Two Genes Required for Meiotic Recombination in Drosophila Are Expressed From a Dicistronic Message

Corresponding author: K. S. McKim, Waksman Institute and Department of Genetics, Rutgers University, 190 Frelinghuysen Rd., Piscataway, NJ 08854., mckim{at}rci.rutgers.edu (E-mail)

Communicating editor: S. HENIKOFF

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have isolated two alleles of a previously unidentified meiotic recombination gene, mei-217. Genetic analysis of these mutants shows that mei-217 is a typical "precondition" gene. The phenotypes of the mutants are meiosis specific. The strongest allele has <10% of the normal level of crossing over, and the residual events are distributed abnormally. We have used double mutant analysis to position mei-217 in the meiotic recombination pathway. In general, mutations causing defects in the initiation of meiotic recombination are epistatic to mutations in mei-41 and spnB. These two mutations, however, are epistatic to mei-217, suggesting that recombination is initiated normally in mei-217 mutants. It is likely that mei-217 mutants are able to make Holliday junction intermediates but are defective in the production of crossovers. These phenotypes are most similar to mutants of the mei-218 gene. This is striking because mei-217 and mei-218 are part of the same transcription unit and are most likely produced from a dicistronic message.

CROSSING over is an important mechanism for segregating homologs at meiosis I. Each crossover matures into a chiasma, which links and orients the homologs on the meiosis I spindle (HAWLEY 1988 ). Crossing over is one outcome of the meiotic recombination pathway(s); the other is gene conversion. One model explaining these events is that meiotic recombination is initiated by a DNA break, which is repaired via a Holliday junction, and then resolved as either a gene conversion or crossover. In Saccharomyces cerevisiae, a double-strand break is responsible for the initiation of meiotic recombination (DE MASSY et al. 1995 ; LIU et al. 1995 ). This mechanism appears to be conserved in other organisms because the candidate for the enzyme that makes the double-strand break in S. cerevisiae, Spo11 (KEENEY et al. 1997 ), has homologs required for meiotic recombination in Drosophila melanogaster (MCKIM and HAYASHI-HAGIHARA 1998 ) and Caenorhabditis elegans (DERNBURG et al. 1998 ).

While several genes in yeast and Drosophila have been suggested to function either in the induction or repair of double-strand breaks, less is known about the genes and mechanisms that control the resolution of the Holliday junction into a crossover or a gene conversion. In yeast and mammals, only members of the mismatch repair protein family have been suggested to be components of late recombination nodules and required for crossover production (HOLLINGSWORTH et al. 1995 ; ROSS-MACDONALD and ROEDER 1995 ; BAKER et al. 1996 ; HUNTER and BORTS 1997 ). In D. melanogaster, several genes required specifically for crossing over have been identified. In mutants of these genes, such as mei-9 and mei-218, crossing over is reduced but the frequency of gene conversion is normal (CARPENTER 1982 , CARPENTER 1984 ).

Crossover-defective mutants in Drosophila have been divided into two classes, the precondition defective and the exchange defective. Precondition-defective mutants have reductions in crossing over that are nonuniform along each chromosome. Thus, these genes are thought to control both the frequency and location of crossover events (SANDLER et al. 1968 ; BAKER and CARPENTER 1972 ). Exchange-defective mutants do not alter the distribution of exchanges as shown by uniform reductions in crossing over along the chromosome. Genetic studies suggest that exchange genes are required later than the precondition genes (BAKER and CARPENTER 1972 ; CARPENTER 1982 , CARPENTER 1984 ; SEKELSKY et al. 1995 ), most likely in the actual resolution reaction. In support of this hypothesis, the mei-9 encodes a protein likely to have an enzymatic role in recombination; it is homologous to the Rad1/XPF family of endonucleases (SEKELSKY et al. 1995 ).

While the evidence from mei-9 suggests that the exchange genes function directly in the resolution reaction, the event(s) in the recombination pathway affected by the precondition genes is not known. In a one-pathway model, precondition genes cause a divergence in the recombination pathway after initiation, one leading to gene conversion and the other leading to crossing over. Alternatively, in a two-pathway model, the precondition genes could function at or before recombination initiation, resulting in two distinct pathways for gene conversion and crossing over. Since the mei-218 gene is not conserved, we are characterizing other genes with a similar phenotype to investigate the nature of the precondition function. We have isolated two alleles of a new precondition gene, mei-217. Surprisingly, this gene maps very close to another precondition gene, mei-218. We have cloned mei-217 and have shown that it is part of the same transcription unit as mei-218. The most likely explanation is that mei-217 and mei-218 are two proteins made from the same transcript.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Screen for X-linked meiotic mutations:
Males of the genotype y/y⁺Y; spa^pol were fed with 25 mM ethyl methane sulfonate for 18 hr and crossed to l(1)15Eb^692-19/FM7c females. Individual y/FM7 females were crossed to FM7/y⁺Y males to generate X chromosome mutagnized lines. To generate homozygotes, in the next generation the y/y⁺Y males were crossed to y/FM7 sisters. In the next generation, y/y females were crossed to their y/y⁺Y brothers to test for nondisjunction. Normally this cross produces yellow females and wild-type males. Nondisjunction in the female germline produces diplo-X and nullo-X eggs, resulting in yellow males and wild-type females. If at least one exceptional progeny was observed, the line was restested either by repeating the brother-to-sister cross or by crossing virgin females to C(1;Y), v f B/O; C(4)RM, ey^R ci/0 males. This latter cross precluded the isolation of mutants that were specific to the male germline, which are often associated with mutations of the rDNA (MCKEE and KARPEN 1990 ). All mutations were tested for allelism to mei-9, mei-38, mei-41, and mei-218 by standard complementation and nondisjunction tests.

Genetic mapping of mei-217:
Low resolution recombination mapping of mei-217 was done relative to pn, cv, m, and f. Both g10 and r1 mapped close to f, which is where mei-218 also maps. To confirm this, both mutants were crossed to deficiencies, duplications, and transgenes in the region (MCKIM et al. 1996 ). mei-217^g10 failed to complement deficiencies Df(1)BK8 (15C1-4; 16C2-7) and Df(1)815-6 (15E1-2; 15E6-7) but complemented Df(1)BK10 (16A2; 16C7-10). For complementation testing with duplications, the fourth chromosome marker spa^pol was used in the crossing scheme. To make mei-217^g10/mei-217^g10; Dp(1;4) females, mei-217^g10/FM7; spa^pol/spa^pol females were crossed to r f B; Dp(1;4)/spa^pol males. The mei-217^g10/Y; Dp(1;4)/spa^pol males were crossed back to mei-217^r1/FM7; spa^pol/spa^pol females to make mei-217^r1/mei-217^r1 Dp(1;4)/spa^pol females. The nondisjunction phenotype of mei-217^g10 was rescued by duplications Dp(1;4)fK7 (14A1-2; 15E6-7) and Dp(1;4)rK20 (14A1-2; 14A4-B1; 15D5-E4; 16A7;B1). This positioned mei-217 between the distal breakpoint of Dp(1;4)fK7 and the proximal breakpoint of Df(1)815-6, which is also the interval that contains mei-218.

Isolation of RNA, RT-PCR analysis, and in situ hybridization:
Total RNA was collected from dissected ovaries or testis by grinding the tissue in 50% RNA lysis buffer (0.3 M sodium acetate, 5 mM EDTA, 50 mM Tris-HCl pH 9.0, 1% SDS)/50% acid phenol followed by two extractions in acid phenol. For Northern blotting, 50–80 µg of total RNA (from 20 ovaries dissected from 10 females) was loaded into each lane and transferred to a nylon membrane. mRNA was prepared from ovaries using the Ambion (Austin, TX) Poly(A)Pure isolation kit. Reverse transcriptase (RT)-PCR was carried out using the single tube methodology and using reagents from Life Technologies or Boehringer Mannheim (Indianapolis). Digoxygenin (DIG)-labeled RNA probes for in situ hybridization were made from linearized mei-218 cDNA clone pH2-15 using the Boehringer Mannheim RNA labeling kit and hybridized as described by TAUTZ and PFEIFLE 1989 .

Sequencing and analysis:
DNA clones for sequencing were prepared by alkaline lysis minipreps followed by polyethylene glycol (PEG) precipitation. PCR products were either directly sequenced following isolation from an agarose gel or first cloned using the Perfectly Blunt cloning system (Novagen). Sequencing was done by the University of Medicine and Dentistry of New Jersey sequencing facility. Sequence analysis was done using the Wisconsin Package Version 9.1 [Genetics Computer Group (GCG), Madison, WI]. Sequences from mutant DNA and another strain of the same genetic background were compared to identify the nucleotide change.

Confocal microscopy:
Stage 14 oocytes were collected from 3–7-day-old females and fixed as described previously (THEURKAUF and HAWLEY 1992 ; MCKIM et al. 1993 ). Oocytes were stained for DNA with propidium iodide or Yo-Pro and for spindles with anti-tubulin (clone DM1A; Sigma, St. Louis) conjugated to either FITC or rhodamine.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Screen for X-linked meiotic mutations:
We screened 2106 chromosomes (MATERIALS AND METHODS) and confirmed 13 mutants with an elevated frequency of X chromosome nondisjunction in females. The rationale is that proper segregation requires chiasmata, which leads to a correlation between nondisjunction rates and defects in crossing over. Mutants with higher nondisjunction frequencies usually have the greatest reductions in meiotic crossing over. Five alleles of mei-218 (j1, j2, g1, g4, and g9) and one allele of mei-9 (j3) were recovered as well as seven mutations in other genes. No alleles of the highly mutable mei-41 gene were recovered. Two possible explanations are (i) we would not expect to isolate strong alleles because they are sterile and (ii) weaker alleles such as the two recovered by BAKER and CARPENTER 1972 have low nondisjunction rates, which may have been at the limit of our screen's sensitivity. Based on genetic mapping and complementation testing, five mutations are alleles of two new genes, gav (three alleles) and mei-217 (two alleles). In both genes the mutants are of the precondition type because they alter the distribution of crossovers (see below and data not shown). mei-217 mutants have a similar effect on crossing over as mei-218, which reduces crossing over to 5–10% of wild type (MCKIM et al. 1996 ). Less severe are the gav mutants that reduce crossing over to ~50% of wild type (data not shown). The last two mutations (g5 and g11) have been characterized less, although g5 had no effect on the frequency of crossing over. Like some other precondition mutants, gav and mei-217 are not DNA repair defective as indicated by their lack of sensitivity to methyl methanesulfonate (MMS; data not shown). Both mutants are also female specific; in hemizygous mutant males there is normal segregation of the X and Y chromosomes (data not shown). The remainder of this article describes the mei-217 gene. A detailed analysis of gav will be published elsewhere.

mei-217, a new X-linked meiotic recombination gene:
The primary defect in mei-217 mutants is a reduction in the frequency of crossing over. Crossing over is reduced by a similar degree on both the X chromosome (Table 1) and the left arm of the second chromosome (Table 2). The severity of the crossover reductions is consistent with the observed frequency of nondisjunction, but it is formally possible that nondisjunction also occurs due to the failure of the chiasmata to ensure segregation at meiosis I, or if there is nondisjunction of sister chromatids at meiosis II. We investigated whether crossover bivalents nondisjoin by simultaneously measuring X chromosome crossing over and disjunction (Table 1). A failure of chiasmata to direct homolog segregation would have been detected by the recovery of females homozygous for X-linked recessive markers. Since there were no such cases observed (r1, n = 534; g10, n = 1354), most or all of the nondisjunction events in mei-217 mutants involved achiasmate chromosomes at the first meiotic division. These data also show an equal number of diplo-X (B⁺ females) and nullo-X (B males) progeny, showing that meiotic chromosome loss was not a significant factor. There is consistently a higher frequency of crossing over in r1 than g10, which suggests that g10 is the stronger allele. While this is not reflected in the X chromosome nondisjunction data of Table 1, this reflects the variation found in nondisjunction tests, since in several other experiments r1/r1 females had significantly less X chromosome nondisjunction (20–25%). The frequency of X chromosome nondisjunction was similar in mei-217^g10/mei-217^g10 and mei-217^g10/Df(1)815-6 females, suggesting that g10 represents a null allele.

Crossing over was substantially reduced in mei-217 mutants, but the reductions were not uniform along each chromosome arm. This is most easily seen for crossing over on the second chromosome (Table 2). In both mei-217 mutants, crossing over was reduced more in distal regions than in proximal regions. For example, in the dp–b region, crossing over was reduced to 9.6% of wild type in mei-217^g10 and to 14.7% of wild type in mei-217^r1. In contrast, in the pr–cn region crossing over was reduced to 62.1% of wild type in mei-217^g10 and increased to 102.1% of wild type in mei-217^r1. A reduction in crossing over that is more severe in distal than proximal regions is the defining feature of the precondition mutants in D. melanogaster, such as mei-218.

The high frequency of X chromosome nondisjunction is consistent with the low level of meiotic crossing over observed in the two mei-217 mutants. We also tested for autosomal nondisjunction by crossing mei-217 mutant females to males carrying second chromosome compound chromosomes. As predicted from the low level of crossing over, second chromosome nondisjunction was elevated (Table 3). Since in these crosses only second chromosome nondisjunction events produce progeny, absolute nondisjunction frequencies cannot be measured. Simultaneous nondisjunction of two chromosome pairs is another characteristic of meiotic mutants. This happens in part because there is a backup system that can direct the segregation of achiasmate chromosomes (HAWLEY et al. 1992 ). While this system operates efficiently when only a single pair of achiasmate chromosomes is present, multiple pairs of large chromosomes cannot be distinguished. In this situation, the chromosomes are evenly segregated regardless of homology, leading to a high frequency of two X chromosomes segregating from two autosomes. The gametic frequency of simultaneous X and second chromosome nondisjunction was >62% for both mutants. Part of this high nondisjunction can be attributed to meioses where the two X chromosomes segregated from the two second chromosomes. These results show that the effects of the mei-217 mutants are specific to chiasmate segregation.

In mei-217 mutants meiotic recombination is initiated but the recombination intermediates are not resolved as crossovers:
Evidence from the following two experiments supports the conclusion that mei-217 mutants initiate meiotic recombination normally, thus making the expected number of chromosomal breakage events (presumably double stranded; MCKIM and HAYASHI-HAGIHARA 1998 ). This conclusion is based on the similar behavior of mei-217 and mei-218 in double-mutant analysis. In mei-218 mutants meiotic recombination is presumed to initiate normally [as judged by the wild-type frequency of gene conversion events (CARPENTER 1982 , CARPENTER 1984 ) and normal number of early recombination nodules (CARPENTER 1989 ) seen in these mutants].

Double mutants with mei-41: In wild-type mature oocytes (stage 14), meiosis arrests at metaphase I (THEURKAUF and HAWLEY 1992 ). We have previously observed that in the absence of chiasmata, such as in recombination-defective mutants, this arrest does not occur and meiosis proceeds through the two divisions (MCKIM et al. 1993 ). Depending on the recombination-defective mutant, precocious anaphase requires the mei-41 gene product (MCKIM et al. 2000 ). mei-41 encodes a homolog of the ataxia telangiectasia mutated (ATM) family of DNA-dependent protein kinases (HARI et al. 1995 ) and has a significant role in regulating the mitotic cell cycle in the presence of double-strand breaks. In double mutants of crossover-defective mutants, such as mei-218, and strong mei-41 alleles, such as D3 or D18, metaphase arrest occurs. In double mutants that fail to initiate meiotic recombination, such as mei-P22 (MCKIM et al. 1998 ) and strong mei-41 alleles, precocious anaphase occurs. It is not known why mei-41 affects precocious anaphase in mutants that can initiate meiotic recombination, but this effect can be used to determine where in the meiotic recombination pathway a gene is required. As expected for mutants that drastically reduce crossing over, in mei-217^g10 and mei-217^r1 mutants precocious anaphase was observed (Fig 1 and Table 4). In mei-41^D18 mei-217^g10 and mei-41^D18 mei-217^r1 double mutants, however, a metaphase arrest was almost always observed, a result that contrasts with that seen in the mei-41^D18; mei-P22 double mutant. Our interpretation of these results is that mei-217 mutants are likely to be proficient at initiating meiotic recombination at normal levels.

View larger version (78K): In this window In a new window Download PPT slide   Figure 1. mei-217 mutations cause a precocious anaphase but are not epistatic to mei-41 mutations. Confocal images of stage 14 oocytes with the chromosomes stained red and the spindle stained green (except mei-41D18 mei-P22P22, which is the reverse). The genotypes of representative oocytes are shown. A and B show the precocious anaphase seen in g10 and r1 mutants. C and D show typical nuclei in double mutants with mei-41D18. For comparison, E is a mei-41D18; mei-P22P22 oocyte.

Double mutants with spnB: spnB encodes a homolog of the RAD51/DMC1 family of double-strand break repair proteins (GHABRIAL et al. 1998 ). Mutations in this gene and others in the same class cause female sterility, most likely a result of the persistence of unrepaired double-strand breaks. Only a few progeny are produced in the first 2 or 3 days of mating (Table 5). Mutations that prevent any genetic recombination and presumably the formation of double-strand breaks, such as mei-W68, suppress the sterility of spnB^BU (Table 5 and GHABRIAL and SCHUPBACH 1999 ). spnB^BU mutants are fertile in the first 2–3 days of mating, so the suppression effects are not readily apparent until after the second day of mating (Table 5). In contrast, in mei-218 mutants where meiotic recombination is initiated normally the sterility of spnB^BU is not suppressed (Table 5). Similar to mei-218, we found that spnB^BU is not suppressed by mei-217^g10 or mei-217^r1. In fact, like mei-218, the phenotype is actually enhanced (Table 5).

Both of these experiments are consistent with the hypothesis that in mei-217 mutants meiotic recombination is initiated normally but crossovers are not produced.

mei-217 and mei-218 are part of the same transcription unit:
Genetic mapping relative to several X-linked loci placed mei-217 close to forked at position 57 of the X chromosome (MATERIALS AND METHODS). This was confirmed by showing that both genes mapped to the same genetic interval defined by Df(1)815-6 and Dp(1;4)fK7. A transgenic fragment containing 15 kb of genomic DNA from this genetic interval was able to rescue mutations in both genes (Fig 2D). These results showed that mei-217 and mei-218 are physically very close and raised the possibility that mei-217 mutations were special alleles of mei-218. Since our original complementation tests were done with the mei-218⁶ allele, the tests were repeated by crossing both mei-217 alleles to 10 existing mei-218 alleles. In all cases the mutations complemented each other. Consistent with these results, a construct containing the mei-218 coding region driven by the hsp83 promoter (Fig 2C and Fig D) rescued mei-218 mutants but not mei-217 mutants. These results confirmed the genetic data that mei-217 and mei-218 are physically close but remain separable genetic units.

View larger version (24K): In this window In a new window Download PPT slide   Figure 2. Restriction map of the mei-217/mei-218 region. (A) The structure of the mei-217/mei-218 transcript and the position of the two ORFs are shown. (B) The primers used in the RT-PCR experiments. (C) The probes used for Northern blots and in situ hybridization. The 1.6-kb fragment is a genomic subclone and the cDNA clone contains the entire mei-218 coding region. Three clones with almost identical start sites were originally isolated (MCKIM et al. 1996 ). (D) The transgenic fragments show the DNA contained in each construct. Solid segments are derived from genomic DNA and gray segments are derived from a cDNA clone. Below each construct is shown the frequency of nondisjunction observed in a strain homozygous for the indicated mutation and carrying one copy of the transgene. The location of the FLAG or HA tag is also shown by a white box. See Fig 4 for amino acid sequence at the junction of the FLAG and HA clones.

Previous work in this region of the genome showed that the mei-218 locus produces a 6–7 kb transcript (MCKIM et al. 1996 ; Fig 3B). This is significantly longer than the largest cDNA, which at 4.2 kb still contains the entire mei-218 coding region (Fig 2C and Fig D). Considering that the 5' end of all three of the longest cDNA clones are at similar positions (Fig 2C), it is possible that the long length combined with strong secondary structure in the region resulted in a failure to recover any full-length cDNA clones. In the region where the cDNA clones begin, there is a direct repeat of the sequence GCCCAC that is located in the loop of a potential hairpin structure (Fig 4A).

View larger version (77K): In this window In a new window Download PPT slide   Figure 3. The mei-217/mei-218 transcript. (A) RT-PCR using primers g10-5 and 4339 (see Fig 2). The spliced product and the expected genomic size (arrow) are shown next to the size markers from the gel. (B) Northern blot of total ovary RNA probed with 32P-labeled mei-218 cDNA. Molecular weight size markers are shown to the left.

View larger version (41K): In this window In a new window Download PPT slide   Figure 4. (A) Predicted stem loop structure found upstream of the mei-218 AUG. (B) The mei-217 cDNA sequence and predicted translation product. The underlined sequences are the primer 6, the predicted leucine zipper, and the repeat sequence that is part of the stem loop structure. Also indicated are the nucleotide changes in mei-217r1 and mei-217g10, and the two base pairs on either side of an intron are shown with bold underline. At the end the mei-217 coding region is the overlap with the mei-218 coding region. The underlined portion of the Mei-217 protein is altered in the FLAG (pHKF218) and HA (pHK3X218) fusion constructs. In pHKF218 this region is ASSASTYQLEAWHASIDLDM while in pHK3X218 this region is ASSLEVDM. In each case the FLAG or HA tag follows the methionine.

To investigate the region upstream of the cDNA clones, we sequenced a 4-kb region that covers the interval between mei-218 and the next upstream gene, rpS5. In addition, a 1.6-kb SacI fragment, which covers the region upstream of the mei-218 cDNA clones (Fig 2), was used to probe two cDNA libraries for longer cDNA clones. No clones were identified, but the existence of a transcript in the region was confirmed by RT-PCR analysis. Using a primer within the mei-218 coding region and primers in the upstream region (g10-5 or g10-6, Fig 2B), a single species of transcript was amplified from ovarian RNA. When spliced, this transcript begins at least 830 bp upstream of the mei-218 AUG. This was shown with the amplification of an ~1400-bp product using the most upstream primer, g10-6, and a mei-218 primer 4339, or a slightly smaller product with g10-5 (Fig 3A). The extensive overlap of this transcript with the 4.2 kb of mei-218 cDNA, combined with the large size of the transcript on Northern blots, suggest that the mature transcript has the entire mei-218 coding region in addition to the upstream sequences. It is likely that the transcript has 5' untranslated region (UTR) sequences extending upstream of the g10-6 primer site. From this site to the 3' end of the cDNA is only ~5 kb, which is at least 1 kb shorter than the transcript observed on Northern blots. Sequence analysis of the RT-PCR products and comparison to the genomic sequence showed the presence of three introns upstream of the mei-218 AUG (Fig 2).

Three lines of evidence support the idea that the region upstream of the mei-218 AUG encodes the MEI-217 protein. First, the upstream splicing pattern maintains an open reading frame (ORF) that begins 662 bp upstream of the mei-218 AUG. This ORF does not end until 19 bp after the mei-218 AUG. Second, this ORF shows codon bias typical of D. melanogaster proteins. Third, the sequence changes in the two mei-217 mutations affect this ORF (Fig 4). The mei-217^g10 mutation is a G > A change causing a glutamate-to-lysine change (GAA to AAA). The mei-217^r1 mutation is an A > T change 42 bp upstream of the AUG. This change does not affect the coding potential of mei-217, and since this chromosome contains a normal mei-218 gene, this mutation may affect translation of MEI-217. The presence of an open reading frame upstream of mei-218 but within the same transcript and the presence of mutations that affect this ORF support the conclusion that mei-217 and mei-218 encode distinct gene products that are made from a dicistronic message.

There are several other possibilities we can eliminate. A fusion protein could be made if the ribosome is able to bypass the stop codon, as occurs in the D. melanogaster kelch gene where a single stop codon separates two long ORFs (ROBINSON and COOLEY 1997 ). This is not the case with the mei-217 ORF because there are several stop codons immediately following the first one. A variant of this concept is that the stop codon is bypassed by ribosome frameshifting. In this case, and also the previous one, we would not expect the mei-217 and mei-218 mutants to complement. Finally, we have not ruled out that the two gene products are made from different transcripts generated by either alternative splicing or initiation sites. For example, the 4.2-kb cDNA clones might represent a mei-218-specific transcript produced by an alternative transcription initiation site. Evidence supporting this hypothesis is lacking, however, since upon probing Northern blots with the mei-218 cDNA or the 1.6-kb SacI genomic fragment upstream of the mei-218 coding region (Fig 2), only one large band at ~6.5 kb was observed (Fig 3B).

The predicted MEI-217 protein is 227 amino acids and 25.8 kD. It has no homologs in the nucleic acid and protein databases, including no similarities to MEI-218, but it has a potential leucine zipper, several phosphorylation sites, and a zinc carboxypeptidase zinc-binding region (Fig 4B). The significance of these motifs has yet to be determined.

The 5' adjacent gene to mei-217/mei-218 is M(1)15D, which encodes a ribosomal protein (rpS5). Comparison of the genomic and cDNA sequences (MCKIM et al. 1996 , and data not shown) shows that the end of the M(1)15D transcript is 960 bp upstream of the mei-217 coding region (Fig 2). This region probably contains the mei-217/mei-218 promoter. We have shown that this region is required for mei-218 expression because a transgene containing the mei-217/mei-218 coding region but lacking the upstream promoter sequences failed to rescue mei-218 mutants (Fig 2). This promoter is probably responsible for the induction of mei217/mei-218 transcription in regions 2 and 3 of the germarium (Fig 5).

View larger version (134K): In this window In a new window Download PPT slide   Figure 5. In situ hybridization using an RNA antisense probe made from mei-218 cDNA showing expression of the mei-217/mei-218 mRNA in the germarium.

A mei-217-mei-218 fusion protein is functional:
In the process of making epitope-tagged mei-218 genes, the mei-217 and mei-218 coding regions were fused. Two constructs were made, resulting in a FLAG (pHKF218) or triple-HA tag (pHK3X218) positioned between the two coding regions, but also resulting in the deletion of MEI-217 amino acids. The HA-tag protein is missing the last 14 amino acids while the FLAG-tag protein is missing only the last 6 amino acids of MEI-217 (Fig 4). Despite these deletions, both constructs rescued mei-218 mutants, but the FLAG-tag construct rescued mei-217^g10 significantly better than the HA-tag construct (Fig 2). We do not know if the reason for the failure of the HA tag construct to rescue is because of the 14-amino-acid deletion or the presence of the HA tag.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have conducted a screen for X-linked mutants that increase X chromosome nondisjunction in female meiosis. In a similar screen by BAKER and CARPENTER 1972 , 189 mutagenized chromosomes were screened and approximately six mutants (excluding very weak ones) similar to ours were found, including two mei-9 alleles and one mei-218 allele. We have screened 10 times the number of chromosomes but only recovered mutations in four new genes. The two strongest new mutations, with levels of crossing over and nondisjunction similar to two of the previously identified genes, mei-9 and mei-218, are members of a new complementation group we have named mei-217. The small number of new mutants suggests that there are only a small number of genes on the X chromosome required for meiotic recombination when mutants are viable and fertile.

mei-217 and mei-218 are part of the same transcription unit:
Although genetic mapping experiments found them to be adjacent, mei-217 and mei-218 are separate genes based on four criteria. First, the mei-217 alleles r1 and g10 complement all alleles of mei-218. Second, genomic and cDNA sequences show another open reading frame upstream of the mei-218 coding region. Third, we have identified the sequence changes in the two mei-217 mutations and found that they are located in the upstream open reading frame. Finally, one of these mutations, r1, is upstream of the mei-217 coding region. If a single protein was made from the transcript, perhaps involving a ribosome frameshift, then r1 should fail to complement mei-218 mutations. Thus, by all standard criteria mei-217 and mei-218 are separate genes. Underlying the genetics, however, is the observation that a single transcript encodes both proteins. As in bacterial operons, this organization provides an efficient mechanism to regulate the transcription or translation of genes with related functions. This makes sense in our case because the similarity in the mei-217 and mei-218 mutant phenotypes suggests that these two genes function in the same pathway toward the generation of crossovers. Unlike prokaryotes, however, we suspect the importance of this organization is at the post-transcriptional level, since mei-217/mei-218 expression is not limited to meiotic cells (see below).

Eukaryotic polycistronic messages are not unusual. Many C. elegans transcripts are polycistronic but are later processed to be monocistronic prior to translation (BLUMENTHAL 1998 ). In mammals, two proteins, Snurf and Snrpn, are made from a dicistronic transcript (GRAY et al. 1999 ). Two dicistronic messages have been reported in D. melanogaster, one at the stoned locus (ANDREWS et al. 1996 ) and another at the Adh locus (BROGNA and ASHBURNER 1997 ). In both of these cases, it appears that the two proteins encoded by the messages have related functions. Our results showing the similar phenotypes of mutations in each coding region are perhaps the clearest demonstration of a dicistronic message where the two genes are required for the same process.

Considering that the conventional mechanism for ribosome binding to the mRNA involves initial contacts with the 5' m7G CAP followed by scanning for the AUG (KOZAK 1989 ), how is mei-218 translated? The second coding region could be translated by reinitiation of the ribosome following the first coding region. This mechanism is unlikely in our case because the mei-217 ORF overlaps the beginning of the mei-218 ORF, and in other cases there is usually a short intercistronic region (15–78 bp; KOZAK 1987 ; LEVINE et al. 1991 ). Another possibility is that translation is initiated at an internal ribosome binding site (IRES; reviewed in SACHS et al. 1997 ). Interestingly, we have identified a direct repeat sequence associated with a hairpin loop in a region that could be an internal entry point for ribosome binding (Fig 2 and Fig 4). Internal ribosome binding sites are associated with hairpin loops in the RNA (SACHS et al. 1997 ).

A mechanism for translating dicistronic messages may exist in Drosophila because it is used in monocistronic messages as well. OH et al. 1992 have argued that a significant fraction of D. melanogaster mRNAs are translated using internal initiation at IRES sites. This assertion was based on the large number of mRNAs with long (>250 bp) 5'UTRs containing upstream AUGs. They showed that the Antp mRNA is translated using an IRES and cited additional evidence that Ubx is as well. We suspect that internal initiation may be used by other meiotic recombination gene messages such as the mei-W68 mRNA, which has a long 5'UTR (860 bp) containing five AUGs (MCKIM and HAYASHI-HAGIHARA 1998 ). The mei-217 coding region is preceded by at least one AUG and may also use an IRES. The r1 mutation is upstream of the coding region and may affect the initiation of translation.

Promoters for meiotic recombination genes:
Meiosis is one of the first tasks undertaken by the oocyte. In region 2 of the germarium pachytene occurs 36–48 hr after the last mitotic division and the zygotene-to-pachytene transition takes <12 hr (CARPENTER 1975 ). Region 2 is also where high levels of mei-218/mei-217 mRNA are first observed. Thus, it might be expected that a common regulatory element might be found for meiotic recombination genes in Drosophila similar to that for S. cerevisiae meiosis genes (MITCHELL 1994 ; GAILUS-DURNER et al. 1996 ). The promoter for mei-217/mei-218 is most likely contained within the 960-bp region defined by the 3' end of the rpS5 gene and the start of mei-217. We have compared the upstream region of mei-217/mei-218 to several other meiotic recombination genes in D. melanogaster [mei-9 (SEKELSKY et al. 1995 ), spnB (GHABRIAL et al. 1998 ), mei-W68 (MCKIM and HAYASHI-HAGIHARA 1998 ), and mei-P22 (H. LIU and K. S. MCKIM, unpublished results)] but have not found sequence similarities suggestive of common promoter response elements. The major similarity between these promoter regions is that they are all small (<1 kb) and probably have a simple structure. We suspect that further analyses will reveal that transcription is not the primary mode of regulating meiotic recombination genes. This idea is based on the observation that mei-217/mei-218 appears to be required only for meiosis (BAKER et al. 1978 ), but its transcription can be detected in embryos, larvae, and in male testis (J. K. JANG and K. S. MCKIM, unpublished data). In addition, RNA levels of numerous other genes increase in germarium region 2, similar to the expression pattern of mei-217/mei-218 (for example, hsp83; DING et al. 1993 ). Perhaps the uniqueness of meiosis is imparted by translational regulation, as is important for many germline differentiation genes (e.g., LANTZ et al. 1994 ; KELLEY et al. 1997 ; WEBSTER et al. 1997 ).

mei-217 and mei-218 are members of a unique class of meiotic recombination genes:
Based mostly on gene conversion analysis, it has been proposed that mei-P22 and mei-W68 are required for initiating meiotic recombination (MCKIM et al. 1998 ), while mei-9 and mei-218 are not required to initiate recombination but are required for crossing over (CARPENTER 1982 , CARPENTER 1984 ). We have used double mutant analysis with mei-41 and spnB to infer the same information. This analysis is based on the hypothesis that mei-41 and spnB are required during the repair of the DNA breaks that initiate meiotic recombination (HAWLEY and FRIEND 1996 ; GHABRIAL et al. 1998 ; SEKELSKY et al. 1998 ). Mutations in mei-P22 and mei-W68 are epistatic to mei-41 and spnB, which is consistent with the essential role of these genes in making the initiating DNA breaks. In contrast, we found that mei-41 and spnB are epistatic to mei-217 and mei-218 mutations. These results place mei-217 into the same class of genes as mei-9 and mei-218. Like mei-218, we propose that mei-217 is not required for the initiation of recombination but is required for recombination intermediates to be resolved as crossovers.

Differences in the phenotypes of mei-9 and mei-218 show that they have different roles in the generation of crossovers. mei-218 may not be required for the exchange process per se but instead is required to determine which recombination sites will be resolved as crossovers (the "precondition" genes). In mei-218 mutants, the crossover frequency is reduced, and the distribution of the remaining events is altered (CARPENTER and SANDLER 1974 ). Even the normally achiasmate fourth chromosome experiences crossing over in a mei-218 mutant (SANDLER and SZAUTER 1978 ). In contrast, the function of mei-9 seems to be specific to crossover resolution (the "exchange" genes). In mei-9 mutants crossing over is reduced uniformly along each chromosome (CARPENTER and SANDLER 1974 ), there is a defect in the repair of mismatches (CARPENTER 1982 ), and fourth chromosome crossing over does not occur (SANDLER and SZAUTER 1978 ). The conclusion from these data is that mei-218 is required earlier than mei-9. Two observations support this two-step model. First, mei-218 mutations are epistatic to mei-9 mutations (SEKELSKY et al. 1995 ). Second, these two mutants have different effects on recombination nodules, which are the organelles at which meiotic recombination occurs. In mei-218 mutants the nodules are greatly reduced in frequency and some are abnormal, while in mei-9 mutants the nodules occur at a normal frequency and look normal (CARPENTER 1979 ). Based on the fact that mei-217 mutants alter the distribution of crossovers, mei-217 and mei-218 probably function in the same process.

There is another important difference between these two groups of genes. Both mei-217 and mei-218 mutants are not sensitive to MMS and therefore are not DNA repair defective. Similar results have been found with mutants in three other precondition genes (E. MANHEIM, R. PATEL and K. S. MCKIM, unpublished results). In contrast, the two known genes in the exchange class, mei-9 (BOYD et al. 1976 ) and mus-312 (J. SEKELSKY, personal communication), are required for DNA repair. Thus, it comes as no surprise that mei-9 encodes a homolog of the DNA repair enzyme Rad1/XPF (SEKELSKY et al. 1995 ). The sequences of mei-217 and mei-218 are not as informative because they lack amino acid sequence homology to known genes. Extrapolating from the existing members of each class, genes required specifically for exchanges encode proteins involved in DNA metabolism, whereas the precondition genes encode a new and as-yet-undescribed group of proteins.

	FOOTNOTES

¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

We are grateful to Robin Ghandi and Eric Chang for technical support and to Elizabeth Manheim for critical reading of the manuscript. This work was supported by grant RPG-98-069-01-DDC from the American Cancer Society to K. McKim.

Manuscript received October 12, 1999; Accepted for publication December 28, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ANDREWS, J., M. SMITH, J. MERAKOVSKY, M. COULSON, and F. HANNAN et al., 1996  The stoned locus of Drosophila melanogaster produces a dicistronic transcript and encodes two distinct polypeptides. Genetics 143:1699-1711[Abstract].

BAKER, B. S. and A. T. C. CARPENTER, 1972  Genetic analysis of sex chromosomal meiotic mutants in Drosophila melanogaster.. Genetics 71:255-286[Abstract/Free Full Text].

BAKER, B. S., A. T. C. CARPENTER, and P. RIPOLL, 1978  The utilization during mitotic cell division of loci controlling meiotic recombination in Drosophila melanogaster.. Genetics 90:531-578[Abstract/Free Full Text].

BAKER, S. M., A. W. PLUG, T. A. PROLLA, C. E. BRONNER, and A. C. HARRIS et al., 1996  Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over. Nat. Genet. 13:336-342[Medline].

BLUMENTHAL, T., 1998  Gene clusters and polycistronic transcription in eukaryotes. Bioessays 20:480-487[Medline].

BOYD, J. B., M. D. GOLINO, and R. B. SETLOW, 1976  The mei-9^a mutant of Drosophila melanogaster increases mutagen sensitivity and decreases excision repair. Genetics 84:527-544[Abstract/Free Full Text].

BROGNA, S. and M. ASHBURNER, 1997  The Adh-related gene of Drosophila melanogaster is expressed as a functional dicistronic messenger RNA: multigenic transcription in higher organisms. EMBO J. 16:2023-2031[Medline].

CARPENTER, A. T. C., 1975  Electron microscopy of meiosis in Drosophila melanogaster females. I. Structure, arrangement, and temporal change of the synaptonemal complex in wild-type. Chromosoma 51:157-182[Medline].

CARPENTER, A. T. C., 1979  Recombination nodules and synaptonemal complex in recombination-defective females of Drosophila melanogaster.. Chromosoma 75:259-292[Medline].

CARPENTER, A. T. C., 1982  Mismatch repair, gene conversion, and crossing-over in two recombination-defective mutants of Drosophila melanogaster.. Proc. Natl. Acad. Sci. USA 79:5961-5965[Abstract/Free Full Text].

CARPENTER, A. T. C., 1984  Meiotic roles of crossing-over and of gene conversion. Cold Spring Harbor Symp. Quant. Biol. 49:23-29[Abstract/Free Full Text].

CARPENTER, A. T., 1989  Are there morphologically abnormal early recombination nodules in the Drosophila melanogaster meiotic mutant mei-218? Genome 31:74-80[Medline].

CARPENTER, A. T. C. and L. SANDLER, 1974  On recombination-defective meiotic mutants in Drosophila melanogaster.. Genetics 76:453-475[Abstract/Free Full Text].

DE MASSY, B., V. ROCCO, and A. NICOLAS, 1995  The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae.. EMBO J. 14:4589-4598[Medline].

DERNBURG, A. F., K. MCDONALD, G. MOULDER, R. BARSTEAD, and M. DRESSER et al., 1998  Meiotic recombination in C. elegans initiates by a conserved mechanism and is dispensable for homologous chromosome synapsis. Cell 94:387-398[Medline].

DING, D., S. M. PARKHURST, S. R. HALSELL, and H. D. LIPSHITZ, 1993  Dynamic Hsp83 RNA localization during Drosophila oogenesis and embryogenesis. Mol. Cell. Biol. 13:3773-3781[Abstract/Free Full Text].

GAILUS-DURNER, V., J. XIE, C. CHINTAMANENI, and A. K. VERSHON, 1996  Participation of the yeast activator Abf1 in meiosis-specific expression of the HOP1 gene. Mol. Cell. Biol. 16:2777-2786[Abstract].

GHABRIAL, A. and T. SCHUPBACH, 1999  Activation of a meiotic checkpoint regulates translation of Gurken during Drosophila oogenesis. Nat. Cell. Biol. 1:354-357[Medline].

GHABRIAL, A., R. P. RAY, and T. SCHUPBACH, 1998  Okra and spindle-B encode components of the RAD52 DNA repair pathway and affect meiosis and patterning in Drosophila oogenesis. Genes Dev. 12:2711-2723[Abstract/Free Full Text].

GRAY, T. A., S. SAITOH, and R. D. NICHOLLS, 1999  An imprinted, mammalian bicistronic transcript encodes two independent proteins. Proc. Natl. Acad. Sci. USA 96:5616-5621[Abstract/Free Full Text].

HARI, K. L., A. SANTERRE, J. J. SEKELSKY, K. S. MCKIM, and J. B. BOYD et al., 1995  The mei-41 gene of D. melanogaster is a structural and function homolog of the human ataxia telangiectasia gene. Cell 82:815-821[Medline].

HAWLEY, R. S., 1988 Exchange and chromosomal segregation in eucaryotes, pp. 497–527 in Genetic Recombination, edited by R. KUCHERLAPATI and G. SMITH. American Society of Microbiology, Washington, DC.

HAWLEY, R. S. and S. FRIEND, 1996  Strange bedfellows in even stranger places: the role of ATM in meiotic cells, lymphocytes,tumors, and its functional links to p53. Genes Dev. 10:2383-2388[Free Full Text].

HAWLEY, R. S., H. A. IRICK, A. E. ZITRON, D. A. HADDOX, and A. R. LOHE et al., 1992  There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology. Dev. Genet. 13:440-467[Medline].

HOLLINGSWORTH, N. M., L. PONTE, and C. HALSEY, 1995  MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair. Genes Dev. 9:1728-1739[Abstract/Free Full Text].

HUNTER, N. and R. H. BORTS, 1997  Mlh1 is unique among mismatch repair proteins in its ability to promote crossing-over during meiosis. Genes Dev. 11:1573-1582[Abstract/Free Full Text].

KEENEY, S., C. N. GIROUX, and N. KLECKNER, 1997  Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88:375-384[Medline].

KELLEY, R. L., J. WANG, L. BELL, and M. I. KURODA, 1997  Sex lethal controls dosage compensation in Drosophila by a non-splicing mechanism. Nature 387:195-199[Medline].

KOZAK, M., 1987  Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol. Cell. Biol. 7:3438-3445[Abstract/Free Full Text].

KOZAK, M., 1989  The scanning model for translation: an update. J. Cell Biol. 108:229-241[Abstract/Free Full Text].

LANTZ, V., J. S. CHANG, J. I. HORABIN, D. BOPP, and P. SCHEDL, 1994  The Drosophila orb RNA-binding protein is required for the formation of the egg chamber and establishment of polarity. Genes Dev. 8:598-613[Abstract/Free Full Text].

LEVINE, F., J. K. YEE, and T. FRIEDMANN, 1991  Efficient gene expression in mammalian cells from a dicistronic transcriptional unit in an improved retroviral vector. Gene 108:167-174[Medline].

LIU, J., T. WU, and M. LICHTEN, 1995  The location and structure of double-strand DNA breaks induced during yeast meiosis: evidence for a covalently linked DNA-protein intermediate. EMBO J. 14:4599-4608[Medline].

MCKEE, B. D. and G. H. KARPEN, 1990  Drosophila ribosomal RNA genes function as an X-Y pairing site during male meiosis. Cell 61:61-72[Medline].

MCKIM, K. S. and A. HAYASHI-HAGIHARA, 1998  mei-W68 in Drosophila melanogaster encodes a Spo11 homolog: evidence that the mechanism for initiating meiotic recombination is conserved. Genes Dev. 12:2932-2942[Abstract/Free Full Text].

MCKIM, K. S., J. K. JANG, W. E. THEURKAUF, and R. S. HAWLEY, 1993  Mechanical basis of meiotic metaphase arrest. Nature 362:364-366[Medline].

MCKIM, K. S., J. B. DAHMUS, and R. S. HAWLEY, 1996  Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E. Genetics 144:215-228[Abstract].

MCKIM, K. S., B. L. GREEN-MARROQUIN, J. J. SEKELSKY, G. CHIN, and C. STEINBERG et al., 1998  Meiotic synapsis in the absence of recombination. Science 279:876-878[Abstract/Free Full Text].

MCKIM, K. S., J. K. JANG, J. J. SEKELSKY, A. LAURENCON, and R. S. HAWLEY, 2000  mei-41 is required for precocious anaphase in Drosophila females. Chromosoma in press.

MITCHELL, A. P., 1994  Control of meiotic gene expression in Saccharomyces cerevisiae. Microbiol. Rev. 58:56-70[Abstract/Free Full Text].

OH, S. K., M. P. SCOTT, and P. SARNOW, 1992  Homeotic gene Antennapedia mRNA contains 5'-noncoding sequences that confer translational initiation by internal ribosome binding. Genes Dev. 6:1643-1653[Abstract/Free Full Text].

ROBINSON, D. N. and L. COOLEY, 1997  Examination of the function of two kelch proteins generated by stop codon suppression. Development 124:1405-1417[Abstract].

ROSS-MACDONALD, P. and G. S. ROEDER, 1995  Mutation of a meiosis-specific MutS homolog decreases crossing over but not mismatch correction. Cell 79:1069-1080.

SACHS, A. B., P. SARNOW, and M. W. HENTZE, 1997  Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell 89:831-838[Medline].

SANDLER, L. and P. SZAUTER, 1978  The effect of recombination-defective meiotic mutants on fourth-chromosome crossing over in Drosophila melanogaster.. Genetics 90:699-712[Abstract/Free Full Text].

SANDLER, L., D. L. LINDSLEY, B. NICOLETTI, and G. TRIPPA, 1968  Mutants affecting meiosis in natural populations of Drosophila melanogaster.. Genetics 60:525-558[Free Full Text].

SEKELSKY, J. J., K. S. MCKIM, G. M. CHIN, and R. S. HAWLEY, 1995  The Drosophila meiotic recombination gene mei-9 encodes a homologue of the yeast excision repair protein Rad1. Genetics 141:619-627[Abstract].

SEKELSKY, J. J., K. C. BURTIS, and R. S. HAWLEY, 1998  Damage control: the pleiotropy of DNA repair genes in Drosophila melanogaster. Genetics 148:1587-1598[Abstract/Free Full Text].

TAUTZ, D. and C. PFEIFLE, 1989  A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.. Chromosoma 98:81-85[Medline].

THEURKAUF, W. E. and R. S. HAWLEY, 1992  Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein. J. Cell Biol. 116:1167-1180[Abstract/Free Full Text].

WEBSTER, P. J., L. LIANG, C. A. BERG, P. LASKO, and P. M. MACDONALD, 1997  Translational repressor bruno plays multiple roles in development and is widely conserved. Genes Dev. 11:2510-2521[Abstract/Free Full Text].

This article has been cited by other articles:

				P. Huang, E. D. Pleasance, J. S. Maydan, R. Hunt-Newbury, N. J. O'Neil, A. Mah, D. L. Baillie, M. A. Marra, D. G. Moerman, and S. J.M. Jones Identification and analysis of internal promoters in Caenorhabditis elegans operons Genome Res., October 1, 2007; 17(10): 1478 - 1485. [Abstract] [Full Text] [PDF]

				E. A. Manheim, J. K. Jang, D. Dominic, and K. S. McKim Cytoplasmic Localization and Evolutionary Conservation of MEI-218, a Protein Required for Meiotic Crossing-over in Drosophila Mol. Biol. Cell, January 1, 2002; 13(1): 84 - 95. [Abstract] [Full Text] [PDF]