a - 1 copyright © the mcgraw-hill companies, inc. permission required to reproduce or display...

A - 1 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

PowerPoint to accompany

Genetics: From Genes to GenomesFourth Edition

Hartwell ● Hood ● Goldberg ● Reynolds ● Silver

ReferenceA

Prepared by Malcolm SchugUniversity of North Carolina Greensboro


Saccharomyces cerevisiaeSaccharomyces cerevisiae: : Genetic Portrait of a YeastGenetic Portrait of a Yeast


Yeast Cells Showing Small and Large Buds Yeast Cells Showing Small and Large Buds Characteristic of Different Phases of Mitotic Cell CycleCharacteristic of Different Phases of Mitotic Cell Cycle

Fig. A. 1


The Life Cycle of Yeast

Fig. A. 2


Outline of Reference AOutline of Reference A

Overview of yeast in laboratoryOverview of yeast in laboratory Current knowledge of the yeast genomeCurrent knowledge of the yeast genome Basic tools for studying yeastBasic tools for studying yeast Significant details of yeast life cycleSignificant details of yeast life cycle

Cell differentiationCell differentiation Molecular mechanisms of determining cell typeMolecular mechanisms of determining cell type

MatingMating How cell-cell communication through pheromones How cell-cell communication through pheromones

promotes conversion of haploid cells to diploid cellspromotes conversion of haploid cells to diploid cells


The Nuclear Genome of YeastThe Nuclear Genome of Yeast 16 linear chromosomes16 linear chromosomes Each chromosome containsEach chromosome contains

One centromereOne centromere Two termini with longer subtelomeric repeats followed by short Two termini with longer subtelomeric repeats followed by short

telomere repeat at very endstelomere repeat at very ends Multiple origins of replication at 30-40 kb intervalsMultiple origins of replication at 30-40 kb intervals Packed into nucleosomes with core histones H2A, H2B, H3, and H4Packed into nucleosomes with core histones H2A, H2B, H3, and H4

Fig. A.3a


Comparison of Yeast and Human Comparison of Yeast and Human GenomesGenomes

YeastYeast HumanHuman

Genome sizeGenome size 12 Mb12 Mb 3200 Mb3200 Mb

Number of Number of ChromosomesChromosomes 1616

22 pairs of autosomes22 pairs of autosomes

1 pair of sex chromosomes1 pair of sex chromosomes

Number of Number of genesgenes 60006000 20,000-30,00020,000-30,000

PloidyPloidy Haploid or DiploidHaploid or Diploid DiploidDiploid


Physical Maps and Complete Physical Maps and Complete Sequence of GenomeSequence of Genome

Karyotypes – pulse-Karyotypes – pulse-field gel electrophoresisfield gel electrophoresis

Fig. A.3 b


Genetic MapsGenetic Maps > 1000 markers determined by tetrad analysis> 1000 markers determined by tetrad analysis 4400 cM – very long compared to other fungi4400 cM – very long compared to other fungi 3 kb/ cM3 kb/ cM Genetic and physical maps proportionalGenetic and physical maps proportional No meiotic recombination in rDNA repeats and around No meiotic recombination in rDNA repeats and around

centromerecentromere

Fig. A.4


Determining Number of Yeast GenesDetermining Number of Yeast Genes

~ 7505 ORFs estimated using arbitrary cutoff of ~ 7505 ORFs estimated using arbitrary cutoff of 100 amino acids/protein100 amino acids/protein

Some ORFs may be shorter, some longerSome ORFs may be shorter, some longer Likely 6000-6500 genesLikely 6000-6500 genes 2000 protein products have no function2000 protein products have no function 20% of genes lethal when disrupted20% of genes lethal when disrupted Introns in 4% - 5% of genesIntrons in 4% - 5% of genes Also genes for rRNAs, 274 tRNA genes, 71 Also genes for rRNAs, 274 tRNA genes, 71

sncRNAs, RNAs of unknown function, 3 RNAs sncRNAs, RNAs of unknown function, 3 RNAs functional subunits of RNase P, endoribonuclease functional subunits of RNase P, endoribonuclease MRP, and telomeraseMRP, and telomerase


Gene Arrangement, Genome Gene Arrangement, Genome Organization, and Genome FunctionOrganization, and Genome Function 75% of genome transcribed into RNAs75% of genome transcribed into RNAs One gene every 1.5 – 2.0 kb of DNAOne gene every 1.5 – 2.0 kb of DNA Some coregulated but not clustered according to function Some coregulated but not clustered according to function

like bacteria operonslike bacteria operons Few reiterated sequencesFew reiterated sequences

100-200 copies of rDNA100-200 copies of rDNA 5-7 copies of tRNA genes5-7 copies of tRNA genes 4-5 copies of subtelomeric repeats4-5 copies of subtelomeric repeats 50 copies of retrotransposons called Ty elements50 copies of retrotransposons called Ty elements

LTRs at either endLTRs at either end 5.4-6.3 kb in length5.4-6.3 kb in length Can pair and recombine even if on different chromosomesCan pair and recombine even if on different chromosomes Source of reciprocal translocations, inversions, and deletionsSource of reciprocal translocations, inversions, and deletions


Genome EvolutionGenome EvolutionEvolved through duplications of large chromosome segmentsEvolved through duplications of large chromosome segments

53 clustered gene duplications among 16 chromosomes53 clustered gene duplications among 16 chromosomes

Fig. A.6


Tools of the Yeast GeneticistTools of the Yeast Geneticist

Mutant Isolation and CharacterizationMutant Isolation and Characterization Transformation and integration of DNA are Transformation and integration of DNA are

very efficientvery efficient Gene replacement creates directed Gene replacement creates directed

mutationsmutations Genome-wide analysisGenome-wide analysis


Mutant Isolation in a Diploid CellMutant Isolation in a Diploid Cell

A mutation on one A mutation on one homolog produces homolog produces 2+ and 2- spores if 2+ and 2- spores if the mutation is:the mutation is: RecessiveRecessive In a nonessential In a nonessential

genegene

Fig. A.7a


Mutant Isolation in a Diploid CellMutant Isolation in a Diploid Cell

If mutation is in If mutation is in gene essential for gene essential for growth:growth: Two live spores are Two live spores are

recovered after recovered after meiosismeiosis

Fig. A.7b


Power of Yeast GeneticsPower of Yeast Genetics

Isolate large numbers of mutants, even rare Isolate large numbers of mutants, even rare mutantsmutants

Easily characterize dominance and Easily characterize dominance and recessivenessrecessiveness

Order gene products in a pathway using Order gene products in a pathway using epistasis testingepistasis testing

Identify interacting gene products via Identify interacting gene products via suppressor analysissuppressor analysis


Transformation and Integration of Transformation and Integration of DNA are Very EfficientDNA are Very Efficient

Transformation of linear or circular DNA Transformation of linear or circular DNA from external sourcesfrom external sources

High level of homologous recombination in High level of homologous recombination in yeast facilitates integration of DNA into yeast facilitates integration of DNA into chromosomechromosome


Gene Gene replacement replacement

makes it makes it possible to possible to alter genes alter genes

without genetic without genetic crosses.crosses.

Fig. A.8


Genome-wide AnalysisGenome-wide Analysis

Basic cell processes are conserved from yeast to humansBasic cell processes are conserved from yeast to humans Large numbers of cells can easily be grown and assayedLarge numbers of cells can easily be grown and assayed Mutations can be isolated in diploids, then analyzed in Mutations can be isolated in diploids, then analyzed in

haploidshaploids Conditional mutants can be isolatedConditional mutants can be isolated Recombination frequencies are high, facilitating Recombination frequencies are high, facilitating

molecular manipulationsmolecular manipulations Genome size is small and number of genes low, so Genome size is small and number of genes low, so

analysis and manipulations of the whole genome are analysis and manipulations of the whole genome are possiblepossible


Cell Differentiation in Yeast: Cell Differentiation in Yeast: Mechanisms for Determining Cell TypeMechanisms for Determining Cell Type

A, a, and a/a cells of yeast are differentiated A, a, and a/a cells of yeast are differentiated cells that express different sets of genescells that express different sets of genes

Differ in how they respond to signals from Differ in how they respond to signals from the environment and developmental the environment and developmental potentialpotential

Different genetic programs establish Different genetic programs establish pathways leading to each cell typepathways leading to each cell type


Sex is Determined by Mating Type Sex is Determined by Mating Type LocusLocus

MAT on chromosome IIIMAT on chromosome III Mating types Mating types aa and and Segregates 2:2 in tetrads derived from Segregates 2:2 in tetrads derived from

MATMATaa/MAT/MAT heterozygous diploids heterozygous diploids MATMATaa/MAT/MAT are sterile are sterile MATMATaa/MAT/MATaa and MAT and MAT/MAT/MAT cells will cells will

mate with cells of opposite typemate with cells of opposite type


MAT Locus Controls Expression of MAT Locus Controls Expression of Genes that Determine Cell TypeGenes that Determine Cell Type

MATMATaa and MAT and MAT share share some DNA sequences and some DNA sequences and some are uniquesome are unique

Presence of YPresence of Yaa, Y, Y , or , or YYaa+Y+Y had different had different consequences on consequences on expression of expression of aa-specific, -specific, --specific, haploid-specific specific, haploid-specific and meiosis (and meiosis (RMERME) genes ) genes due to activator and due to activator and repressor differencesrepressor differences

Fig. A.9


Mem1 is Both a Positive and Negative RegulatorMem1 is Both a Positive and Negative Regulator

In a In a cell, Mcm1 acts together with cell, Mcm1 acts together with 1 to 1 to activate transcription of activate transcription of -specific genes -specific genes

but acts to repress a-specific genesbut acts to repress a-specific genes

Fig. A-10a


Mem1 is Both a Positive and Negative RegulatorMem1 is Both a Positive and Negative Regulator

In an In an aa cell, Mcm1 alone is the transcriptional cell, Mcm1 alone is the transcriptional activator of activator of -specific genes.-specific genes.

Fig. A-10b


Mating Type SwitchMating Type Switch

Yeast strains differ Yeast strains differ according to stability according to stability of mating typeof mating type

Heterothallic strainsHeterothallic strains stable haploid stable haploid

mating typesmating types Homothallic strains Homothallic strains

(HO)(HO) switch mating typeswitch mating type

Fig. A. 11 a


Cassette Model of Mating Type SwitchCassette Model of Mating Type Switch

HML, MAT, and HML, MAT, and HMRHMR Mating information Mating information

on chromosome IIIon chromosome III MAT – information MAT – information

expressedexpressed HML, HMR – silent HML, HMR – silent

information information Can be copied to Can be copied to

MATMAT

Fig. A.11 b


Roles of HML, HMR, and HO in Roles of HML, HMR, and HO in Mating Type SwitchMating Type Switch

Wild-type strainsWild-type strains HML contains HML contains information information HMR contains HMR contains aa information information

aa// do not switch even in HO strains do not switch even in HO strains aa1 and 1 and 2 regulators repress transcription of 2 regulators repress transcription of

HO geneHO gene Cell can receive a cassette of mating type Cell can receive a cassette of mating type

information located in HML or HMR and information located in HML or HMR and insert it into the MAT playback locusinsert it into the MAT playback locus


Steps for Retrieving Information Steps for Retrieving Information from Storage Locusfrom Storage Locus

Product of HO gene initiates processProduct of HO gene initiates process HO-encoded enzyme makes double-HO-encoded enzyme makes double-

stranded cut in chromosome III at a specific stranded cut in chromosome III at a specific 18 bp sequence to right of Y segment in 18 bp sequence to right of Y segment in MATMAT

Double-stranded break is repairedDouble-stranded break is repaired Results in replacement of MAT DNAResults in replacement of MAT DNA


Gene Products that Function to Gene Products that Function to Keep Storage Loci SilentKeep Storage Loci Silent

Loss-of-function mutation in four SIR genes Loss-of-function mutation in four SIR genes cancel repression and activate the cancel repression and activate the 1, 1, 2, or 2, or aa1 genes located at HML or HMR1 genes located at HML or HMR

Haploid cellsHaploid cells Phenotype resembles that of Phenotype resembles that of aa// diploid because diploid because

MATMATaa and MAT and MAT alleles are expressed alleles are expressed sir sir -- lines – HO-mediated transposition can lines – HO-mediated transposition can

occur from MAT to storage locioccur from MAT to storage loci Wild-type cells – mating type information flows Wild-type cells – mating type information flows

only from storage loci to MATonly from storage loci to MAT


Role of HO in Mating Type Determination and Role of HO in Mating Type Determination and Stability Serves as Paradigm for DevelopmentStability Serves as Paradigm for Development

Pedigrees of cells Pedigrees of cells undergoing HO-undergoing HO-mating-type switch mating-type switch reveal rules reveal rules governing mitotic governing mitotic cell lineages during cell lineages during developmentdevelopment

Rule 1 - cells always Rule 1 - cells always switch in pairsswitch in pairs

Fig. A.12 a,b


Rule 2 - Cells Rule 2 - Cells must gain must gain competence to competence to switch switch Do so through Do so through

experience of experience of switching at least switching at least onceonce

Fig. A.12c

Role of HO in Mating Type Determination and Role of HO in Mating Type Determination and Stability Serves as Paradigm for Development Stability Serves as Paradigm for Development

(cont.)(cont.)


Cell-to-cell Communication Through Pheromones Cell-to-cell Communication Through Pheromones Triggers Conversion of Haploid Triggers Conversion of Haploid aa and and cells to cells to aa//

DiploidsDiploids

Pheromones bind to receptors and activate a signal transduction pathway

Fig. A.13


Sterile mutants that produce no zone of growth Sterile mutants that produce no zone of growth inhibition either fail to secrete mating pheromone, or inhibition either fail to secrete mating pheromone, or

secrete mating pheromone but are unresponsive to secrete mating pheromone but are unresponsive to pheromone from opposite cell type.pheromone from opposite cell type.

Fig. A.14


Signal transduction Signal transduction cascade in mating cascade in mating responseresponse

Binding of Binding of pheromone to pheromone to receptor causes receptor causes conversion of GDP to conversion of GDP to GTPGTP Activation of MAP Activation of MAP

kinase cascadekinase cascade Activation of STE12 Activation of STE12

transcriptional transcriptional activatoractivator

STE12 turns on genes STE12 turns on genes involved in G1 arrest involved in G1 arrest of cells, cell fusion, of cells, cell fusion, and nuclear fusionand nuclear fusion

Fig. A.15

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