Cloning of the Zhos @ hiZu melunogaster Meiotic Recombination Gene mei - 218 : A Genetic and Molecular Analysis of Interval 15 E

The mei-218 gene product is required for both meiotic crossing over and for the production of recombination nodules, suggesting that these organelles are required for meiotic exchange. In this study the null phenotype of mei-218 was defined through the analysis of three preexisting and five new alleles. Consistent with previous studies, in homozygous mi-218 mutants meiotic crossing over is reduced to 90% and the residual exchanges are abnormally distributed, suggesting a defect in both the completion of genetic recombination and in the mechanism by which the sites for crossing over are chosen ( SANDLER et al. 1968; BAKER and CARPENTER 1972; CARPENTER and SANDLER 1974). Electron microscopic analysis of mei-218 oocytes revealed the presence of normal synaptonemal complexes between the chromosome arms, indicating that the chromosomes are able to pair (CARPENTER 1979a). Thus the mi-218 mutant defect is intimately associated with recombination itself and not a secondary consequence of the failure of homologues to pair. A clue to the cellular defect in mei-218 mutants was the observation that the recombination nodules, organelles believed to facilitate crossing over, are reduced in number and often have abnormal morphology (CARPENTER 1979a) . Analysis of intragenic recombination at the rosy locus has shown that gene conversion events occur at least as frequently as in wild type (CARPENTER 1982,1984). These observations suggest that recombination events initiate in meZ-218 oocytes but are not resolved into crossovers. Since mei-218 mutants have no defects in somatic cells (BAKER et al. 1978), it appears that the mei-218 gene product is a meiosis-specific component required for correct assembly and function of the recombination nodule. Here we report the isolation of five new mei-218 alCorresponding author: R. Scott Hawley, Section of Molecular and Cellular Biology, University of California, Davis, CA 95616. E-mail: [email protected] Genetics 144: 215-228 (September, 1996) leles isolated in an unbiased screen that did not select against lethal or sterile mutants. Although all mez-218 alleles exhibit strong reductions in the frequency of meiotic recombination, none display a discernible effect on viability, fertility, or mitotic chromosome segregation. By analyzing the phenotypes of these mutants both as homozygotes and as D f/mei-218 heterozygotes, we were able to assess the null phenotype of the mei218 gene. To precisely map mei-218 and facilitate cloning, we constructed a genetic and molecular map based on chromosome rearrangements and restriction fragment length polymorphisms (RFLPs). The mi-218 gene was found to produce a transcript predicted to encode a 1186-amino acid, 134kD protein. The protein sequence is not similar to any in the protein or translated nucleotide databases. We also characterized the region flanking mei-218. Within this region is M ( 1 ) 150, a member of a class of genes (Minutes) that are recessive lethal but have dominant effects on both growth rate and female fertility ( LINDSLEY and ZIMM 1992). Consistent with the recent analysis of two other Minute genes, we found that M(l )15D encodes a Drosophila homologue of ribosomal protein S5. MATERIALS and IETHODS Drosophila stocks All cultures were raised at 23-25' on corn meal-molasses yeast media. With the exception of new mutations and aberrations reported here, all mutants are described in LINDSLEV and ZIMM ( 1992). The mei-218' mutation was referred to in MCKIM et al. 216 K. S. McKim, J. D. Dahmus and R. S. Hawley (1993) as mei-218"4. FM7is a multiply inverted Xchromosome that does not crossover with the normal X chromosome. FM7a is homozygous fertile, while FM7c is homozygous female sterile, The duplication Dp( 1;4)r+ (Figure 1 ) was isolated by GREEN ( 1963) as a derivative of T( 1;4) B". Dp (l;?) f + 71 b is the duplication element of Tp (1;3) f + 71b separated from the deficiency by making Dp(1;3) f +71b/+, Df(1) f +71b/FM7a, f females and then recovering the f ( + ) B progeny from these females ( i . e . , + /Dp(l;3)f+ 716; FM7, f / Y ) . Screen for mei-218 alleles: y cu u f car/y+ Y males were fed 0.025 M EMS and then crossed to y me~218'/FM7a females. Individual y cu u f car/ y mei-218' virgin females were collected and tested by crossing to YsX.YL, u f B/ 0; C(4)M, ci t$/Omales. If a new me-218 mutation was present on the mutagenized chromosome, then diplo-X ( y cu u f car/y mei-218') and nullo-X ( Y'X.YL, u f B/O) exceptions were recovered. The putative new mutants were confinned by crossing the diplo-Xfemale exceptions to the same Y'X.Y", u f B/O males. The new mutations were recovered linked to the forked marker by crossing the y cu u f car/y mei-218' females to FM7a, f/y+Y males. In a screen of 7010 EMS-treated chromosomes, 25 were recovered, which upon retesting, gave high levels of nondisjunction in trans to mei-218'.. Twenty of these, however, were dominant and caused nondisjunction in trans to a mei-218+ chromosome. Although not proven, most of these mutations were likely translocations, as many of them were either male sterile or male lethal. In addition, the dominant mutants did not increase the frequency of fourth chromosome nondisjunction, as occurs in mei-218 mutants. The remaining five chromosomes carried new mei-218 mutations. Measuring nondisjunction: In most crosses, the X chromosome nondisjunction frequency was calculated as 2 (exceptional progeny) / 2 (exceptional progeny) + ( regular progeny) . For mei-218/Df (1 ) BK8 females, the nondisjunction frequency was calculated as (exceptional males) / (exceptional males + regular females). The female progeny heterozygous for the deficiency, which is the exceptional females and half of the regular progeny, were not included in the calculations because they were Minute, slow developing, and sporadically represented. Bar reversion and deletions of Dp (1;4) r+: We screened for deletions among the progeny of irradiated males carrying both a normal X chromosome marked with the dominant Bar mutation and the Dp(l;4)r' duplication. The presence of Dp(l;4)rf did not affect the expression of the Bar phenotype, thus reversion of Bar could be scored in the presence of the duplication in males or females. r f B; Dp( 1;4)r+ males were treated with 4000 rad of X-irradiation and crossed to r f B/r f B; Dp( 1;4)r+ females (Figure 2 ) . Deletions of B could be recognized because B / B females have a more severe eye phenotype than B/+ or B/D ffemales. All Barreversions were initially recovered as r f B/r f Df( 1)B; D p ( 1 ; 4 ) r f / + females. If the deficiency deleted a Minute locus, the dominant slow-growth phenotype was rescued by the duplication. The deficiencies were subsequently balanced as r f Df(l)B/FM7c; Dp(l;4)r+/+; with the duplication being retained if the deficiency was Minute. In the same cross, new deletions of Dp(I;4)r+ could be recognized by either a rudimentary or forked phenotype. The r + f duplications were stable as r f B/r f B; Dp(l;4)rt f -/+ stocks because r females are sterile. The rf + duplications were kept in stock with a lethal mutation covered by the duplication (i.e., y cu u l(1) f/ y'Y; Dp(l;4)rK20/+). Isolation of new lethal mutations: In a screen of 5000 25 mM EMS-treated y cu u f chromosomes, 80 new lethal mutations covered by Dp(l;4)r+ were isolated (R. FRENCH, D. HADDOX and R. S . HAWLEY, unpublished data). The same screen was also done with 4000 rad of X-rays; 5000 chromosomes were screened and 30 mutations were recovered. The stocks were maintained either with C(1)DX (and y cu u f/t Dp(1;4)r+/+ males) or balanced by FM7c. All of the X-ray-induced mutations and the EMS mutations not covered by Dp (1;4) fK4 (Figure 1 ) were mapped by complementation crosses to duplications and deficiencies. An additional collection of 16 EMS-induced lethal mutations mapping within the 15A-16A region (Figure 1 ) were generously provided by S. RUTHERFORD (personal communication ) . Complementation test crosses: Each new derivative of Dp(l;4)r+ that was either r + f or rf + could have carried either a new point mutation in forked or rudimentary or a deletion including either locus. To determine if the new derivatives carried deletions, they were tested for the ability to rescue a deletion covered by the original Dp(1;4)rt chromosome. The rf + duplications were crossed to D f (l)rDl, u f/FM7c; Dp(l;4)r+/spap"i females and the r + f duplications were crossed to D f (1 ) BKl6/FM7c; Dp (1;4) r+ /spapD1 females. If the resulting ror fmutant males ( i . e . , D f ( l ) / t Dp/spa""l) were lethal, the duplication carried a deletion. One of the rduplications (Dp(1;4)rK20) and four of the f duplications were found to be deletions of Dp (1;4)r+ (Table 3 ) . The breakpoints of the new duplications were determined by complementation crosses to lethal mutations. y cu u I( 1) f/FM7c females were crossed to r f B / Y ; Dp(l;4)r+ f -/+ males. The presence of y cu u 1 (1)J Dp (1;4)r+ f /+ male progeny indicated that the duplication carried the wild-type allele of the essential gene. Complementation tests between lethal mutations [designated I( 1)-x and 1( 1 ) y 1 were done by crossing y cu u l (1) -x f / K Dp(1;4)r+/+ males to y cu u V l ) y f/ FM7cfemales. If the two mutations complemented, fmked females [y cu u l(1)-x f/ y cu u l(1)y J + / + I were observed. Complementation tests between Minute loci were based on their dominant phenotype, since in some cases the double Minute heterozygotes were subviable. A typical experiment was D f (1)BK16/FM7; e ( l ; 4 ) r + / + Molecular Genetics of mei-218 217