Su(Ste) Diverged Tandem Repeats in a Y Chromosome of Drosophila melanogaster Are Transcribed and Variously Processed

Corresponding author: Vladimir A. Gvozdev, Institute of Molecular Genetics, Kurchatov Sq. 46, Moscow 123182, Russia, gvozdev{at}img.ras.ru (E-mail).

Communicating Editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We report the organization and transcription of diverged tandemly repeated Y-linked Su(Ste) genes that are considered as suppressors of testis-expressed X-linked-repeated Stellate genes that encode a protein sharing extensive homology with ß-subunit of casein kinase 2. Clustering of restriction variants is confirmed. Size variants of Su(Ste) repeats appeared to be nonhomogeneously distributed among the P1 phage clones. Different ways of Su(Ste) RNA processing because of the appearance of new splice sites and polyadenylation signals were detected. The high extent of homology between Stellate and Su(Ste) repeats suggested a possibility of Stellate suppression by antisense transcription of Su(Ste) elements. The detection of only "sense" Su(Ste) cDNAs in testis cDNA library allows us to reject this proposal. The genomic and cDNA clones are shown to be equally diverged. This indicates widespread rather than restricted transcription capacity of these repeats.

THE X-linked Stellate (Ste) gene clusters located in euchromatin as well as in heterochromatin (LIVAK 1990 ; SHEVELYOV 1992 ; PALUMBO et al. 1994 ) are represented by tandem repeats encoding a protein with significant identity to the casein kinase 2 ß-subunit (LIVAK 1990 ; BOZZETTI et al. 1995 ). Ste genes are overexpressed in testes of males lacking a Y-chromosome or a specific region of the Y-chromosome carrying the Su(Ste) locus (HARDY et al. 1984 ). Ste overexpression leads to abnormalities of gametogenesis and male sterility. Expression of Ste is inhibited by the Y-linked Su(Ste) locus comprising repeats highly homologous to Ste units (LIVAK 1990 ; BALAKIREVA et al. 1992 ). Male fertility is restored in the presence of the Su(Ste) locus. The evolution of these bizarre gene arrays is discussed as an example of meiotic drive (HURST 1992 ; HURST 1996 ).

A current model of the Ste-Su(Ste) interaction based on the molecular analysis by LIVAK 1990 suggests that the suppression of Ste transcription and aberrant splicing of Ste mRNAs depend on a higher affinity of Su(Ste) genes for limited amounts of transcription and splicing factors. However, transcription of Su(Ste) repeats was not demonstrated. Here we report studies of Su(Ste) transcription in testes using a cDNA testis library. We detected alternative ways of RNA processing of Su(Ste) transcripts caused by their divergence. This phenomenon is without precedent for tandem arrays. The extended region of homology between the X-linked Ste and the Y-linked Su(Ste) repeats suggested a hypothesis that antisense Su(Ste) transcription may be involved in the suppression of Ste genes (DANILEVSKAYA et al. 1991 ). However, only sense Su(Ste) cDNA variants were detected. Thus, antisense mechanism of suppression seems to be untenable.

The clustering of Su(Ste) restriction variants was recently revealed (MCKEE and SATTER 1996 ). However, the small size of genomic DNA in phage does not allow one to demonstrate clustering of repeat length variants. Molecular studies of P1 phage, comprising ~100-kb genomic fragments and containing up to 30 copies of Su(Ste) units, extend the knowledge concerning diversification of Su(Ste) repeats. We present striking confirmation of the restriction site clustering described earlier (MCKEE and SATTER 1996 ) and demonstrate that length variants are nonuniformly distributed throughout the locus.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genomic libraries:
Using a Drosophila melanogaster genomic cosmid pHC79 library from pn2a stock (provided by V. ALATORTSEV, MOSCOW, INSTITUTE OF MOLECULAR GENETICS) and a bacteriophage P1 library from the y; bw cn sp stock (SMOLLER et al. 1991 ) were screened as described (SAMBROOK et al. 1989 ) by hybridization to a Y-specific Su(Ste) probe (probe 2, Figure 1A). A 2.5-kb XbaI fragment from G12 phage, as well as 1.8- and 1.6-kb XbaI fragments from cosmids 1 and 5, was subcloned for further analysis. The subcloned fragments were partially sequenced from double-stranded plasmid DNA templates using standard M13 and specific primers. Sequencing was carried out using 7-deaza-dGTP reagent kit and Sequenase version 2.0 T7 DNA polymerase (Amersham, Arlington Heights, IL).

View larger version (36K): [in this window] [in a new window]   Figure 1. —Structural polymorphism of Su(Ste) repeats. (A) Two thick horizontal arrows indicate size and direction of transcription of Su(Ste) repeats. Open and shaded boxes represent the Stellate-like and Y-specific regions, respectively. Vertical arrows designate invariant (X or H) and variable (X* or H*) XbaI and HindIII sites, respectively. The insertion of 1360 element with inverted repeats is indicated (out of scale). Hybridization probes are shown as blackened rectangles. The stippled region in the 1.7-kb unit indicates 165-bp insertion (Ins.). (B) Southern blot analysis of genomic and P1 cloned DNA. 5 µg of DNA from gt wa males (lanes 1 and 3) and females (lanes 2 and 4) and 0.5 µg of P13 (lane 5) and G12 (lane 6) phage DNA were digested with Xba1, electrophoresed, blotted, and hybridized. The sizes of the fragments are given in kb.

cDNA library:
A lambdaZAPII cDNA library (Stratagene, La Jolla, CA) from testes (Canton S strain) was provided by T. HAZELRIGG. The library was plated and screened according to the Stratagene protocol. After screening, using a Y-specific Su(Ste) probe (probe 2, Figure 1A), three independent clones, designated as cDNA 1512, 13 and 15 (out of 1,000,000 screened), were isolated. Screening using the Ste probe 1 (Figure 1A) did not reveal additional signals. To detect an extreme 5' end of Su(Ste) transcripts, a second round of screening was performed using probe 3 (Figure 1A) specific to the 5' end of Ste transcripts. As a result, the cDNA clone 511 was isolated. After in vivo excision, the pBluescript SK- plasmids containing cDNA inserts were sequenced bidirectionally.

Southern analysis:
Male, female, and phage DNAs were digested and electrophoresed on 0.8% agarose gels, transferred to HyBond-N filters (Amersham) and probed with labeled fragments. Fragments for hybridization were obtained using PCR (Figure 1A). Probes were labeled by random-primed reaction in the presence of alpha P³²-dATP.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Polymorphism and internal arrangement of diversified Su(Ste) repeats:
It was shown earlier (BALAKIREVA et al. 1992 ) that the 2.8-kb Su(Ste) repeats are represented by three main elements: a region of homology to X-linked Ste genes, a Y-specific segment, and a mobile element 1360 inserted in the Ste-homologous sequence (Figure 1A). A high level of Su(Ste) sequence variability and variation of repeat length was demonstrated (BALAKIREVA et al. 1992 ; LIVAK 1990 ; MCKEE and SATTER 1996 ). Using XbaI digested genomic DNA and hybridization of Southern blot to the Ste-probe (probe 1, Figure 1A), abundant male-specific fragments of 2.8, 2.5, 1.8, and 1.6 kb in size (Figure 1B) were detected in all the studied stocks (data not shown). The 1.25- and 1.15-kb fragments detected in male as well as in female DNA correspond to euchromatic and heterohromatic X-linked Ste variants, respectively (LIVAK 1990 ; SHEVELYOV 1992 ).

Using a genomic cosmid library, we cloned and sequenced the fragments carrying the 1.6- and 1.8-kb XbaI repeats. The larger fragment carries a 165-bp insertion in the Y-specific region flanked by duplication of the target TTAAA sequence (Figure 1A and Figure 3); this 165-bp insertion sequence is also present in the 2.8-kb fragments described by BALAKIREVA et al. 1992 , but it is absent in the 1.6-kb copy subcloned from the cosmid. The variable XbaI site in the 1360 element is responsible for the 1.8-kb fragments carrying insertion. The diagram of the Su(Ste) repeat polymorphism is presented (Figure 1A). The results allow us to conclude that the variable XbaI site detected in the 1360 element is presented in both types (2.5 and 2.8 kb) of units.

View larger version (108K): [in this window] [in a new window]   Figure 2. —Southern analysis of genomic Su(Ste) variants. Genomic DNA (3 mkg) of males of y; bw cn sp stock and DNA of P13 and G12 phage were digested with the indicated enzymes (X - XbaI, B - BamHI, H - HindIII), electrophoresed, blotted to HyBond-N filter and probed with a labeled Y-specific fragment (Figure 1A). The 1.6- and 0.9-kb fragments designated by asterisks are the products of HindIII digestion of 2.5-kb repeat. Fragment lengths are indicated in kb.

View larger version (56K): [in this window] [in a new window]   Figure 3. —Sequences of Stellate and Su(Ste) genes and four Su(Ste) cDNAs. The numbering begins from 352 bp of the Stellate pSX83.4 (LIVAK 1990 ) and from 169 bp of the Su(Ste) pSY61.2 (BALAKIREVA et al. 1992 ) variants. A hyphen means that the base is the same as in the Ste sequence, and an asterisk means that the base is missing. The GT of splice donors (D) and the AG of splice acceptors (A) in the Ste and Su(Ste) transcripts are indicated by bold italics. Arrows indicate duplications referred to slippage events. The sequences upstream of the start ATG codon are boxed. The stop codons in conceptual ORFs are underlined. The putative intron branchpoint is indicated by bold letters. The cases of the Su(Ste) nucleotide sequence diversification not detected previously in the Su(Ste) variants (BALAKIREVA et al. 1992 ) are circled. The insertion in the Y-specific region from 1259 to 1424 is underlined. The sequences of cDNAs 13, 1512, 511 have been deposited in GenBank under accession numbers L42286, L42287, and L42288, respectively.

To study the internal organization of Su(Ste) units in the cluster, we used a recombinant P1 phage library constructed from the DNA of the y; bw cn sp stock containing typical genomic pattern of the Su(Ste) XbaI restriction fragments (compare Figure 1B, lane 3; Figure 2, lane 1). Two recombinant phage comprising ~80–100 kb of genomic DNA were isolated using the Y-specific probe 2. Southern hybridization (XbaI digestion, hybridization with probe 2) revealed only the 2.8- and 2.5-kb Y-specific fragments in both phage (Figure 1B). Using a whole phage as a hybridization probe, we have not detected additional bands, apart from those mentioned above and a vector-related fragment (data not shown). Thus, these phage carry clusters of 20–30 Su(Ste) units uninterrupted with other sequences. The G12 phage comprises predominantly 2.5-kb repeats, whereas P13 contains equal numbers of both length variants (Figure 1B). The repeats without the variable XbaI site are clustered, since no 1.8- and 1.6-kb fragments were detected in the P1 phage comprising up to 30 Su(Ste) units. These results confirm strikingly the restriction site clustering of Su(Ste) repeats (MCKEE and SATTER 1996 ). The size variants are not uniformly distributed throughout the Su(Ste) locus. The genomic DNA used for construction of the P1 library contains an excess of the 2.5-kb copies as compared to the 2.8-kb ones. Nevertheless, the P13 phage contains practically an equal number of the 2.5- and 2.8-kb variants.

The clustering of repeats with similar restriction sites was also demonstrated using BamHI and HindIII digestion of both phage. Southern hybridization with the Y-specific probe demonstrates that the Su(Ste) repeats cloned in P13 and G12 phage carry no sites for BamHI that are detected elsewhere in the Su(Ste) locus (Figure 2). Sequencing of Su(Ste) copies revealed HindIII site in 1360 element as well as a variable one in the Y-specific region (Figure 1A). Thus, HindIII digestion may produce the fragments of the same sizes (2.5 and 2.8 kb) as XbaI digestion as well as 1.7-, 1.6-, 1.1- and 0.9-kb fragments (Figure 1A and Figure 2). Overexposure of genomic Southerns reveals the corresponding fragments in genomic DNA (not shown). The appearance of the 1.6-kb fragment is a result of a small deletion in the 5' region of the 1.7-kb fragment (data of PCR analysis, not shown). At the same time, Southern analysis indicates the presence of Su(Ste) copies lacking HindIII sites (~5.6-kb fragment in P13 phage). The 2.5-kb HindIII fragment is easily detected in P13 phage indicating that it contains preferentially the copies lacking the variable HindIII site in the Y-specific region. The detection of 1.6- and 0.9-kb fragments may be a result of HindIII digestion of a part of the 2.5-kb copies carrying variable HindIII site in P13 phage and all of the 2.5-kb repeats in phage G12. Hybridization probe 2 overlaps predominantly with the 0.9- and 1.1-kb fragments (Figure 1A) and does not allow us to make a quantitative evaluation of fragments abundance. Thus, all the 2.5-kb Su(Ste) repeats in G12 phage carry variable HindIII site in the Y-specific region, whereas it is absent in a lot of the 2.5-kb repeats in P13 phage. Thus, clustering of restriction variants throughout the regions of ~100 kb of Su(Ste) locus was demonstrated.

Transcripts of Su(Ste) repeats and their processing:
The cDNA testis library was screened using the Y-specific fragment of Su(Ste) gene as a probe (probe 2, Figure 1A). Four different Su(Ste) cDNAs (13, 1512, 511, 15) were identified by the detection of Su(Ste) diagnostic nucleotide substitutions and two deletions as compared to homologous Ste sequences. The Su(Ste) divergence was shown earlier (BALAKIREVA et al. 1992 ), but further evidence of diversification represented by nucleotide substitutions, slippage events and nucleotide insertions and/or deletions (Figure 3) were detected. Four Su(Ste) cDNAs differ at 6.4% of sites (considering both point differences and small insertions and/or deletions). The same value was calculated (MCKEE and SATTER 1996 ) using the previously published data for five genomic Su(Ste) copies (BALAKIREVA et al. 1992 ). Thus, the divergence of the cDNA sequences shows that different diversified repeats are transcribed. No Ste cDNAs were isolated using probe 1 (Figure 1A). This result corroborates silencing of Ste transcription in XY males.

The Su(Ste) cDNA sequences (Figure 3) allow us to define the location of introns and sites of polyadenylation. The sequencing revealed the same direction of transcription as was shown (LIVAK 1990 ) for the X-linked homologous Ste units. This observation is not consistent with the suggestion that antisense Su(Ste) transcription (DANILEVSKAYA et al. 1991 ) is a mechanism for inhibition of Ste expression. All the Su(Ste) transcripts proceed into the Y-specific region. The sequences of the cDNAs 13 and 1512 indicate the usage of different sites of polyadenylation (Figure 3). The strategy of the cDNA library construction (Stratagene protocol), using random as well as oligo(dT) primers, resulted in the origination of some cDNA clones lacking poly(A) tracts. Su(Ste) repeats do not contain a region of the Ste poly(A) signal sequence substituted by the Y-specific segment carrying its own putative polyadenylation signals, one of which may participate in RNA 13 maturation (Figure 3 and Figure 4).

View larger version (17K): [in this window] [in a new window]   Figure 4. —Diagram of Stellate and Su(Ste) genes and patterns of Su(Ste) processing. The Stellate ORF is indicated by open rectangles, fragments of two other putative ORFs are presented by shaded and blackened rectangles. Deletions are indicated by triangles pointing away from the lines; ciphers show the number of deleted nucleotides.

We compared the observed patterns of the Su(Ste) RNA splicing to the previously detected Ste RNA processing (Figure 4), resulting in elimination of two introns. All the detected Su(Ste) cDNA sequences demonstrate precise elimination of the second intron peculiar to Ste gene (Figure 3 and Figure 4). The 3' splice site for the first Ste intron is damaged in the Su(Ste) repeats as a result of G to C transversion of the canonical last G nucleotide of the intron (BALAKIREVA et al. 1992 ). The cDNA 511 sequence revealed the usage of an alternative 3' splice site located four nucleotides downstream. This 3' splice site matches better the consensus for Drosophila small introns (MOUNT et al. 1992 ) than the nearby 3' site used for the Ste intron processing. Excision of this intron leads to the disruption of the Ste-like ORF, but it is again restored as a result of two single nucleotide deletions.

The cDNA 1512 sequence indicates the presence of a new processed intron with a deduced 5' splice site (position 370, Figure 3). This splice site may be considered as a canonical GU, taking into account that diverged transcribed Su(Ste) sequences may contain GT nucleotides at this position (see sequence of cDNA 511). The detected 5' splice site (TGGTGTG) matches well with the canonical AGGTA/GAG consensus for short introns (MOUNT et al. 1992 ). The 3' splice site sequence is also in accordance with the previously detected ones (MOUNT et al. 1992 ). A putative branchpoint CTAAT consensus also matches well with the detected CGAAT sequence near the 3' splice site (Figure 3).

The sequence of cDNA13 allows us to suppose an excision from the primary transcript of the extremely short 28-nt intron. The 3' splice site TGAAGAT matches adequately to the canonical sequence TA/GCAGA/GT. The 5' splice site may be deduced taking into account that the second nucleotide of the donor splice site of this putative intron sequence is quite variable in Su(Ste) repeats (BALAKIREVA et al. 1992 ). For example, CT nucleotides start the intron sequence in the Dm12 Su(Ste) variant (BALAKIREVA et al. 1992 ). Thus, the putative 5' splice site may be deduced as the AGCTATG. The use of CT nucleotides as the 5' splice site in Drosophila Gpdh gene has been reported previously (MOUNT et al. 1992 ). The cDNA 13 may be related to a diverged but as yet undetected Su(Ste) repeat. The vestige of a branchpoint in this extremely short intron may be attributed to the GGCAAGT sequence. A possibility of translation of transcripts is considered in DISCUSSION.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The idea of concerted evolution of multigene and tandem repeated genes in eukaryotes is widely discussed. However, direct experimental data concerning the details of proposed mechanisms of their homogenization are rare. The high level of divergence of Su(Ste) repeats has been shown earlier (BALAKIREVA et al. 1992 ) and was recently discussed and evaluated (MCKEE and SATTER 1996 ). Su(Ste) repeats differ at 6.4% of nucleotide sites as compared to 2.5% of divergence for homologous Ste repeats (MCKEE and SATTER 1996 , calculated from the previously reported data; BALAKIREVA et al. 1992 ; SHEVELYOV 1992 ).

Our results concerning restriction and length polymorphism of Su(Ste) repeats indicate a very high degree of clustering of repeats with similar restriction sites. The evidence of such clustering was shown earlier as a result of analysis of as many as four tandem Su(Ste) repeats cloned in a lambda phage (MCKEE and SATTER 1996 ). We extended this conclusion demonstrating clustering of Su(Ste) units with similar restriction sites throughout the region comprising up to 30 copies. The region of Su(Ste) cluster may be completely homogeneous for a restriction site variant (XbaI restriction of P13 and G12 phage), but remains heterogeneous for 2.5- and 2.8-kb size variants.

The Su(Ste) length variants were shown to be nonrandomly distributed throughout the locus. Phage P13 contains an approximately equal amount of the 2.5- and 2.8-kb variants, whereas phage G12 is comprised predominantly of the 2.5-kb size variant. This observation confirms the earlier published data that Su(Ste) size variants appear to be clustered in particular subintervals of the locus (MCKEE and SATTER 1996 ). The gene conversion events were detected earlier between Su(Ste) repeats (BALAKIREVA et al. 1992 ), and we suppose that it is involved in large scale homogenization of heterochromatic repeats.

The function of heterochromatic tandem-repeated genomic structures in eukaryotes is often under question. In this context, a special interest in Y-linked Su(Ste) repeats arose, because these repeats are thought to be involved in suppression of the homologous X-linked Ste genes transcription. Testis-specific overexpression of the Ste genes in males, lacking a Y chromosome or its fragment comprising the Su(Ste) locus (HARDY et al. 1984 ; PALUMBO et al. 1994 ), leads to the formation of crystals in primary spermatocytes and to abnormalities of meiosis. Thus, repeated units of the Su(Ste) locus interacting with Ste genes restore male fertility. The mechanism of suppression remains unknown. Here we report the first attempts to understand these interactions as well as our observations concerning molecular diversification of Su(Ste) repeats. Transcription of Su(Ste) repeats was detected. The calculated sequence divergence of the four Su(Ste) cDNA (6.4%) exactly matches the calculated Su(Ste) repeats divergence (MCKEE and SATTER 1996 ). This implies that the expression is a general property of the Su(Ste) locus and it is not restricted to definite Su(Ste) variants. There is little direct evidence of transcription of genes embedded in the large block of heterochromatin of the Y chromosome, such as a dynein gene (GEPNER and HAYS 1993 ), a histone-like gene (RUSSEL and KAISER 1993), and satellite sequences (BONACCORSI et al. 1990 ). We present a new example of transcription of Y-linked genes.

We demonstrated that the Su(Ste) and Ste genes are transcribed in the same direction. Attempts to detect Su(Ste) antisense transcription in testes and elsewhere using a riboprobe complementary to the Ste noncoding strand was unsuccessful. Thus, the hypothesis of Su(Ste) antisense transcription (DANILEVSKAYA et al. 1991 ) suggested to explain Ste suppression seems to be erroneous.

Transcription of different divergent Su(Ste) copies was demonstrated. The most intriguing feature of Su(Ste) repeat processing is the origination of new splice sites as compared to the Ste pre-mRNA fate. The cDNA 1512 sequence revealed the origin of a new 5' splice site (Figure 3) as a result of C to T substitution in a corresponding Su(Ste) genomic copy. Sequencing of the cDNA 511 revealed the usage of the newly formed 3' splice site instead of the damaged Ste-specific one.

Different translation products may be formally predicted, taking into consideration the existence of differential splice sites in Su(Ste) transcripts (Figure 4, see also Figure 3). The Ste ORF starts 11 nucleotides upstream of the 5' splice site of the first intron and ends four nucleotides downstream of the 3' splice site of the second Ste intron (LIVAK 1990 ). The cDNA 511 demonstrates a shift of the Ste ORF because of usage of the alternative 3' splice site for processing of the first intron. However, the Ste ORF is restored downstream owing to two single nucleotide deletions, and it is then shifted again as a result of a 10-bp deletion and stops upstream of the 5' splice site of the second intron. The sequence of the cDNA 1512 allowed us to propose the altered position of the initiation codon because of a G to A substitution (position 183, Figure 3) resulting in a Val to Met replacement. The CTGGCAAC sequence immediately upstream of ATG codon in the Ste gene is quite similar to the CTGCCAC sequence upstream of the putative start codon in the cDNA 1512. These observations strengthen a suggestion that cDNA 1512 demonstrates the origination of a novel start codon in the open reading frame (ORF) homologous to the Ste one.

Three different cDNA clones of Su(Ste) repeats have potential possibility to encode three different conceptual polypeptides comprising fragments of Ste ORFs (Figure 4, open rectangles) and segments of two other ORFs (Figure 4, presented by shaded and blackened rectangles). We have no evidence that Su(Ste) repeats may represent functional protein coding genes. The analogous observation of Y-linked diverged gene copies in Drosophila melanogaster genome, supposed to be able to encode different functioning polypeptides (histone-like proteins), was reported (RUSSEL and KAISER 1993). However, in the latter case all the supposed differences of conceptual peptides were attributed to microdeletions or nucleotide substitutions.

It is not easy to attribute different ways of transcript processing and translation to the mechanism of suppression. Possibly, suppression of Ste transcription accomplished by Su(Ste) repeats may be attained through their competition for positively acting transcription factor(s), taking into account the similarity of putative promoter regions in the Ste and Su(Ste) repeats. This explanation disregards the coding potential of Su(Ste) repeats. However, all of the predicted Su(Ste) proteins as well as Ste protein are similar to the ß-subunit of casein kinase 2 (CK2). CK2 subunits may be involved in transcription control of corresponding genes via DNA-protein interaction (ALLENDE and ALLENDE 1995 ). CK2 was shown to be a predominantly nuclear protein in human cells (KREK et al. 1992 ). The human -subunit of CK2 (CK2) is capable of activating transcription of the ß-subunit of CK2 (ßCK2) gene (ROBITZKI et al. 1993 ). A possibility of ßCK2 gene product interaction at the level of gene transcription was also discussed (ROBITZKI et al. 1993 ). Thus, coordinate regulation of CK2 and ßCK2 synthesis may be attained at the level of gene transcription. The Ste gene product as well as the predicted Su(Ste) cDNA 511 encode products contain the conservative zinc finger domain peculiar to ßCK2 genes (ALLENDE and ALLENDE 1995 ). This domain is encoded by the nucleotide stretch from position 417 to position 512 (Figure 3) and was considered as a putative DNA recognition element (BERG 1990 ). It is tempting to speculate that Ste protein and its isoforms also take part in DNA-protein interactions; the Su(Ste) genes may encode polypeptides capable of suppressing Ste transcription. The truncated variants of Su(Ste) cDNAs lacking the region encoding the zinc finger domain may participate in protein-protein interaction affecting Ste transcription. A precedent of a crucial role of short polypeptides ensuring dramatic silencing of transcription complexes has been reported (MOLINA et al. 1993 ).

We speculate that selection constraints at the translational level may underlie Su(Ste) diversification, although it was supposed that selection may well be reduced at Su(Ste) repeats, taking into account a high level of Su(Ste) repeat dissimilarities (MCKEE and SATTER 1996 ). It is tempting to attribute differential splice sites in Su(Ste) transcripts to novel functional properties of polypeptide products but this proposal needs to be corroborated by the direct detection of predicted polypeptides.

	ACKNOWLEDGMENTS

We are grateful to T. HAZELRIGG for providing the cDNA testis library and to YU. SHEVELYOV for discussion, and anonymous reviewers for their helpful comments. This work was supported by Russian Foundation for Basic Research (grants 96-04-49026 and 96-15-98072) and the Russian Program "Frontiers in Genetics" to V.A.G.

Manuscript received May 21, 1997; Accepted for publication September 18, 1997.

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