSSSQKDFDIEEGPITsKSWEEGTRQSSRNFDTHENYIQDDDFPLDDAGTI---GWDLGITEKXXXX

GNDFKFNY-QGDELVLNFENDNENNSNGGEDTSVENEGPVANLKDYELGL-EAQASEEENXXXXXXSVEQGRRLGESIMS ––EEPTDFGFDLDIEKEAPAGNIDTITDAMTESQPKQTGT

DLQQKL-NTRRRNSKLLNTK

QQNLQQDKT-NFQDVILDYQTKKFYDYIKERSIV-VGRTTRSNPPFKRKMLLVDIIPSRMEENIIDAKTRNEQTTIQTEKVRPTPGEVASKAIvº MAKILRKELSEEKEVIFTDVLKSQ

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* GR225 205SDH1 240SDc 20 23.8P-FZR 150X-FZR 165SE—cDC20 371

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291289

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GEAQTGANFDDVERGVSRQIAASAFLSLLNLATKGMVKLNEYPVADAVTKDLK- - - - - -

ANTEP--ENITKREASRGFFDILSLATEGCIGLSQTEAFGNIKIDAK

QRPAKRVKSHIPYRVLDAPCLRNDFYSNLISWSRTINNVLVGLGCSVYIWSPGKQFRQIAKVPYRVLDAPSLADDFYYSLIDWS-STDVLAVALGKSIFLTD--IKLRKININPERILDAPGFQDDFYLNLLSWSK-KNVLALALDTALYLWNPRKATRKISRIPFKVLDAPELQDDFYLNLVDWS-SQNVLAVGLGSCVYLWSPRKPTRKISKIPFKVLDAPELQDDFYLNLVDWS-SLNVLSVGLGTCVYLWSPRKPIRKVPKNPYKVLDAPELQDDFYLNLVDWS-SQNQLSVGLAACVYLWS

EKEGAVSILDHQYLSEKRDLVTCVSFCPYNTYFIVGTKFGRILLYDNNTGI D - – TENEYTSLSWIGAGSHLAVGQANGLVEIYDATTGDVSLLTD – F – ENTTICSVTAJSDDDCHISIGKEDGNTEIWD

ACTSQVTRLCD––LSPDANTVTSVSWNERGNTVAVGTHHGYVTVººDACTSQVIRLCD––LSVEGDSVTSVGWSERGNLVAVGTHKGFVQIYDATTSQVIKLCDLGQTNEQDQVTSVQWCDKGDLLAVGTSRGVTQIAD

º,*

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93

cells have no vegetative phenotype. Under sporulation conditions, these

mutants undergo both meiotic divisions but do not form a spore wall (datanot shown). The expression kinetics and mutant phenotype of YDR104 are

very similar to that of SPS1, another middle gene (Friesen et al., 1994). We

designate YDR104 as SWM1 (spore wall maturation).

Proposed roles for genes induced during sporulation

One challenge is to understand more specifically what contribution

each of the induced genes makes to the efficiency of sporulation or the fitness

of the resulting spore. Fortunately, the detailed expression profiles tend to

ideratify genes with related functions. This allows us to suggest possible roles

of genes of unknown function by a "guilt-by-association" argument. We next

describe subsets of genes induced during sporulation and propose functionsfor them during the different steps of meiosis and spore formation.

Chromosome behavior in prophase. The meiosis-specific events of

hornologous chromosome pairing and recombination must be coordinatedWith DNA replication and sister chromatid cohesion. We find that genesknown to be involved in these different processes cluster as early genes.

Many of the previously identified early class I genes are involved in

Shromosome pairing and recombination. Examples include ZIP1 and RED1,

Which encode coiled-coil proteins of the synaptonemal complex (reviewed byRoeder, 1997). It is likely that some of the novel early class I genes, for

example, YOR177, which encodes a putative coiled-coil protein, may also beinvolved in chromosome synapsis and meiotic recombination.

Many genes involved in DNA replication (RNR1, CDC21, RFA2, RFA3,

POL1, POL30, MCM3, CDC47, and DBF4; Schild & Byers, 1978) or sister

chromatid cohesion (SCC2, PDS1, PDS5, SMC1, and SMC3) are also induced

=***

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94

early during sporulation. Unlike other genes involved in sister chromatid

cohesion, we find that the mitotic cell cycle-regulated gene, SCC1 (Guacci et

a 1-, 1997), is not induced during meiosis. Because sister chromatid cohesion

must be maintained in anaphase I, the requirements for sister chromatid

cohesion in the first meiotic division differ from those in meiosis II or

mitosis (reviewed by Miyazaki & Orr-Weaver, 1994). It is possible that a

novel sporulation-induced protein, for example, Spo69, or YER106, which

shares homology with the condensin, Smc3, performs this function duringmeiosis.

Exit from prophase. Meiotic prophase ends after recombination with

fully synapsed homologous chromosomes and duplicated but unseparatedSPEs. Mutants lacking Ndt80 arrest at precisely this point, suggesting that

Nolt&O is important for triggering progression from prophase to the firstmeiotic division (Xu et al., 1995). Several putative targets of Ndt80 may be

involved in desynapsis and later steps. For example, UBC9, which is induced

nici-way through sporulation, encodes a functional homolog of theºn armmalian ubiquitin-conjugating enzyme, Hsubcº. This protein has been

implicated in disassembly of the synaptonemal complex during

*Permatogenesis (Kovalenko et al., 1996). We also identified another gene

With homology to ubiquitin-conjugating enzymes, YOR339, to be a middleSPOrulation gene strongly induced by ectopic Ndt80 expression. Therefore,

new transcription at the end of prophase, perhaps mediated by Ndt80, may

initiate desynapsis by stimulating degradation of components of theSyriaptonemal complex.

Meiotic nuclear division: machinery involved in chromosome

distribution. Chromosome distribution during mitosis requires the

95

functioning of cyclin-dependent kinases, motor proteins, and kinetochore

proteins. Genes encoding proteins in each of these classes are induced duringmeiosis.

B-type cyclins, in particular, five of the six CLB genes, are induced just

before cells initiate meiotic division (Chu & Herskowitz, 1998). Genes for

several known and presumptive kinesin-like (Cin8, Kip3, Kar3, and YGL075),

dynein-like (Pac11), and myosin-like (Tid3 and YGR179) motor proteins are

also expressed at this time. These proteins may be involved in chromosome

movement, or possibly in cytoskeletal remodelling, or organelle movement.

Other genes required for chromosome segregation in vegetative cells

(for example, CSE4 and IPL1) are also induced during sporulation. CSE4 codes

for a protein similar to a mammalian kinetochore protein, CENP-A, which is

involved in centromere organization during spermatogenesis (Palmer et al.,1990; Stoler et al., 1995). Cse4 may play a similar role. IPL1 encodes aSerine/threonine kinase essential for chromosome segregation in vegetative

Cells. It is striking that a mammalian homologue of IPL1, AYK1, is induced

just prior to meiosis I (Yanai et al., 1997), similar to the timing of IPL1

**Pression during sporulation.

Anaphase of the meiotic divisions. Exit from mitosis is governed by

Proteolysis mediated by the APC and its specificity factors, Cdc20 and Hot1 (39).We observe that CDC20, HCT1, and many APC components (APC4, APC5,

APC9, APC11, CDC16, CDC23, CDC26, and CDC27) are induced mid-waythrough sporulation, thus implicating APC-mediated proteolysis in exit fromOne or both of the meiotic divisions. Because the meiotic divisions, especially

the first, have important differences from mitosis, regulation of Clbassociated kinase levels must be altered from the mitotic program to allow for

96

consecutive nuclear divisions. It is possible that a novel sporulation-induced

protein, such as the sporulation-specific Cdc20/Hot1 homolog, YGR225, mightalso be required for exit from meiotic division.

We also observe several other genes involved in exit from mitosis,

including CDC14 and CDC5, to be induced during sporulation. A

recTuirement for these genes in meiosis has previously been observed (51),

and a mammalian homolog of CDC5 is reported to be induced specifically

during spermatogenesis (52). Two genes, YDR219 and YLR368, encoding F

box proteins (53) are also induced mid-way through sporulation, raising the

in triguing possibility that an SCF-like ubiquitin-conjugating complex may be

necessary for progression through meiosis.

Spore morphogenesis. During prospore membrane formation, vesicles

normally bound for the plasma membrane are thought to be redirected to the

incipient prospore membrane (Neiman, 1998). In addition to induction of

SPO20, which codes for the sporulation-specific SNAP25 homolog necessaryfor prospore membrane growth (Neiman, 1998), we observe increased

**pression by 7 h of several other genes which may be necessary for vesicle

fusion during sporulation: SEC12, SED4, ROM2, YPT1, YPT10, and YPT32.

Reorganization of the cytoskeleton may also be required to redirect vesicles to

the prospore membrane. To this end, several genes which encode actininteracting proteins (e.g. CDC42, BNR1, SPA2, and PEA2) are observed to beinduced at the time of prospore membrane formation (see Figure 3-3D-F).Their proteins may localize to the leading edge of the prospore membrane, ashas been observed for several of the septins (Fares et al., 1996), and may

comprise the coated bulb-like lip structure (see Figure 3-3E).

97

The two leading edges of each prospore membrane grow around the

nuclear membrane, engulfing organelles and cytoplasm, and eventually fuse

to encapsulate each haploid nucleus (see Figure 3-3F). Genes involved in

membrane fusion (e.g. FLIS2 and KEL2) are induced at this time (7 h) and may

be required for fusion of the two leading membranes. We also observe

induction midway through sporulation of STE5, which encodes a scaffold

protein for the MAP kinase signalling module used during mating (reviewed

by Levin & Errede, 1995). Ste5 may be redeployed during sporulation to hold

together components of the proposed sporulation MAP kinase cascade (see

Friesen et al., 1994; Krisak et al., 1994).

Several genes corresponding to putative GPI-proteins (Caro et al., 1997)

are also induced at the time of spore formation and are candidate components

of the prospore membrane or spore wall. These include the proposed

membrane proteins encoded by SPS2, YCL048, GAS2, and GAS4, and the

proposed cell wall proteins corresponding to SPR2, YPL130, CWP1, and TIR2.

Other potential structural components of the spore wall include

transmembrane proteins such as those predicted to be encoded by YDR104,

YOLO47, YAL018, and YCR061.

Organelle morphogenesis. Changes in organelle structure have been

noted to correlate with prospore membrane encapsulation (Miyakawa et al.,

1984). Based on the corresponding gene expression and cytology data,

candidate proteins for mitochondrial and vacuolar morphogenesis were

identified. Several genes involved in mitochondrial function are induced by

7 h, including the yeast homolog (YBR179) of the Drosophila FZO gene

required for mitochondrial fusion (Hales et al., 1997). In addition, VAM7 and

VAC8, which are required in vegetative cells for vacuolar morphogenesis and

**-ºs.=º

f ºsº

ºf *L-->■

º|l/CS

98

inheritance, respectively (Wada et al., 1992; Wang et al., 1998), are middle

sporulation genes. Their expression may facilitate the synthesis of new

vacuoles in a maturing spore (Roeder and Shaw, 1996).

Transcription factors and divergently transcribed genes. Our

understanding of the sporulation transcriptional cascade comes from

knowledge of two key transcriptional activators (see Figure 3-1), Imel/Ume6,

which activates early genes (reviewed by Kupiec et al., 1997), and Ndt80,

which activates middle genes (Chu and Herskowitz, 1998). The expression

analysis provides candidates for other regulators which may participate in the

transcriptional cascade (see also Mitchell, 1994; Kupiec et al., 1997). For

example, the FKH1 gene is induced early during sporulation. Fkh1 contains a

forkhead DNA-binding domain and is homologous to Meiq, the fission yeast

forkhead protein required for transcription during sporulation (Horie et al.,

1998).

How the mid-late and late genes are transcribed is not known. Middle

genes that encode proteins with DNA-binding domains may be responsible.

These include the gene products corresponding to SPS18, which has a Zn

finger domain (Ireland et al., 1994), YGL183, which has an HMG box, and

YPRO78, which has homology to the C-terminal domain of the transcription

factor, Mbp1.

One mechanism for coordinate transcription of two genes with related

functions is for them to share a regulatory region and be divergently

transcribed. DIT1 and DIT2, which encode enzymes involved in crosslinking

tyrosines in the yeast ascospore wall are regulated in this manner (Briza et al.,

1990; 1994). We identified other pairs of divergently transcribed genes which

are induced during sporulation and whose products may function in the

º

/

-

º

99

same step of meiosis or spore formation: YBR063/YBR064, YHR184/YHR185,

YJL037/YPL038, YOR297/YOR298, YPL033/YPL034, and YPL200/YPL201.

GENOME-WIDE STUDIES OF A DEVELOPMENTAL PATHWAY

Before we were able to study all the genes of yeast simultaneously, we

already knew that gametogenesis in yeast was a developmental pathway and

that there was a transcriptional cascade, and even that Imel and Ndt80 were

key regulators. What we have found here, however, is the degree and extent

Of the regulatory pattern. Literally hundreds of genes turn out to be under the

control of these and additional regulators yet to be studied in detail. We are

now in a position to survey essentially every gene in the genome with a

Particular pattern of expression for cis-acting regulatory sites.

The finding that Ndt80 regulates more than 100 genes induced during

SPOrulation encourages a global view of the developmental pathway beyond

that of a transcriptional cascade. The many steps of sporulation can be seen astwo rnajor phases -- first, meiotic prophase, and second, meiotic division and

8amete morphogenesis -- with Ndt80 controlling entrance into the second

Phase, possibly in response to the meiotic recombination checkpoint (65, 3). Itis tempting to draw parallels with both spermatogenesis (66, 3) and human998;enesis, where a natural arrest between these two stages can last for decades(67).

Our analyses are readily applicable to genome-wide surveys of other

developmental processes, even in systems less experimentally tractable than

Yeast. For virtually any ordered process, potential regulators and sites can be

identified in a similar manner. In addition, analogous genetic manipulationsof key regulators can be carried out by use of ectopic expression or dominant

=sº

sº****sº º

3

s

100

negative derivatives. Our studies also serve as a reminder that multiple

layers of compexity may be uncovered -- for example, we have found that

additional proteins may modulate Ndt80 function (either to assist or inhibit

it) or be functionally redundant with Ndt80.

Last, but not least, the method that we have employed allows us to

assign potential functions for genes heretofore uncharacterized. By relating

expression to detailed knowledge of landmark events (e.g. morphogenesis),

we can make inferences about function. Clustering provides another means

for inference (16). Not only can we find functions for genes but also for

repeated sequence motifs that serve as cis-regulatory sites. Best of all, of

course, one can readily test all of these inferences by making suitable

mutations, as we have done here in several instances.

THE NEXT STEP

We have alluded to the conservation of genes expressed during

meiosis between yeast and mammals, and thus the potential usefulness ofthis analysis in furthering our understanding of gametogenesis in highereukaryotes. It is also vital to point out, however, that half of the genes thatare induced during sporulation do not yet have enough known about them

even to have names. Little is known about the details of spore

morphogenesis and the biology of the spores themselves. In this respect, use

of microarray analysis as an unbiased genetic screen should prove useful as aSource of candidate genes that may play roles in these processes. Further

characterization of these genes must be approached with careful observationand an open mind. To facilitate the full exploitation of the observationsreported here, we have provided on-line both the primary expression data for

=_sºprº

.ºS|-|

101

all yeast genes during sporulation (http://rana.stanford.edu/sporulation) and

the cluster analysis of these data (http://www.sciencemag.org).

Materials and Methods

RNA Preparation

Cells from the wild-type SK1 background, YSC328 (a/o ura■ leu2 lys2

ho::LSY2), or the Ndt80-deficient SK1 background, YSC330 (a/o ura■ leu2 lys2ho:::LYS2 ndt&0::LEU2) were transfered to sporulation medium (SPM) at t=0using the protocol described in Chu & Herskowitz (1998) to maximize the

Synchrony of sporulation. For YSC328, RNA was harvested at time t = 0, 0.5,2, 5, 6, 7, 9, and 11.5 hours after transfer to SPM. (RNA from t=0.5 h was

harvested from a separate time course using the same strain background andidentical experimental conditions.) For YSC330, RNA was harvested at 0, 2,

and 6 h after transfer to SPM. Polyadenylated [poly(A)t) RNA was prepared byPurification with an oligo (dT) cellulose column as described in (DeRisi et al.,1997.

Cells from the wild-type W303 background (a ura■ leuz his3 trp 1)

Containing either the GAL1-10 promoter integrated at UIRA3 (YSC552) or the

GAL1-10 promoter fused to HA-tagged NDT80 (YSC553) were induced with17% galactose for three hours as described in Chu & Herskowitz (1998) beforeRNA was harvested. [Poly(A)t) RNA was prepared following the protocol ofRapid RNA Isolation from Invitrogen(3).

9.

.| --

S.-º

*

102

Northern Analysis

Aliquots from each time point were assayed by Northern analysis

following the protocol described in Chu & Herskowitz (1998). Primers from

Resgen'8) were used to make probes corresponding to the open reading frames

of DMC1, SPS4, DIT1, and SPC100. RNA expression patterns as assayed by

microarray analysis were confirmed by Northern analysis for representative

genes, including YAL018 and YDR104.

Microarray Analysis

Microarray analysis of [poly(A)*] RNA was carried out as described in

DeRisi et al., 1997.

NMeme Analysis

An unbiased search for motifs in each of the temporal classes using

NMENME (68) resulted in identification of sequence motifs matching the

Previously MSE and URS1 sites (Chu et al., 1998; Hepworth et al., 1995;

Ozsarac et al., 1997, Buckingham et al., 1990). For Figure 3-2, differing degreesof degeneracy, based on the output of MEME, were used to search thePromoter regions (600 bp upstream of the presumptive translation initiation

Sodon, on both strands) for each gene. The most degenerate sequence used forthe NMSE was 5 HDVKNCRCAAAWD, where letters refer to IUPAC codes for

PNA nucleotides; the least degenerate consensus sequence was 5'

HDVGNCACAAAAD. Likewise, for the URS1 sequence, the most degenerateSequenced was 5' DSGGCGGC; the least degenerate was 5 TCGGCGGCTDW.

MEME is available on the World Wide Web at http://www.sdsc.edu/MEME

Clustering AnalysisGenes included in the clustering analysis were chosen by the following

criteria (Eisen et al., submitted: The standard deviation of the log expression

~sº

ºsº **

grºw****

*

º

103

value exceeded 1.62 between any two timepoints for a given gene. For any

given measurement to be considered, the correlation coefficient, on a per

pixel basis, must have exceeded 0.2.

Microscopy

During the sporulation time course of YSC328, samples of cells were

prepared every 30 min for microscopy. Cells were examined by Nomarski

and fluorescent microscopy as described in (3). For electron microscopy, cells

were immediately fixed in 4% glutaraldehyde (in PBS, pH 6.7) for 1-2 h. Fixed

cells were then rapidly frozen in 20% glycerol in liquid nitrogen and held at

–70°C for 48 h. At time of use, cells were thawed on ice and processed for

electron microscopy as described by B. Byers and L. Goetsch, J. Bacteriol. 124,511 (1975).

Gerne DisruptionsGenes corresponding to YDR104, YPRO07, and YGR225 were deleted by

Precise disruptions of each open reading frame with the hisG-LIRA3 cassette.NAethods were from M. Rose, F. Winston, P. Hieter Methods in Yeast Genetics

(Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press) (1990); R.Rothstein, Meth. Enzymol. 194, 281 (1991); J. Sambrook, E. Frisch, T. Maniatis,

Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring

Harbor, NY. Cold Spring Harbor Laboratory Press) (1989); and F. Sherman, G.

Fink, J. Hicks, Methods in Yeast Genetics: A Laboratory Manual (Cold Spring

Harbor, NY: Cold Spring Harbor Laboratory Press) (1986).

AcknowledgementsWe are indebted to Mike Eisen for his advice on the clustering analysis,

and Vishy Iyer for help with genome array production. We also thank Paul

104

=sº

sºº

.ºº

º *

º- 1

Spellman and Sue Biggins for their help; Flora Banuett, Nancy Kleckner, and

Daryl Meling for useful discussion; Breck Byers, Sue Klapholz, Yona Kassir

and Aaron Mitchell for comments on the manuscript. Strains of the SK1

background and the hisG-LIRA3 disruption cassette were kind gifts from

Nancy Kleckner. S.C. and I.H. are supported by NIH grant AI18738. J.D.R. was

supported by the HHMI and NIH grant HG00450. P.O.B. is an Associate

Investigator of the HHMI. J.M. and D.B. are supported by NIH grantGM4.6406.

2rºº ºsº

->pesº

3

105

CHAPTER FOUR

CONCLUSIONS

g

106

Characterization of Ndt80 and the transcriptional program of

sporulation has led to a deeper understanding of how sporulation is regulated

at both the molecular level -- based on our comprehensive analysis of gene

expression during sporulation-- and at the organismal level in the context of

gametogenesis in other eukaryotes. Before we began our studies, it was

known that the transition from meiotic prophase to the first meiotic division

was a carefully monitored event involving checkpoint proteins such as Radl?

(Lydall et al., 1996), and was likely to depend on Cdc28 (Shuster & Byers, 1984).

My studies have synthesized these two lines of study by showing that Ndt80 is

required for the transcriptional activation of the regulatory subunits of Cdc28

during sporulation and may be a target of the meiotic recombinationcheckpoint.Gametogenesis is a two-stage process

Based on microarray analysis, we have shown that approximately 500

Senes are induced during sporulation, about three times the previous

estimate of others (Burns et al., 1994). We found that Ndt80 is a major

regulator of over 100 of these genes which are expressed midway through

SPOrulation. This finding has led us to the view that sporulation is a two

Stage process, in terms of both cytological events and transcriptional

regulation. Many of the genes responsible for events of meiotic prophase,

Such as homologous chromosome pairing and recombination, are activated

by the transcription factor, Imel. Subsequently, many of the genes responsible

for events of meiotic division and spore formation are regulated by Ndt80

(See Figure 4-1). This two-stage format minimizes the chances of progression

107

=º 3-

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º

*º|º -

-

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º

-- a

Figure 4-1. The Two Stages of Gametogenesis

Gametogenesis can be divided into two stages: meiotic prophase and

meiotic division. In sporulation, two key transcription factors, Imel and

Ndt80, regulate these two stages step and constitute a transcriptional cascade.

A checkpoint monitors the status of recombination intermediates before

allowing passage from the first to the second stage. In spermatogenesis, there

are also two key stages. Progression into the second may also be under a

recombination checkpoint. Oogenesis is also divided into two stages which

are separated by a natural arrest point in late prophase. However, oogenesis

differs from sporulation and spermatogenesis in several respects. First,

external cues (i.e. progesterone), instead of internal monitoring (i.e. meiotic

recorrubination checkpoint), trigger progression into the second stage. Second,

the transition between stages involves translational control of the kinase,Mos, which initiates a MAP kinase signalling cascade. Third, the meioticdivisions themselves result in an asymmetrical distribution of cytoplasmicTrlaterial.

108

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SPORULATIONSPERMATOGENSISOOGENESISSTAGEONE:Imel2 MIOTICPROPHIAG TRANSITION:recombinationnatural LAPºCPC-WACScheckpointrecombination point

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*,–ºs*-*-L--Yº—6.y-2:º*

--->º-

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into meiotic divisions before recombination is complete, which would be a

lethal event. We propose that the molecular mechanism for such regulation

during sporulation is to place Ndt80 activity under control of the meiotic

recombination checkpoint.

Gametogenesis in general can also be viewed as a two-stage process.

Spermatogenesis shares many parallels with sporulation -- both are

continuous differentiation processes in which a wave of transcription occurs

just prior to meiotic division (Iino et al., 1995; Lin et al., 1996; and Chu &

Herskowitz, 1998). In both, meiosis yields four gametes, each of which are

packaged and functional. A striking similarity is that in each process mutants

exist which cause an arrest at pachytene (see Figure 4-2). We propose that

there may also be internal monitoring of recombination in spermatogenesis.

Oogenesis, although mechanistically distinct from either sporulation

or spermatogenesis, can also be divided into two stages with a natural arrest

point between meiotic prophase and the meiotic divisions. This inherent

arrest period gives the oocyte time to grow and also to repair any accumulated

DNA damage due to incomplete recombination. Therefore, an internal

checkpoint may not be necessary to monitor meiotic recombination during

oogenesis. In this case, progression into the second stage of oogenesis is

under control of external cues. A burst of progesterone secreted by cells

surrounding the oocytes triggers translation of the kinase, Mos, which

initiates meiotic maturation (see review, Page & Orr-Weaver, 1996).

Thus there is a striking parallel among these three forms of

gametogenesis. Our work provides a molecular explanation for the transition

s

110

Figure 4-2. Parallels between Sporulation and Spermatogenesis

There are several parallels between sporulation and spermatogenesis.

Both are continuous processes in which the meiotic divisions are

coordinately regulated with gamete formation. One level of this regulation is

transcriptional (White-Cooper et al., 1998; Chu & Herskowitz, 1998). In

addition, in both processes mutations which trigger an arrest at the end of

meiotic prophase (see review Clancy, 1998; Sassone-Corsi, 1997). In yeast, this

arrest is dependent on the meiotic recombination checkpoint of which Ndt80

activity may be a target. It remains to be determined whether this arrest is

checkpoint-regulated in spermatogenesis.

3

7/7 1,

* Q_*

111

SPORULATION checkpoint –P- Ndt80

YeastCellin MeioticProphase SPERMATOGENESIS checkpoint? -D-Primary Spermatocyte

waveof

\}\}\}!Lllj:\i\\\|

Spermatozoa

É

into the second stage of sporulation and may also provide a framework for

understanding the analogous event in spermatogenesis. In yeast, a

demarcation between the two stages has also been functionally defined as the

point of commitment to sporulation (reviewed by Kupiec et al., 1997). It will

be of interest to determine what role, if any, Ndt80 might have in

commitment and whether analogous functions exist in spermatogenesis. For

example, one reason why a mutation in one of several genes results in an

arrest in pachytene of spermatogenesis (reviewed by Sassone-Corsi, 1997) is

that each gene may be involved in activation of a "commitment" molecule

which drives the spermatocyte into the second phase of meiosis. Without the

necessary function of these genes, progression cannot continue.

Additional characterization of the sporulation transcriptional cascade

Our analysis of gene expression during sporulation is only the first step

to understanding the regulation of sporulation. As with any experiment, the

time course should be repeated with improvements -- such as better

sporulation synchrony, shorter time intervals, and using control

backgrounds, such as an a■ a diploid strain. Although Imel and Ndt80 appear

to be the major activators, other regulators must be involved. Candidate

proteins, for example, and potential regulatory sites, for example, have been

identified. The next step is to determine the exact role of these proteins and

sites by examining the sporulation efficiency and transcriptional kinetics in

cells mutated for such genes or sites. Classical biochemical means can be used

to identify proteins which bind to the identified sites.

.º º --

=

3

-7.

113

Molecular characterization of meiosis and spore formation

Returning to the original question of how meiosis differs from mitosis

at the molecular level, our preliminary studies provide some leads as to what

these distinctions might be, at least at the level of regulated expression (see

Figure 4-3). First, novel genes are expressed during meiosis (for example,

HOP1 and NDT80) which may be required to carry out the unique steps of

meiosis, such as synaptonemal complex formation, and the coordination of

meiotic divisions and spore morphogenesis. Second, vegetatively expressed

genes are redeployed at precise times during meiosis. This may account for

certain differences between the two processes. For example, specific

expression at the end of meiotic prophase of CLB5, which codes for the B-type

cyclin involved in initiating DNA synthesis, might actually serve to inhibit

re-replication between the two meiotic divisions. Third, expression of

meiosis-specific homologs of known vegetative genes may modify the basic

cell cycle machinery to accommodate meiotic events. For example, Spo69

may replace the function of Scol in sister chromatid cohesion in anaphase I.

It will be of interest to assay sister chromatid cohesion in Spo69-deficient cells

under sporulating conditions to determine whether sister separation occurs.

As another example, SpoZ0 may alter the timing or levels of cyclin

destruction during the meiotic divisions. It will also be of interest to follow

the levels of the B-type cyclins (or Pds1) in SpoZ0-deficient cells under

sporulating conditions. Additional characterization of these and other novel

genes identified by our comprehensive screen of gene expression during

sporulation seems likely to further our understanding of meiosis.

114

Figure 4-3. Meiosis is a Specialized Form of Mitosis

We have found one major difference between meiosis and mitosis to

be at the level of regulated gene expression. Taking advantage of this

observation, we and others have identified candidate genes which may be

responsible for the molecular differences between meiosis and mitosis. First,

there are many novel genes, such as NDT80, which are expressed only during

sporulation. These are candidates for participitating in the unique steps of

meiosis (such as meiotic recombination, synaptonemal complex formation,

reductional division, and coordination of meiosis with spore

morphogenesis). Second, certain vegetative genes, such as CLB5, are also

expressed during meiosis, but under different regulatory control. The timing

of their expression during meiosis, may influence their function. Third,

meiosis-specific homologs of vegetative genes, such as SPO69 and SPO70, may

be required for modifying the basic cell cycle machinery for meiotic division.

Fº*

*-

pºss

à

3

115

(s) (e)Prophase ~ 2^ G1

SPO69 is 3

116

APPENDIX ONE

Clb2 CAN FUNCTIONALLYSUBSTITUTE FOR C1b1

IN MEIOSIS

3

117

Abstract

In order to test whether Clb1 has a meiosis-specific role which cannot

be substituted functionally by another B-type cyclin, Clb2, I ectopically

expressed Clb2 during sporulation by placing it under control of CLB1

regulatory regions. I found that when Clb2 is expressed during meiosis in

such a manner, it can functionally complement the absence of Clb1. These

observations indicate that Clb1 and Clb2 have overlapping activities whichcan function in both the mitotic and meiotic divisions.

Introduction

Clb1- and Clb2-associated kinase activities have different relative levels

in meiosis and mitosis. In mitosis, Clb2 is the predominant B-type cyclin

(Surana et al., 1991), whereas in meiosis, Clb1 is the predominant cyclin

(Grandin & Reed, 1993). A molecular explanation for this difference in kinase

levels is that CLB1 but not CLB2 message is induced during meiosis (Chu and

Herskowitz, 1998).

One mechanism which might distinguish meiosis and mitosis is

differential recognition by the basic cell cycle machinery of appropriate targets.

This specificity might come from meiosis-specific expression of a given target.

However, the observation that CLB1 and CLB2 are differentially expressed

during meiosis suggests that specificity might also come from the cell cycle

machinery itself. I wondered whether Clb1 and Clb2 might not be

functionally redundant, but instead that Clb1 might have a meiosis-specific

function which could not be performed by Clb2 (see Figure A1-1). If

Clb1/Cdc28 but not Clb2/Cdc28 recognizes a meiosis-specific substrate, this

predicts that Clb2 would be able to functionally substitute for Clb1 in meiosis.

118

Figure A1-1. Models for Clb1 and Clb2 Functions in Meiosis

In vegetative cells, both CLB1 and CLB2 are expressed (Fitch et al., 1992).

Although Clb2 is the predominant B-type cyclin, Clb1 and Clb2 are

functionally redundant (Surana et al., 1991). Either Clb1/Cdc28 or Clb2/Cdc28

can recognize a proposed mitotic s for Appendix Oneorulating cells, only

CLB1 and not CLB2 message is induced (Chu & Herskowitz, 1998). In Model

1, Clb1/Cdc28 has a specific function, such as recognition of a meiosis-specific

substrate, Z, which cannot be carried out by Clb2/Cdc28. In Model 2, Clb1 and

Clb2 are can both function in meiosis, as in mitosis. Both Clb1/Cdc28 and

Clb2/Cdc28 complexes can recognize Z.

3

119

VEGETATIVE CELL

Clb1 and Clb2 can both function in mitosis

GTOs

SPORULATING CELL

MITOSIS

Model 1: Clb1 has a meiosis-specific role

Gºº –P- MEIOSIS

CLB2@

Model 2: Clb1 and Clb2 can both functionin meiosis

GºOr 2% N. MEIOSIS

CLB2 @

Or ;

120

Precedent for a meoisis-specific function of a differentially-expressed isoform

comes from work with O-tubulin during spermatogenesis. The mitotic and

meiotic isoforms of O-tubulin, O.84B and oS5E, respectively, are 98% similar.

However, 0.85E is not functionally equivalent to 084A, even when expressed

under the control of the O.84A regulatory region (Hutchens et al., 1997).

I have carried out some initial experiments which suggest that Clb2 can

in fact function during meiosis in place of Clb1.

Materials and Methods

Plasmids constructions

Two constructs were made in which the open reading frames for either

CLB1 or CLB2 were placed under control of the 5' and 3' regulatory regions of

CLB1 (see Figure A1-2). pSC429 (abbreviated 5 CLB1-CLB1-CLB13) refers to

the construct containing the 5' CLB1 promoter followed by the CLB1 open

reading frame and the CLB1 3'UTR region. pSC446 (abbreviated 5 CLB1

CLB2-CLB13) refers to the construct containing the 5' CLB1 promoter

followed by the CLB2 open reading frame and the CLB1 3'UTR region.

pSC429 and pSC446 were generated by PCR amplification of yeast

genomic DNA, following the conditions recommended by Boehringer

Mannheim for use of Expland Polymerase(B). Oligos OSC74 and OSC216 (see

Table A1-1) were used to amplify 675 bp of the CLB1 promoter. This construct

was then dropped into a pKS305 vector (Sikorski and Hieter, 1989) digested

with Not■ and Xbal to generate pSC385 (5'CLB1 promoter in pRS305). The

open reading frames for CLB1 and CLB2 were then amplified with

OSC77/OSC217 and OSC83/OSC232, respectively. The PCR fragment

corresponding to CLB1 was inserted into pSC385 by cutting with Xbal and

2--a

º

;

;

121

Figure A1-2. CLB1 and CLB2 under Control of CLB15 and 3' Regulatory

Regions

Plasmids (pSC429 and pSC446) were constructed by inserting the CLB1

or CLB2 open reading frames, respectively, under the control of 675 bp of the

CLB1 promoter and 845bp of the CLB1 3'UTR.

-)*

122

YIJL[],8IºITO

■■■■■■■■ ***A

***\f\f\r\dIgTO,G

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LAITO-ZATO-LATO„G:9ï■ oSd

YIJL[]■ 8IºITO

NÊNI■ T■ T■19

LATO-LATO-LATO„G:6Z■ OSd

123

BamhI to generate pSC400 (5 CLB1-CLB1). The PCR fragment corresponding

to CLB2 was digested with Nhel and BamhI and inserted into pSC385 cut

with Xbal and BamhI to generate pSC401 (5 CLB1-CLB2). 845bp of the CLB1

3'UTR region was amplified using OSC78/OSC141. This PCR product was

dropped into the BamhI and XhoI site of pSC400 and pSC401, respectively, to

generate pSC429 and pSC446 respectively. pSC429-5 and pSC429-6 refer to

different minipreps 5 and 6, respectively. pSC446-4 and pSC446-7 indicate

minipreps 4 and 7, respectively. DNA manipulations were performed as

described in Sambrook et al. (1989).

Strain construction

pSC429 and pSC446 were integrated into YSC007 and YSC699 (see Table

A1-2) at the LEU2 locus by digesting with BstEII, as described in Rose et al.

(1990). It should be noted that the transformation efficiency of YSC007 was

very low. (About 16 integrants were obtained per transformation. This may

have been due to reduced BstEII activity.) In contrast, for transformation of

YSC699, BstEII was shown to be active, and a normal integration frequency

was obatined. The sporulation efficiencies of three to four transformants

were tested for each experiment.

Results

To test whether Clb2 can functionally substitue for Clb1 in meiosis, I

constructed a plasmid, pSC446 (5'CLB1-CLB2-CLB13), containing the CLB2

open reading frame under control of the 5' and 3' regulatory regions of CLB1.

This promoter region includes all three of the putative MSE motifs

implicated in regulating middle gene expression for CLB1 (Chu and

Herskowitz, 1998). In parallel, a control plasmid, pSC429 (5 CLB1-CLB1-CLB1

:

124

3), was also made which contained the CLB1 open reading frame under

control of the same 5' and 3' regulatory regions.

Ectopic expression of Clb2 does not affect sporulation

I first wanted to test whether ectopic expression of CLB2 might affect

the ability of a wild-type cell to progress through sporulation. If Clb1 and Clb2

have activities specific to meiosis and mitosis, respectively, then the presence

of Clb2 might interfere with normal meiosis. However, I found that

expression of Clb2 did not affect progression through sporulation (see Table

A1-3). The sporulation efficiency of wild-type cells (YSC007) expressing either

pSC429 or pSC446 is normal.

Clb2 can functionally substitute for Clb1 in sporulation

I next wanted to test whether ectopic expression of Clb2 could

functionally complement for the role of Clb1 in sporulation. Although a

Clb1-deficient strain in the W303 background has no sporulation phenotype,

Clb1 is required for efficient tetrad formation in a strain also lacking Clb3 and

Clb4 (Dahmann & Futcher, 1995). A wild-type strain expressing pKS305 forms

tetrads at 47% efficiency (line 1, Table A1-4). In contrast, a clb1 clb3 clb4 strain

does not form any tetrads, but does produce monads and dyads (line 2, Table

A1-4), presumably because only one of the two meiotic divisions are

completed (Dahmann & Futcher, 1995). Ectopical expression of Clb2 under

control of the CLB1 regulatory region in a clb1 clb3 clb4 background restores

the sporulation efficiency to greater than 50% (lines 5-6, Table A1-4).

Unfortunately, the control construct of CLB1 expressed under its own

regulatory region failed to complement in the clb1 clb3 clb4 background (lines

3-4, Table A1-4). This failure is most likely due to the introduction of a

mutation in the CLB1 open reading frame during PCR amplification.

:

125

Discussion

My initial findings that ectopic expression of Clb2 can functionally

complement for Clb1 in meiosis suggest that meiosis does not require the

specific activity of the Clb1-Cdc28 complex and favors Model 2 of Figure A1-1.

Molecular specificity for progression through meiosis may instead be at the

level of expression of a meiosis-specific substrate.

In addition to repeating the work presented above, several additional

experiments should be done to complete this analysis. First, a construct of the

CLB1 open reading frame under its own regulatory regions should be remade

and shown to functionally complement tetrad formation of a clb1 clb3 clb4

strain. Second, the ectopic expression of CLB2 during meiosis in both wild

type and clb1 clb3 clb4 strains should be verified using antibodies specific to

Clb2 protein.

Besides CLB1 and CLB2, there are other examples of isoforms with

opposite expression levels in meiosis and mitosis. For example, genes

encoding the kinase homologs, Dbf2 and Dbf20, are oppositely expressed in

mitosis and meiosis, and may not be functionally redundant. In vegetative

cells, DBF2 levels are cell cycle-regulated, peaking at the G2/M transition,

whereas DBF20 levels remain low and constant (Toyn et al., 1991). During

sporulation, DBF20 levels are induced midway through, whereas DBF2 levels

remain low and unchanged (Chu, DeRisi, et al., 1998). Dbf20 interacts with

another protein, Spol2, and each is essential only in a dbf2A background

(Malavasic and Elder, 1990; Toyn and Johnston, 1993). It has been proposed

that Spol2 and Dbf20 together carry out an essential function that is

redundant with one for Dbf2 (Toyn and Johnston, 1993). Although Spol2 is

dispensible in vegetative cells (which have high Dbf2 levels), a spo12/sp012

:

i:

126

diploid can only undergo a single meiotic division (Klapholz and Esposito,

1980). One explanation for this differential necessity for Spol2 in mitosis

versus meiosis is that Dbf2 and Dbf20 are not functionally redundant. In the

presence of Dbf2, Spol2 is not required. But in the absence of Dbf2 function --

such as in vegetative dbf24 cells (Toyn and Johnston, 1993), or wild-type

sporulating cells -- Spol2, presumably together with Dbf20, has an essential

function in exit from nuclear division. Experiments similar to ones proposed

in this appendix may further clarify whether other developmentally

expressed isoforms are functionally redundant.

:

127

Table A1-1. Oligonucleotides used to generate plasmids for Appendix One

OSC74 Not■ site 675 bp upstream of CLB1 promoter region5'GCC ACT ATG CGG CCG CTC ACT ACC GTTTTC GAG TAG GGG CTCTCACAA AAC G3'

OSC77 BamhI site at the end of the CLB1 open reading frame (noncoding strand)5'CGC GGA TCC CTC ATG CAA TGT CAT AAT ATCATA TCC3'

OSC78 BamhI site at the end of the CLB1 open reading frame (codingstrand)5'CGC GGA TCC TGA AGG TGC TAG CCT ACA CAG AAA ACC3'

OSC83 BamhI site at the end of the CLB2 open reading frame (noncoding strand)5'CGC GGA TCC TTC ATG CAA. GGT CAT TATATCATA GCC G3'

OSC141 XhoI site 845bp downstream of the CLB1 open reading frame(non-coding strand)5'CCG ACT ACT CGA GTC ATC TGC CTG TTC ATT GCC3'

OSC216 Xbal site at the start of the CLB1 open reading frame (non-coding-

strand)5'GCC GCG TCT AGA TAT GAG AAG ATT AAA GCTTCCTTT GAT GG3'

OSC217 Xbal site at the start of the CLB1 open reading frame (codingstrand)5'GCCTCT AGA ATG TCA CGA TCC CTTTTG G3'

OSC232 Nhel site at the start of the CLB2 open reading frame (codingstrand) 5'GCC AGAGCT AGC ATG TCC AACCCA ATA GAA AAC 3'

Table A1-2. Yeast Strains for Appendix Onestrain mating strainnumber type relevant genotype backgroundYSC007 a/O. W303

YSC699 a/O. Clb1::UIRA3 clb3:TRP1 clb4::HIS3 W303The W303 genotype is ade2-1 can1-100 leu2-3,112 his3-11,15 ura■ trp1-1 psi

s

i

i

128

Table A1-3. Clb2 Does Not Affect Progression through SporulationIn O total # cells

Spores dyads tetrads countedYSC007 58% 4% 38% 203

ySC007 + pSC429 (5 CLB1- CLB1- CLB13)45% 3% 52% 200

YSC007+ pSC446 (5 CLB1-CLB2-CLB13)44% 3% 53% 200

Table A1-4. Clb2 Can Functionally Complement for Clb1 during SporulationInO total # cells

Spores monads dyads tetrads counted1. YSC007 4 pKS305 49%

-4% 47%. 303

2. YSC699 + pKS305 58% 18% 24%-

403

3. YSC699 + pSC429-5.68% 6% 26%-

4004. YSC699 + pSC429-6 63% 13% 24%

-400

5. YSC699 + pSC446-434% 1% 9% 55%, 3006. YSC699 +pSC446-7 23%

-13% 64% 200

.i

129

APPENDIX TWO

POTENTIAL REGULATORS OF NDT80:

Ime4, Ime2 AND Ids2

.

130

Abstract

Ime4 has been implicated to have a function at approximately the same

time of meiosis as Ndt80. To characterize a possible role for Ime4 in

regulation of Ndt80 activity, Ndt80 function was assayed in Ime4-deficient

strains under sporulation conditions. In the absence of Ime4, only a basal

level of NDT80 message is transcribed and CLB1 is not induced. We observed

that expression of NDT80 in vegetative cells is sufficient to induce

transcription of IME2. These results suggest that Ime4 is required for Ndt80

function and that Ndt80 may be part of an Ime2-dependent positive feedback

loop for early gene expression.

Introduction

Both Ime4 and Ime2 function in transcription of the early sporulation

genes (reviewed by Kupiec et al., 1997). Ime4 is essential for sporulation and

is required for accumulation of IME1 transcript (Shah & Clancy, 1992; see

Figure A2-1). IME2 encodes a meiosis-specific serine-threonine protein

kinase and is itself an early gene (Yoshida et al., 1990; Kominami et al., 1993).

Once synthesized, Ime2 can replace Imel function in activating early gene

expression, possibly by phosphorylating and activating an unknown

transcription factor (Bowdish and Mitchell, 1993).

Intriguingly, both Ime4 and Ime2 have other functions in addition to

early gene transcription. Ime4-deficient cells manipulated to synthesize Imel

(either through a dominant RES1-1 mutation or by deletion of RME1) do not

progress through sporulation but instead arrest at pachytene (Kao et al., 1990;

Shah & Clancy, 1992). Ime2 is required for middle gene expression, perhaps

:

131

Figure A2-1. Initiation of Sporulation

Diploid a/o cells under starvation conditions initiate the program of

sporulation by inhibiting expression of RME1. The absence of Rmel, in

conjunction with the presence of Ime4, induces IME1 expression. Imel is the

key transcription factor for the early genes, which include IME2. Once

synthesized, Ime2 regulates expression of both early and middle genes.

Expression of NDT80, the key transcription factor for the middle genes, is

dependent on both Imel and Ime2.

.

132

Ime4p

starvationT--Y a1-02—RME1—IME1

Ime1p/ Ume6p

==IME2/

EARLYGENESIME2\-HOP1HOP1 SPO11SPO11 REC114 NDT80?NDT80—-Ndt80p ***---

!

MIDDLEGENESSPS1 CLB1

g

indirectly via the IDS2 gene product, which is required for Ime2 activation of

middle but not early genes (Sia and Mitchell, 1995). Thus Ime4, like Ndt80,

is necessary for progression through pachytene (Xu et al., 1995). Ime2 and

Ids2, like Ndt80, are necessary for middle gene transcription. I have found

that Ime4 is necessary for Ndt80 transcriptional activity and that Ndt80 itself

can activate IME2 expression.

Materials and Methods

Yeast strains

Yeast strains (see Table A2-1) of the MCD1 and W303 background were

grown and sporulated according to the protocol of Shah & Clancy (1992) and

Chu & Herskowitz (1998), respectively.

RNA analysis

Northern analysis was done following the protocol of Chu &

Herskowitz (1998). RNA samples were taken at t=0, 3.75, 8, 9.5 and 11 h in

sporulation medium. Microarray analysis was done following the protocol of

DeRisi et al. (1997).

Results

Ime4 is necessary for Ndt80 activity

To assay whether Ime4 is required for Ndt80 function, we first

determined whether NDT80 is expressed in the absence of Ime4. Strains of

the MCD1 background were sporulated, and RNA samples were periodically

harvested over an 11 hour time course. In a wild-type strain undergoing

sporulation, NDT80 message is first detectable by 3.75 hours and then

increases in expression by 8 hours (see Figure A2-2). In an ime4 background,

:

134

Figure A2-2. Ime4 is Necessary for Ndt80 and NDT80 Transcript Levels

Homozygous, a/O diploid yeast strains were placed under sporulation

conditions: wildtype (+/4; YSC912), ime4 RES1-1 (YSC916), RES1-1 (YSC913),

and ime4 (YSC915). RNA was periodically harvested, and equal amounts

were loaded on a gel for Northern analysis. Vertical bars demarcate each time

point. The first and last bars represent t=0 and 11 h in sporulation medium,

respectively. The intermediate time points are 3.75, 8, and 9.5 h. Probes used

were NDT80 and CLB1. TCM1 served as a loading control. After each

hybridization and exposure, the blot was stripped following the protocol ofChu & Herskowitz, 1998.

|:

135

-------

|||

||||||||||||

||1|1—]

WAISu■UILI

||||

|

■ aul■ L-IS3IXIL-IS3IXI■ ºlu■ +/+

136

NDT80 message is not detectable. This result was expected since in the

absence of Ime4, IME1 cannot accumulate, and therefore NDT80 is not

transcribed (Shah & Clancy, 1994; Chu & Herskowitz, 1998). However, we

found that in an ime4 RES1-1 double mutant background, a basal level ofNDT80 is now detectable. RES1-1 is a semi-dominant mutation which allows

full levels of Imel expression (and therefore sporulation) to occur even when

Rmel is present (Kao et al., 1990). In a RES1-1 strain background, NDT80 is

expressed with kinetics and induction levels comparable to a wild-type strain.

Thus Ime4 is not required for initial transcription of NDT80, but is necessary

for further induction of NDT80 transcription.

Transcription of NDT80 appears to be under control by a positive

feedback loop. Ndt80 can act on MSE sites in its promoter to up-regulate its

own expression (Chu & Herskowitz, 1998). Thus the observation that Ime4 is

required for maintenance but not initation of NDT80 transcription suggests

that Ime4 may be required for Ndt80 activity itself. To test this hypothesis,

Ndt80 activity, as assayed by CLB1 transcription, was also examined (see row 2,

Figure A2-2). Although CLB1 is expressed in the RES1-1 background, it is not

transcribed in the ime4 RES1-1 double mutant background. This observation

is consistent with a requirement of Ime4 for Ndt80 transcriptional activity.

Synthesis of Ndt80 is sufficient to induce IME2 expression

Ectopic expression of Ndt80 in vegetative cells results in increased

expression of many genes, many of which are middle genes (Chu, DeRisi et

al., 1998). We found that synthesis of Ndt80 induces IME2 message by greater

than six-fold as assayed by microarray analysis (Chu, DeRisi et al., 1998).

Similar results were also obtained by Northern analysis (see Figure A2-3). We

searched the promoter region of IME2 and found an Ndt80-binding site

137

Figure A2-3. Ndt80 Induces Transcription of IME2

Asynchronously growing strains, YSC552 (pGAL) and YSC554 (pGAL

NDT80), were grown in the presence of galactose. RNA was harvested and

equal amounts loaded on a gel for Northern analysis. Blots were probed withIME2 and TCM1.

138

08LCIN-TV5)d-TV5)d–

eN[I]>|-

TCM1 e

139

located 552 bp upstream of the IME2 open reading frame. Ndt80 may

recognize this MSE and induce IME2 expression. It remains to be determined

whether the induction observed in vegetative cells also occurs during

sporulation. The data from microarray analysis for IME2 do, in fact, show a

second boost in IME2 expression midway through sporulation (Chu, DeRisi et

al., 1998). Although the largest induction of IME2 message occurs between t=0

and t=0.5 h (from a red-to-green ratio of 0.67 at t=0, to 21.8 at 0.5 h), there is

still almost a two-fold induction from 13.9 at 2 h, to 24.8 at 5 h. It is possible

that this second induction at 5 h is Ndt80-dependent.

Discussion

Ime4 is necesssary for further induction but not initiation of NDT80

transcription. Since Ndt80 is capable of stimulating its own transcription in a

positive feedback loop (Chu & Herskowitz, 1998), Ime4 may be required for

Ndt80 function as a transcriptional activator. Consistent with this hypothesis,

I have found that CLB1 is not expressed in the absence of Ime4. Ndt80 activity

is also dependent on the meiotic recombination checkpoint (Chu &

Herskowitz, 1998). It remains to be determined whether these similar

requirements of completed recombination and Ime4 are overlapping or

independent. We have developed a simple assay for Ndt80 autoinduction:

ectopic synthesis of Ndt80 in vegetative cells induces transcription of a tagged

form of Ndt80 under control of its native promoter (Chu & Herskowitz, 1998).

Therefore, an initial test of whether Ime4 is required for Ndt80 activity is to

see whether Ndt80 autoinduction can occur in Ime4-deficient strains. (IME4

is not a meiosis-specific gene.)

140

Figure A2-4. Potential Regulators of NDT80: Ime4, Ime2, and Ids2

In addition to Imel and Ime2 (Chu & Herskowitz, 1998; Friesen et al.,

in press), there are several other potential regulators of NDT80. (1) Ime4 is

necessary for Ndt80 activity. (2) Ime2 is necessasry for initiation of NDT80

transcription and expression of the middle genes in an Ids2-dependent

manner. Therefore, Ids2 may be required for Ime2-mediated transcription of

NDT80. (3) The kinase activity of Ime2 may be directly responsible for post

translational modification and thus activation of Ndt80. (4) Ndt80 can

induce IME2 expression.

141

Ime4p

starvation~\ a1-02—RME1—IME1=>IME2

Ime1p/(4)? UmebpEARLYGENES

IME2(1)2HOP1\-OP1 SPO11SPO11(3)? REC114(2) NDT30?Ids2?TNDT80-*sº

MIDDLEGENESSPS1 CLB1

§

The interactions between Ime2 and Ndt80 are complex. Ime2 is

necessary for both early and middle gene expression, including that of NDT80

itself (Friesen et al., in press). Ids2 is required for Ime2-mediated middle gene

expression. Thus initiation of transcriptional activation of NDT80 may be

dependent on both Ids2 and Ime2. Ime2 may also be required for activating

Ndt80 protein. I have observed Ndt80 to be post-translationally modified

both when ectopically synthesized in vegetative cells and during sporulation.

Assuming this modification is due to phosphorylation, Ime2 may be the

responsible kinase. Taken together, there are at least three levels of NDT80

expression (see Figure A2-5). NDT80 is not expressed in vegetative cells and

in early meiotic prophase. Initiation of NDT80 expression occurs just prior to

exit from pachytene dependent on Imel and Ime2 (Xu et al., 1995; Chu &

Herskowitz, 1998; Friesen et al., in press). An induced level of NDT80

expression then occurs. This induction may be dependent on Ndt80 itself,

Ime4, and the meiotic recombination checkpoint.

The observation that Ndt80 can induce expression of IME2 in

vegetative cells suggests that there may be a positive feedback loop betweenNdt80 and Ime2. It remains to be determined whether Ndt80 also induces

IME2 message during sporulation. The most direct way to address this

question would be to mutate the Ndt80-binding site found in the IME2

promoter and subsequently characterize IME2 expression during sporulation.

It is intriguing that two of the three early sporulation genes with MSE sites in

their promoters (IME2 and SLZ1) both are significantly induced by GAL

NDT80 (6.3 and 10.3-fold, respectively) and are induced midway through

sporulation. SLZ1 increases from a red-to-green ratio of 8.9 at 2 h to 31 at 5 h.

Thus a subset of early genes may be regulated by both Imel and Ndt80.

143

Figure A2-5. A Model for the Different Levels of NDT80 Expression

At least three different levels of NDT80 expression have been observed.

A. NDT80 is not expressed in vegetative cells and in cells prior to meiotic

pachytene. B. A basal level of NDT80 expression occurs just prior to middle

gene expression and is dependent on both Imel and Ime2. C. There is a

second increase in NDT80 expression. This boost may be due to Ndt80

recognition of an MSE in its own promoter. In addition, this induction is

dependent on Ime4 and the meiotic recombination checkpoint.

144

fQ■ )H■ ºil>Pil|-l

<Z Cp:F.§R

A | | |I | | Ç

EARLY MID MID- LATELATE

time in SPM

Imel■ —-Ime2 –Umeb

Ndt80, Ime4,meiotic recombination

checkpoint

145

Both Ime2 and Ime4 have homologs in higher eukaryotes (Renee

Reijo, personal communication; Mary Clancy, personal communication).

Ime2 is similar to several human kinases. Ime4 is 51% identical (over 285

amino acids) with the human gene MT-A70 which encodes a

methyltransferase. Although the extent of the homology with yeast

counterparts remains to be determined, some aspects of transcriptional

regulation of meiosis may be conserved in higher eukaryotes.

146

Table A2-1. Yeast Strains for Appendix Twostrain mating strainnumber type relevant genotype backgroundYSC912 a/O. MCD1

YSC913 a/O. RES1-1 MCD1

YSC915 a/O. ime4::LEUI2 MCD1

YSC916 a/O. ime4::LEUI2 RES1-1 MCD1

YSC917 a/O. ime4::LEUI2 rme1::LEUI2 MCD1

YSC552 d pGAL W303

YSC553 d pGAL-Ndt80-HA W303

The MCD1 genotype is leu2 his3 trplura■ ho::HIS3 sprä:lacz.The W303 genotype is ade2-1 can1-100 leu2-3,112 his3-11,15 ura■ trpl-1 psi

Acknoweldgements

I would like to thank Mary Clancy for reagents and thoughtful

discussions regarding these experiments and Renee Reijo for her insight and

patience.

147

APPENDIX THREE

A ROLE FOR Clb5IN MEIOSIS

148

Abstract

In vegetative cells, Clb5 and Clb6 are required at G1/S for DNA

synthesis and at G2/M for inhibiting re-replication. I have found that in

meiosis, Clb5 is also required for DNA synthesis. In addition, based on our

observations that CLB5 and CLB6 messages are induced midway through

sporulation (after the time of DNA replication), Clb5 and Clb6 may have

additional roles in meiosis. The protein levels of Clb5 during meiosis were

characterized and shown to be present at the time of the meiotic divisions.

Models for a dual role of Clb5 in meiosis are presented.

Introduction

S. cerevisiae has six B-type cyclins. Although their functions overlap,

there is some specificity. Clb1 and Clb2 are involved in mitotic division, Clb3

and Clb4 function in spindle pole body separation (SPB) and short spindle

formation, and Clb5 and Clb6 are necessary for DNA synthesis (reviewed by

Nasmyth, 1996). During meiosis, Clb1-, Clb3-, Clb4-, but not Clb2-associated

kinase activities are detectable (Grandin & Reed, 1993). We observed that the

messages for five of the six B-type cyclins (CLB2 again being the exception) are

induced midway through meiosis (Chu & Herskowitz, 1998). This

observation with respect to CLB5 and CLB6 is surprising in several respects.

First, the simulatenous expression of these five cyclins in meiosis is notably

different than their staggered expression in mitosis (reviewed by Koch and

Nasmyth, 1994; see Figure A3-1). Second, the induction of CLB5 and CLB6

midway through sporulation (after the time of premeiotic DNA replication)

raises the questions of whether their respective gene products function

during premeiotic DNA synthesis and/or later during the meiotic divisions.

149

Figure A3-1. B-type Cyclins in Meiosis and Mitosis

The expression of the B-type cyclins is notably different between

mitosis and meiosis. In the vegetative cell cycle, CLB5 and CLB6 are expressed

in early G1 and are necessary for DNA replication. CLB3 and CLB4 are

expressed in late G1 and are required for SPB duplication and for short spindle

formation. CLB1 and CLB2 are expressed at the G2/M transition and are

required for mitosis. In sporulation, CLB2 is not expressed, and the messages

for CLB1, CLB3-CLB6 are all induced at the end of meiotic prophase, at which

time SPB separation occurs and the meiotic divisions proceed.

150

VegetativeGrowth=

Sporulation=

Mitosis+

Meiosis+

BudMorphogenesisSporeMorphogenesis

IsotropicBudGrowthSporeMorphogenesis MitoticDivisionMeioticDivisions CLB1/2_yCLB1—C-

CLB3/4

\CLB5/62.MIMII

GºGºG2/M G1G1

~CLB5/6Clb5/6S

CLB3/4

g

Based on their expression pattern, several models for a possible

function for Clb5 (and also Clb6) in meiosis can be drawn (see Figure A3-2).

Although Cdc28-associated kinase activity is predicted to drop as cells exit

meiosis I (Grandin & Reed, 1993), it must also be maintained high enough to

prevent re-replication before meiosis II (Picard et al., 1996). The additional

activities contributed by Clb5 and Clb6 might be part of a meiosis-specific

regulation of Cdc28 activity. In G2/M of the vegetative cell cycle, Clb5 and

Clb6 maintain kinase levels high enough so that DNA synthesis cannot occur

(Dahmann et al., 1995; see review Stern & Nurse, 1996). They may have a

similar function after meiosis I. It is also possible that Clb5 and Clb6 may be

the targets of a meiosis-specific mechanism which lowers cyclin-associated

kinase activity between meiosis I and II. To address these issues, I

characterized the requirement for Clb5 during different stage of meiosis and

made reagents to examine Clb5-associated kinase activity during meiosis.

Material and Methods

Yeast stains used are listed in Table A3-1. Protocols for sporulation

followed that of Chu & Herskowitz (1998).

FACS analysis

Two ml of synchronized cells in SPM were harvested, washed with 1

ml 50 mM Tris, pH 7.5, and fixed in 1 ml 70% ethanol (in Tris, pH 7.5) for 1 h.

Fixed cells were subsequently washed twice in 1 ml 50 mM Tris before RNase

treatment (100 pil 1 mg/ml RNase in 20XTE) for 1 h at 37°C, followed by an

overnight incubation at 4°C. Proteinase K was then added to a final

concentration of 0.4 mg/ml and incubated for 1 h at 55°C, followed by two

washes in 1XPBS. The pellet was resuspended in 100 pil 50 pg/ml propidium

152

Figure A3-2. Models for Clb5 Function during Meiotic Division

The specific induction of CLB5 and CLB6 message at the prophase to

metaphase I transition suggests that they may function during the meiotic

divisions. An inhibitor, X, of Clb5 (and also Clb6) activity in meiosis may

lower cyclin-associated kinase activities enough to allow for anaphase I

(Model A). Another possibility is that activity contributed by Clb5/Cdc28 (and

Clb6/Cdc28) maintains kinase levels high enough to prevent DNA

replication between meiosis I and II and thus allow progression into meiosis

II (Model B).

153

2. MI MII

Prophase

154

iodide and incubated for 1 h at 23°C or overnight at 4°C. The samples were

then diluted 1:10 in PBS, sonicated for 5 sec, and analyzed.

Western Analysis

Our protocol is based on that of Hann and Walter (1991). Exponentially

growing cells were harvested and resuspended in 500 pil of cold TCA buffer

(20 mM Tris, pH 8.0, 50 mM NH4OAc, 0.5 mM EDTA) with protease inhibitors

(0.2 mM PMSF in isopropanol, 2% aprotinin, 2 mM benzamidine, 2 mM

leupeptin). All subsequent steps were performed at 4°C. Lysates were made

by bead-beating 4 x 30 sec with glass beads. The supernatant was saved, and

the remaining extract was resuspended in 500 pil of 1:1 TCA buffer : 20%TCA

and bead-beat twice more. Supernatants were pooled and spun for 10 min at

15 krpm. Pelleted protein was resuspended in 300 pil freshly made TCA

sample buffer, boiled for 10 min, and centrifuged for 10 min at 15 krpm. The

final supernatant was saved. Proteins were separated on a 10%

polyacrylamide gel and transferred to nitrocellulose filters in 150 mM glycine,

20 mM Tris (pH 7.5), 20% methanol, 0.05% SDS. Blots were blocked for 1 h in

buffer containing 10% dry milk, 0.1% Triton X-100 in TBS (0.5M NaCl, 0.2 mM

TrisCl, pH 7.5). The filter was subsequently washed in TBST (0.1% Triton X

100 in TBS), incubated with a 1:1000 dilution of the primary antibody, 12CA5

(in TBST with 2% dry milk, 0.1% Triton X-100), washed again in TBST, then

incubated with a 1:2000 dilution of the secondary antibody (horseradish

perxidase-coupled goat anti-mouse). Final washes with TBST with 0.3%

Triton X-100, then TBST with 0.1% Triton X-100, were performed before

developing using the Amersham ECL protein detection kit.

Immunopreciptiation

Yeast lysates were harvested from 50 ml of vegetatively growing cells of

155

O.D. 0.8 and frozen imediately in liquid nitrogen for subsequent storage at

-80°C. Upon time of use, lysates were thawed on ice and resuspendend in 0.5

ml lysis buffer with inhibitors (20 mM Tris, 7.4, 100 mM NaCl, 50 mM NaF, 50

mM fl-glycerophosphate, 5 mM EDTA, 0.2% Triton X-100). All subsequent

steps were done at 4°C. Extracts were made by bead-beating twice for 1 min,

with 1 min ice in between. An aliquot from each sample following the bead

beating was taken for Western analysis (see "load" lanes, Figure A3-6) and for

measuring protein concentration by the Bradford assay, BioFad(R). For each

sample, 1 mg of protein was used in 0.5 ml lysis buffer and an equal volume

of HA-slurry. After incubation by rotation for 2 h, aliquots were again taken

(see "supe" lanes, Figure A3-6), and samples were washed three times in lysis

buffer, and once in washing buffer (50 mM HEPES, 1 mM DTT). Samples

were equally loaded onto a 10% gel (see "I.P" lanes, Figure A3-6) and

processed for Western anlaysis as described above.

156

Results

Clb5 is required for premeiotic DNA synthesis

Having observed that CLB5 and CLB6 transcript levels are induced

midway through meiosis, we wondered whether their gene products might

have a role in meiotic progression as has been shown for Clb1-Clb4 (Grandin

and Reed, 1993; Dahmann and Futcher, 1995). We first observed that clb5 cells

sporulate at levels only 8% that of wild-type, with an equal distribution of

tetrads, dyads, and monads (see Table A3-2, part I). In contrast, wild-type cells

produced 96% tetrads and 4% dyads. To characterize the Clb5-deficient

mutants further, we analyzed their ability to undergo premeiotic DNA

synthesis by FACS analysis. After transfer to sporulation medium at time

zero, wild-type cells initiated DNA synthesis by three hours (Figure A3-2b).

By nine hours a majority of cells had replicated, as evidenced by a 4n DNA

content (Figure A3–2d). In contrast, clb5 mutant cells were severely

compromised in their ability to undergo DNA synthesis (Figure A3-2e-g).

Only after nine hours had some replication occurred (Figure A3-2h). Because

of this earlier requirement for Clb5 in premeiotic DNA synthesis, a later role

for Clb5 during meiotic division could not be determined by a simplecharacterization of a clb5 mutant.

A role for Clb5 in meiotic division

The observation that a clb1 clb3 clb4 strain can still form dyads (see

Table A3–2 part II; Dahmann & Futcher, 1995), suggests that other B-type

cyclins, such as Clb5 and Clb6, may be sufficient for carrying out a single

meiotic division. I found that a clb1 clb3 clb4 clb6 strain also sporulates to

produce dyads with a similar efficiency (see Table A3-2, part II). Assuming

157

Figure A3-3. Clb5 is Required for Normal Progression through Premeiotic

DNA Synthesis.

Wild-type a/o (ySC328, a-d) or clb5/clb5 a/o (ySC752, e-h) cells were

sporulated in a synchronous manner. Cells were harvested at three-hour

intervals, fixed, and stained with propidium iodide for analysis by FACS to

determine cellular DNA content. (a, e) t-0, (b,f) t-3, (c, g) t-6, and (d, g) t-9

hours in sporulation medium. A CLB5 plasmid complemented the

sporulation phenotype of a clb5 strain (data not shown).

158

clb5/clb5+/--

=0e. t=0

110

55

a. t

-4f. t-3

7

3

b. t-3

=6g. t=6C. t

100

h. t-9=9d. t

4n2n4n2n

159

that Clb2 protein is not present in meiosis in the W303 background, as is

reported in the strain background of Grandin & Reed (1993), we conclude thata cell can exit

prophase and go through at least one nuclear division relying solely on Clb5

activity.

Clb5 and Clb6 are essential for sporulation

In seeking a possible role for Clb6 in meiosis, I found that a Clb6

deficient strain had no sporulation phenotype in a wild-type, single mutant

(clb1, clb3, or clb4), double mutant (clb1 clb3, clb1 clb4, or clb3 clb4), or triple

mutant (clb1 clb3 clb4) background (see Table A3-2, parts II and III). A clb5 clb6

strain is more severely compromised for sporulation than a clb5 strain (see

Table A3-2), suggesting that Clb6 may also function in premeiotic DNA

syntehsis.

Spore morphogenesis is independent of the destruction box of Clb5

To test the hypothesis that normal progression through meiosis

requires destruction of Clb5 protein, I examined the sporulation phenotype of

a homozygous mutant in which the destruction box of Clb5 was deleted

(Clb5Adb). The B-type cyclins share a destruction box domain which each

protein for ubiquitination and subsequent degradation in a cell cycle

dependent manner (see review King et al., 1996). In the vegetative cell cycle,

Clb5Adb cells are viable and have a mild morphological defect of multiple,

elongated buds, similar to that of cdc34-2's at non permissive temperature

(Schwob et al., 1994; see Figure A3-4A). I found that Clb5Adb mutants

sporulate with normal efficiency, but up to 15% of all tetrads are of linear

morphology and are multiply connected (see Figure A3-4B). This striking

160

Figure A3-4. A role for Clb5 in Sporulation

The phenotypes of Clb5Adb cells (YSC817) in rich medium (A) and

under sporulation conditions (B) were examined. Clb5Adb cells are viable but

form multiple, elongated buds. Clb5Adb homozygous mutants sporulate with

normal efficiency but form linear tetrads at a frequency of 15% (see Table A3

3). clb1 clb5 GAL-CLB5 strains (YSC683) were also characterized when grown

in rich medium (C) or under sporulation conditions (D). clb1 clb5 GAL-CLB5

(or clb1 clb5) vegetatively growing cells are elongated, compared with wild

type, clb1, or clb5 single mutants. Upon sporulation, clb1 clb5 GAL-CLB5

strains form linear tetrads with a frequency of about 10% (see Table A3-3).

161

** *

*

1/14 7.

A R_*

162

phenotype may be secondary to a requirement for destruction of Clb5 in

vegetative bud morphogenesis and cytokinesis or may reflect a role for Clb5

in SPB rotation during sporulation. The finding that Clb5Adb cells still

produce four spores suggests that use of the destruction box of Clb5 is not

necessary for normal progression through meiosis. However, a more

detailed characterization of this phenotype -- such as assaying the viability of

each spores of a tetrad -- remains to be done.

GAL-Clb5 can complement the sporulation defect of a Clb5-deficient strain

The question remains as to the origin of Clb5 activity during

premeiotic DNA synthesis. Only a basal level of CLB5 message is detectable

prior its induction midway through sporulation. Therefore, either this

minimal amount of CLB5 is sufficient for DNA replication, or Clb5 protein is

inherited from the previous vegetative cell cycle, as has been observed with

other proteins (Keller & Young, 1997). Because Clb5 is required for

sporulation, it was not suprising to observe that a clb1 clb5 strain does not

form spores (see Table A3-2, Part I). However, I also found that the presence

of a GAL-CLB5 plasmid can complement the sporulation defect of both clb5

and clb1 clb5 strains (see Table A3-3), even without galactose

supplementation. (This may be due to a leakiness of the GAL1,10 promoter.)

Interestingly, a clb1 clb5 GAL-CLB5 strain under sporulation conditions forms

linear tetrads with an increased frequency (see Figure A3-4D and Table A3-3).

This phenotype may be secondary to a vegetative requirement, since both clb1

clb5 and clb1 clb5 GAL-CLB5 cells are more elongated than wild-type, clb1, or

clb5 cells (see Figure A3-4C). Intriguingly, a clb1 clb5 strain which carries two

copies of GAL-CLB5 has an increased frequency of dyads (see Table A3-3).

This preliminary observation is consistent with the hypothesis that elevated #S_Y

*

163

levels of Clb5 may prevent two successive meiotic divisions from occuring(see Figure A3-2, Model A). However, it remains to be determined whether

this phenotype is dependent on Clb5 in specific, or on the clb1 clb5

background.

Characterization of Clb5

To determine whether Clb5 protein levels have kinetics similar to

CLB5 mRNA during sporulation, we followed a tagged form of Clb5 (see

Figure A3-5). Clb5-HA is detectable in vegetatively growing tagged (+, lane 1),

but not untagged (-, lane 2) strains. A small amount of protein is detectable at

t=0 of the sporulation time course (lane 3). The amount of Clb5-HA increases

slightly by 2 h (lane 4) and accumulates from 4-6 h in SPM (lanes 5-7), before

decreasing in levels by 7 h (lane 8). CLB5 message is induced midway through

sporulation (approximately 4-5 h, depending on the synchrony of a given

timecourse) (Chu & Herskowitz, 1998). Thus the timing and expression

levels of Clb5 protein during sporulation correlates with that observed for

CLB5 message. The earlier detection of protein versus message at 2 h may be

due to a greater sensitivity of Western over Northern analysis, or possibly

due to an accumulation of the basally expressed CLB5 message.

As a first step towards following Clb5-associated kinase activity during

sporulation, I showed that an HA-tagged Clb5 protein can be

immunoprecipitated from the SK1 background (see Figure A3-6). Subsequent

analysis following both Clb5 protein and kinase activity during meiosis cannow be done.

Discussion

Our preliminary characterizations implicate a role for Clb5 in both

164

■ /■ t tº

* Q_Y

Figure A3-5. Clb5 Expression during Sporulation JExtracts were made from strains of the SK1 background which express

HA-tagged Clb5 (YSC949; lanes 1, 3-8), and untagged Clb5 (YSC328, lane 2). ºLanes were equally loaded, and the 12CA5 antibody was used to detect the HA * } {

epitope. Lanes 1 and 2 are extracts from vegetatively growing cells. Lanes 3-8 / ºare extracts made at hourly intervals during sporulation (lane 3, t-0; lane 4, 2 º

h; lane 5, 4 h; lane 6, 5 h; lane 7, 6 h; lane 8, 7 h in sporulation medium). Lane

2 contains a cross-reacting band that migrates with slightly slower mobility º

than the band of interest and is present only in extracts from vegetative cells. *

165 sº

veg sporulationI I I I

+ - 0 2 4 5 6 7 h in SPM

*** ** **** * * * * * * ~ * .*-- ~~ ->

†- * - ==E=- - -

- --~~~Zºº* &&. *

lane 1 2 3 4 5 6 7 8

166 sº

Figure A3-6. Immunoprecipitation of Clb5-HA (in SK1 Background)

Strains used for this analysis include HA-tagged CDC45 (YSC965; lanes

1, 4, 7), wild-type SK1 strain (YSC328; lanes 2, 5, 8); and HA-tagged CLB5 SK1

strain (YSC949; lanes 3, 6, 9). Crude extracts before immunoprecipitation (or

"load" were assayed for the tagged protein of interest (lanes 7-9). The

supernatant following extract binding with the HA-matrix (or "supe") were

assayed for the tagged protein of interest (lanes 4-6). The immunoprecipitate

(or "IP") corresponding to each original extract were assayed for the tagged

protein of interest (lanes 1-3). The rightward pointing arrow indicates a band

corresponding to Cdc45-HA (lanes 1, 4, and 7). The leftward pointing arrow

indicates a band corresponding to Clb5-HA (lanes 3 and 9).

167

I/I ■ º

* Q_Y

~l~_º

->

-

*** º

, Rº

sºlane 1

º,

168

premeiotic DNA synthesis and meiotic division. The observation that a clb1

clb3 clb4 clb6 strain can still sporulate to form dyads suggests that Clb5 is

sufficient to provide B-type cyclin activity to drive a single meiotic division.

In addition, the finding that Clb5Adb cells can still sporulate to form tetrads

suggests that normal progression through meiosis does not require timely

degradation of Clb5 protein via its destruction box. However, the

hypothesis that Clb5 is specifically regulated during the meitoic divisions

cannot be ruled out. A destruction box-independent means of lowering Clb5

associated kinase activity may exist (as has been found for other proteins). In

addition, the relatively poor synchrony of sporulation may confound

detection of any slight drop in Clb5 protein or associated kinase activity

between meiosis I and II. Several steps can now be taken to address this issue

fruther. First, if Clb5 is specifically targeted during meiosis, then there may be

a difference in the protein and associated kinase levels of Clb5 versus other B

type cyclins (such as Clb1) during sporulation. To this end, the SK1 strain,

YSC949, which contains both HA-tagged Clb5 and myc-tagged Clb1, may be

useful. Second, one way to avoid the asynchrony problem of sporulation is to

examine Clb5 expression at a single cell level. Immunofluorescence of tagged

Clb5 protein and DAPI staining of nuclei can be done to determine whether

Clb5 protein is continuously present throughout the meiotic divisions.

To examine a potential role for Clb5 during meiotic division, it would

be optimal to allow CLB5 expression during vegetative cell growth and

premeitoic DNA synthesis, but to prevent its induction specifically midway

through sporulation. Such alterations in regulation might be feasible by

mutating the two putative MSE sites found within the first 600 base pairs of

the CLB5 promoter (Chu & Herskowitz, 1998). If in such a CLB5AMSE

I/7, ■ º

* Q_Y

... [* *

{} | .

169

·■-"*·■ |-~■#=

**■|-#·■*|×****|-„“■■

mutant, CLB5 message were no longer induced midway through sporulation,-

closer characterization of such a mutant might clarify a role for Clb5 in

meiotic division.

170 º

mutant, CLB5 message were no longer induced midway through sporulation, _*

closer characterization of such a mutant might clarify a role for Clb5 in

meiotic division.

l.*º

S.*

■ %

Z

º––

º- 7.

y

º-- *

- * * * ,

*º->170º

- º

\ |\ \

Table A3-1. Yeast Strains for Appendix Three ! sstrain mating strain .

number type relevant genotype backgroundYSC328 a/O. SK1

YSC752 a/O. clb5::UIRA3 SK1

YSC949 a/O. CLB1-(myc)3 CLB5(HA)3 SK1YSC007 a/O. W303

YSC648 a/O. clb 1::UIRA3 W303

YSC657 a/O. clb5::UIRA3 W303

YSC658 a/O. clb6::LEUI2 W303

YSC659 a/O. clb5::URA3 clb6::LEUI2 W303

YSC682 a/O. clb1::UIRA3 clb5::URA3 W303

YSC683 a/O. clb1 clb5 TRP1::GAL-CLB5/+ W303

YSC684 a/O. Clb1 clb5 TRP1::GAL–CLB5 W303 º

YSC686 a/O. Clb5 TRP1::GAL–CLB5 W303

YSC699 a/O. clb1::UIRA3 clb3::TRP1 clb4::HIS3 W303 º, ■ º

YSC724 a/O. clb3::TRP1 clb4::HIS3 W303 R_Y

YSC753 a/O. clb1::URA3 clb6::LEUI2 W303 |YSC754 a/O. Clb3::TRP1 clb6::LEUI2 W303 *

YSC755 a/O. clb4::HIS3 clb6::LEUI2 W303 -

YSC756 a/O. clb3::TRP1 clb4::HIS3 clb6::LEUI2 W303 * ,YSC759 a/O. clb1::URA3 clb3::TRP1 clb6::LEUI2 W303

YSC760 a/O. clb1::URA3 clb4::HIS3 clb6::LEUI2 W303 & I -YSC761 a/O. clb1::UIRA3 clb3::TRP1 clb4::HIS3 clb6::LEUI2 W303 yº

YSC817 a■ 0. clb5Adb W303 A 2.

YSC965 3. CDC45(HA)3 CDC7(myc)9 bar:LEu2 W303s

All a/o diploid strains are homozygous unless otherwise noted.The SK1 genotype is ura■ ho::LYS2 leu2 lys2 ~

The W303 genotype is ade2-1 can1-100 leu2-3,112 his3-11,15 ura■ trpl-1 psi- sº

k

171

A Q_\

Table A3-2. B-type Cyclins in Meiosis !immature/ !

no spores monads dyads tetrads Il º,Part I T -+ 46

-2 51 1300 * | |

clb1 66 1 8 25 200 -Clb5 92 3 3 2 900 ^,Clb1 clb5 93 3 4

-100

Part IIclb3 clb4 48

-6 46 200

clb1 clb3 clb4 78 9 13-

400clb1 clb3 clb4 clb6 74 13.5 12 0.5 600Part IIIclb6 53

-3 44 800

clb1 clb6 65 2 8 25 700clb3 clb6 41 2 12 45 400clb4 clb6 44 2 10 44 800

clb3 clb4 clb6 44 2 11 43 600 ºr -ºclb5 clb6 98 1 1

-700 !' .

clb1 clb3 clb6 44 4 19 31 800 ºnclb1 clb4 clb6 69 15 15 1 700

Numbers given are percentages of total number of cells (n). | sº-tº

Table A3-3. Gal-Clb5 Can Complement the Sporulation Defect of a Clb5- º,Deficient Strain. | º

In O linearr IIlOIl ds tetra tr Il {} | .

clb5 ºGAL–CLB5 22 1 17 60

-103

-

*

Clb1 clb5 35 1 13 36 13 100 -

GAL-CLB5/+ º

clb1 clb5 26 5 31 35 3 100

GAL-CLB5/GAL-CLB5 4.Numbers given are percentage of total number of cells (n). º

º

Acknowledgements:I thank Julia Owens for the gift of YSC965, and Adam Rudner, Julia 1.

Owens, Julia Charles, and Sue Jasperson for guidance on theimmunoprecipitation protocol.

172 sº

APPENDIX FOUR

NDT80 AND ITS ºREGULATION º

173 º

Abstract

Ndt80 is responsible for the transcription of over 100 genes during

sporulation. Given this extensive regulatory domain, it is not surprising that

Ndt80 function may be regulated at several levels. In addition to studies of

transcriptional regulation (see also Appendix Two), I identified a post

translationally modified form of Ndt80 and further characterized a possible

role for Ndt80 as a target of the meiotic recombination checkpoint.

Introduction

Ndt80 activity is known to be regulated at several levels (see Figure A4

1). Transcription of NDT80 itself is slightly delayed relative to canonical early

genes (Xu et al., 1995) but is dependent on the early gene regulators, Imel and

Imez (Chu & Herskowitz, 1998; Friesen et al., in press). The promoter of

NDT80 contains not only an Imel binding site (URS1 motif) but also two MSE

sites. Ndt80 can activate its own expression, presumably through recognition

of these sites (Chu & Herskowitz, 1998). In addition, Ndt80 activity is

dependent on the meiotic recombination checkpoint (Chu & Herskowitz,

1998; also see Introduction).

There are likely to be other means of regulating Ndt80 activity. For

example, Lee & Honigberg (1996) identify a nutritional requirement at the end

of prophase necessary for sporulation. Perhaps, initiation of NDT80

transcription is subject to nutritional regulation. In addition, Ndt80 may be

regulated at a post-transcriptional level such as protein localization or posttranslational modification.

174

tºy

Figure A4-1. Modes of Regulation of Ndt80 Activity

At least three modes of regulating Ndt80 activity are known. Initiation

of NDT80 transcription is dependent on the key transcription factors for the

early genes, Imel and Imez (A). Further induction of NDT80 expression may

be due to a positive feedback loop in which Ndt80 activates its own expression

through recognition of the Ndt80 binding site (MSE motif), (B). Ndt80

activity is also dependent on the meiotic recombination checkpoint. In

backgrounds where the checkpoint is activated (such as in dmc1 strains),

putative targets of Ndt80, such as CLB1, are not transcribed (C).

|

175

* Imel HºrIme? 80

Ndt80B.

-HO

NDT80 H.

C.

defective meioticrecombination

e.g. Ydmc1/ |dmc1 | Meiotic Recombination

CheckpointRad24, Mec1, Radl'7

Ndt80

—TMSETHFCLB1

176

**,

Materials and Methods

Yeast strains are listed in Table A4–1.

Immunofluorescence

Our protocol is based on that of Pringle et al. (1991). Five ml of

synchronized, sporulating cells were harvested per timepoint and fixed in

formaldehyde. Spheroplasted cells were attached to polylysine-coated slides

and treated with methanol (6 min at -20°C) and then acetone (30 sec at -20°C).

Permeabilized cells were blocked (0.1% Triton X-100, 1% BSA in PBS) before

incubation with primary antibody, 9E10 (Kolodziej and Young, 1991; a kind

gift from Joachim Li, UCSF), diluted 1:80 (1% BSA in PBS) and secondary

antibody (rhodamine-conjugated goat anti-mouse antibody; Jackson

Immunoresearch) diluted 1:100. Mounting medium (Fluoromount G,

Southern Biotechnology Associates, Inc.) containing DAPI (20 pil 1 ug/ml) was

applied before cells were flattened. Cells were visualized with an Olympus

BX60 microscope.

Western analysis

Extracts for Figures A4-3 and A4-4C were made as described in

Appendix Three. Extracts for Figure A4-6 were made by the "rapid yeast

protein extracts" protocol which follows. 1.5 ml of mid-log culture were

harvested and incubated on ice for 5 min. Pellets were then resuspended in

0.15 ml soultion I (1.85 M NaOH, 7.4% ■ º-mercaptoethanol) and incubated on

ice for 10 min. A second 10 min incubation was repeated after the addtion of

0.15 ml 50% TCA. Extracts were then spun in the microfuge (4°C, 2 min).

Pellets were washed with 1 ml acetone and resuspended in the appropriate

volume of sample buffer. Samples were boiled before use. All samples,

I/I 1.

A Q_N

º~º-*

<--

177

independent of how the extracts were prepared, were processed for Western

analysis as described in Appendix Three.

Results

When I first began this work in July of 1996, I initially took several

approaches to try to understand the function of Ndt80. In order to screen for

interacting proteins by two-hybrid analysis, I fused the entire Ndt80 coding

sequence to the DNA binding domain of LexA. (Incidentally, it was use of

this construct which revealed a transcriptional activity associated with

Ndt80.) In addition, in order to identify genetic interactors with Ndt80, I

planned to high copy suppress the sporulation defect (more spec■ icially, the

lack of fluorescence) of an Ndt80-deficient cell under sporulation conditions.

Because I was less interested in suppressors that bypass Ndt80 function (as

opposed to suppressors that act by mass action), I chose not to use an nat30A

background for my screen. Instead, I planned to use the ndt&0-1 background

(Xu et al., 1995). However I found that the sporulation defect of the original

ndt80-1 strain (NKY2171) could not be complemented by a full-length Ndt80

construct, sufficient to complement an nat30A strain (NKY2296). (The

NKY2171 strain probably had acquired additional mutations which prevented

sporulation such as mitochondrial mutations.) Therefore, I rescued the

ndt80-1 allele by gap repair (Rothstein, 1991). Several constructs, each

containing fragments of the rescued ndt&0-1 allele, were made and sequenced

to identify alteration(s) in the ndt&0-1 sequence which might localize regions

important to Ndt80 function. Because of progress in other aspects of my work

with Ndt80, this allele was never reintroduced into a wild-type background.

Fortunately, Kirsten Benjamin has been able to make use of this information

*

& ",-,º

178

…***

and these reagents and information in her studies on the autoregulation ofNdt80.

Ndt80 is nuclearly localized and detectable throughout meiosis I and II

Since Ndt80 is a transcriptional activator, we expected it to be present in

the nucleus. In addition, Ndt80 contains a putative nuclear localization

signal (amino acids 176-179; Xu et al., 1995). Because NDT80 message is

detectable during later time points of sporulation, I was curious whether

Ndt80 protein is also present beyond prophase. Ndt80 was tagged with a Myc

epitope and localized to the nucleus in sporulating wild-type cells both during

prophase, as assayed by a single DAPI-staining body (Figure A4-2b), and also

throughout meiosis I and meiosis II, as assayed by two (Figure A4-2c-f) and

four (Figure A4-2g-h) DAPI-staining bodies, respectively. I also found Ndt80

HA protein to be present beyond prophase as assayed by Western analysis

(Figure A4-3). These observations raise the possibility that Ndt80 function is

required not only to exit prophase and enter meiosis I but also for later eventsduring sporulation

GAL-NDT80 inhibits vegetative growth

The pachytene arrest point of homozygous mutants suggests that

Ndt80 is required for initiation of meiosis and spore formation. In addition,

since commitment to sporulation occurs around the time of pachytene, I was

curious as to whether Ndt80 activity might be sufficient for commitment to

sporulation. To test these hypotheses, I made a construct containing an HA

tagged Ndt80 fused to the GAL1,10 promoter (GAL-NDT80-HA). Wild-type

diploid cells expressing GAL-NDT80-HA do not in fact form spores (asvisualized by Nomarski microscopy and by assaying for fluorescence of the

mature spore wall). However, I found that there is a general correlation Q_Y

179

Figure A4-2. Ndt80 Co-Localizes with DNA throughout Meiosis

Cells from SK1 strains containing untagged (ySC484) (a) or myc-epitope

tagged (ySC446) Ndt80 (b-i) were grown under sporulation conditions, fixed,

and processed for indirect immunofluorescence microscopy using the 9E10

antibody, which recognizes the myc epitope (row 1). DAPI staining was

performed in parallel to visualize the nucleus (row 2). The stages of meiosis

as inferred by DAPI-staining are (a-c) early meiosis to prophase, (d-f) prophase

to completion of meiosis I, (g-h) meiosis II, (i) tetrad of four spores. Ndt80

(myc)3 colocalized with DAPI-staining throughout all stages of meiosis (b-h)

until spore formation was complete (i). The absence of an Ndt80-(myc)3

signal in spores may be due to difficulties in immunofluorescence staining

once a spore wall has formed (Fares et al., 1996).

180

--rººt

e m

-E

Q)©■ )\ºm

ed

181

n**

Figure A4-3. Ndt80-HA is Detectable beyond Meiotic Prophase

Yeast extracts were harvested from SK1 strains containing untagged

Ndt80 (YSC484, -, lanes 1, 3, 5, and 7) and HA-tagged Ndt80 (YSC407; +, lanes 2,

4, 6, and 8) at times t-0, 4, 6, and 7.5 hours (h) after transfer to sporulation

medium (SPM). Equal amounts of extract were loaded into each lane.

Western analysis was performed using the 12CA5 antibody, which recognizes

the HA epitope. Arrow indicates HA-epitope tagged Ndt80 protein. Asterisk

marks a slower migrating form of Ndt80. (Meiosis I and meiosis II DAPI

staining bodies were present at 4 h and beyond.)

182

t=0 4 6 7.5 h in SPM| | | | | I | |

- + - + - + - + HA tag

>}. ->

<!- -

-

Zºº,

lanes 1 2 3 4 5 6 7 8

183 sº

there is a general correlation between strains expressing high levels of GAL

NDT80-HA and a slow vegetative growth phenotype (see Figure A4-4). An

integrant expressing high levels of Ndt80 protein at 1.2 hours, and even more

at 3 hours (integrant #1, Figure A4-4C) grows very slowly on galactose

supplemented plates after 2 days (Figure A4-4A, (ii)). A slower migrating

form of Ndt80-HA is also detectable in this strongly expressing integrant

(asterisk, Figure A4-4C). In contrast, an integrant that expresses low levels of

Ndt80 even after 3 hours (integrant #2, Figure A4-4C), does not have a slower

migrating form and does not slow cell growth on galactose plates (Figure A4

4B, (ii)).

Although no morphological differences were detectable at a gross level

in cells expressing high levels of Ndt80, these cells were found to have

modifications at the ultrastructural level. The central and outer plaques of

the spindle pole bodies (SPB) in strains expressing GAL-NDT80-HA are much

larger than those of normal vegetative cells and resemble the modified SPBs

of cells undergoing sporulation (John Kilmartin, personal communication).

We now know that Ndt80 activates transcription of over 100 middle

sporulation genes, which include genes involved in SPB function, such as

SPC42 and MPS1. In light of these observations, the slow growth phenotype

may be due to the detrimental effects of these changes that can be seen

cytologically.

The slower migrating band detectable in both wild-type sporulating

cells and in vegetative cells ectopically expressing Nd8t■ ) (see asterisk, Figures

A4-3 and A4-4) may be a post-translationally modified form of Ndt80-HA. It

remains to be determined whether this form is due to phosphorylation and

184

Figure A4-4. GAL-NDT80-HA Inhibits Vegetative Growth

Plasmids containing the GAL1,10 promoter (pCAL) (i), or NDT80-HA

under control of the GAL1,10 promoter (pCAL-NDT80) (ii), were integrated

into the wild-type W303 strain, YSC007. Two different integrants each of

pGAL and pCAL-NDT80-HA were streaked on galactose-supplemented plates

(A and B), or grown in liquid culture containing galactose (C). Integration of

pGAL alone (A and B, ii) did not affect cell growth. However, integrant #1 of

pGAL-NDT80 (A, ii) did not grow on galactose plates, whereas integrant #2

(A, ii) did grow on galactose. All integrants grew equally well in medium

containing glucose instead of galactose (data not shown). Extracts were made

from integrant #1 (lanes 1-4) or integrant #2 (lanes 5-7) and analyzed by

Western analysis (C). Cells were either untreated (lanes 1, 3, and 7) or

induced with galactose for 1.2 h (lanes 2 and 5) or 3 h (lanes 4 and 6) before

harvesting. Arrow indicates Ndt80-HA protein. Asterisk marks a slower

migrating form of Ndt80-HA.

185

•*•

C. integrant #1F-1

1.2 3

H-, +

integrant #2H

1.2 3 h in GALI-1

+ + - galactose

lane 5 6 7

186

* *

* *

==

-*

- -

*

* - -

* * *s

-- - * :

whether this is an active form of Ndt80. However, it is worth noting that

there is a general correlation between strains expressing the slower migrating

form of Ndt80 and those with increased Ndt80 activity (compare integrants 1

and 2, Figure A4–4).

Ndt80 activity is a target of the meiotic recombination checkpoint

Preliminary characterization has implicated Ndt80 as a target of the

meiotic recombination checkpoint. Strains in which the checkpoint isactivated due to accumulation of recombination intermediates were found to

lack Ndt80 activity for up to 8 h after transfer to sporulation medium (Chu &

Herskowitz, 1998). These conclusions were based on using CLB1 transcription

as an assay for Ndt80 activity. Consistent with this working model, I observed

that the prophase arrest of an nat30 mutant is epistatic to the radl? dmc1

phenotype. To characterize the role of Ndt80 in the meiotic recombination

checkpoint further, I used another assay for Ndt80 activity -- SMK1

transcription (see Figure A4-5A). The results with CLB1 or SMK1 are similar,

suggesting that a general function of Ndt80 is inactivated by the checkpoint.

In addition, I repeated the original experiment, this time assaying Ndt80

activity for up to 10 h after transfer to sporulation medium (see Figure A4–

5B). I found that by 10 h, a slight amount of CLB1 transcription occurs,

suggesting that Ndt80 eventually becomes active.

It has been shown that the absence of Rad17 allows the prophase arrest

of a dmc1 mutant to be bypassed without the repair of any recombination

intermediates (Lydall et al., 1996). A checkpoint role has also been attributed

to Red1 and Mek1 (Xu et al., 1997). However, it remains controversial

whether mutations in Red1 or Mek1 result in repair of the recombination

intermediates triggering the checkpoint. I found that CLB1 is transcribed in

'', ''-

■ º ºn

->

187

Figure A4-5. Ndt80 is a Target of the Meiotic Recombination Checkpoint(A) RNA was harvested from SK1 strains YSC907, YSC927, and YSC928

at hourly intervals after transfer to sporulation medium (SPM) for up to eight

hours. The Northern blot was hybridized with a probe to SMK1. See Figure

6, Chu & Herskowitz (1998) for hybridizations of this same blot with probes

against NDT80, CLB1, and TCM1. (B) RNA was harvested from strains

YSC907 and YSC926 at intervals of two hours after transfer to SPM for up to 10

hours. The Northern blot was hybridized with probes to either NDT80 or

CLB1. (To ensure that YSC907 had not acquired additional mutations, such as

alterations in the mitochondrial genome, I showed that a plasmid carrying

the wild-type DMC1 gene could complement the sporulation defect of the

strain.)

188

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L–|—||—||—||—|

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*LXIVNS8Z99ff£ZL08/99ff£ZL08/99†9ZL0=}

|||||||||||||||||||

|_|_|_|_|_|_|_|ZIppu

LoupLoupZIppu*'C'.

189

-º-º:

both dmc1 red1 and dmg 1 mek1 double mutants (data not shown), which

shows that Ndt80 is active in these backgrounds.

An intriguing question is how the meiotic recombination checkpoint

may impinge on Ndt80 activity. Regulation may occur at any of several

levels -- stability of NDT80 message, translation of Ndt80 protein, post

translational modification of Ndt80, localization of Ndt80 protein, association

of Ndt80 with interacting proteins, or Ndt80 activity itself. In an initial

attempt to address this question, I followed Ndt80 levels in different

backgrounds. I found that Ndt80 is detectable in checkpoint-arrested cells

(dmc1 mutants; see Figure A4-6B), as well as in unarrested cells (radly or

dmc1 radly mutants; see Figure A4-6A). Although there are differences in

the kinetics and levels of Ndt80 in these various backgrounds, the fact that

Ndt80 protein is synthesized at all suggests that the checkpoint regulates a

step after Ndt80 translation. To pursue this preliminary observation, both

Ndt80 protein and activity should be followed in the same time course,

examining levels at closer time intervals.

Discussion

To determine whether the putuative post-translational modification

identified for Ndt80 is due to phosphorylation, one can test whether this

modification is sensitive to various phosphatases. If Ndt80 is a

phosphoprotein, it will be of interest to determine whether this reflects the

state of Ndt80 function. For example, does the phosphorylation state

correspond to activation of the meiotic recombination checkpoint?

(Unfortunately the "rapid protein yeast extract" protocol used to make extracts

in Figure A4-6 cannot detect the putative post-translationally modified form

ºa

190

A

Figure A4-6. Ndt80-HA is Detectable in dmc1 Strains JPreviously described HA-tagged and untagged forms of Ndt80 were º

integrated at the URA3 locus of SK1 strains YSC907, YSC935, and YSC936. ºExtracts were harvested at 6 or 12 hours after transfer to sporulation medium

(SPM) and analyzed by Western blotting using the 12CA5 antibody against the

HA epitope. (A). Ndt80 protein (as indicated by arrow) is detectable after 6 h

of transfer to SPM in the radl 7 background and after 12 hours in SPM in the

dmc1 radl 7 background. (B). Ndt80 protein is also detectable in arrested dmg 1

cells at 6 and 12 h after transfer to SPM.

º--

191 º

WAISu■lIZL9ZL9L—L–13e}-VH--+TLoup

■ sòIIeaua■ Á■ oedIºg

ZIppuLoup■ s3.J.IeON*V…vv

192■

of Ndt80, even in a wild-type background. Thus, to determine whether there

is any correlation between Ndt80 activity and protein mobility, extracts

should be made using the more traditional protocol used in Figures A4-3 and

A4-4.) In addition, it will be of interest to identify the relevant kinase.

Several candidates include the serine/threonine kinases, Ime2, Cdc28, and

CacS. Ime2 has been found to be necessary for expression of the middle genes

(Sia and Mitchell, 1995). I identified at least one site in Ndt80 (S-P-I-K, aa 391

394) which fits the consensus Cdc28 phosphorylation site (S/T-P-X-K/R). In

addition, both Cdc28- and Cdc5-deficient cells have arrest points similar to

that of Ndt80-deficient cells (Shuster & Byers, 1984; Xu et al., 1995; G.

Simchen, personal communication). It would also be interesting to

determine whether activation of the checkpoint affects the nuclear

localization of Ndt80 (see Figure A4-2).

One step towards understanding the function of Ndt80 would be to

localize the domains of Ndt80 necessary for MSE recognition or

transcriptional activation. This could be done by assaying different fragments

of Ndt80 for the ability to gel shift a MSE probe or activate transcription from

an MSE-lacz reporter. The N-terminal three-quarters of Ndt80 is reported to

contain several ■ º-rich domains which possibly form a ■ º-propeller structure

necessary for protein interactions. The C-terminal quarter of Ndt80 has a

three-helix signature motif -- a variation of the standard helix-loop-helix

motif known to bind DNA (C. Bazan, personal communication).

The question remains whether Ndt80 is involved in the commitment

of cells to sporulation. I found that ectopic expression of Ndt80 in vegetative

cells is not sufficient to trigger sporulation. Other inputs, such as nutrient

conditions, may be necessary. Another question is which, if any, of the many

193

*…*…

targets of Ndt80 are sufficient to bypass an nat30 mutant arrest. I tried to

express CLB1 under control of the promoter of either NDT80 or IME1 (the

latter reagent was a generous gift from Yona Kassir). However, I found that

these cells still did not undergo meiotic division or form spores. A closer

characterization of these strains -- such as assaying sister chromatid separation

-- may be worthwhile (suggested by A. Murray).

194

- **

. . "

*

Table A4-1. Yeast Strains for Appendix Four

strain mating strainnumber type relevant genotype backgroundYSC007 a/O. W303

YSC407 a/o NDT80-untagged SK1YSC446 a/o NDT80-myc SK1YSC484 a/O. NDT80-HA SK1

YSC907 a/o dmc1::ARG4 trp 1::hisG arga-Bgl SK1YSC926 a/O rad17::his G-URA3 SK1

YSC927 a/O rad17::his G-URA3 SK1

YSC928 a/o radl 7:hisG-URA3 dmc1::ARG4 trpl.::hisG argº-Bgl SK1YSC935 a/O rad 17::his G SK1

YSC936 a/o radl?::hisG dmc1::ARG4 arga-Bgl SK1

The W303 genotype is ade2-1 can1-100 leu2-3,112 his3-11,15ura3 trp 1-1 psiThe SK1 genotype is ura■ lys2 HO::LYS2 leu.2

195

*-

** *

---

REFERENCES

196

Amon, A., Tyers, M., Futcher, B., and Nasmyth, K. (1993). Mechanisms that

help the yeast cell cycle clock tick: G2 cyclins transcriptionally activate G2

cyclins and repress G1 cyclins. Cell 74,993-1007.

Bailey, T. and Elkan, C. (1994). Fitting a mixture model by expectation

maximization to discover motifs in biopolymers. Proceedings of the Second

International Conference on Intelligent Systems for Molecular Biology, AAAI

Press, Menlo Park, 28-36.

Bell, L. R., Maine, E. M., Schedl, P., and Cline, T. W. (1988). Sex-lethal, a

Drosophila sex determination switch gene, exhibits sex-specific RNA splicing

and sequence similarity to RNA binding proteins. Cell 55, 1037-46.

Bishop, D. K., Park, D., Xu, L., and Kleckner, N. (1992). DMC1: a meiosis

specific yeast homolog of E. coli recA required for recombination,

synaptonemal complex formation, and cell cycle progression. Cell 69, 439-56.

Bowdish, K. and Mitchell, A.P. (1993). Bipartite structure of an early meiotic

upstream activation sequence from Saccharomyces cerevisiae. Mol. Cel. Biol.

13, 2172–81.

Briza, P., Breitenbach, M., Ellinger, A., and Segall, J. (1990). Isolation of two

developmentally regulated genes involved in spore wall maturation in

Saccharomyces cerevisiae. Genes Dev. 4, 1775-89.

Buckingham, L., Wang, H., Elder, R., McCarroll, R., and Esposito, R.E. (1990).

197

Nucleotide sequence and promoter analysis of SPO13, a meiosis-specific gene

of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. 87, 9406-10.

Burns, N., Grimwade, B., Ross-MacDonald, P., Choi, E., Finberg, K., Roeder,

G.S., and Snyder, M. (1994). Large-scale analysis of gene expression, protein

localization, and gene disruption in Saccharomyces cerevisiae. Genes Dev. 8,1087–105.

Byers, B. and Goetsch, L. (1975). Behavior of spindles and spindle plaques in

the cell cycle and conjugation of Saccharomyces cerevisiae. J. Bacteriol. 124,511–523.

Byers, B. (1981). Cytology of the Yeast Life Cycle. In The Molecular Biology of

the Yeast Saccharomyces. Life Cycle and Inheritance. J.N. Strathern, E.W.

Jones, and J.R. Broach, eds. (Cold Spring Harbor, NY: CSH Laboratory Press),

pp. 59-96.

Cao, L., Alani, E., and Kleckner, N. (1990). A pathway for generation and

processing of double-strand breaks during meiotic recombination in S.cerevisiae. Cell 61, 1089-101.

Caro, L., Tettelin, H., Vossen, J., Ram, A., van den Ende, H., Klis, F. (1997). In

silicio identification of glycosyl-phosphatidylinositol-anchored plasma mem

brane and cell wall proteins of Saccharomyces cerevisiae. Yeast 13, 1477-89.

198

Chan, C. and Botstein, D. (1993). Isolation and characterization of chromo

some-gain and increase-in-ploidy mutants in yeast. Genetics 135, 677-91.

Chapman, D. L., and Wolgemuth, D. J. (1993). Isolation of the murine cyclin

B2 cDNA and characterization of the lineage and temporal specificity of

expression of the B1 and B2 cyclins during oogenesis, spermatogenesis and

early embryogenesis. Development 118, 229-40.

Chu, S. and Herskowitz, I. (1998) Gametogenesis in yeast is regulated by a

transcriptional cascade dependent on Ndt80. Mol. Cell 1, 685-696.

Clancy, M.J. (1998). Meiosis: Step-by-step through sporulation. Current

Biology 18, R461-R463.

Collart, M. and Struhl, I. (1994). NOT1(CDC39), NOT2(CDC36), NOT3, and

NOT4 encode a global-negative regulator of transcription that differentiallyaffects TATA-element utilization. Genes Dev. 8, 525-37.

Covitz, P., Herskowitz, I., Mitchell, A.P. (1991). The yeast RME1 gene encodes

a putative zinc finger protein that is directly repressed by a1-02. Genes Dev. 5,1982–9.

Cross, F. R., and Tinkelenberg, A. H. (1991). A potential positive feedback loop

controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle.Cell 65,875–83.

199

|-

Dahmann, C., Diffley, J. F., and Nasmyth, K. A. (1995). S-phase-promoting

cyclin-dependent kinases prevent re-replication by inhibiting the transition of

replication origins to a pre-replicative state. Curr. Biol. 5, 1257-69.

Dahmann, C., and Futcher, B. (1995). Specialization of B-type cyclins for

mitosis or meiosis in S. cerevisiae. Genetics 140, 957–63.

DeRisi, J.L., Iyer, V.R, and Brown, P.O. (1997). Exploring the metabolic and

genetic control of gene expression on a genomic scale. Science 278, 680-86.

DeVirgilio, C., DeMarini, D., Pringle, J.R., (1996). SPR28, a sixth member of

the septin gene family in Saccharomyces cerevisiae that is expressed

specifically in sporulating cells. Microbiol. 142,2897-905.

Dubois, E. and Messenguy, F. (1997). Integration of the multiple controls

regulating the expression of the arginase gene CAR1 of Saccharomyces

cerevisiae in response to differentnitrogen signals: role of Gln2p, Argkp

Mcm1p, and Umeåp. Mol. Gen. Genet. 253, 568-80.

Echols, H. and Green, L. (1971). Establishment and maintenance of repression

by bacteriophage lambda; the role of the cI, cII, and cIII proteins. Proc. Natl.

Acad. Sci. U.S.A. 68,2190-4.

Eddy, E. and O'Brien, D. (1998). Gene expression during mammalian meiosis.

Curr. Top. Dev. Biol. 37, 141-200.

200

Eisen, M.B., Spellman, P.T., Brown, P.O., and Botstein, D. (1998). Cluster

analysis and display of gene expression patterns. Submitted.

Engles, F. and Croes, A. (1968). The synaptinemal complex in yeast.

Chromosoma 25, 104-110.

Epstein, C. B., and Cross, F. R. (1992). CLB5: a novel B cyclin from budding

yeast with a role in S phase. Genes Dev. 6, 1695-706.

Esposito, R.E., Frink, H., Bernstein, P., and Esposito, M.S. (1972). The genetic

control of sporulation in Saccharomyces. II. Dominance and

complementation of mutants of meiosis and spore formation. Mol. Gen.

Genetics 114, 41-8.

Esposito, R.E. and Esposito, M.S. (1974). Genetic recombination and

commtiment to meiosis in Saccharomyces. Proc. Natl. Acad. Sci. 71, 3172-8.

Esposito, R.E., and Klapholz, S. (1981). Meiosis and Ascospore Development.

In The Molecular Biology of the Yeast Saccharomyces. Life Cycle and

Inheritance. J.N. Strathern, E.W. Jones, and J.R. Broach, eds. (Cold Spring

Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 211-287.

Fares, H., Goetsch, L., and Pringle, J. (1996). Identification of a

developmentally regulated septin and involvement of the septins in spore

formation in Saccharomyces cerevisiae. J. Cell Biol. 132, 399–411.

201

Fitch, I., Dahmann, C., Surana, U., Amon, A., Nasmyth, K., Goetsch, L., Byers,

B., and Futcher, B. (1992). Characterization of four B-type cyclin genes of the

budding yeast Saccharomyces cerevisiae. Mol. Biol. Cell. 3, 805-18.

Friesen, H., Lunz, R., Doyle, S., and Segall, J. (1994). Mutation of the SPS1

encoded protein kinase of Saccharomyces cerevisiae leads to defects in

transcription and morphology during spore formation. Genes Dev. 8, 2162-75.

Friesen, H., Hepworth, S.R., and Segall, J. (1997). An SSnó-Tup1-dependent

negative regulatory element controls sporulation-specific expression of DIT1

and DIT2 in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 123-34.

Garber, A. T., and Segall, J. (1986). The SPS4 gene of Saccharomyces cerevisiae

encodes a major sporulation-specific mRNA. Mol. Cell. Biol. 6, 4478-85.

Ghiara, J. B., Richardson, H. E., Sugimoto, K., Henze, M., Lew, D.J.,

Wittenberg, C., and Reed, S. I. (1991). A cyclin B homolog in S. cerevisiae:

chronic activation of the Cdc28 protein kinase by cyclin prevents exit from

mitosis. Cell 65, 163-74.

Glotzer, M. (1995) The cell cycle. The only way out of mitosis. Current

Biology 5, 970-2.

Gönczy, P., Thomas, B.J., and DiNardo, S. (1994). roughex is a dose-dependent

regulator of the second meiotic division during Drosophila spermatogenesis.Cell 77, 1015-25.

202

Goutte, C., and Johnson, A. D. (1988). al protein alters the DNA binding

specificity of 02 repressor. Cell 52,875.

Grandin, N., and Reed, S. I. (1993). Differential function and expression of

Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis. Mol. Cell.

Biol. 13, 2113–25.

Guacci, V., Koshland, D., and Strunnikov, A. (1997). A direct link between

sister chromatid cohesion and chromosome condensation revealed through

the analysis of MCD1 in S. cerevisiae. Cell 91, 7-57.

Hagan, I., Hayles, J., and Nurse, P. (1988). Cloning and sequencing of the

cyclin-related cac13+ gene and a cytological study of its role in fission yeast

mitosis. J. Cell Sci. 91, 587-95.

Hales, K. G., and Fuller, M. T. (1997). Developmentally regulated

mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell

90, 121-129.

Hepworth, S. R., Ebisuzaki, L. K., and Segall, J. (1995). A 15-base-pair element

activates the SPS4 gene midway through sporulation in Saccharomyces

cerevisiae. Mol. Cell. Biol. 15, 3934-44.

Horesh, O., Simchen, G., Friedmann, A. (1979). Morphogenesis of the

synapton during yeast meiosis. Chromosoma 75, 101-10.

203

Horie, S., Watanabe, Y., Tanaka, K., Nishiwaki, S., Fujioka, H., Abe, H.,

Yamamoto, M. and Shimoda, C. (1998). The Schizosaccharomyces pombe

mei 4+ gene encodes a meiosis-specific transcription factor containing a

forkhead DNA-binding domain. Mol. Cell. Biol. 18, 2118-2129.

Hutchens, J., Hoyle, H., Turner, F., Raff, E. (1997). Structurally similar

Drosophila alpha-tubulins are functionally distinct in vivo. Mol. Biol. Cell,

8,481-500.

Iino, Y., Hiramine, Y., and Yamamoto, M. (1995). The role of cdc2 and other

genes in meiosis in Schizosaccharomyces pombe. Genetics 140, 1235-45.

Ireland, L., Johnston, G., Drebot, M., Dhillon, N., DeMaggio, A., Hoekstra, M.,

and Singer, R. (1994). A member of a novel family of yeast 'zn-finger'

proteins mediates the transition from stationary phase to cell proliferation.

EMBO J., 13,3812-21.

Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983). Transformation of

intact yeast cells treated with alkali cations. J. Bacteriol. 153,163-168.

Jackson, J. and Lopes, J. (1996). The yeast Umeå gene is required for both

negative and positive transcriptional regulation of phospholipid biosynthetic

gene expression. Nucl Acids Res. 24, 1322-9.

204

Kadosh, D. and Struhl, K. (1997). Repression by Ume6 involves recruitment

of a complex containing Sin3 corepressor and Rpd3 histone deacetylase to

target promoters. Cell 89, 365-71.

Kassir, Y., Granot, D. and Simchen, G. (1988). IME1, a positive regulator gene

of meiosis in S. cerevisiae. Cell 52, 853-62.

Keller, A. and Young, E. (1997). Meiotic inheritance of functional Gal&0S gene

product in Saccharomyces cerevisiae. Yeast 13, 441-7.

King, R., Deshaies, R., Peters, J., and Kirschner, M. (1996). How proteolysis

drives the cel cycle. Science 274, 1652-9.

Klapholz, S. and Esposito, R.E. (1980). Isolation of SPO12-1 and SPO13-1 from

a natural variant of yeast that undergoes a single meiotic division. Genetics,

96, 567-88.

Klaphoz, S., Waddell, C., and Esposito, R. (1985). The role of the SPO11 gene

in meiotic recombination in yeast. Genetics 110, 187-216.

Kleckner, N. (1996). Meiosis: how could it work? Proc. Natl. Acad. Sci. 93,

8167-74.

Koch, C., and Nasmyth, K. (1994). Cell cycle regulated transcription in yeast.

Curr. Opin. Cell Biol. 6,451-9.

205

Kominami, K., Sakata, Y., Sakai, M. and Yamashita, I. Protein kinase activity

associated with the IME2 gene product, a meiotic inducer in the yeast

Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 57, 1731-5

Kovalenko, O, Plug, A., Haaf, T., Gonda, D., Ashley, T., Ward, D., Radding C.,

and Golub, E. (1996). Mammalian ubiquitin-conjugating enzyme Ubc9

interacts with RadS1 recombination protein and localizes in synaptonemal

complexes. Proc. Natl. Acad. Sci. 93,2958-63.

Kratzer, S. and Schuller, H. (1997). Transcriptional control of the yeast acetyl

CoA synthetase gene, ACS1, by the positive regulators CAT8 and ADR1 and

the pleiotropic repressor LIME6. Molec. Microbiol. 26, 631-41.

Krisak, L., Strich, R., Winters, R. S., Hall, J. P., Mallory, M. J., Kreitzer, D.,

Tuan, R. S., and Winter, E. (1994). SMK1, a developmentally regulated MAP

kinase, is required for spore wall assembly in Saccharomyces cerevisiae. Genes

Dev. 8, 2151-61.

Kupiec, M., Byers, B., Esposito, R. E., and Mitchell, A. P. (1997). Meiosis and

Sporulation in Saccharomyces cerevisiae. In The Molecular and Cellular

Biology of the Yeast Saccharomyces. Cell Cycle and Cell Biology, J.R. Pringle,

J.R. Broach, and E.W. Jones, eds. (Cold Spring Harbor, New York: Cold Spring

Harbor Laboratory Press), pp. 889-1036.

206

Lashkari, D., DeRisi, J., McCusker, J., Namath, A., Gentile, C., Hwang, S.

Brown, P., and Davis, R. (1997). Yeast microarrays for genome wide parallel

genetic and gene expression analysis. Proc. Natl. Acad. Sci. 94, 13,057-62.

Law, D.T. and Segall, J. (1988). The SPS100 gene of Saccharomyces cerevisiae is

activated late in the sporulation process and contributes to spore wallmaturation. Mol. Cell. Biol. 8, 912-22.

Lee, R. and Honigberg, S. (1996). Nutritional regulation of late meiotic events

in Saccharomyces cerevisiae through a pathway distinct from initiation.

Mol. Cell. Biol. 16, 3222-32.

Leem, S.-H., Chung, C.-N., Sunwoo, Y., and Araki, H. (1998). Meiotic role of

SWI6 in Saccharomyces cerevisiae. Nucl. Acids Res. 26, 3154-3158.

Levin, D. and Errede B. (1995). The proliferation of MAP kinase signaling

pathways in yeast. Cur. Op. in Cell Biol. 7, 197-202.

Li, X. and Cai, M. (1997). Inactivation of the cyclin-dependent kinase Cdc28

abrogates cell cycle arrest induced by DNA damage and disassembly of the

mitotic spindle in Saccharomyces cerevisiae. Mol. Cell Biol. 17, 2723-34.

Lin, T. Y., Viswanathan, S., Wood, C., Wilson, P. G., Wolf, N., and Fuller, M.

T. (1996). Coordinate developmental control of the meiotic cell cycle and

spermatid differentiation in Drosophila males. Development 122, 1331-41.

207

Lydall, D., Nikolsky, Y., Bishop, D. K., and Weinert, T. (1996). A meiotic

recombination checkpoint controlled by mitotic checkpoint genes. Nature 383,840-3.

Maher, M., Cong, F., Kindelberger, D., Nasmyth, K., and Dalton, S. (1995). Cell

cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a

ternary complex factor. Mol. Cell. Biol. 15, 3129-37.

Marschall, L.G., and Stearns, T. (1997) Cytoskeleton: Anatomy of an

organizing center. Curr. Biol. 7, R754-6.

Malavasic, M. and Elder, R. Complementary transcripts from two genes

necessary for normal meiosis in the yeast Saccharomyces cerevisiae. Mol. Cell.

Biol. 10, 2809-19.

Malone, R. E. (1990). Dual regulation of meiosis in yeast. Cell 61, 375-8.

Matsubara, N., Yanagisawa, M., Nishimune, Y., Obinata, M. and Matsui, Y.

(1995). Murine polo like kinase 1 gene is expressed in meiotic testicular gem

cells and oocytes. Mol. Rep. and Dev. 41, 407-15.

Michaelis, C., Ciosk, R., Nasmyth, K. (1997). Cohesins: chromosomal

proteins that prevent premature separation of sister chromatids. Cell 91, 3545.

208

Miller, J. H. (1972). Experiments in Molecular Genetics (Cold Spring Harbor,

New York: Cold Spring Harbor Laboratory).

Mitchell, A. P. (1994). Control of meiotic gene expression in Saccharomyces

cerevisiae. Microbiol. Rev. 58, 56-70.

Moens, P. B., and Rapport, E. (1971). Spindles, spindle plaques, and meiosis in

the yeast Saccharomyces cerevisiae (Hansen). J. Cell Biol. 50, 344-61.

Miyakawa, I., Aoi, H., and Sando, N. (1984). Fluorescence microscopic studies

of mitochondrial nucleoids during meisois and sporulation in the yeast,

Saccharomyces cerevisiae. J. Cell Sci. 66, 21-38.

Miyazaki, W.Y. and Orr-Weaver, T.L. (1994). Sister-chromatid cohesion inmitosis and meiosis. Annual Review of Genetics 28, 167-87.

Moens, P.B. and Rapport, E., (1971a). Spindles, spindle plaques, and meiosis

in the yeast Saccharomyces cerevisiae (Hansen). J. Cell. Biol. 50, 1344.

Moens, P.B. and Rapport, E. (1971b). Synaptic structures in the nuclei of

sporulation yeast, Saccharomyces cerevisiae (Hansen). J. Cell Sci. 9, 665.

Molnar, M., Bahler, J., Sipiczki, M., and Kohli, J. (1995). The recô gene of

Schizosaccharomyces pombe is involved in linear element formation,

chromosome pariing and sister-chromatid cohesion during meiosis. Genetics141, 61-73.

209

Moore, D., Page, A., Tang, T., Kerrebrock, A., Orr-Weaver, T. (1998). The

cohesion protein MEI-S332 localizes to condensed meiotic and mitotic

centromeres until sister chromatids separate. J. Cell Biol. 140, 1003-12.

Murray, A.W. and Hunt, T. (1993). The Cell Cyle, an Introduction. (W.H.

Freeman and Company, New York).

Nasmyth, K. (1996). At the heart of the budding yeast cell cycle. Trends Gen.

12, 405-412.

Nasmyth, K. (1996). Viewpoint: putting the cell cycle in order. Science 274,1643–5.

Neiman, A. (1998). Prospore membrane formation defines a

developmentally regulated branch of the secretory pathway in yeast. J. CellBiol. 140, 29–37.

Ozsarac, N., Straffon, M. J., Dalton, H. E., and Dawes, I. W. (1997). Regulation

of gene expression during meiosis in Saccharomyces cerevisiae: SPR3 is

controlled by both ABFI and a new sporulation control element. Mol. Cell.

Biol. 17, 1152-9.

Pagano, M., Pepperkok, R., Verde, F., Ansorge, W., and Draetta, G. (1992).

Cyclin A is required at two points in the human cell cycle. EMBO J. 11,961-71.

210

Page, A. W., and Orr-Weaver, T.L. (1997). Stopping and starting the meiotic

cell cycle. Curr. Op. Genet. Dev. 7, 23-31.

Palmer, D., O'Day, K., and Margolis R. (1990). The centromere specific histone

CENP-A is selectively retained in discrete foci in mammalian sperm nuclei.Chromosoma 100, 32-6.

Patton, E., Willems, A., and Tyers, M. (1998). Combinatorial control in

ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis. Tr. in

Genetics 14, 236-243.

Percival-Smith, A. and Segall, J. (1986). Characterization and mutational

analysis of a cluster of three genes expressed preferentially during sporulation

of Saccharomyces cerevisiae. Mol. Cell. Biol. 6, 2443-51.

Picard, A., Galas, S., Peaucellier, G., and Doree, M. (1996) Newly assembed

cyclin B-cdc2 kinase is required to suppress DNA replication between meiosis

I and meiosis II in starfish oocytes. EMBO J. 15, 3590-8.

Reichardt, L., and Kaiser, A. D. (1971). Control of lambda repressor synthesis.

Proc. Natl. Acad. Sci. USA 68,2185-9.

Richardson, H., Lew, D.J., Henze, M., Sugimoto, K., and Reed, S. I. (1992).

Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and inG2. Genes Dev. 6, 2021-34.

211

Roeder, A.D. and Shaw, J.M. (1996). Vacuole partitioning during meiotic

division in yeast. Genetics 144, 445-458.

Roeder, G. S. (1997). Meiotic chromosomes: it takes two to tango. Genes Dev.11, 2600-2621.

Rose, M. D., Winston, F., and Hieter, P. (1990). Methods in Yeast Genetics

(Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

Rothstein, R. (1991). Targetting, disruption, replacement, and allele rescue:

integrative DNA transformation in yeast. Meth. Enzymol. 194, 281-301.

Ruggiu, M., Speed, R., Taggart, M., McKay, S.J., Kilanowski, F., Saunders, P.,

Dorin, J., and Cooke, H. J. (1997). The mouse Dazla gene encodes a cytoplasmic

protein essential for gametogenesis. Nature 389, 73-7.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning. A

Laboratory Manual, Second Edition (Cold Spring Harbor, New York: Cold

Spring Harbor Laboratory Press).

Sagata, N. (1997). What does Mos do in oocytes and somatic cells? Bioessays,

19, 13–21.

Sassone-Corsi, P. (1997). Transcriptional checkpoints determining the fate of

male germ cells. Cell 88, 163-6.

212

Schild, D. and Byers, B. (1978). Meiotic effects of DNA-defective cell division

cycle mutation of Saccharomyces cerevisiae. Chromosoma 70, 109-130.

Schild, D. and Byers, B. (1980). Diploid spore formation and other meiotic

effects of two cell-division-cycle mutations of Saccharomyces cerevisiae.

Genetics 96, 859–876.

Schultz, L. D., and Friesen, J. D. (1983). Nucleotide sequence of the TCM1 gene

(ribosomal protein L3) of Saccharomyces cerevisiae. J. Bacteriol. 155, 8-14.

Shah, J. and Clancy, M.J. (1992). IME4, a gene that mediates MAT and

nutritional control of meiosis in Saccharomyces cerevisiae. Mol. Cell Biol.

12, 1078–86.

Sherman, F., Fink, G., and Hicks, J. B. (1986). Methods in Yeast Genetics: A

Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor

Laboratory Press).

Shuster, E. O., and Byers, B. (1989). Pachytene arrest and other meiotic effects

of the start mutations in Saccharomyces cerevisiae. Genetics 123, 29-43.

Sia, R. and Mitchell, A.P., (1995). Stimulation of later functions of the yeast

meiotic protein kinase Ime2p by the IDS2 gene product. Mol. Cell. Biol. 15,5279-87.

213

C

Simchen, G. (1974). Are mitotic functions required in meiosis? Genetics 76,745-753.

Sikorski, R.S., and Hieter, P. (1989). A system of shuttle vectors and yeast host

strains designed for efficient manipulaiton of DNA in Saccharomyces

cerevisiae. Genetics 122, 19–27.

Stoler, S., Keith, K., Curnick, K. and Fitzgerald-Hayes, M. (1995). A mutation

in CSE4, an essential gene encoding a novel chromatin-associated protein in

yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis.Genes Dev. 9, 573-86.

Soderstrom, K. O., and Suominen, J. (1980). Histopathology and ultrastructure

of meiotic arrest in human spermatogenesis. Arch. Pathol. Lab. Med. 104,47682.

Stern, B. and Nurse, P. (1996). A quantitative model for cdc2 control of S

phase and mitosis in fission yeast. Tr. Genet. 12, 345-50.

Stillman, B. (1996). Cell cycle control of DNA replication. Science 274, 165964.

Surana, U., Robitsch, H., Price, C., Schuster, T., Fitch, I., Futcher, A. B., and

Nasmyth, K. (1991). The role of CDC28 and cyclins during mitosis in the

budding yeast S. cerevisiae. Cell 65, 145-61.

214

Sweeney, C., Murphy, M., Kubelka, M., Ravnik, S. E., Hawkins, C. F.,

Wolgemuth, D. J., and Carrington, M. (1996). A distinct cyclin A is expressed

in germ cells in the mouse. Development 122, 53-64.

Thomas, B. J., Gunning, D. A., Cho, J., and Zipursky, L. (1994). Cell cycle

progression in the developing Drosophila eye: roughex encodes a novel

protein required for the establishment of G1. Cell 77, 1003-14.

Toscani, A., Mettus, R.V., Coupland, R., Simpkins, H., Litvin, J., Orth, J.,

Hatton, K.S. and Reddy, E.P. (1997). Arrest of spermatogenesis and defective

breast development in mice lacking A-myb. Nature 386, 713-7.

Townsley, F.M. and Ruderman, J.V. (1998). Proteolytic ratchets that control

progression through mitosis. Trends in Cell Biol. 8, 238-244.

Toyn, J.k, Araki, H., Sugino, A., and Johnston, L. (1991). The cell-cycle

regulated budding yeast gene DBF2, encoding a putative protein kinase, has a

homologue that is not under cell-cycle control. Gene 104, 63-70.

Toyn, J. and Johnston, L.H. (1993). Spol2 is a limiting factor that interacts

with the cell cycle protein kinases Dbf2 and Dbf20, which are involved in

mitotic chromatid disjunction. Genetics 135,963-71.

Toyn, J. and Johnston, L.H. (1994). The Dbf2 and Dbf20 protein kinases of

budding yeast are activated after the metaphase to anaphase cell cycle

transition. EMBO J. 13, 1103-13.

14'■ ,

Q_º

Y

*

215 ySº

Tsui, K., Simon, L., and Norris, D. (1997). Progression into the first meioticdivision is sensitive to histone H2A-H2B dimer concentration in

Saccharomyces cerevisiae. Genetics 145, 647-59.

Wada, Y. and Y. Anraku (1992). Genes for directing vacuolar morphogenesis

in Saccharomyces cerevisiae. II. VAM7, a gene for regulating morphogenic

assembly of the vacuoles. J. Biol. Chem. 267, 18671-75.

Wang, Y., Catlett, N., and Weismann, L. (1998). VacSp, a vacuolar protein

with armadillo repeats, functions in both vacuole inheritance and protein

targeting from the cytoplasm to vacuole. J. Cell Biol. 140, 1063-74.

White-Cooper, H., Schafer, M. A., Alphey, L. S., and Fuller, M. T. (1998).

Transcriptional and post-transcriptional control mechanisms coordinate the

onset of spermatid differentiation with meiosis I in Drosophila. Development

125, 124–34.

Xu, L., Ajimura, M., Padmore, R., Klein, C., and Kleckner, N. (1995). NDT80, a

meiosis-specific gene required for exit from pachytene in Saccharomycescerevisiae. Mol. Cell. Biol. 15, 6572–81.

Xu, L., Weiner, B. M., and Kleckner, N. (1997). Meiotic cells monitor the status

of the interhomolog recombination complex. Genes Dev. 11, 106-18.

Yoshida, M., Kawaguchi, H., Sakata, Y., Kominami, K., Hirano, M., Shima, H.,

Akada R., and Yamashita I. (1990). Initiation of meiosis and sporulation in216

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