Molecular Characterization of Mutant Alleles of the DNA Repair/Basal Transcription Factor haywire/ERCC3 in Drosophila

Corresponding author: Margaret T. Fuller, Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5427., fuller{at}cmgm.stanford.edu (E-mail)

Communicating editor: R. S. HAWLEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The haywire gene of Drosophila encodes a putative helicase essential for transcription and nucleotide excision repair. A haywire allele encoding a dominant acting poison product, lethal alleles, and viable but UV-sensitive alleles isolated as revertants of the dominant acting poison allele were molecularly characterized. Sequence analysis of lethal haywire alleles revealed the importance of the nucleotide-binding domain, suggesting an essential role for ATPase activity. The viable hay^nc2 allele, which encodes a poison product, has a single amino acid change in conserved helicase domain VI. This mutation results in accumulation of a 68-kD polypeptide that is much more abundant than the wild-type haywire protein.

THE haywire locus of Drosophila encodes the fly homolog of ERCC3, a human gene associated with the DNA repair deficiency disease xeroderma pigmentosum group B (XP-B) (MOUNKES et al. 1992 ). Mutations in the Saccharomyces cerevisiae homolog SSL2/RAD25 are defective in overall nucleotide excision and transcription-coupled repair (SWEDER and HANAWALT 1994 ). In addition, these proteins appear to play a role in transcription. The human ERCC3 (SCHAEFFER et al. 1993 ) and S. cerevisiae SSL2/RAD25 (FEAVER et al. 1993 ) gene products are found to be associated with the basal transcription factor TFIIH. Purified TFIIH complements the excision repair defect of lysates from cells lacking ERCC3 function (DRAPKIN et al. 1994 ). Consistent with a function in transcription, SSL2/RAD25 is an essential gene (PARK et al. 1992 ), and a temperature-sensitive allele of SSL2/RAD25 inhibits transcription at the restrictive temperature (QUI et al. 1993 ). Different alleles of SSL2/RAD25 show defects that can separate the roles of the protein in excision repair and transcription (GUZDER et al. 1994 ). As for SSL2/RAD25 of yeast, extreme alleles of haywire are lethal (REGAN and FULLER 1990 ), whereas viable mutant alleles are UV sensitive (MOUNKES et al. 1992 ). Only a few cases of XP-B are known to date, suggesting that ERCC3 is also essential in humans, and that only patients carrying special weak mutant alleles of ERCC3 survive to birth (WEEDA et al. 1990 ; VERMEULEN et al. 1994 ). A viable point mutation in ERCC3 now defines a new trichothidystrophy complementation group which, however, does not cause XP-B (WEEDA et al. 1997 ).

The fly, yeast, and human ERCC3 homologs show hallmark conserved domains characteristic of the superfamily of DNA/RNA helicases (GORBALENYA et al. 1989 ; LINDER et al. 1989 ). Two of these, the predicted nucleotide- and magnesium-binding domains, are responsible for the essential ATPase activity of the SSL2/RAD25 (PARK et al. 1992 ) and ERCC3 (MA et al. 1994A ) products. Helicase activity is associated with the TFIIH transcription complex (SCHAEFFER et al. 1993 ; DRAPKIN et al. 1994 ), and helicase activity of the ERCC3 protein has been demonstrated in vitro (MA et al. 1994B ). Helicase activity of ERCC3 requires the remaining conserved helicase domains, which have been shown by mutational analysis to function in excision repair of UV-sensitive cell cultures (MA et al. 1994A ).

The Drosophila ERCC3 homolog haywire was originally identified through an unusual mutant allele, hay^nc2, that acted as a dominant enhancer of mutations in the testis-specific ß-tubulin B2t. Males heterozygous for either hay^nc2 or B2t^null and wild type are fertile at 25°. However, flies transheterozygous for one copy of hay^nc2 and one copy of B2t^null are male sterile at 25°. The genetic interaction between hay^nc2 and B2t^null suggested the hay^nc2 allele encoded a poison product because flies transheterozygous for B2t mutations and a deficiency of haywire were male fertile (REGAN and FULLER 1988 ). Revertant alleles of hay^nc2 abolish the genetic interaction between hay^nc2 and B2t^null, but still result in novel abnormal phenotypes with respect to male fertility (REGAN and FULLER 1990 ), suggesting that a poison product encoded by the hay^nc2 allele is responsible for the failure to complement tubulin mutations.

In this article, we report the molecular characterization of mutations that identify regions of haywire that are important either for viability or DNA repair. In addition, we show data indicating that the hay^nc2 allele causes production of a 68-kD polypeptide that accumulates to much higher levels than wild-type haywire protein. Mutant haywire alleles that revert the genetic interaction between hay^nc2 and B2t^null abrogate production of the stable, truncated protein, suggesting that the same defect causes accumulation of the 68-kD polypeptide and the poison product activity.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fly strains and culture:
Fly stocks and crosses were maintained using standard fly media at 25°. Balancer chromosomes, visible markers, Df(1)w, B2t^null, and Df(3L)E(z)7e¹¹ (67E1-4;67F1-3) are described in LINDSLEY and ZIMM 1992 . The following haywire alleles were used: hay^nc2, a recessive male sterile mutation that fails to complement mutations in B2t (REGAN and FULLER 1988 ), and six alleles recovered as revertants of the failure of hay^nc2 to complement B2t^null (REGAN and FULLER 1990 ). Fertility of mutant combinations of alleles was determined by testis dissections, as described (MOUNKES et al. 1992 ).

Sequencing of haywire alleles:
The coding region of haywire was divided into halves by two sets of PCR primers, each amplifying ~1.5-kb fragments from haywire. Primers used to amplify the 5' half of haywire were 5'-GGAAGCTTGCCATACGTGCTGTGTTAC-3', and 5'-CCAAGCTTCCAGCACCATGATGCCCC-3'. Primers used to amplify the 3' half of haywire were 5'-GGGCTGTGGAATTCTTGTGACCACATAC-3' and 5'-GGAAGCTTGGGTTACTGCAGTGGTAAAG-3'. For the viable alleles hay^nc2 and hay^nc2rv8, templates for the PCR reactions were genomic DNA made from flies heterozygous for the viable haywire allele and a deficiency for haywire, Df(3L)E(z)7e¹¹. For lethal haywire alleles, template DNA for the PCR reactions was made from flies heterozygous for the lethal revertant allele and the parent chromosome, hay^nc2. PCR products were purified and sequenced as described (SANGER et al. 1977 ; BIGGIN et al. 1983 ; KRETZ et al. 1989 ). One strand of the revertant alleles was sequenced, except in areas where there were changes. Changes were verified by sequencing the complementary strand.

Northern analysis:
Total RNA was isolated by RNAzol extraction according to Cinna Scientific. RNA (30 µg) was run on 0.1 M sodium phosphate gels, blotted onto a Hybond membrane (Amersham, Arlington Heights, IL), and hybridized as described (SAMBROOK et al. 1989 ). Blots were probed with a haywire riboprobe made from a HindIII-linearized cDNA construct (MOUNKES et al. 1992 ) and a random-primer-labeled 1.5-kb EcoRI-SalI fragment from the plasmid containing rp49 (O'CONNELL and ROSBASH 1984 ).

haywire antibody production and purification:
A 2.5-kb HindIII-NotI fragment from the haywire cDNA plasmid was treated with Klenow (MOUNKES et al. 1992 ), inserted into pQE30 (Qiagen) digested with SmaI, and phosphatase treated to make a construct encoding full-length haywire protein tagged with six histidine residues at the N terminus of haywire. Induction of fusion protein expression with 2 mM IPTG (Sigma, St. Louis) and harvesting of fusion protein were performed as described (MOUNKES and FULLER 1998 ) with the following differences. haywire fusion protein was not soluble in 8 M urea and was solubilized in 6 M guanidine/TN1. Supernatants with soluble protein were applied to a Ni²⁺–NTA column (Qiagen) equilibrated in 6 M guanidine/TN1. The column was washed with 6 M guanidine/TN1, and fusion protein was eluted in TN1/8 M urea and 60 mM imidazole. Antiserum to purified protein was raised in rabbits (Josman Laboratories, Napa, CA). Antibodies were affinity purified using the pure, original antigen coupled to Affi-gel 10 (Bio-Rad, Richmond, CA) essentially as described (MOUNKES and FULLER 1998 ).

Western analysis:
Protein samples were made by grinding 20 adult flies in an Eppendorf tube in 0.15 ml of solution A (0.1 M Tris, pH 9.0, 0.1 M EDTA, 1% SDS, 0.5% DEPC) plus 1 mM PMSF. Samples were spun in the microfuge for 30 sec to pellet large body parts. Supernatants were transferred to a new tube, 10 µl each were brought to 1x sample buffer (LAEMMLI 1970 ), and proteins were separated by 10% SDS-PAGE. Gels were blotted to nitrocellulose (Schleicher & Schuell, Keene, NH) as described (TOWBIN et al. 1979 ). haywire was visualized by enhanced chemiluminescence (ECL) staining as described by the manufacturer (Amersham). Affinity-purified primary antibody was used at 1:1500. Horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (Amersham) was used at 1:4000.

Germline transformations:
PCR mutagenesis of a 380-nucleotide BglII-SacI fragment of the 3' end of the haywire cDNA was performed to introduce the same C-to-T transition found in the hay^nc2 allele. The mutagenic oligonucleotide had the sequence 5'-CCAGATCTCTTCGCATGGCGGCTCTTGTCGTCAGGAGG-3'. The mutagenized BglII-SacI fragment was subcloned into a 4.5-kb BamHI fragment of the 3' half of a genomic haywire clone (MOUNKES et al. 1992 ). The resulting BamHI fragment was then used to replace the corresponding wild-type fragment by cloning into a 5.2-kb BamHI fragment obtained by partial digestion of the haywire genomic clone. The entire 7.8-kb haywire genomic fragment with the C-to-T change was then excised with HindIII and cloned into the pCaSpeR transformation vector (PIROTTA 1988 ) previously linearized with HindIII and treated with calf intestinal phosphatase (CIP). All cloning junctions within haywire and the mutated site were sequenced to verify sequence integrity at the cloning junctions and introduction of the mutation. Restriction enzymes, CIP, and Taq DNA polymerase were purchased from Boehringer Mannheim (Indianapolis). DNA (200 µg/ml) twice purified by cesium chloride banding was injected by standard methods (RUBIN and SPRADLING 1982 ) into Df(1)w embryos with p[ry⁺, 2–3] (ROBERTSON et al. 1988 ) at 200 µg/ml as a source of transposase. One transformant linked to the X chromosome was isolated.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Molecular lesion in the hay^nc2 allele responsible for the dominant genetic interaction with B2t:
The original hay^nc2 mutation caused replacement of arginine 652 with a cysteine (Table 1) in conserved helicase domain VI (Figure 1). This missense mutation was present in all the revertant alleles of hay^nc2, as expected. As hay^nc2 homozygotes are viable, arginine 652 is not absolutely required for haywire function in basal transcription, but it may be important in DNA repair since the hay^nc2 allele is UV sensitive (MOUNKES et al. 1992 ).

View larger version (43K): In this window In a new window Download PPT slide   Figure 1. Alterations in lethal and viable haywire alleles. Amino acid changes are indicated above the appropriate residues. X indicates sequences deleted in the e11 wild-type strain; $ indicates a mutation to a stop codon. The conserved helicase domains are defined by dashed lines underneath the sequences. The asterisk indicates the conserved lysine within the P loop of the nucleotide-binding domain. WT, wild type; rv2, haync2rv2; rv3, haync2rv3; rv8, haync2rv8; rv12, haync2rv12; rv7, haync2rv7; rv1, haync2rv1. Alleles in boldface type are homozygous viable. All other mutant haywire alleles are lethal.

The hay^nc2 mutation resulted in accumulation of a polypeptide that migrated on SDS polyacrylamide gels as 68 kD, cross-reacted with anti-haywire antisera, and was present at much higher levels in the mutant than in the wild type (Figure 2). The 68-kD apparent molecular weight polypeptide was 26 kD less than expected for the full-length protein, suggesting the possibility that the hay^nc2 allele results in accumulation of a shortened form of the haywire protein. Although truncation at the site of the hay^nc2 mutation (Arg652-to-Cys) would produce a predicted 68-kD N-terminal peptide, the region of haywire protein present in the 68-kD polypeptide is not known. It is also formally possible that the 68-kD polypeptide does not correspond to a fragment of the haywire protein, but instead is an unrelated polypeptide that is overproduced specifically in the hay^nc2 mutant background and that cross-reacts with the anti-haywire antisera. The 68-kD polypeptide did not accumulate in either the wild-type background strain (red e, Figure 2) or in flies carrying revertant alleles of hay^nc2 (Figure 2), indicating that accumulation of the 68-kD product results from the hay^nc2 allele. The affinity-purified, anti-haywire antibodies recognized many bands smaller than the expected wild-type size in Western blots. Although some of these smaller peptides could be proteins with cross-reacting epitopes, some could also be degradation products of haywire.

View larger version (59K): In this window In a new window Download PPT slide   Figure 2. The haync2 mutation results in accumulation of a 68-kD polypeptide (nc2) not seen in the wild type. Western analysis of whole-fly homogenates from haywire alleles. WT in first lanes (A and B) indicates homogenates from the wild-type background strain, red e, on which the haync2 mutation and subsequent revertant alleles were made. Positions of molecular weight markers are indicated on the left in kilodaltons. (A) Mutant haywire alleles over a wild-type balancer. (B) Comparison of mutant haywire alleles in combination with a deficiency (Df) or wild-type balancer chromosome (+).

To demonstrate that the Arg652-to-Cys mutation was responsible for the production of the 68-kD polypeptide in hay^nc2 animals, we constructed a transgenic animal with the hay^nc2 C-to-T transition introduced into an otherwise wild-type haywire genomic fragment. The mutated haywire gene was introduced into a background wild type for haywire, and the resulting transformants were tested for production of the truncated hay^nc2 product. Several transformant lines established from a single G_o fly all made and accumulated the 68-kD polypeptide seen in the original hay^nc2 flies (Figure 3).

View larger version (48K): In this window In a new window Download PPT slide   Figure 3. The haync2 missense mutation is responsible for production of the 68-kD polypeptide (nc2 trunc). Western analysis of haync2 transgenic animal made by site-directed mutagenesis of the wild-type gene. Molecular weight markers (kilodaltons) are indicated on the right. WT, wild-type haywire protein; nc2 trunc, truncated product accumulating in haync2 flies. The lanes are as follows: +, wild-type background strain (red e); nc2/+, haync2 over a wild-type balancer (TM3); P[A3,w+], transgenic animal with mutated copy of haywire carrying the haync2 CT transition introduced into an otherwise wild-type haywire genomic fragment; w; ScO/CyO, genetic background in which the transgene was propagated; P[A, ry+], wild-type copy of haywire introduced into flies. Note: Both P[A3, w+] and P[A, ry+] have two wild-type endogenous copies of haywire.

Transformants carrying the Arg652-to-Cys change also showed the genetic interaction with B2t^null characteristic of the original hay^nc2 mutation. One copy of the altered gene carried on the insert plus one wild-type endogenous copy of haywire resulted in male sterility in flies heterozygous for the B2t^null allele (Table 2, line 5). (The flies shown in Table 2, line 5, have one of the two endogenous copies of haywire deleted.) One copy of the altered transgene also caused reduced male fertility in B2t^null/+ heterozygotes, even in the presence of two wild-type endogenous copies of haywire (Table 2, line 7), although these flies were not as sterile as hay^nc2 +/+ B2t^null. Thus, the genetic interaction appeared to be sensitive to the ratio between mutant and wild-type haywire (Table 2, compare lines 5 and 7). Similar behavior was observed in the failure of a missense mutation in -tubulin to complement B2t^null (HAYS et al. 1989 ), and it is consistent with hay^nc2 encoding an antimorphic poison that can compete with the wild-type protein. The Arg652-to-Cys altered transgene caused both accumulation of the 68-kD polypeptide and failure to complement B2t mutations, demonstrating that the single mutation was responsible for both phenotypes associated with hay^nc2.

The high levels of the 68-kD possible fragment of haywire accumulating in the hay^nc2 mutant did not appear to result from increased transcription or stability of the haywire mRNA encoded by hay^nc2. Northern blot analysis of all the alleles sequenced revealed relatively normal levels of haywire transcript in all cases (Figure 4), with the possible exception of the hay^nc2rv3 and hay^nc2rv7 alleles. hay^nc2rv3/TM3 and hay^nc2rv7/TM3 flies appeared to have reduced levels of haywire mRNA compared to other alleles tested over the same balancer chromosome. As both of these alleles introduce stop codons early in the haywire protein-coding region (below), they may cause destabilization of the mRNA encoding the mutant allele (TALESA et al. 1995 ). In addition, accumulation of the 68-kD polypeptide in hay^nc2 did not appear to be caused by translation of an abnormally processed message, as no alterations in haywire message size were detected in flies carrying the hay^nc2 allele.

View larger version (80K): In this window In a new window Download PPT slide   Figure 4. Flies carrying the haync2 allele accumulate normal levels of haywire transcript. Northern analysis of flies hemi- or heterozygous for mutant haywire alleles. Molecular weight markers (in kilobases) are indicated to the left of the blot. A 2.5-kb haywire transcript (hay) was observed in all haywire alleles. The blot was reprobed for rp49 (20) as a loading control. Lanes: 1, red e; 2, E(z)7e11/TM3; 3, haync2/E(z)7e11; 4, haync2rv8/E(z)7e11; 5, haync2/TM3; 6, haync2rv8/TM3; 7, haync2rv1/TM3; 8, haync2rv2/TM3; 9, haync2rv3/TM3; 10, haync2rv7/TM3; 11, haync2rv12/TM3.

Lethal alleles isolated as revertants of hay^nc2:
Several mutations that revert the failure of hay^nc2 to complement mutations in B2t introduce stop codons into the haywire coding region, supporting the hypothesis that hay^nc2 fails to complement B2t because of the production of a poison product (Table 1). hay^nc2rv1 introduces a stop codon in helicase domain VI, hay^nc2rv3 results in a stop codon in the conserved nucleotide-binding domain, and hay^nc2rv7 has a stop codon six amino acids before the predicted magnesium-binding domain (helicase domain II, Figure 1). These nonsense mutations are all lethal, supporting an essential role in flies for the haywire gene product, probably in basal transcription.

A missense mutation (hay^nc2rv2) and small rearrangement (hay^nc2rv12) indicate that the nucleotide-binding domain of haywire is essential for viability. The hay^nc2rv2 mutation replaced cysteine 377 with a tyrosine residue (Table 1). This cysteine, which lies eight amino acids downstream of the P loop (WALKER et al. 1982 ) in the nucleotide-binding domain (Figure 1), is conserved among the Drosophila, human, mouse, and yeast homologs. The hay^nc2rv12 lesion was a small chromosomal rearrangement located at the `GAGKS' P loop of the nucleotide-binding site (brackets in Figure 1). All these revertants of the genetic interaction of hay^nc2 also reverted the defect that leads to accumulation of the 68-kD polypeptide (Figure 2).

A viable revertant of hay^nc2:
The hay^nc2rv8 viable mutation changed glutamic acid 278 to lysine, introducing a charge change and possibly affecting protein structure or stability. hay^nc2rv8 homozygotes are viable, indicating that glutamic acid 278 is not essential for basal transcription. Glu278 could be important for the repair of UV damage, since this allele also showed UV sensitivity (MOUNKES et al. 1992 ). Alternatively, the UV sensitivity could be caused by the still-present hay^nc2 lesion, while the hay^nc2rv8 reversion merely alleviated the poison nature of the hay^nc2 product. Again, the hay^nc2rv8 missense mutation also reverted the defect leading to accumulation of the 68-kD polypeptide characteristic of hay^nc2.

Altered amino acids encoded by wild-type haywire alleles:
The sequences of three wild-type alleles from different genetic backgrounds revealed regions of haywire/ERCC3 that are not essential (Figure 1). A wild-type cDNA from a dp cn bn strain and two different wild-type genomic sequences (red e and e¹¹) differed at several positions within the amino acid coding region. Alanine residues at positions 238 and 244 in the dp cn bn and red e backgrounds were changed to threonine and proline, respectively, in wild-type haywire from the e¹¹ chromosome. Amino acid 663 was a methionine in the e¹¹ strain but a leucine in both the red e and dp cn bn strains. In addition, the e¹¹ wild-type allele of haywire had a four-amino-acid deficiency at positions 253–256 compared with haywire on both the red e and dp cn bn chromosomes. The four consecutive amino acids missing in the e¹¹ background are valine/valine/alanine/alanine. Most of the wild-type polymorphisms occur in regions that are poorly conserved between the Drosophila, yeast, and mammalian homologs, and may identify residues of haywire not essential for function.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The single nucleotide mutation in the hay^nc2 allele that causes an arginine-to-cysteine change is responsible for both the production of a shortened polypeptide associated with the hay^nc2 allele and the genetic interaction observed between hay^nc2 and B2t mutations. Several molecular mechanisms of reverting the genetic interaction between hay^nc2 and B2t^null are suggested by sequence analysis of the haywire alleles obtained as revertants of hay^nc2. Destruction of the P loop of the nucleotide-binding domain reverted the genetic interaction between hay^nc2 and B2t^null (hay^nc2rv12, Table 1) possibly by destroying the ATP-binding or helicase function of the hay^nc2 primary product. Two other revertant alleles, hay^nc2rv2 and hay^nc2rv8, caused missense mutations that could alter protein conformation or stability. hay^nc2rv2, which caused a change from a cysteine to tyrosine, altered a potential disulfide bond partner. hay^nc2rv8, which caused a change from a glutamic acid to lysine, caused a change from a negatively charged residue to a positively charged residue. Finally, nonsense mutations hay^nc2rv1, hay^nc2rv3, and hay^nc2rv7 reverted the genetic interaction between hay^nc2 and Bt2^null. The finding that at least four out of six revertants of hay^nc2 (the three stop codons and the P-loop mutation) are mutations that should knock out haywire function supports the idea that the original hay^nc2 allele encodes a poison product.

The shortened polypeptide that accumulates in hay^nc2 flies is likely to be either responsible for or a direct result of the poison nature of the hay^nc2 allele, as the 68-kD polypeptide no longer accumulated in six out of six mutants that reverted the dominant enhancer effect of hay^nc2 (Figure 2). The 68-kD polypeptide that accumulates in hay^nc2 flies is not likely to be caused by a simple truncation of the haywire protein near the site of the nc2 mutation. The stop codon mutation in hay^nc2rv1 (Table 1) suggests that a simple truncation event near amino acid 652 results in neither production of a poison product nor accumulation of the 68-kD polypeptide (Figure 2). Furthermore, the hay^nc2rv1 allele is lethal, perhaps because of truncation of the protein within the last conserved helicase domain, whereas the hay^nc2 allele is viable (Table 1).

The hay^nc2 allele encodes a product that must contain at least some wild-type function, as hay^nc2/Df(hay) flies were fully viable. As the mutation responsible for causing accumulation of the truncated product is not a stop codon, full-length haywire protein could be initially expressed from the hay^nc2 allele. It is possible that this full-length but mutant haywire protein could provide sufficient haywire function for viability when initially expressed. However, defects in functioning or processing of the defective protein because of the Arg652-to-Cys mutation could alter or disrupt normally occurring processing of the full-length haywire protein, resulting in accumulation of a shortened form of the protein in hay^nc2. The observation that the 68-kD shortened polypeptide accumulated to much higher levels than the wild-type haywire product also suggests the possibility of turnover of haywire/ERCC3 as a normal part of its function. We would like to raise the possibility that the haywire protein might normally be degraded as part of the functional cycle of TFIIH in transcription initiation. It is also possible that the 68-kD polypeptide that accumulates in hay^nc2 flies is the product of some other gene that is upregulated in this particular mutant background. If so, however, the product of this hypothetical upregulated gene would have to cross-react with the affinity-purified, anti-haywire antiserum. In addition, upregulation of the other hypothetical gene appears to depend strictly on the poison product effect of the hay^nc2 allele, as the polypeptide was not detected in any of the six revertants of hay^nc2.

The hay^nc2 allele genetically interacts with mutations in the B2t locus (REGAN and FULLER 1988 ), which encodes a testis-specific ß-tubulin isoform (KEMPHUES et al. 1979 ). Levels of ß₂-tubulin are critical for spermatogenesis in flies. Flies carrying a deficiency for B2t are already close to a threshhold requirement of ß₂-tubulin, since B2t^null heterozygotes are fertile at 25° but sterile at 18° (FULLER et al. 1989 ). Further decreasing the level of transcription by altering the function of a basal transcription factor might bring the level of expressed ß₂-tubulin below the critical threshold required for the many microtubule-mediated tasks necessary for spermatogenesis. It is feasible that either full-length haywire protein with the Arg652-to-Cys mutation or accumulation of a 68-kD fragment of the haywire protein in hay^nc2 flies could cause such a deleterious effect on transcription, causing the hay^nc2 mutation to act as a dominant enhancer of B2t mutations.

	FOOTNOTES

¹ Present address: California Pacific Medical Center Research Institute, Stern Building, 2330 Clay St., San Francisco, CA 94115.

	ACKNOWLEDGMENTS

We thank Barbara Robertson and Kim Schuske, who conducted genetic screens to identify new revertant alleles of hay^nc2, and Cricket Wood for technical assistance. We thank Stig Hansen and Robert Tjian for help and advice in producing affinity-purified antibodies to the haywire protein. We acknowledge the protein and nucleic acid (PAN) facility, Stanford University, for synthesizing oligonucleotides used in this work. This work was supported by National Institutes of Health grants HD-18127 and HD-29194 to M.T.F.

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