the drosophila maternal effect locus deadhead encodes a ... · sophila thioredoxin homolog....

Copyright 0 1994 by the Genetics Society of America

The Drosophila Maternal Effect Locus deadhead Encodes a Thioredoxin Homolog Required for Female Meiosis and Early Embryonic Development

Helen K. Salz,' Thomas W. Flickinger, Elizabeth Mittendorf, Alexandra Pellicena-Palle, Jane P. Petschek* and Elizabeth Brown Albrecht

Department of Genetics, Case Western Reserve University, Cleveland Ohio 44106-4955

Manuscript received September 10, 1993 Accepted for publication November 26, 1993

ABSTRACT This study describes the identification, function and molecular characterization of deadhead, a Dro-

sophila thioredoxin homolog. Although in vitro studies have shown that thioredoxin can post- translationally regulate the activity of many different proteins, we find that this homolog is not essential for viability. The phenotypic analysis of two different mutations which eliminate function suggests that dhd is essential for female meiosis. The majority of eggs laid by females homozygous for null mutations are fertilized but fail to complete meiosis. A small number of escaper embryos initiate development and display a range of phenotypes suggesting functions in both preblastoderm mitosis and head development. Our analysis of deadhead's RNA expression pattern is consistent with its maternal effect function: the RNA is predominately expressed in the nurse cells of the ovary, is maternally deposited into the egg, but does not appear to be zygotically expressed during embryogenesis. Thus both our genetic and molecular data are consistent with a function during meiosis and preblastoderm mitosis. Whether the head defect indicates an additional function or is an indirect consequence of earlier defects remains to be determined.

T HE transformation from egg to embryo requires a change in environment which permits the oocyte

nucleus to complete meiosis and the resulting zygotic nuclei to begin a series of rapid mitotic divisions. In Drosophila, mature oocytes are arrested at metaphase of meiosis I and are reactivated within the oviduct (reviewed in GLOVER 1991). After fertilization, which also occurs in the oviduct, the female and male pronuclei enter S phase and begin a series of 13 rapid mitotic division cycles. These first 13 division cycles occur syn- chronously with neither cytokinesis nor gap (G) phases. Following mitosis 13, the nuclei are cellularized and a G, phase is added to the cell cycle. The 14th, 15th and 16th rounds of mitosis are no longer synchronous, but instead enter mitosis in a spatially and temporally regulated fashion. After mitosis 16 only the neural cells continue to divide with G,, S, G2 and M phases.

Although recent studies have demonstrated a remark- able amount of similarity between cell cycle regulation after cellularization and the prototypic cell cycle of yeast and cultured mammalian cells, regulation prior to cellularization is fundamentally different (reviewed in GLOVER 1991). First, there must be sensors which detect both the successful completion of meiosis and fertilization before allowing the entry into the first mitosis. Moreover, since the completion of meiosis occurs in the absence of fertilization, the "sensing" mechanisms are

EMBL/GenBank Data Libraries under the accession number L27072. The sequence data presented in this article have been submitted to the

' Present address: Department of Zoology, Miami University, Oxford, Ohio ' To whom correspondence should be addressed.

45056.

Genetics 136: 1075-1086 (March, 1994)

likely to be different. Second, the initial 13 nuclear divisions lack an important cell cycle checkpoint which normally insures completion of DNA synthesis before mitosis is initiated. Whether these divisions also lack a checkpoint which insures the completion of mitosis prior to the reinitiation of S phase remains to be determined. Third, both the machinery and the regulatory components required for these early divisions must be supplied by the mother because these events occur before the initiation of zygotic transcription. Conse- quently, the cell cycle dependent regulation necessary to insure normal early development must occur at the post- transcriptional level. As a first step toward understanding the regulation of

the reactivation of meiosis and the initiation of embryonic mitosis, genetic studies have begun to identify genes required for these early events. Some of the genes, such as stringthe cdc 25 homolog, were found to encode products required for all cell divisions (EDGAR and O'FARRELL 1989, 1990). Other genes encode products which are essential only for female meiosis and/or the nuclear cell divisions. For instance, nod encodes a kinesin-like protein which is required only for female meiosis (ZHANG and HAWLEY 1990; ZHANG et al. 1990). a-Tub67C is a specialized form of a-tubulin which is only required for female meiosis and early embryonic mitosis ( M A ~ H E W S et al. 1993). gnu, pan gu and plutonium are required to repress premitotic S phase before fertilization and may also be required to suppress the premature entry into premitotic S phase in subsequent divisions as well (FREEMAN and GLOVER 1987; SHAMANSKI and

1076 H. K. Salz et al.

ORR-WEAVER 1991). Similarly, f s ( l ) Y a , is a nuclear envelope protein which is required for the initiation of the embryonic mitotic cell cycle (LIN and WOLFNER 1991).

In this reportwe present the identification, initial phenotypic characterization and cloning of deadhead ( d h d ) . We find that dhd is a thioredoxin homolog required for female meiosis. In addition, the analysis of a small number of embryos which do complete meiosis indicates that d h d is also required for the early nuclear divisions. Thioredoxins are small proteins which function, in vitro, to reduce disulfide bonds (reviewed in HOLMGREN 1989; BUCHANAN 1991). Since redox reactions, like phosphorylation, can be used to regulate protein activity, these results suggest that dhd is essential for post-translational modification of protein activity. The finding that d h d is a maternally provided gene product whose expression is limited to oogenesis and early development suggests that dhd does not encode a general protein disulfide reductase but is instead required for specific development processes.

MATERIALS AND METHODS

Drosophila stocks and culture conditions: Mutations and rearrangements not described in the text are described in LINDSLEY and ZIMM (1992). Flies were reared at room tempera- ture which ranged from 22" to 25" on a standard cornmeal, yeast, molasses and agar medium.

Isolation of P element-induced dhd mutations and their revertants A collection of -1,500 X-linked Pelement insertion lines was generated and screened for female-sterile mutations within Df(l)DEB4D. Df(l)DEB4D is an X chromosome deficiency which is deleted for polytene bands 4E 1,2-4F 11-12 (SALZ 1992). The P element used in this screen is the P-lacW enhancer trap element described in BIER et al. (1989) located on the second chromosome balancer CyO. To destabilize the P element insertion on the second chromosome, w/w; Cy0 P-lacW/+ virgins were mated to males which carry a source of transposase, 82-3 (ROBERTSON et al. 1988) of the genotype w; Sb A2-3/TM6. The F, males ( w ; Cy0 P-lacW/+; Sb A2-3/+) were mated to C(1)DXywf females. It is in the germline of the F, males which carry the source of transposase that the P element can transpose to any of the chromosomes. Single male progeny of this cross which carry a new insertion event (recognized because the males are w+ Cy') without a source of transposase (Sb') were crossed to Df(l)DEB4D/FM7 virgins. The progeny from each vial were scored simultaneously for X linkage of the P element insertion and a female-sterile mutation within the deficiency. Of the approximately 6,700 established lines, 1,588 were found to contain X-linked P element insertions, two of which were found to be sterile over Df(l)DEB4D. Both insertions, P2 and P8 were found to be alleles of an unidentified gene which we named deadhead (dhd). Only P8 is described in detail in the text.

Wild-type revertants and deletion derivatives of dhdp8 were isolated as follows. w dhdP8/FM7virgins were crossed to males which carry a source of transposase (w; Sb A2-3/TM6.). The F, males ( w dhdP8; Sb A2-3/+) were crossed to C(I)DX ywf females and the male progeny in which the P element has remobilized (recognized because the males have white eyes) were crossed to Df(l)DEB4D/FM7 virgins and scored simultaneously for loss of the P element (as judged by the loss of the w+ eye marker) and reversion to wild type. Five wild-type revertants were isolated. In addition, the lines in which the P

element was lost but were still sterile were screened for deletions of genomic DNA by genomic Southern blotting. One deletion derivative, dhdj5 was chosen for further analysis. The deficiency Df(l)dhd8I was generated in a similar screen that allowed the recovery of lethals.

Molecular analysis Unless otherwise noted, standard molecular techniques used are described in SAMBROOK et al. (1989). Genomic DNA adjacent to the insertion in dhdP8 was isolated by plasmid rescue as described in (PIRROTTA 1986). We were pleased to find that the genomic DNA adjacent to the P element insertion was located within a -50-kb walk previously initiated in our laboratory from clones generously provided by K. VALGEIRDOTTIER and M. L. PARDUE. In situ hybridization to polytene chromosomes has shown that this walk is entirely con- tainedwithin 4F1,2. Only the central 14 kb are described in this report.

Genomic DNA was prepared and Southern blots were carried out as described in SALZ et al. (1987). Total RNA was isolated as described in CHOMCZYNSIU and SACCHI (1987) and poly(A+) RNAwas prepared and Northern blots carried out as previously described in ALBRECHT and SALZ (1993).

cDNA clones were isolated from a Drosophila ovarian cDNA library (STEINHAUER et al. 1989). A 0.8-kb cDNA clone from this library was completely sequenced on both strands by the dideoxy chain termination method (SANGER et al. 1977). The location of the P element insertion was determined by sequencing the genomic sequence adjacent to the P element insertion in dhdp8. Data base searches and alignments were generated by the BLAST programs at NCBI using the BLAST network service (ALTSCHUL et al. 1990). P element-mediated germline transformation: Germline

transformations were carried out essentially as described by SPRADLING and RUBIN (1982). The XhoI/XhoI and XbaI/ BamHI genomic fragments were inserted directly into the pCaSpeR4 transformation vector (PIRROTTA 1988) and coin- jected with the helper plasmid, phsT (STELLAR and PIRROTTA 1986), into yw"" embryos.

Immunohistochemistry, in s i tu hybridizations and cuticle preparations: Whole mount in situ hybridization to ovaries and embryos were carried out according to TAUTZ and PFEIFLE (1989) with the modifications introduced by COOLEY et al. (1992). Polytene chromosome in situ hybridizations were carried out according to the method of ENGELS et d . (1986). The fixation and staining of embryos were carried out as described in BOPP et al. (1991) with modifications suggested by CRON- MILLER and CUMMINGS (1993). Embryos were stained for engrailed protein with a monoclonal antibody diluted 1:lO (DI- NARDO et al. 1985), stained with a biotinylated goat anti-mouse secondary antibody diluted 1:5000 (Chemicon) and visualized with the Vectastain ABC elite horseradish peroxidase signal detection system according to the manufacture's instructions. The anti-sperm antibody (AX-D5; KARR 1991) was diluted 1:5 and visualized by staining with a rhodamine conjugated goat anti-mouse secondary antibody uackson Laboratories) diluted 1200. DNA was visualized by staining with 4,6diamidinc-2- phenylindole (DAPI) at 1 pg/ml in 1 X phosphate-buffered saline. Cuticle preparations were prepared and mounted in Hoyer's medium as described in WIESCHAUS and NUSSLEIN- VOLHARD (1986).

RESULTS

Isolation and genetic characterization of deadhead The d e a d h e a d ( d h d ) locus was identified during a Pel- ement screen for recessive female-sterile alleles that mappedwithin the 4E1,2-4F11,12 region on the Xchro- mosome (see MATERIALS AND METHODS). To demonstrate

TABLE 1

Distribution of embryonic stages seen in a 4-hr collection of embryos from dhd and wild-type mothers

Pcl-rrnt;lgc o f cml,l~os ;I1

I.rss l l l> l l l x I’olc crII so. 01

(:rnot!.pc o f nurlc;1l- fornl;1tion c l l l l , t~o~ Inotllel- divisions and later sc;~ges counted

M ’ i M tvpc 11 X 9 d h r l ” ~

20(i 90 I O

dhr l” 224

I):< 7 222

Eml)tTos frotn a 4-hr collrction wcre stained w i t h DAPI and csi~ln- inrtl. Emlx-\.os in whirh 110 nuclr;~r structure-s could l w tlctrccc*tl wcrc n o t inclutlrtl.

that the single Pelement insertion, located within polytene band 4F1,2 is the cause of the female-sterili?, we asked whether there was a correlation between reversion of the mutant phenotype with the loss of the Pelement insertion. Indeed, of the five independently isolated wild-type revertants of c l l~d”’~ , all appeared to be precise or near precise excisions of the I’ element insertion a s judged by Southern blot analysis (data not shown).

Because all o f the known mutations within polytene band 4F1,2 complement dlzd”~’ (data not shown; see S..u:z 1992 for a complete genetic map of this region) we initiated a screen to isolate additional alleles of dhd. This was done by remobilization of the P element insertion within dhci”” (see SIATERlhIS ASI) vF..n~o~)s). Each derivative line was then screened for the presence of small deletions of flanking genomic DNA. Two derivatives were chosen for further study: dltd’’ a small (- 1.3 kb) deletion which does not affect the function of the neigh- boring genes and I l f ( l ) d l ~ d 8 1 a larger (greater than 16 kb) deletion which removes the dhd transcription rmit as well as several additional genes (see below).

Both the genetic and molecular characterization suggest that dhd”” and d h d 5 are n u I I alleles. Both mutations eliminate the d h d RNA (see molecular analysis below) and have similar maternal-efrect mutant phenotypes when homozygous or hemizygous. Mutant females lay eggs which are morphologically normal but do not hatch. Neither allele has any effect on viability or male fertility, nor is the mutant phenotype rescuable by a wilcl- type allele provided by the father.

Eggs from dhd mutant mothers fail to complete meiosis: Females homozygous for d h d mutations lay eggs that appear to be morphologically normal but most fail to initiate development. Embryos from both mutant and wild-type mothers were collected during a 4-hr time pe- riod, fixed and stained with DAPI to visualize the nuclei. Of the wild-type embryos 89% had completed at least 10 nuclear divisions, as evidenced by the formation of pole cells and the migration of the nuclei to the cortex (Table 1 and Figure 1A). In contrast to wild type, only 7-10% of the embryos from mutant mothers had completed at least 10 nuclear divisions. Even when the embyos from

r b

mutant mothers were aged for an additional 2 hr (a 2-4-hr collection), there was no significant change in the distribution o f phenotypes (data not shown). Simi- larly, in a 4-f-hr collection, only -10% of the embryos developed past blastoderm (data not shown).

Because most mutant embryos fail to advance through the nuclear cleavage stages, we examined earlier stages in zygotic development in greater detail. 0-4-hr collec- tions of embryos were stained with DAPI to visualize the nuclei (Figure 1) . To veric that we were only examining fertilized eggs, we double stained with a monoclonal antibody that recognizes the sperm tail ( K A K K 1991). We found that although the rate of fertilization between mutant and wild-type embryos was similar, in about 90% of the embryos from mutant mothers we failed to detect any signs of development nor did we observe any evidence of a female pronucleus (Figure l , C , D and E). Moreover, i n these embryos we rarely observed normal polar body structures. In wild-type eggs, meiosis takes placewithin the uterus, consequently by the time the egg is laid, female meiosis is normally completed and the female polar nuclei which are located in the anterior- dorsal region of the embryo often fuse together and form a starlike structure called the polar body (Figure 1 B; see also CASIPOS-OKTEGA and HAKTESSTEIS 1985). For example, the mutant embryo in Figure 1E contains a polar body-like structure which resembles wild type although it appears to have too many chromosome arms. The most common mutant phenotype, however, is lack of a star-like structure (Figure 1, C and D). Instead, they have a structure that resembles a nucleus arrested in anaphase with some evidence of chromosome fragmen- tation. Taken together with the absence of develop ment, these observations suggest that eggs from dhd mutant mothers do not complete meiosis.

dhd mutations cause defects in preblastoderm nuclear cleavage divisions: Interestingly, about 10% of the embryos from d h d mothers initiate development; however development is not normal. In contrast to wild- type embryos in which the initial nuclear divisions are synchronous (Figure lA) , the nuclear divisions in the mutant embryos are asynchronous (Figure 1, F and G). Moreover, in stage 10 embryos, there are regions of the cortex that are devoid of nuclei, suggesting that not all nuclei have migrated to the periphery. Interestingly, these “holes” appear to be seen most often at the anterior end of the embryo. In addition, a subset of mutant embryos display a different phenotype in which the nuclei vary in size and show differential amounts of staining, suggesting different levels of ploidy (Figure 1 H).

Effect of dhd mutations on head development: Although embryos from mutant mothers do display a range of abnormalities early in development, about 5% develop far enough to secrete cuticle. Examination of the cuticle indicates that head involution had not occurred during gastrulation (Figure 2). In contrast to the head re-

FIGL‘RE 2.-(:uticlcs o f ‘ clnbryos from wild-type and dl~d’’’~ mothers. (A) Dark field photograph of a cuticle from ;I wild- type embryo. Anterior is t o the left. Thc head is involuted and there are 3 thoracic and 8 abdominal segments. (B) Dark field photograph of a cuticlr from a mutant embryo. Antrrior is to the left. Head i n \ o l u t i o n was n o t completed but the remainder of the cuticle appears to be normal. ((:) Dark field photograph of a cuticle from a mrltant emhryo. Anterior is t o the left. Head involution failed t o occur. The remainder of the cuticle ( n o t shown) appears normal. (D) Phasr contrast photograph of the head region o f a cuticle from the wiltl-Tpr embryo shown i n (A). Anterior is up. Portions of the cephalophaly1gral skeleton are labcltd: 1;Itcl-algraten ( L C ; ) , vertical plates (VT) vcn- tral arms (VA). The postcriorwall o f the pha lyn is also labeled (ppw). (E) Phase contrast photograph of the hc;Itl region of a cuticle from the mutant rmlx-yo shown in (B). Anterior is up.

gion, the remainder of the cuticle appears to have differ- entiated normally. No defects were obsemed i n either the abdominal or thoracic denticles or in the tail region.

In those embryos which have no involuted head, it is difficult to determine which head structures are present or absent (Figure 2C). However, i n those embryos which have a partially involuted head, i t is possible to identic some of the individual structures (Figures 2, R and E). In these embryos, most of the structures that make up the cephalopharyngeal skeleton are present but disorganized (JC‘RGESS rt nl. 1986). The most prominent defects are the shortening of the lateralgraten and the fusion of the ventral arms. The fusion of the ventral arms suggests that the posterior wall of the pharynx is absent. The shortening of the lateralgraten could be the result of a deletion ofcertain cell types or alternatively it could be secondary to the Failure to complete head involution. Several other components of the cephalopharyngeal skeleton (the ventral plate, the dorsal bridge and the dorsal arms) are more easily seen in different planes of focus (data not shown). Similarly, the mouth hooks, H piece and cirri are visible, as well as the maxillary, an- tennal and labial sense organs.

Role of dhd Thioredoxin Homolog 1079

The phenotype of these embryos resembles the phenotype caused by mutations in the head gap genes. Since maternal effect genes cause phenotypes that often re- semble the phenotypes of mutations in genes that they regulate, it is possible that d h d , like the gap genes are required for the specification of cell fate in the embryonic head. To examine this possibility directly, we examined the expression of three genes orthodentical, empty spiracles and cap 'n collar, which are all expressed in the head region during blastoderm (DALTON et al. 1989; FINKELSTEIN and PERRIMON 1990; MOHLER et al. 1991). We found that the RNA expression pattern of these three genes, examined by in situ hybridization to whole mount embryos, appeared normal in embryos from dhd mothers which reached blastoderm (data not shown).

While specification of cell fate does not seem to be affected by d h d mutations during blastoderm, further examination of gastrulating embryos, using the expression of the Engrailed protein as a molecular marker ( DI- NARDO et al. 1985), reveals a range of abnormalities in the head region (Figure 3). During germband extension, Engrailed protein expression is detected in the posterior of every segment. In embryos from dhd mutant mothers, expression in the intercalary segment is never detectable (Figure 3, B, C and D) and is often not detectable in the labrum (Figure 3, B and D). In addition, expression in the mandibular and maxillary segments suggests these segments are either fused or disorganized (Figure 3, B and C).

Although expression of the Engrailed protein appears to be normal in the remainder of the embryo, a com- parison of the expression pattern in the posterior and the anterior of the embryo suggests that the timing of germband extension in the posterior of the embryo is not synchronized with the complex morphological movements which take place in the head region. The extent to which the germband has extended is usually determined based on the positions of parasegments 8 and 9 in the posterior end of the embryo. Using this criterion, the embryo in Figure 3B appears to have extended its germband farther than the embryo in Figure 3C. However, by examining the morphology of the head region, we find that the cephalic furrow is still visible in the embryo in Figure 3B whereas the cephalic furrow in the embryo in Figure 3C has already fused and the stomodeum invagination has formed. A similar situation is evident in the embryo in Figure 3D where the position of parasegments 8 and 9 indicates that the embryo is at the maximum germband extension stage, yet in the head region the stomodeum invagination, which normally would have been formed by this stage, has only begun to invaginate.

dhd encodes a maternally expressed RNA which is absent in dhdPx and d h d 5 females: The DNA flanking the P element insertion site in dhdPx was cloned and

A B d M

. ."

CI dbd

FIGURE 3.-Expression of Engrailed protein in wild-type and dhd"" embryos. (A) Wild-type embryo at maximal germband extension. The Engrailed protein is detected in the posterior compartment of every segment. The head segments that can be visualized in this plane of focus are labeled: labrum (Ir) intercalary (ic) mandibular (md) maxillary (mx) and labial (li) . Parasegments 8 and 9 are labeled to indicate how far the germband has extended. Note the position of the stomodeum invagination located between the labrum and intercalary segments. (B) dhd embryo at early germband extension as indicated by the positions of parasegments 8 and 9. The cephalic furrow is still visible between the mandibular and maxillary segments. Although present, the expression pattern within the mandibular and maxillary segments is disorganized. More- over, expression within the labrum and the intercalary segments is not detectable. ( C ) dhd embryo which appears to be at an earlier stage of germband extension than the embryo in (B) based on the position of parasegments 8 and 9. However, the cephalic furrow has fused and the stomodeum invagination (located between the labrum and intercalary segments) has formed. In addition, the staining pattern within the mandibular and the maxillary segments is disorganized and expression within the intercalary segment is not detectable. (D) dhd embryo at maximal germband extension based on the position of parasegments 8 and 9 yet the position of the st@ modeum invagination appears to be located more anterior than in wild type suggesting that it hasjust begun to be formed. Although the expression pattern in the mandibular, maxillary and labial segments appears to be normal, expression in the labrum and intercalary segments is not detectable.

found to be within a -50-kb walk previously isolated in 4F1,2 (see MATERIALS AND METHODS). To identify candi- dates for the dhd transcriptional unit, genomic fragments from the 14 kb surrounding the insertion site were used as probes on Northern blots. We reasoned that as a maternaleffect locus, dhd should be tran- scribed in females and therefore a female-specific RNA would be a good candidate for the dhd transcription unit. The results of this analysis are summarized in Fig- ure 4B. None of the RNAs detected appeared to be female-specific, however a 1-kb RNA adjacent to the P element insertion is more abundant in females than in males and is not expressed in dhdPx females (Figure 4A and Figure 5A). Instead, a weakly hybridizing 4kb RNA is detectable (Figure 4A). Several lines of evidence demonstrate that the 1-kb RNA eliminated in dhdP8 females is the dhd transcriptional unit. First, in all five wild-type revertants of dhdP*, the 1-kb RNA is again expressed in females (data not shown). Second, the P element insertion associated with dhdPx interrupts the open reading frame at the 5' end of the gene (Figures 4B and 7).

1080 13. K. Salz P l n l .

probes: RNAs detected:

dhd -@

transformation constructs:

wild type dhd P8

XbaBam

I Xho/Xho I

Barn Xho E xba E m Xho Xho

I ’ \

1.6, 1 . 0 9 0 1.4 d a n d 1.0 Q d 1 l.OQ(d) 2.30 3.8. 3.5,l.E andl.5 90 / I \ 1 kb -

li \ 1 kb

dhd FIGURE 4.4dentification of the dhd transcriptional unit. (A) Northern blot of 5 pg of poly(A’) RNA prepared from sexed adults

from either wild-type or d / ~ d ” , ~ homozygous animals probed with the 2.7-kh E.:roRI/Xbnl genomic probe which surrouncls the site of the P element insertion. In wild-type adults two RNAs arc detected, a I-kh RNA is detected in both males and females antl a 2.3kb RNA is detected only in males. In dhd”“ homozygous females the I-kb RNA is missing and a weakly hybridizing 4-kb RNA is visible. In males the abundance of the I-kb RNA is reduced and the expression of the 2.J-kh RNA appears to be rlnafrected. (R) A molecular map of the region surrounding dhd. Genomic probes from this region detect five groups of RNAs. Only the I-kb RNA acljacent to the P element insertion is altered in dhd mutations. A more detailed map of the dhd genomic region and the approximate position of the cDNA (established by comparing the restriction map of the cDNA and genomic clones) is intlicatcd at the bottom. The position of the Pclement insertion associated with dhd”” is indicated by a vertical line attached to a triangle. The region of genomic DNA deleted in dhdI5 and Dj( I )dhdX/ was determined by Southern blot analysis antl is indicated by a solid line above the map. The extent of the genomic DNAs used for germline transformation are also indicated above the map.

Third, this RNA is also absent in dI2dI5 females (data not piece (the 12.7-kl1 XhrrI/RrrmHI transformation con- shown and Figure 5A). Finallv, rescue of the mutant phe- struct) does not (Figure 4B). notype via germline transformation demonstrates that Although a 1-kb RNA is still detectable i n dhd” males, the 4.8-kb XhoI/XIzoI genomic transformation construct we have not pursued its origin because it has no appar- includes the dhd gene, whereas an overlapping genomic ent function. Males deficient for 5 kb of genomic DNA

A wild typeQ dhd ovaries -0v OV P8 J5

_IT" .- - - -

dhd +

Ir m

B wild type embryos

5C Actin + dhd +

FIGCRF. 5.-Stage and tissue specific RNA expression pattern of dhd. (A) Northern blot of 30 pg of total RNA prepared from ovaries (ov) or female carcasses in which the ovaries had been removed (-ov) probed with a 1.7-kb EcoRI/XbnI genomic fragment which only detects the dhd transcript (see Figure 4). To control for the amount of RNA loaded onto each lane, the blot was subsequently stripped and reprobed with rpAl DNA ( Q I m

nl . 1987). (€3) Northern blot of .5 pg of polV(A+) RNA prepared from staged wild-type embryos and probed with the 1.7-kb EcoRI/XDnI genomic fragment. To control for the amount of RNA loaded onto each lane, the blot was also probed with 5C actin DNA (FRYBERG rt 01. 1983).

surrounding dAd (from the KpnI to XbnI sites see Figure 4B) are both viable and fertile. These deficiency males were constructed by combining the X-linked deficiency Df(1)dhdcJI with an autosome that carried the 12.7-kb XbnI/RamHI genomic transformation construct. In the absence of the transformation construct, Df(l)dhd81 males are lethal suggesting that a vital gene resides within the XbaI/BamHI genomic region. These data also confirm that dhd is strictly a maternaleffect gene.

Further examination of the expression pattern of d h d by Northern analysis is consistent with its maternaleffect function. We find that dhd is predominately expressed in the ovary, although a low level of expression can be detected in female carcasses from which the ovaries have been removed (Figure 5A). Furthermore, this RNA is present at high levels in 0-I-hr embryos and thereafter the level of RNA steadily declines until it is no longer detectable in 4-5-hr embryos (Figure 5B).

To determine whether d/zd expression is tissue specific, we examined its RNA pattern by i n .si/?r hybrirl- ization. I n ovaries, no expression is ohsenred before stage 9 (Figure 6A and data not shown). But by stage 1 OB there is a high level of expression in the nurse cells of the developing egg chamber (Figure GB). In contrast, no d h d RNA is detected in the somatically derived follicle cells. Furthermore, the transfer of the RNA from the nurse cells to the oocyte is visible, indicating that the RNA is being transported into the oocyte (Figure GB). The maternallv provided dhd RNA is uniformly distrib- uted throughout the embryo and is easily detected through the svncytial nuclear cleavage stage (Figure 6C). Thereafter dhd RNA declines in abundance until i t is no longer detected after the cellular blastoderm stage (Figures 6D and data not shown).

dhd encodes a thioredoxin: Sequencing of a 0.8-kb cDNA revealed a small open reading frame of 107 amino acids (see MATERIALS AND METHO1)S and Figure 7). The translation of the longest open reading frame results in a protein with extensive similarity to thioredoxins from a number of different organisms. As illustrated in Figure 8, the amino acid identity between d h d and the human thioredoxin is 36% over the entire length of the protein. Ifconservative amino acid substitutions arc included the similarity increases to 65%. dhd and the yeast thioredoxin exhibit 32% identity and 53% similarity. The overall similarity behveen dhd and thioredoxin extends throughout the whole molecule. Although other proteins, such as protein disulfide isomerase, have thioredoxin-like domains, they tend to have multiple re- peats of this domain and have a higher molecular weight, thus suggesting that dhd is a thioredoxin ( BARDMZLL and BECKM'ITH 1993).

DISCUSSION

We have identified and characterized the maternal effect gene dhd which encodes a thioredoxin homolog. Thioredoxin is one of several known proteins which which can reduce specific disulfide bonds in targeted proteins (reviewed in HOI.W;RES 1989 and BLKI IXS.AS

1991). Whether dhd actually encodes a protein capable of catalyzing the reduction of disulfide bonds wil l require biochemical confirmation.

Extensive biochemical studies have determined that thioredoxin can reduce disulfide bonds of many different proteins. For instance in 71ifro studies have shown that thioredoxin can reduce ribonucleotide reductase (reviewed in HOI.MRF.S 1989), convert the glucocorticoid receptor to its steroid binding confirmation (GRIPPO ~t nl. 1985), convert NF-KB to its DNA binding confirmation (MATTIIEWS ~f nl. 1992) and control microtubule assembly (KHAS and Lvncl;.st\ 1991). However, because other proteins can clearly function in a similar capacity, it remains to be determined whether all the diverse functions attributed to thiore-

c d l ~ ~ l a r bl;\stodcrm.

doxin in 7)i/ro are actually carried out by this molecule it1 71i71o.

"hile Iiochemical studies have determined that thioredoxin is capable of reducing disulfide bonds in many different targetctl proteins, i t remains to be determined whether it serves a s a ubiquitous protein disulfide reductase i n 71iuo. Indeed, the fhctional studies that have been carried out thus far suggest that thioredoxin is not a general protein disulfide reductase. Yeast mutations which eliminate thioredoxin function are viable, but have abnormal cell cycles in which S phase is %fold longer than in wild type (MUI.I.ER 1991). The finding that these mutant strains are not lethal, suggests that the rate of DNA synthesis is reduced but not eliminated. Similarly, in Xenopus the inhibition of thioredoxin activity by the injection of spinach thioredoxin into early emblyos also results in the inhibition of DNA synthesis (HAK-I'SIAN rt ( I / . 1993). Interestingly, E. coli thioredoxin also plays an important role in the DNA synthesis of T7 where it is an essential protein subunit of the T7 DNA polymerase yet it rlors n o / utilize thioredoxin for its own DNA synthesis; i n fact thioredoxin is completely dispens- able for P;.schrrirhin coli growth (HLWX r / rrl. 198'7; Ti\- ROK rt nl. 198'7; HISL\\VAS and RICII.-\KI~SOS 1992).

Our examination of the i n 7 ~ i 7 m consequences of the elimination of a thioredoxin homolog in Drosophila suggests that dhd does not encode a product essential for all cells, but is instead required only for spccific functions. Both the tnrltant phenotype and thc RSA expression pattern of the dhd gene clearly demonstrate that dhrl's essential functions are restricted to oogenesis and the maternal-support of embryonic development. Mu-

tant females lay eggswhich are morphologically normal, are fertilized but do not hatch. The most prevalent defect obsewed in these eggs is an abnormal polar body structure.

Although a more detailed cytological analysis wil l be required to determine the cause of these abnormal structures, the homolocgy to thioredoxin suggests the possibility that d h d is required for DNA synthesis. In- deed, the mutant phenotype could be due to the inability of the spindle to separate unreplicated chroma- tin. However, we fail to detect any RNA expression during the time when premeiotic S phase takes place. In oogenesis, premeiotic S phase takes place in the 16 cell germline cyst which is located at the tip of the germarium i n region 1 ( c r \ R P E : T l X K 1981). On the other hand, it is possible that the level of dhd RNA in the germarium is below the level of detection.

Given the number of fhct ions attributed to thioredoxin, i t is equally likcly that d h d has another function during meiosis. Indeed, its RNA expression pattern is more consistent with a later function. dhrl RNA is first detected within the oocyte cytoplasm just before meiosis I begins, during stage 10. I n wild type, the first overt signs of meiosis 1 are detected during stage 11 when the chromosomes condense (THH'KKAL'F and H , \ w . E Y 1992). By stage 13 the nuclear envelope has broken down and the meiotic spindle has assembled. Meiosis is then arrested in the mature oocyte (stage 14) i n metaphase of the first meiotic division. Meiosis remains arrested until the oocyte enters the oviduct, at which point meiosis is completed and fertilization occurs. In embryos from dhd mutant mothers, a polar body structure with condensed


GGTGTAGTTC ACAATTTGCA GCGAAAGCGC CAACAGCCCC ATCACGTCAA GTTCTTTAAT 60

TTAAAAAAAA GAAGAAAAAC ACTTTGCTAA TCCTCGCGAC ATAAAA ATG GCA TCC GTA 1 1 8 M A S V

CGC ACC ATG AAC GAC TAT CAC AAG CbC ATC GAG GCG GCC GAC GAC AAG 166 R T M N D Y H K R I E A A D D K

CTA ATC GTG CTG GAT TTC TAT GCG ACA TGG TGT GGT CCC TGC AAG GAA 214 L I V L D F Y A T W C G P C K E

ATG GAG AGC ACC GTC AAA TCG CTG GCC AGA AAA TAC TCC AGC AAG GCG 262 M E S T V K S L A R K Y S S K A

GTG GTG CTC AAG ATC GAT GTG GAC AAA TTC GAG GAG CTG ACG GAG CGC 310 V V L K I D V D K F E E L T E R

TAC AAG GTG CGC AGC ATG CCA ACG TTT GTC TTT TTG CGC CAA AAT CGA 358 Y K V R S M P T F V F L R Q N R

CGC CTG GCC TCC TTT GCC GGC GCC GAC GAG CAC AAG CTG ACC AAC ATG 406 R L A S F A G A D E H K L T N M

ATG GCC AAG CTG GTG AAG GCG TAA GCAGCAATCC TGCCCAGCAA TTAGCAATCA 460 M A K L V K A *

GCAGCGCATC TTTTTTTTTT TACTTATTTA ACTCATCTTT TAAACATGTT CGCCTCATTT 520

GTGTTCGTTT ATGTATTCGA TGTTATGTGT ATGCTCATGT GATGTTTAGC TTGTAAGCGC 580

GAGATGTGGG TAGCAGGAGA TGCAGTGCAG CCAACAGCAG TGACCAGATG ATATATGCTA 640

CTACTACTAC TTATATGCTA TGATTTGTGG CGCGGAGGCT GTCTGCGACA CATAACCCGC 700

CCATTAGCTT TAAGATTCAG GCACTAAGAA GCAATTCGAT CAATAAATTA TTGTAACCAC 160

TCTGGACATT TAAATGTTAA CATGGAAAAA AAAAAA 791

FIGURE 7.-DNA sequence of dhd cDNA. The nucleotide sequence of a full length dhd cDNA is shown along with its predicted translational open reading frame. The site of the P element insertion associated with dhdp8 is indicated by a triangle.

chromosomes is always observed, indicating that the early steps of meiosis, chromosome condensation and nuclear envelope breakdown, do not require dhd function. In contrast, the aberrant polar body suggests that meiosis is blocked by anaphase. Thus our analysis suggests that dhd is required for the completion but not the initiation of meiosis.

Thioredoxin has also been implicated in the control of microtubule assembly (KHAN and LUDUENA 1991). In- deed, the mutant phenotype could be suggestive of a defect in the assembly and/or function of the meiotic spindle. Because aspects of both the assembly and the components of the spindle are specialized for oocyte meiosis, it is certainly conceivable that their assembly and/or function might require a specialized molecule (THEURKAUF and HAWLEY 1992; h " H E W S et al. 1993).

On the other hand, it is also possible that the reactivation of meiosis is prevented. Recent studies have suggested that the release from metaphase arrest (and pos sibly for all metaphase to anaphase transitions) is mechanistically similar to the transition from a resting cell to a cell committed to mitotic cycling (reviewed in HUNT 1992). These findings in conjunction with the finding that thioredoxin plays a role in the transformation of resting lymphocytes to immortalized cells by either EBV or HTLV-1 suggests the possibility that dhd is

required for the reactivation of meiosis and/or the activation of the egg (WOLLMAN et al. 1988; TAGAYA et al. 1989; WAKASUGI et al. 1990; YODOI and T u ~ s z 1991). In Drosophila, meiosis is arrested in metaphase I and begins anaphase at ovulation. The recent studies of MCKIM et al. (1993) have suggested that reactivation requires the release of forces that hold bivalents at the metaphase plate. Thus one might anticipate a mutation that interferes with the release from meiotic arrest and/or activation of the egg to have a similar phenotype. Since maternally derived gene products within the egg are used to control and construct the machinery required for these early events, their activation in response to ovulation must be controlled at the post-transcrip tional level. The possibility that a protein disulfide reductase could be required for this process is intriguing.

In addition to defects in meiosis, mutant embryos also display two distinct types of escaper phenotypes: a mitotic defect is observed in preblastoderm embryos and a head defect is observed in older embryos. The small percentage of embryos which do initiate development display a range of defects in preblastoderm mitosis which is also suggestive of the inability of the spindle to separate chromosomes. Although it is possible that the preblastoderm defect is simply a consequence of the meiotic defect, we find this unlikely because normal po-

1084 H. K Salz et al.

Yeast MVTQLKSASEYDSALASGDKLVWDDFFATWCGPCKMIAPMIEKFAEQYSD

Fly MASVRTMNDYHKRIEAADDKLIVLDFYATWCGPCKEMESTKYSS + + + + + + I 1 1 1 1 + 1 + 1 1 I I I I I I I I ++ I I + I l l +

Human MVKQIESKTAFQEALDAAGDKLVWDDFSATWCGPCKMIKPFFHSLSEKYSN

I I + 1 1 1 + 1 + 1 1 + 1 l 1 1 1 1 1 l + ++ I + I I

Yeast AA-FYKLDVDEVSDVAQKAEVSSMPTLIFYKGGKEVTRWGANPAAIKQAIASNV 104

Fly KAWLKIDVDKFEELTERYKVRSMPTFVFLRQNRRLASFAGADEHKLTNMMAKLVKA 107 I I + l I I + + + + + + I I l l 1 + I + + + I l + + + I I

+ I + + l I I +++ + I + I I I I I ++ +++ 1 + 1 I++ I 1 + + I I Human V-IFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV 105

FIGURE 8.-The predicted protein product of dhd has homology to thioredoxins. The predicted dhd protein is aligned with the sequence of the human thioredoxin (WOLLMAN et al. 1988) and the yeast TRXl gene (MULLER 1991). Identical amino acids are indicated by vertical lines and similar amino acids are indicated by a plus (+). The three proteins are of similar size and show extensive overall homology throughout the protein including the presumed active site, WCGPCK, indicated in bold face type.

lar body structures are observed in developing embryos. Instead, we feel it is more likely that d h d is required for preblastoderm mitosis as well as meiosis.

Since regulation of mitosis prior to cellularization may be fundamentally different from regulation after cellularization, it is tempting to speculate that a specialized molecule is required to regulate these cell divisions. Moreover, since these divisions are likely to be regulated at the post-transcriptional level, it is particularly satisfy- ing to find that d h d encodes a factor known to be required in post-translational modification of protein activity. However, since thioredoxin has also been shown to play arole in premitotic DNAsynthesis (MULLER 1991; HARTMAN et al. 1993), it is equally likely that d h d is also required for premitotic DNA synthesis. Yeast mutations which eliminate thioredoxin function are not lethal, but do have abnormal cell cycles in which S phase is %fold longer than in wild type (MULLER 1991). The finding that the absence of thioredoxin is not a lethal in these mutant yeast suggests that rate of DNA synthesis is reduced but not eliminated. Similarly, in Xenopus the inhibition of thioredoxin activity in early embryos results in the inhibition of DNA synthesis (HARTMAN et al. 1993). In contrast to most cell cycles, however, the early mitotic divisions in Xenopus are lacking the cell cycle checkpoint which verifies that DNA synthesis is completed before mitosis can begin (reviewed in MURRAY 1992). Thus instead of lengthening S phase to accommodate this defect, as occurs in yeast, mitotic divisions continue for several rounds in spite of the fact that DNA replication is not completed. Consequently, the chromosomes are fragmented and the embryo arrests before gastrulation. Interestingly, this type of defect strongly resembles the mitotic mutant phenotype observed in d h d mutant embryos, further suggesting a role for d h d in DNA synthesis during the nuclear divisions.

The second escaper phenotype observed in d h d mutant embryos is the lack of head involution. Although d h d does not appear to have a direct effect on the organization of the presumptive head region during blastoderm, as shown by the normal expression pattern of

three head specific genes, we do observe that many escaper embryos have a reduced number of nuclei in the anterior end of the embryo. Perhaps the failure to in- volute the head is a consequence of the reduction in the number of nuclei. Previous studies have shown that the number of cell divisions, not the actual timing of the cell divisions, is critical for many aspects of normal development (EDGAR and O'FARRELL 1990). Alternatively, it is possible that d h d has a more direct effect on head development. If this is the case, then we would expect that the protein from the maternal transcripts to perdure until gastrulation because the maternal d h d RNAs are no longer detectable by this time. Experiments to determine whether this is the case are underway.

The existence of escaper phenotypes in known null mutations suggests a partial functional redundancy which allows some embryos to complete meiosis and at- tempt to proceed through the early division cycles. Whether this putative redundancy is carried out by another protein disulfide reductase is not known; however, it is unlikely that the putative redundant function is carried out by a closely related protein because we have been unable to detect other thioredoxin genes by low stringency hybridization to genomic DNA (A. PELLICENA- PALLE and H. K SALZ, unpublished data). Indeed, the notion of an unrelated protein with a similar function is further supported by biochemical studies which have shown that at least one other unrelated protein, glutaredoxin, can replace thioredoxin in many reactions (HOLMGREN 1989).

In summary, we have identified and characterized mutations in a thioredoxin homolog which is essential for female meiosis and early embryonic development. Al- though further studies will be required to elucidate the exact role of this thioredoxin homolog in oogenesis and maternal control of early development, this study clearly indicates the importance of thioredoxins in regulating specific developmental processes.

We thank C. CRONMLLER for helpful discussions and commentS on the manuscript. This work was supported by a grant from the National


Science Foundation (H.RS.), a Young Investigators Award from the Mathers Charitable Foundation (H.K.S.), the Markey Foundation (T.W.F. and J.P.P.) and the National Research Service Awards 5T32GMO8056 1E.B.A) and HD07104 (J.P.P and T.W.F.).

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Communicating editor: T. SCH~JPBACH

the drosophila maternal effect locus deadhead encodes a ... · sophila thioredoxin homolog....

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