Genetic and Molecular Characterization of sting, a ... - Semantic Scholar

Copyright  1999 by the Genetics Society of America

Genetic and Molecular Characterization of sting, a Gene Involved in Crystal Formation and Meiotic Drive in the Male Germ Line of Drosophila melanogaster Armin Schmidt,* Gioacchino Palumbo,† Maria P. Bozzetti,† Patrizia Tritto,‡ Sergio Pimpinelli‡ and Ulrich Scha¨fer* *Institut fu¨r Genetik, Heinrich-Heine-Universita¨t, D-40225 Du¨sseldorf, Germany, †Istituto di Genetica, Universita` di Bari, I-70216 Bari, Italy and ‡Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Genetica e Biologia Molecolare, Universita` di Roma “La Sapienza,” I-00185 Rome, Italy Manuscript received June 5, 1998 Accepted for publication November 6, 1998 ABSTRACT The sting mutation, caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility is correlated with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry) locus. In addition, their morphology is needle-like in Ste1 flies and star-shaped in Ste flies, once again as observed in cry2 males. The sti mutation leads to meiotic drive of the sex chromosomes, and the strength of the phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene was cloned and shown to code for a putative protein that is 866 amino acids long. A C-terminal domain of 82 amino acids is identified that is well conserved in proteins from different organisms. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry2 males, a 0.7-kb mRNA is produced.

T

HE Y chromosome of Drosophila is entirely heterochromatic and totally dispensable for viability, but necessary for male fertility (Hess and Meyer 1968). Mutational analysis has revealed only nine Y-chromosomal elements in Drosophila melanogaster, six male fertility factors, two required for normal meiosis, and the rDNA containing bobbed locus (Pimpinelli et al. 1986). One of the Y-linked genes necessary for correct meiosis is the crystal (cry) locus (Pimpinelli et al. 1986), also known as Suppressor of Stellate [Su(Ste); Livak 1990]. Males that lack the cry locus show multiple meiotic alterations that also affect their fertility. The spectrum of the meiotic abnormalities includes, abnormal chromosome condensation and segregation. Moreover, prominent crystal formation, a phenomenon first observed in X/0 males (Meyer et al. 1961), is also present in primary spermatocytes. Previous work demonstrated that the abnormal meiotic phenotype reflects an unusual negative regulatory interaction that normally occurs between crystal and the X-linked Stellate (Ste) locus located in the 12E1,2 region of the polytene map (Palumbo et al. 1994). An interesting notion is that the Ste and cry loci are composed of homologous repetitive sequences. Additional Ste elements outside the Ste locus have also been

Corresponding author: Ulrich Scha¨fer, Max-Planck-Institut fu¨r biophysikalische Chemie, Abt. Molekulare Entwicklungsbiologie, D-37070 Go¨ttingen, Germany. E-mail: [email protected] Genetics 151: 749–760 (February 1999)

found in the X heterochromatin (Shevelyov 1992; Palumbo et al. 1994). Ste and cry loci are normally silent, but deletion of cry induces the derepression of the Ste elements in the male germ line and leads to the mutant phenotype. The Ste sequences that contain two introns are transcribed abundantly and processed correctly only in the testes of cry2 males, producing a 750-bp poly(A)1 (Livak 1990). This transcript encodes for a 19,500-D protein with striking homology to the b subunit of casein-kinase 2 (CK2) (Livak 1990). We recently generated an antibody against the Ste protein and showed that this protein is absent in X/Y males, but that it is the main, if not the sole, component of the crystalline aggregates in X/Y cry2 males. Moreover, we have shown by in vitro assays that the Ste protein can interact with the a subunit of CK2 to form a complex that exhibits some properties of an active a2b2 holoenzyme. The functional homology of the Ste protein with the b subunit of CK2 has suggested that the Ste protein may compete in vivo with the normal b subunit by binding to the a-catalytic subunit, impairing the normal CK2 regulatory functions. The presence of topoisomerase II among the nuclear CK2 targets has suggested that decreased activity of topoisomerase II could be a major factor contributing to the spectrum of meiotic chromosome alterations induced by crystal deletions (Bozzetti et al. 1995). Although all experimental evidence has shown that the Ste protein is absent in normal males, it is still an open question whether the Ste-cry system is of any

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functional importance or completely dispensable. Independent of its nature, however, the genetic dissection of the Ste-cry system should allow one to identify genetic elements involved in its regulation that also probably play an important role in controlling fundamental aspects of male meiosis in Drosophila. Here we report the results of the first approach to this problem: a genetic and molecular analysis of a mutation that derepresses the X-linked Ste sequences in spermatocytes of cry1 males.

MATERIALS AND METHODS Chromosomes, crosses, and cultures: The original insertion line as well as other sti alleles were kept over the balancer chromosome CyO (Lindsley and Zimm 1992). The B Scry1Yy1 chromosome (abbreviated as cry1Y in the text) carries an almost complete deletion of the crystal locus. The ywf and wildtype X chromosomes, all characterized for the Ste sequence contents, are described in Palumbo et al. (1994) and in this article. For more complete information on these chromosomes and markers, see Lindsley and Zimm (1992). All fly cultures and crosses were grown on standard fly medium at 258. For remobilization of the P{lacW} element (Bier et al. 1989), the transposase source P{ry1 D2-3}(99B) (Robertson et al. 1988) was introduced. The progeny was screened for loss of the w1 phenotype, and lines were established from single events. P-element-mediated germ-line transformation (Rubin and Spradling 1982) was done according to standard protocols (Spradling 1986). All P-element vectors had the white gene as a selectable marker, and w1118 embryos were consequently injected. Microscopy and histology: For the analysis of spermatogenic stages, testes were dissected in Ringer’s solution and immediately visualized under phase-contrast optics with a Zeiss photomicroscope. Antibody staining was performed according to the methods of Palumbo et al. (1994) and Bozzetti et al. (1995). The histochemical assay for b-galactosidase activity in larval and adult flies essentially followed the protocol of Glaser et al. (1986), and the assay in embryos was performed as described in Kla¨mbt et al. (1991). Staining was allowed overnight at room temperature. Cloning methods: DNA cloning and screening procedures followed standard protocols (Sambrook et al. 1989). The genomic library used for the screening was generated from DNA isolated from 0, to 12-hr embryos of the D. melanogaster wildtype strain Canton-S and cloned in the vector l FIX II (Stratagene, La Jolla, CA). For cDNA isolation, a library of cDNAs to RNA from adult D. melanogaster (Novagen) in the phage vector lEXlox(2) (Palazzolo et al. 1990) was screened. To get a full-length cDNA clone, a 59 RACE experiment (Frohmann et al. 1988) using the 59 RACE system of GIBCO BRL (Gaithersburg, MD) was performed. Plasmid DNA sequencing (Chen and Seeburg 1985) was done by the dideoxy chain terminating method (Sanger et al. 1977). DNA slot blot analysis: The DNA slot blot analysis was performed as described in Palumbo et al. (1994). However, the precise estimation of the copy number in Ste alleles was difficult because of dilution problems resulting from the high number of repeats. Total RNA extraction: Fifty pairs of adult testes from different strains were hand-dissected in modified Ringer’s solution (Ashburner 1989). After the addition of 0.9 ml of 0.1 m NaCl, 0.1 m Trisbase, 0.03 m Na2EDTA, and 1% Sarkosyl, the testes were homogenized and extracted with phenol:chloroform:

isoamylalcohol (50:49:1). The RNA was precipitated by adding 2.5 ml ethanol and was stored at 2208. The samples were dissolved in 15 ml of mix (470 ml deionized formamide, 157 ml 37% formaldehyde, 98 ml 103 MOPS, and 275 ml sterile water) and heated at 608 for 15 min. Two milliliters of loading buffer for RNA (Sambrook et al. 1989) was added to each sample. Electrophoresis was performed on a 1% agarose 3-[Nmorpholino]propanesulfonic acid (MOPS) formaldehyde gel, as described in Sambrook et al. (1989). Northern blot analysis: After electrophoresis, the gel was photographed and washed in sterile water for 10 min and then in 203 SSC for 1 hr. Transfer was performed on a Hybond-N nylon membrane overnight. Baking was for 45 min at 808 with vacuum. Hybridization was performed in 25 ml of 50% formamide, 53 SSC, 53 Denhardt’s, 0.53 SDS, 10 mm EDTA, pH 8, and 100 mg/ml salmon sperm DNA at 428 overnight. After hybridization, the filter was washed at 658 for 15 min in 23 SSC and 0.1% SDS and then in 13 SSC and 0.13 SDS for 15 min. Autoradiography was performed on AGFA CURIX 100 film. Computer analyis: The nucleic acid sequence was analyzed with the PC/GENE program package. Databank searches were performed with the BLAST programs (Altschul et al. 1990, 1997) using the worldwide web server of the National Center for Biotechnology Information (NCBI; Washington, DC). The CLUSTAL W program (Thompson et al. 1994) was used for alignments. Estimation of meiotic parameters and statistical analysis: For the evaluation of both the frequency of disjunctional failures and the level of meiotic drive, we have followed the model of McKee (1984) and McKee and Lindsley (1987), as described in Palumbo et al. (1994). Briefly, if the probability of disjunction of the X and Y chromosomes is PXY, and the recoveries of the X and Y chromosomes are RX and RY, respectively, the frequencies of the four sex chromosome gamete types are [X] 5 1⁄2PXYRX, [Y] 5 1⁄2PXYRY, [XY] 5 1⁄2(1 2 PXY)RXRY, and [0] 5 1⁄2(1 2 PXY). Each cross yields three independent observations, and there are three parameters. Hence, these equations have unique solutions for any one cross. Those solutions are as follows: PXY 5 1/(1 1 √([X][XY])/([Y][0])); RX 5 √([X][XY])/([Y][0]); and RY 5 √([Y][XY])/([X][0]). Similar parameters can be used to describe autosomal behavior. In our experiments, we followed the second chromosomes by crossing males to C(2)EN-bearing females. All ova produced by these females are disomic or nullisomic for the second chromosome. Thus, all normal haplo-2 sperm yield aneuploid, lethal zygotes, as do exceptional nullo-2 and diplo-2 sperm that happen to fertilize eggs of the noncomplementary karyotype. Since all survivors are necessarily nondisjunctional, scoring of survivors only would not permit estimation of the frequency of autosomal nondisjunction, but counting the number of lethal eggs does. If nullo-2 and diplo-2 ova are equally frequent, for P2 is the probability of disjunction of the second chromosomes and R2 is the probability of survival of a sperm bearing a second chromosome, we have [22] 5 1⁄2(1 2 P2)R 22, [0] 5 1⁄2(1 2 P2), and [dead zygotes] 5 P2R2 1 1⁄2(1 2 P2)R22 1 1⁄2(1 2 P2). The three observed numbers (two of which are independent) again give unique solutions for the parameters. They are: P2 5 ([dead zygotes] 2 [22] 2 [0])/ (4[0] x √[22]/[0] 2 [22] 2 [0] 1 [dead zygotes]) and R2 5 √[22]/[0]. The effect of Stellate copy number on fertility was assessed using conventional linear regression, while maximum likelihood procedures (Bishop 1975) were used to evaluate the relationship of nondisjunction and meiotic drive to Stellate copy number and to each other. Maximum likelihood estimates were numerically approximated using the computer

sting Mutation and Meiotic Drive

Figure 1.—(A) Phase-contrast photographs of living spermatogenic stages of X/0 males and (B and C) homozygous sti males. Needle-like crystals can be seen both in spermatocytes and spermatids. Note the existence of Y-chromosomal lampbrush loop structures (arrows in B), whereas only nucleolar structures can be seen in X/0 spermatocytes. program MLIKELY.PAS (version 2.2, 1992, by L. G. Robbins). A description of this analysis is reported in Palumbo et al. (1994).

RESULTS

Phenotype of the insertional mutation: The original mutation was isolated in a screen for male sterile inser-

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tions (U. Scha¨fer, M. Hollmann, A. Schmidt and M. A. Scha¨fer, unpublished results) among 2225 P{lacW} insertion lines that were generated by A. Beermann and C. Schultz in the laboratory of J. A. CamposOrtega at the University of Cologne and generously given to us. The male sterile phenotype displayed variable penetrance, leading sometimes to progeny from homozygous males (see also below). The P{lacW} insertion was localized by in situ hybridization to polytene region 32D on the left arm of chromosome 2 (Schmidt et al. 1996), giving the mutation the provisional name ms(2)32D. Analysis of the spermatogenic stages in homozygous mutant males revealed the existence of needlelike structures in spermatocytes and spermatids (Figure 1). These needles strongly resemble those crystals found in X/0 males of Drosophila melanogaster (Meyer et al. 1961; see also Figure 1A), although the mutant males clearly have an X/Y genotype, as visualized by the presence of Y-chromosomal lampbrush loops. Since we have generated an antibody against the Ste protein (Bozzetti et al. 1995), we first tested whether these needles consist of the Ste protein despite the fact that they are produced in X/Y male germ cells. This is indeed the case, as demonstrated in Figure 2. Furthermore, the crystal morphology is dependent on the Ste genotype. In mutant male flies carrying a Ste1-containing X chromosome, the crystals have a needle-like shape (Figure 2, A and B), whereas in a Ste background, they are similar to the star-shaped crystals found in Ste/ cry2 males (Figure 2C). Thus, the crystals’ phenotype is determined by the Ste locus in both the X/Y, cry1 homozygous sting mutant males and in X/Y, cry2; sting1 males. Because the processed Stellate transcript can also be detected in homozygous mutant males by Northern analysis (see below), the gene was renamed sting (sti), as an acronym for Stellate-interacting gene and for the production of sting-like crystals. sting is a negative regulator of Ste sequences: To test the hypothesis that the phenotypes shown by the sti homozygous mutants are related to the Stellate system,

Figure 2.—An antibody against Ste protein recognizes the crystals in sti males. (A) Young and (B) mature primary spermatocytes of Ste1 testes showing needle-shaped crystals. (C) Star-shaped crystals are found in primary spermatocytes of the Ste male.

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we analyzed the expression of the Stellate RNA in these individuals by using different Ste alleles. Total RNA was extracted from the testes of X/Y males homozygous and heterozygous for the sti mutation carrying different Stellate alleles. The patterns of Stellate expression were compared with those of X/Y or X/0 testes by Northern hybridizations. As shown in Figure 3, the same smeared 750-nt Stellate RNA, typically present in X/Y, cry2 testes (Figure 3A, lane 1), is also always present in testes of homozygous sti males independently of the Ste allele present (Figure 3A, lane 3; Figure 3B, lanes 2 and 4; Figure 3C, lane 2). It is important to note that a high-molecular-weight RNA (of z8 kb) is present in the testes of X/Y regular males (Figure 3A, lane 2) and in X/Y, heterozygous sti males (Figure 3A, lane 5; Figure 3B, lanes 1 and 3; Figure 3C, lane 3). This fragment, however, appears reduced or almost absent, concomitantly with the production of the 750-base fragment, in the testes of X/Y, cry1 homozygous sti males or X/Y, cry2; sti1 males, respectively. This suggests that, like the crystal locus (Livak 1990), sting also seems to control Stellate expression, mainly at post-transcriptional levels. The reduction of the high-molecular-weight RNA

seen in the testes of X/0 males (Figure 3, A and C, lanes 1) is observed in sting homozygous males only in combination with the mutant Stellate alleles (Figure 3B, lanes 2 and 4; Figure 3C, lane 2), while it is not seen with the Ste1 alleles (Figure 3A, lane 3), unless the crystal deletion is also present (Figure 3A, lane 4). Intriguingly, we have seen that the same high-molecular-weight Ste RNA fragment and another 1.2-kb fragment also are present in other tissues of males and females, such as the larval brains, where they are very abundant (Figure 3C, lanes 4 and 5; Figure 3D, lanes 1 and 2), salivary glands (Figure 3D, lanes 3 and 4), and ovaries (Figure 3D, lane 5), irrespective of the sti genotypes (see Figure 3C for an example) or the absence of the Y chromosome (data not shown). To determine the origin of these two types of constitutive transcripts, we analyzed the Ste transcription pattern in the testes of males carrying the W12 X chromosome, which lacks the euchromatic Ste cluster (Palumbo et al. 1994). Figure 3E shows the Ste transcription pattern in W12 X/Y, cry1 (lane 1) and W12 X/Y, cry2 (lane 2) males. The presence of the 8-kb Ste RNA transcripts in Figure 3E, lane 1, and the absence of the 1.2-kb Ste RNA in Figure 3E, lanes 1 and

Figure 3.—Ste transcription patterns in testes and somatic tissues from males and females of the indicated genotypes. In both the Ste1 (A) and Ste mutant alleles (B and C), a 750to 850-nt RNA is present when the males are homozygous for the sti mutation, as in the case of cry2 males. In the last two lanes in (C), the presence of high-molecular-weight Ste transcripts in the brains is evident. In this tissue, however, the sti mutation does not induce the 750- to 850-nt transcript (compare the first two lanes). (D) The high-molecular-weight Ste transcripts plus the 1200 nt are also present in the brains and salivary glands (s. g.) of Oregon-R wild-type males and females, as well as in the ovaries (ov.) of the latter. (E) The transcription pattern in the testes of cry1 and cry2 males lacking the euchromatic Ste cluster. The presence of the high-molecularweight Ste transcripts indicates their heterochromatic origin, and the absence of the 1200nt transcript indicates its euchromatic origin. As a control in all the sections except D, where an actin probe was used, the ribosomal protein 49 (rp49 ) DNA probe (O’Connell and Rosbash 1984) was used for loading quantitation.


2, indicate that the higher-molecular-weight transcripts correspond to the heterochromatic sequences, while the other transcript has an euchromatic origin. From lane 2 of the same figure, it is also evident that in cry2 male testes, the 8.0-kb transcripts decrease concomitantly with the production of the 750- to 850-base Ste transcripts. This suggests that these transcripts are processed in testes of cry2 males, producing the 750-nt RNA. Finally, we tested the possibility that the high-molecularweight band was not RNA. We extracted total RNA from .200 testes of wild-type males, and different aliquots were produced; these aliquots were treated with RNAsefree DNAse for different testing times before loading on the gel for Northern blotting with a control aliquot without the DNAse treatment. DNAse was tested for its activity in the same conditions on genomic DNA. The high-molecular-weight band was not affected by these treatments. Therefore, these data show that the Ste sequences are also normally transcribed in somatic tissues, and that the sti gene negatively controls the Ste expression only in male spermatocytes. Ste copy number and chromosome behavior in sti mutant males: It is known from previous experiments that the X/Y, cry2 males show meiotic chromosome nondisjunction and meiotic drive (Hardy et al. 1984; Palumbo et al. 1994). To test if the sti homozygous mutant males show the same spectrum of meiotic abnormalities correlated with the number of Ste repeats, we chose three Ste1 alleles with low numbers and seven Ste alleles with different high numbers of Ste repeats, as estimated by

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slot blots (Palumbo et al. 1994). We analyzed fertility and sex chromosome behavior in sti homozygous or heterozygous males carrying Ste1 or Ste alleles. The derivation of the disjunctional parameter PXY (the probability of disjunction of the X and Y), the drive parameters RX (the recovery of X-bearing sperm) and RY (the recovery of Y-bearing sperm), and the statistical methods used have been described in detail (Palumbo et al. 1994; see also materials and methods). The data presented in Table 1 lead to the following conclusions: (1) The sting mutation affects male fertility, and this is inversely correlated with the Ste repeat copy number. (2) The sting mutation induces meiotic nondisjunction, as seen in the high number of XY and 0 sperm; this is directly correlated with the Ste repeat copy number. (3) The sting mutation affects meiotic drive, as there is an excess of X over Y and 0 over XY sperm. Moreover, it is interesting to note that the spectrum of these defects is present also in the meiosis of heterozygous sti males carrying the Stellate X chromosomes. Taken together, the results of the statistical analysis described above show that sti affects fertility, disjunction, and drive, and that it is semidominant. As with cry1 (see Palumbo et al. 1994), however, PXY and fertility are much more sensitive to the Ste state (Ste vs. Ste1 or Ste copy number) than RX or RY are. A clear correlation between fertility and the total number of Stellate copies was also found. The effects of Ste copy number on autosomal behavior were also assessed. sti homozygous males carrying four different Ste alleles were crossed to females carrying the

TABLE 1 Progeny produced by crosses of single males carrying X chromosomes with different Ste alleles in combination with B SYy1 and sti to homozygous y w f females

Chromosomes S2 A S2 B F11A F11B ywfA ywfB S3 A S3 B Va A Va B Q-A A Q-A B G2 A G2 B S10 A S10 B G3 A V3 A

a

No. of Ste copies 15 16 29 200 200 200 300 300 400 400

Sperm genotype Male no.

X

Y

0

XY

Total F1

Progeny/ male

PXY disj.

RX drive

RY drive

21 10 22 17 20 15 22 26 24 22 32 25 24 15 20 20 49 50

1332 667 1885 1925 772 1155 516 3591 348 1787 132 3776 28 2041 11 3952 0 0

770 601 1127 1260 450 1264 139 1735 80 904 44 1905 12 1020 0 2007 0 0

35 0 144 2 106 2 170 39 102 7 60 37 9 5 1 40 0 0

4 0 19 0 7 0 10 9 1 1 4 7 1 1 0 8 0 0

2162 1268 3197 3187 1335 2421 835 5374 531 2698 240 5725 50 3067 12 6007 0 0

102.95 126.8 145.00 187.47 66.75 161.40 37.95 206.69 22.12 122.64 7.50 229.00 2.08 204.47 0.60 300.00

0.988 — 0.965 0.999 0.956 0.999 0.866 0.992 0.943 0.998 0.831 0.994 0.859 0.998 — 0.994

0.444 — 0.470 0.874 0.336 0.676 0.467 0.691 0.206 0.531 0.447 0.612 0.509 0.633 — 0.627

0.257 — 0.280 0.572 0.196 0.740 0.126 0.334 0.047 0.269 0.149 0.434 0.218 0.316 — 0.319

a The last letter indicates whether sti is in homozygous (A) or heterozygous (B) condition; Sammichele 2 (S2), Fairfield-11 (F11), and y w f are Ste1 strains, whereas Sammichele 3 (S3), Valdemarskiv (Va), Qiryat-Anavin (Q-A), Giovinazzo 2 (G2), Salve 10 (S10), Giovinazzo 3 (G3), and Valenzano 3 (V3) are Ste strains. For further explanations, see materials and methods.

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compound second chromosome C(2)EN, and the results are shown in Table 2. These crosses show the same basic pattern reported in previous work (Hardy et al. 1984; Palumbo et al. 1994) for Ste1 combined with synthetic, translocation-generated cry deficiencies or with the cry1Y chromosome. The main features of this pattern are the following: (1) the recovery of numerous diplo-2 and nullo-2 sperm, (2) a strong disruption of sex chromosome disjunction among these sperm, and (3) an excess recovery of nullo-2 sperm, which, as discussed earlier (Palumbo et al. 1994), probably reflects an autosomal meiotic drive that is not correlated with Ste copy number. The observed drive of chromosome 2 when sex chromosome disjunction is regular (Table 2; first two rows) leads to the expectation of a similar preference for 0 over 22 sperm in those cases where the disjunction is destroyed. The lack of such an excess of 0;0 over 0;22 sperm (Table 2, row 4) suggests, therefore, that the disjunction of the sex chromosomes and autosomes is not completely independent in frequency, but that it is biased, to some extent, toward opposite poles. Cloning of the sti gene: To be certain that the insertion of the P{lacW} element causes the mutant phenotype, the transposon was remobilized by the introduction of a transposase source (Robertson et al. 1988). Among the lines that had lost the w1 phenotype, many had reverted to the wild type. This demonstrates that the observed male sterility is a consequence of the P{lacW} element inserted into the 32D region. Thus, a molecular analysis seemed feasible by the isolation of flanking sequences. Using the inherent feature of the P{lacW} element, a plasmid rescue clone (Figure 4) containing sequences from the insertion site was isolated. This clone was used to screen a genomic library in l phage, and two overlapping phages containing a total of 35 kb in genomic sequences were isolated (data not shown). Identification of the sti gene was hampered by the fact that the isolated region was rich in transcription units (Schmidt 1996). Preliminary analysis, however, pointed to a transcribed region 39 to the integration site.

A corresponding cDNA clone was isolated and found to be incomplete, with a length of 1345 nt that included 24 A residues at the 39 end. As a result, the sequence was extended by a 59 RACE (Frohmann et al. 1988), and an apparently full-length clone was generated. Both genomic and cDNA clones were sequenced (sequence data are deposited in the EMBL databank under accession number X94613). The sting mRNA has a length of 2790 nt, assuming that the start site is identical to the beginning of the cDNA sequence. If this assumption is correct (see also below), then the P{lacW} is inserted 58 bp upstream of the transcriptional start site, i.e., in the 59 flanking region of the transcript, a common site for P insertions (Spradling et al. 1995). The transcript is composed of nine exons and eight mostly short introns, where all exon-intron boundaries obey the Drosophila consensus sequences (Mount et al. 1992). The untranslated regions are rather small, with 48 and 120 nt for the 59 and 39 regions, respectively. A canonical poly(A) addition signal (AATAAA, Proudfoot and Brownlee 1976) is located 33 bp upstream of the 39 end. The putative translation product of the open reading frame of 2598 nt is a polypeptide with 866 amino acids and a molecular weight of 99 kD. There is a possible bipartite nuclear localization signal (Dingwall and Laskey 1991) starting at amino acid 415. Since the spacer length is 8 instead of the common 10 amino acids, it is an open question whether this signal is functional. A databank search revealed 28 significant matches in the nonredundant GenBank (probability for a chance match ranging from 6 3 1025 to 1 3 102115), as well as additional ones in the expressed sequence tag (EST) database. The vast majority of these sequences are derived from genome projects and therefore cannot provide any hint about a possible function of the Sti protein. The two exceptions are recently isolated genes from the plant Arabidopsis thaliana. The argonaute (Bohmert et al. 1998) and zwille genes (Moussian et al. 1998) are involved in the regulation of the self-perpetuation of the shoot meristem. How this is achieved, however, is unknown, and

TABLE 2 Progeny produced by crosses of sti homozygous males carrying the B SYy1 chromosome and different X-linked Ste alleles to C(2)EN-bearing females Sperm genotype of survivors Chromosome

Total eggs

ywf

2149

Valde-marskiv

2721

Qiryat-Anavin

2925

Salve 10

1405

2/2 0 2/2 0 2/2 0 2/2 0

X

Y

XY

0

Disjunction: P2

Drive: R 2

6 18 6 69 3 27 2 4

4 7 14 4 2 6 0 0

0 0 0 0 0 0 0 0

3 4 17 20 15 13 3 1

0.964

0.669

0.913

0.631

0.958

0.659


755 Figure 4.—Restriction map of the sting region. Only that part from the two overlapping l phages that is relevant to the work described is shown (top); the position and orientation of the P{lacW} insertion are indicated. The two transcription units from this region, RpL9 and sting, are shown; open boxes indicate translated regions, and black boxes indicate untranslated ones. The size and position of the plasmid rescue clone and of the three mutant rescue clones, MR-1– MR-3, are shown; the plasmid rescue clone does not contain the XbaI site that is present in the l phage from which the mutant rescue clones were derived. The two fragments used for lacZ fusions are also shown. The BamHI site, indicated in brackets, in sti:lacZ1 and in MR-3, is derived from the multiple cloning site of one of the l phage clones. The HindIII site in sti:lacZ-2 is the one close to the 39 end in the P{lacW} element.

the molecular function of sting-related proteins is, thus, still an enigma. The most striking sequence similarity is localized in the C-terminal region of all the related proteins from the databank (see also Bohmert et al. 1998; Moussian et al. 1998). This domain is homologous to the 82 amino acids close to the C terminus of Sti (amino acids 763– 844). A multiple alignment and the consensus sequence of the hereafter-called “sting domain” are presented in Figure 5. At least 13 different genes that code proteins with a sting domain have already been identified in the almost completely sequenced Caenorhabditis elegans genome (Wilson et al. 1994). In less-advanced genome projects, three (Arabidopsis) and two (Drosophila) have been found so far. Although the totally sequenced Saccharomyces cerevisiae genome does not code any protein with a sting domain, the fission yeast Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum do code for at least one sting domain-containing protein, indicating that this domain is not unique to metazoa. All but one of the putative C. elegans proteins found in the databank search contain blocks of sequence identities to the sting protein outside the sting domain. These regions vary in size and are preferentially found in the C-terminal part of the proteins (data not shown). Two proteins coded by genes D2030.6 and C01G5.2 display sequence identities throughout the Sti protein. The best alignment could be generated with the D2030.6 gene product (Figure 6). The high percentages of sequence identity (31.9%) and sequence similarity (52.1%) over the entire length of the proteins indicate that the product of gene D2030.6 could be the C. elegans orthologue of the Drosophila Sti protein. It is noteworthy that these two C. elegans proteins also have the smallest number of amino acid replacements within the sting domain. As proof that the gene that was characterized is in

fact sting, a mutant rescue experiment was performed. Three genomic fragments (MR-1, MR-2, and MR-3) that differed in the length of their 59 upstream region were reintroduced into the Drosophila genome (Figure 4). Even the shortest construct was able to rescue the male sterile phenotype of the original insertion line, thus demonstrating that we have identified the sting gene. Since we only checked for restored fertility in the transgenic flies, we cannot say anything about rescue of the other phenotypes of the sti mutation. It is likely, however, that the semidominant phenotype (see Table 1) is also present in these transgenic flies. Expression pattern of sti: Northern analysis revealed a polyadenylated RNA of z3.0 kb length in accordance with the 2790 bp of the cDNA clone. In wild-type flies, the sti RNA accumulates exclusively in the gonads (Figure 7). Because of a more abundant expression in the ovaries, the sti transcript can be detected easily in total RNA isolated from whole females, but rarely in total RNA isolated from whole males. In homozygous mutant males and females, the expression is deregulated. As a consequence, the 3.0-kb RNA is also intensely transcribed outside the gonads. A larger 4.5-kb RNA also accumulates exclusively in the testes. The fact that the 3.0-kb RNA species continues to be transcribed is compatible with our previous theory that the integration site is most likely 59 to the transcriptional start site. The misexpression outside the gonads can already be seen in heterozygous flies (data not shown). All our attempts to clarify the structure of the new 4.5-kb testis-specific transcript were unsuccessful. In the remobilization experiments mentioned above, new mutant alleles were also found. Most informative for the function of the sting gene was the mutation sti3a, a putative null allele. Because of a large deletion, not only the sti, but also the neighboring RpL9 expression (see Schmidt et al. 1996) was affected, resulting in se-

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Figure 5.—Multiple alignment of the C terminus (amino acids 763–844) of the D. melanogaster Sti protein with related C-terminal regions from mostly hypothetical proteins identified in various genome projects (the first two letters indicate the species: Dm, Drosophila melanogaster ; Ce, Caenorhabditis elegans ; Pp, Pristionchus pacificus ; Dd, Dictyostelium discoideum; Sp, Schizosaccharomyces pombe ; At, Arabidopsis thaliana; Os, Oryza sativa). Partial sequences found, e.g., from corn, rat, or human cDNA clones, are not included. The sequences are ordered by the increasing probability of a chance match in the BLAST search (Altschul et al. 1997), and the alignment was performed with CLUSTAL W (Thompson et al. 1994). The very few insertions and deletions are not indicated. The GenBank accession numbers for the sequences are AA978826 (Dm-stir, sting-related cDNA LD33002); Z69661 (Ce-1, gene C01G5.2); Z73906 (Ce-2, gene D2030.6); AA193873 (Pp-1, cDNA rs06d11.r1); C90322 (Dd-1, clone SSI205); AF016682 (Ce-3, gene not defined); Z69661 (Ce-4, gene T07D3.7); Z29121 (Ce5, gene ZK757.3); AL023705 (Sp-1, Argonaute-like protein); AC003033 (At-al, Argonaute-like protein); U91995 (At-arg, Argonaute protein; Bohmert et al. 1998); AJ223508 (At-zw, Zwille protein; Moussian et al. 1998); AA751529 (Os-1, cDNA 96AS0254); Z82085 (Ce-6, gene ZK218.8); AF068711 (Ce-7, gene not defined); Z81128 (Ce-8, gene T23D8.7); Z49886 (Ce-9, gene C06A1.4); Z81556 (Ce-10, gene F58G1.g); U29244 (Ce-11, gene ZK1248.7); Z71266 (Ce-12, gene R06C7.1); and U80443 (Ce-13, gene C24A11.3). The consensus for the sting domain is defined by amino acids that are identical or related at least in 17 out of the 22 aligned proteins (77.3%). The amino acids are in the one-letter code; the structurally similar amino acids (according to Karlin and Ghandour 1985) are abbreviated a (acidic), b (basic), h (hydrophobic), and p (polar), respectively.

vere impairment of vitality. The few escapers had minute bristles, were male and female sterile, and crystal needles could be found in spermatogenic stages. Heterozygous sti/sti3a flies were only male sterile and produced needles. Northern analysis with RNA isolated from a few homozygous sti3a escapers indicated that sti was not expressed at all. This result was confirmed by the analysis of flies transgenic for a fragment that restored viability by introducing one copy of the RpL9 gene (Schmidt et al. 1996). These flies were still female sterile, thus demonstrating that the sterility was separable from the neighboring Minute gene. Males of the corresponding genotype were sterile needle producers. Hence, the null phenotype of the sti mutation is most likely sterility in both sexes, accompanied by the production of needles in spermatocytes and spermatids. In Northern analysis, the 3.0-kb sti RNA could only be detected in 0- to 6-hr-old embryos, but not in older ones (Schmidt 1996). Taking into account the large accumulation of transcripts during oogenesis, this could indicate that only maternally provided RNA is present in the embryos. We hypothesized, therefore, that the observed female sterility instead represents a maternal lethality. To distinguish between these alternatives, homozygous sti3a females transgenic for the RpL9 gene were mated to wild-type flies. Those females laid eggs that started development. Embryogenesis, however, stopped at various stages, starting with late blastoderm (stage 5 according to Campos-Ortega and Hartenstein 1997). Only very few embryos survived gastrulation (stage 8), but they died before germ band retraction (stage 12). This proves that the maternal contribution of sti mRNA is essential for early development; i.e., the observed female sterility is indeed a consequence of a maternal lethal effect. Two different sting-lacZ fusion genes (see Figure 4) were introduced into the germline by P-element-mediated transformation to monitor the expression pattern in the gonads. For comparison, the lacZ expression driven by enhancers close to the P{lacW} insertion of the original male sterile enhancer trap line is also presented (Figure 8A). The large construct should contain all regulatory elements because it starts in the neighboring gene. Furthermore, the 59 upstream regulatory sequences are identical to clone MR-3, a clone that can fully restore the wild type. The b-galactosidase activity produced by the large construct should, therefore, reflect the normal activity of the sti gene. In the testes, enzymatic staining could be detected only at the very tip, i.e., in spermatogonia and early spermatocytes (Figure 8B). This activity can also be demonstrated in the larval testis (Figure 8C). This is almost complementary to the expression pattern in the original enhancer trap line, where the staining does not start at such early stages, but is observed only in late spermatocytes (Figure 8A). If the enhancer trap expression pattern faithfully mirrors the expression of the endogenous sting gene,


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Figure 6.—Alignment of the sti protein from D. melanogaster and its putative orthologue from C. elegans gene D2030.6 (GenBank accession number Z73906). Identical amino acids are connected by vertical lines, and dots indicate structurally similar amino acids (according to Karlin and Ghandour 1985).

the apparent loss-of-function phenotype of the sting mutation can be explained easily. The gene is not transcribed, or at least not at the correct level, in the cells where it should be, i.e., early germ cells. Instead, the 3.0-kb mRNA accumulates primarily in late spermatocytes and at later stages, where the crystal needles can be observed also. The only synthesis in late spermatocytes might also be true for the 4.5-kb RNA, whose structure and function is, however, still unknown. The misexpression shown by the original enhancer trap line indicates that the P{lacW} insertion has separated or destroyed important regulatory elements. In accordance with this interpretation, the short construct with sequences from the 39 end of the P{lacW} to the transcriptional start site does not support any expression in testes. In females transgenic for the large construct, expression was restricted to the ovary. There was a short, transient expression in the oocyte around stage 6 of oogen-

esis (Figure 8D; stage according to King 1970). At later stages, from stage 9 on, b-galactosidase activity was found in the nurse cells (Figure 8E). Later on, with the dumping of the nurse cell contents, this activity was detected in the oocyte and egg. Such a maternal contribution had to be postulated from the observed maternal lethal effect of a sti null allele (see above). Although the ovary could not be reproducibly stained in the original male sterile enhancer trap line, the short construct does show b-galactosidase activity in the ovary. Whether this difference is caused by activating or repressing elements that cannot function in one of the two transgenic lines is still an open question. DISCUSSION

In this work, we have described a new autosomal gene, sting, which is localized in polytene interval 32D on

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Figure 7.—Northern analyses with a sti cDNA probe. Twenty micrograms of total RNA was loaded into each lane. The RNA was isolated from whole males (M), from male carcasses (Cm), i.e., without testes, from testes (T), from whole females, from female carcasses (Cf ), i.e., without ovaries, and from ovaries (O). All six RNA types were isolated from wildtype and sti flies. The arrow points to the 3.0-kb sti mRNA, whereas the arrowhead points to the additional 4.5-kb RNA in the mutant testes.

chromosome 2 and, when mutated, leads to a combination of a male sterile and maternal lethal effect in Drosophila melanogaster. In particular, the male sterility depends on a spectrum of meiotic abnormalities that closely resemble those produced by the deficiency of the Y-linked crystal locus. Our cytological and genetic analyses show that the sti mutation induces the same three phenomena as in X/Y, cry2 males: (1) the formation of crystalline aggregates in primary spermatocytes; (2) meiotic nondisjunction of sex chromosomes and autosomes, and (3) meiotic drive of sex chromosomes and autosomes. As in the case of X/Y, cry2 males, the meiotic defects induced by the sti gene result from the activation of both the heterochromatic and euchromatic Ste sequence clusters, leading to the production of a protein that, depending on its amount, self-aggregates in needle- or star-shaped crystals (Hardy et al. 1984; Palumbo et al. 1994; Bozzetti et al. 1995). The similarity of the meiotic effects to the situation in X/Y, cry2 males goes even further. In both cases, sex chromosome nondisjunction is correlated to Ste copy number, but this is not found with respect to drive. Thus, the Ste-sti and Ste-cry interactions are a system in which sex chromosome disjunction and drive are uncoupled, in contrast to the deficiencies of the X-Y pairing sites of the X heterochromatin that cause strongly correlated nondisjunction and meiotic drive (McKee and Lindsley 1987).

Figure 8.—b-Galactosidase activity in the original insertion line (A) and in flies transgenic for the sti:lacZ-1 construct (B–F). In A, staining is detectable throughout the whole testis from an adult male, with the exception of the tip (arrows). In contrast, the testis tip is the only stained area in sti:lacZ-1 transgenic flies (arrows in B). The same holds true for the larval testis (C, arrows; the arrowheads point to fat body tissue with some background staining). (D) Transient expression in early oocytes (arrows). (E) Strong b-galactosidase staining in the nurse cells of later stages during oogenesis.


The Northern analysis of the Ste expression in sti mutant flies led us to discover that the Ste sequences are basically transcribed in several tissues of males and females that produce high-molecular-weight transcripts. Our observations that the high-molecular-weight Ste transcripts are present in males carrying the W 12 X chromosome and lacking the euchromatic Ste cluster strongly suggest that these transcripts correspond to the heterochromatic Ste cluster. Unfortunately, at the present time, a viable X chromosome with a deletion of the heterochromatic Ste cluster is not available, so we cannot determine whether transcripts from the euchromatic cluster are also part of the high-molecularweight fraction. Interestingly, we show a concomitant decrease of this fraction with the production of the 750to 850-nt Ste transcripts, which is more evident in the testes of cry2 males independent of the Ste allelic status or in the testes of cry1 homozygous sti males when they carry a Ste allele. This indicates that at least for the heterochromatic Ste sequences, the regulation of their expression in X/Y, cry2 testes could be post-transcriptional. It has already been shown that crystal can regulate the expression of Ste sequences at the transcriptional and splicing levels (Livak 1990). With the present results, it is not unreasonable to suggest that these two different levels of regulation reflect the fact that the heterochromatic and euchromatic clusters are under different regulatory mechanisms, namely that the euchromatic Ste sequences are under transcriptional control, while the heterochromatic sequences are mainly regulated at the post-transcriptional level. The data presented in this article clearly show that we have identified a gene that somehow interferes with the Ste-cry interaction system. Alternative hypotheses can be proposed to explain the interference of sti with the Ste-cry system. It is possible that both cry and sti are part of the same machinery that exerts control of Ste expression and, perhaps, additional genes in meiotic cells. On the other hand, it is possible that sti misfunction affects chromosome organization in meiotic cells, leading to the inactivation of the crystal locus, thus triggering Ste expression. In this case, there are at least two ways in which chromosomal alterations formally inactivate crystal. The sti mutation may induce Y chromosome loss or produce an alteration in the chromatin conformation of the crystal region. Our careful analysis of homozygous sti male testes revealed the presence of the Y loops in all mature spermatocytes, thus ruling out a sti-induced chromosome loss. Regarding the possible alteration in chromatin conformation, it is very difficult to perform a significant assay because the chromosomes are already altered as a result of the action of Ste protein. Therefore, at this time, we do not have strong indications favoring one of the two alternative hypotheses. The interaction shown here between the autosomal sti and the X-chromosomal Ste gene opens the question of how these two partners have evolved. It is known, by Southern hybridizations, that the repetitive X-chromosomal Stel-

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late gene exists only in D. melanogaster and its closest relatives, D. mauritiana and D. simulans (Livak 1984). On the other hand, sequences homologous to at least parts of the Sti protein could be detected in a variety of organisms, ranging from unicellular ones to higher plants and animals (see above). Therefore, we expected to detect sti sequences in more Drosophila species than Ste sequences are found. This was indeed the case. The sting gene hybridizes even under stringent conditions with DNA from all species of the melanogaster subgroup, i.e., D. erecta, D. mauritiana, D. simulans, D. teissieri, and D. yacuba (Schmidt 1996). Thus, the Ste-sti interaction is apparently a rather recent acquisition in some species of the melanogaster subgroup. It is tempting to speculate that the primary sti function is related to the observed maternal lethality, and that this essential function is the one responsible for the conservation of Sti protein. Its function during spermatogenesis would then only be a secondary one. In favor of this hypothesis is the modular structure of the upstream controlling regions, where the sequences necessary for expression in the ovary can clearly be separated from those that regulate expression in the male gonad. What, then, could be the primary function of the Sti protein? One possibility is that the wild-type function represses general or specific transcript processing. This is strongly supported by the Northern analysis of Ste expression, where processed Ste mRNA is found in mutant males. By this view, an induced out-of-phase gene expression should be responsible for the observed lethal maternal effect. Unfortunately, the primary structure of the Sti protein does not provide any clue to elucidate the sting function. It does not contain any of the known domains that could function in gene regulation, transcript processing, or protein-protein interactions. Because a large number of genes that code for proteins with the sting domain have been identified in C. elegans, and three and two of those genes have already been found in Arabidopsis and Drosophila, respectively, it is safe to postulate that all higher metazoa will harbor a sting domain gene family. Further experiments are needed to elucidate the function of this gene family during the life cycle of various organisms. We thank A. Beermann, C. Schultz, and J. Campos-Ortega for making the P{lacW} insertion lines available to us for the screening. We thank D. David and M. Mu¨ller-Borg for their expert technical assistance and M. Scha¨fer for support and discussion. We are very grateful to L. Robbins for his invaluable help in statistical analysis and L. Fanti for helping in immunofluorescence experiments. This work was supported by grants from the Deutsche Forschungsgemeinschaft to U.S. (Scha278/7-1) and from the Italian Ministero dell’Universita` e della Ricerca Scientifica e Technologica (40%) to G.P.

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