Use of a Yeast Site-Specific Recombinase to Produce Female Germline Chimeras in Drosophila

We describe an efficient method for generating female germline mosaics by inducing site-specific homologous mitotic recombination with a yeast recombinase (FLP) which is driven by a heat shock promoter. These germline mosaics are produced in flies heterozygous for the agametic, germline-dependent, dominant female sterile (DFS) mutation ovoD1, where only flies possessing germline clones are able to lay eggs. This method, the "FLP-DFS" technique, is very efficient because more than 90% of females with germline clones can be recovered. We show that this heat-inducible, site-specific mitotic recombination system does not affect viability and that the germline clones recovered are physiologically the same as those created by X-ray induced mitotic recombination. We describe the parameters of FLP-recombinase induced germline mitotic recombination and the use of the "FLP-DFS" technique to analyze the maternal effect of X-linked zygotic lethal mutations.     

Sex chromosomes in flowering plants

Sex chromosomes in dioecious and polygamous plants evolved as a mechanism for ensuring outcrossing to increase genetic variation in the offspring. Sex specificity has evolved in 75% of plant families by male sterile or female sterile mutations, but well-defined heteromorphic sex chromosomes are known in only four plant families. A pivotal event in sex chromosome evolution, suppression of recombination at the sex determination locus and its neighboring regions, might be lacking in most dioecious species. However, once recombination is suppressed around the sex determination region, an incipient Y chromosome starts to differentiate by accumulating deleterious mutations, transposable element insertions, chromosomal rearrangements, and selection for male-specific alleles. Some plant species have recently evolved homomorphic sex chromosomes near the inception of this evolutionary process, while a few other species have sufficiently diverged heteromorphic sex chromosomes. Comparative analysis of carefully selected plant species together with some fish species promises new insights into the origins of sex chromosomes and the selective forces driving their evolution.     

Isolation and Characterization of Dominant Female Sterile Mutations of Drosophila Melanogaster. II. Mutations on the Second Chromosome

Twenty-four, second chromosome, dominant female sterile (Fs) mutations in Drosophila are described. Fs(2) were isolated at a frequency of approximately 1 per 1000 EMS-treated chromosomes screened. In comparison the isolation of frequency for second chromosome zygotic recessive lethal mutations was approximately 550 per 1000. Complementation analysis of the Fs(2) revertants showed that the 24 Fs(2) mutations identify 13-15 loci, calculated to be about 65-75% of the second chromosome genes EMS mutable to dominant female sterility. Two of the Fs(2) mutations are useful tools for the dominant female sterile technique: Fs(2)1 for induction and detection of germ-line clones and Fs(2)Ugra for follicle cell clones. Several of the Fs(2) mutations bring about novel mutant phenotypes. Seven of them alter egg shape, whereas the others arrest development primarily at two stages: around fertilization by five Fs(2) and during cleavage divisions [by Fs(2) in three loci]. The remaining that allow development to the larval stage of differentiation include four new dorsal alleles and one dominant torso allele. Analysis of germ-line chimeras revealed that with two exceptions all the Fs(2) mutations are germ-line dependent. The Fs(2) mutations were mapped mainly on the basis of mitotic recombination induced in the female germ-line cells of adult females. That most of the Fs(2) may be gain-of-function mutations is indicated by the unusual behavior of the Fs+ germ-line clones and also by the fact that 90% of the could be induced to revert.     

Recombination and disjunction in female germ cells of Drosophila depend on the germline activity of the gene sex-lethal

Gametogenesis in males and females differs in many ways. An important difference in Drosophila is that recombination between homologous chromosomes occurs only in female meiosis. Here, we report that this process relies on the correct functioning of Sex-lethal (Sxl) which is primarily known as the master gene in somatic sex determination. Certain alleles of this gene (Sxl(fs)) disrupt the germline, but not the somatic function of Sxl and cause an arrest of germ cell development during cystocyte proliferation. Using dominant suppressor mutations that relieve this early block in Sxl(fs) mutant females, we discovered additional requirements of Sxl for normal meiotic differentiation of the oocyte. Females mutant for Sxl(fs) and carrying a suppressor become fertile, but pairing of homologous chromosomes and formation of chiasmata is severely perturbed, resulting in an almost complete lack of recombinants and a high incidence of non-disjunction events. Similar results were obtained when germline expression of wild-type Sxl was compromised by mutations in virilizer (vir), a positive regulator of Sxl. Ectopic expression of a Sxl transgene in premeiotic stages of male germline development, on the other hand, is not sufficient to allow recombination to take place, which suggests that Sxl does not have a discriminatory role in this female-specific process. We propose that Sxl performs at least two tasks in oogenesis: an 'early' function in formation of the egg chamber, and a 'late' function in progression of the meiotic cell cycle, suggesting that both events are coordinated by a common mechanism.     

Sister-Chromatid Misbehavior in Drosophila Ord Mutants

In Drosophila males and females mutant for the ord gene, sister chromatids prematurely disjoin in meiosis. We have isolated five new alleles of ord and analyzed them both as homozygotes and in trans to deficiencies for the locus, and we show that ord function is necessary early in meiosis of both sexes. Strong ord alleles result in chromosome nondisjunction in meiosis I that appears to be the consequence of precocious separation of the sister chromatids followed by their random segregation. Cytological analysis in males confirmed that precocious disjunction of the sister chromatids occurs in prometaphase I. This is in contrast to Drosophila mei-S332 mutants, in which precocious sister-chromatid separation also occurs, but not until late in anaphase I. All three of the new female fertile ord alleles reduce recombination, suggesting they affect homolog association as well as sister-chromatid cohesion. In addition to the effect of ord mutations on meiosis, we find that in ord2 mutants chromosome segregation is aberrant in the mitotic divisions that produce the spermatocytes. The strongest ord alleles, ord2 and ord5, appear to cause defects in germline divisions in the female. These alleles are female sterile and produce egg chambers with altered nurse cell number, size, and nuclear morphology. In contrast to the effects of ord mutations on germline mitosis, all of the alleles are fully viable even when in trans to a deficiency, and thus exhibit no essential role in somatic mitosis. The ord gene product may prevent premature sister-chromatid separation by promoting cohesion of the sister chromatids in a structural or regulatory manner.     

Analysis of novel alleles of brother of tout‐velu,the drosophila ortholog of human EXTL3 using a newly developed FRT42D ovoD chromosome

The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovo^D dominant female‐sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini‐white (w ^{+ mW.hs}), or rosy (ry⁺) and neomycin (neo^R) transgenes are in common use. Parallel ovo^D lines were developed using w ^{+ mW.hs} FRT insertions on the X and chromosomes 2R and 3L, as well as ry⁺, neo^R FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry⁺, neo^R FRT background, are not amenable to germline clonal analysis without labor‐intensive recombination onto chromosome arms containing a w ^{+ mW.hs} FRT. Here we report the creation of a new ovo^D line for the ry⁺, neo^R FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent we characterized the maternal‐effect phenotypes of novel brother of tout‐velu alleles generated on an FRT42D chromosome in a somatic mosaic screen. We find that an apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages.     

Isolation of autosomal mutations in Drosophila melanogaster without setting up lines

Summary Mutants of Drosophila melanogaster are being used increasingly for studying different biological mechanisms. However, most attempts to identify new mutations have been restricted to the X-chromosome. It has been very difficult to identify new loci on the autosomes, as recessive mutations have to be made homozygous by setting up independent cultures for each mutagenized chromosome. We introduce a mutagenesis scheme which does not require setting up independent cultures. It uses meiotic recombination in compound autosomes to make recessive mutations homozygous and allows the screening of tens of thousands of mutagenized chromosomes with relatively little effort. In a pilot experiment, we tested about 33,300 chromosomes for temperature-sensitive paralytic mutations. We obtained 62 independent paralytic mutations and a large number of other mutations. Eight out of 25 of the paralytic mutations are on the autosomes. This method makes autosomes, which constitute about 80% of the Drosophila genome, more accessible for mutational analysis of various biological mechanisms.     

Female Sterile Mutations on the Second Chromosome of Drosophila Melanogaster. I. Maternal Effect Mutations

In mutagenesis screens for recessive female sterile mutations on the second chromosome of Drosophila melanogaster 529 chromosomes were isolated which allow the homozygous females to survive, but cause them to be sterile. In 136 of these lines, mutant females produce morphologically normal eggs which cannot support normal embryonic development. These "maternal-effect" mutations fall into 67 complementation groups which define 23 multiply hit and 44 singly hit loci. In eggs from 14 complementation groups development is blocked before the formation of a syncytial blastoderm. In eggs from 12 complementation groups development is abnormal before cellularization, 17 complementation groups cause abnormal cellularization, 12 complementation groups cause changes in cellular morphology in early gastrulation stages, and 12 complementation groups seem to affect later embryonic development.     

Chromosome Rearrangements in CAENORHABDITIS ELEGANS

A method for selecting unlinked duplications of a part of the X chromosome of C. elegans is described. Five such duplications have been identified. One of them, Dp (X;V)1, is translocated to linkage group V, where it suppresses crossing over along the left half of linkage group V. Dp(X;V)1 homozygotes grow slowly and are sterile. The other four duplications are associated with chromosome fragments, as observed cytologically by fluorescence microscopy, and tend to be lost. Their frequency of loss is higher in strains homozygous for a mutation that promotes nondisjunction of X chromosomes. The recombination frequencies between two of these duplications and the X have been measured: the frequencies are at least 50 times less than for X-X recombination in the same region. The duplications may prove useful as balancers of recessive lethal mutations.     

Analysis of male recombination in third chromosomes of Drosophila melanogaster

Data on male recombination in twenty third-chromosomal lines of Drosophila melanogaster are presented. Frequencies of female and male recombination have been calculated in seven intervals along the third chromosome. The influence on male recombination (M.R.) exercised by different factors such as population origin (cellar, vineyard), the presence of heterozygous inversions and recessive lethal chromosomes, is analyzed. The results obtained lead to the main conclusion that M.R. is not affected by the presence of heterozygous inversions which reduce female recombination in the same lines. In the light of this effect, the possible mechanism operating on male recombination is discussed. Lethal chromosomes reduce significantly the number of male recombination events as compared with wild chromosomes. We have not obtained significant differences in male recombination frequencies between the cellar and the vineyard lines.     

Gene targeting and gene trap screens using embryonic stem cells: new approaches to mammalian development.

Mouse embryonic stem cell lines offer an attractive route for introducing rare genetic alternations into the gene pool since the cells can be pre-screened in culture and the mutations then transmitted into the germline through chimera production. Two applications of this technique that seem ideally suited for a genetic analysis of development are enhancer and gene trap screens for loci expressed during gastrulation and production of targeted mutations using homologous recombination. These approaches should greatly increase the number of mouse developmental mutants available and help to elucidate the genetic hierarchy controlling embryogenesis.     

Seventeen Complementation Groups of Mutations Decreasing Meiotic Recombination in Schizosaccharomyces Pombe

We have analyzed 43 recessive mutations reducing meiotic intragenic recombination in Schizosaccharomyces pombe. These mutations were isolated by a screen for reduced plasmid-by-chromosome recombination at the ade6 locus. Sixteen of the mutations define 10 new complementation groups, bringing to 17 the number of genes identified to be involved in meiotic recombination. The mutations were grouped into three discrete classes depending on the severity of the recombination deficiency in crosses involving the ade6-M26 recombination hotspot. Class I mutations caused at least a 1000-fold reduction in M26-stimulated intragenic recombination at the ade6 locus. Class II mutations reduced M26-stimulated recombination approximately 100-fold. Class III mutations caused a 3-10-fold reduction in either M26-stimulated or non-hotspot recombination. We obtained multiple alleles of class I and class II mutations, suggesting that we may be nearing saturation for mutations of this type. As a first step toward mapping, we used mitotic segregation to assign fourteen of the rec genes to chromosomes. Mutations in the six rec genes tested also caused a decrease in intragenic recombination at the ura4 locus; five of these mutations also reduced intergenic recombination between the pro2 and arg3 genes. These results indicate that these multiple rec gene products are required for high level meiotic recombination throughout the S. pombe genome.     

Introducing defined chromosomal rearrangements into the mouse genome

Chromosomal rearrangements have been instrumental in genetic studies in Drosophila. Visibly marked deficiencies (deletions) are used in mapping studies and region-specific mutagenesis screens by providing segmental haploidy required to uncover recessive mutations. Marked recessive lethal inversions are used as balancer chromosomes to maintain recessive lethal mutations and to maintain the integrity of mutagenized chromosomes. In mice, studies on series of radiation-induced deletions that surround several visible mutations have yielded invaluable functional genomic information in the regions analyzed. However, most regions of the mouse genome are not accessible to such analyses due to a lack of marked chromosomal rearrangements. Here we describe a method to generate defined chromosomal rearrangements using the Cre--loxP recombination system based on a published strategy [R. Ramirez-Solis, P. Liu, and A. Bradley, (1995) Nature 378, 720--724]. Various types of rearrangements, such as deletions, duplications, inversions, and translocations, can be engineered using this strategy. Furthermore, the rearrangements can be visibly marked with coat color genes, providing essential reagents for large-scale recessive genetic screens in the mouse. The ability to generate marked chromosomal rearrangements will help to elevate the level of manipulative mouse genetics to that of Drosophila genetics.     

Correlation between recombination frequency and fitness in Drosophila melanogaster

The origin and maintenance of genetic recombination are unsettled evolutionary issues. Genetic variation affecting recombination frequency appears to be pervasive in nature, suggesting that natural selection must increase recombination frequency under some circumstances. However, theoretical arguments and experimental evidence indicate that the frequency of recombination should be reduced by natural selection.A hypothesis not previously explored is that recombination modifiers may directly affect the fitness of their carriers; rather than only indirectly (through the production of recombinant progeny) as generally assumed. We have tested this hypothesis by examining three fitness components (viability, male fertility, and female fecundity) in Drosophila melanogaster homozygous for second chromosomes isolated from a natural population. Then, we have measured the frequency of recombination in flies heterozygous for each wild second chromosome and a chromosome carrying five recessive alleles.The results indicate that genes modulating the frequency of recombination have direct effects on fitness as proposed by the hypothesis. However, the correlation between frequency of recombination and fitness is negative. Thus, the riddle of recombination remains unexplained and, in fact, more puzzling that ever.     

Genetic Characterization of Small Ovaries, a Gene Required in the Soma for the Development of the Drosophila Ovary and the Female Germline

The small ovary gene (sov) is required for the development of the Drosophila ovary. Six EMS-induced recessive alleles have been identified. Hypomorphic alleles are female sterile and have no effect on male fertility, whereas more severe mutations result in lethality. The female-sterile alleles produce a range of mutant phenotypes that affect the differentiation of both somatic and germline tissues. These mutations generally produce small ovaries that contain few egg cysts and disorganized ovarioles, and in the most extreme case no ovarian tissue is present. The mutant egg cysts that develop have aberrant morphology, including abnormal numbers of nurse cells and patches of necrotic cells. We demonstrate that sov gene expression is not required in the germline for the development of functional egg cysts. This indicates that the sov function is somatic dependent. We present evidence using loss-of-function and constitutive forms of the somatic sex regulatory genes that sov activity is essential for the development of the somatic ovary regardless of the chromosomal sex of the fly. In addition, the genetic mapping of the sov locus is presented, including the characterization of two lethal sov alleles and complementation mapping with existing rearrangements.     

Interacting Genes That Affect Microtubule Function in Drosophila Melanogaster: Two Classes of Mutation Revert the Failure to Complement between Hay(nc2) and Mutations in Tubulin Genes

The recessive male sterile mutation haync2 of Drosophila melanogaster fails to complement certain beta 2-tubulin and alpha-tubulin mutations, suggesting that the haywire product plays a role in microtubule function, perhaps as a structural component of microtubules. The genetic interaction appears to require the presence of the aberrant product encoded by haync2, which may act as a structural poison. Based on this observation, we have isolated ten new mutations that revert the failure to complement between haync2 and B2tn. The revertants tested behaved as intragenic mutations of hay in recombination tests, and fell into two phenotypic classes, suggesting two functional domains of the hay gene product. Some revertants were hemizygous viable and less severe than haync2 in their recessive phenotype. These mutations might revert the poison by restoring the aberrant product encoded by the haync2 allele to more wild-type function. Most of the revertants were recessive lethal mutations, indicating that the hay gene product is essential for viability. These more extreme mutations could revert the poison by destroying the ability of the aberrant haywirenc2 product to interact structurally with microtubules. Flies heterozygous for the original haync2 allele and an extreme revertant show defects in both the structure and the function of the male meiotic spindle.     

Complex recombination events at the hypermutable minisatellite CEB1 (D2S90).

Some minisatellite structures are the site of high rates of DNA recombination in non-pathological situations, with an excess of motif insertion events and a locus-dependent sex-specific mutation bias. We previously reported the cloning of the hypermutable minisatellite locus CEB1 (D2S90), remarkable for its 13% mutation rate in the male germline (compared to approximately 0.4% in female). We have sought to analyse the mechanisms underlying the addition or deletion of motifs at this locus using the minisatellite variant repeat mapping technique. This is possible with a high precision due to the extreme sequence polymorphism seen between different motifs. No crossing-over event was observed among 38 informative neomutations. Four of the 19 informative mutant alleles with an addition of motifs are interallelic events, the others are intra-allelic. Overall, the insertion and deletion mutations are spread along the alleles, although the subset of interallelic events shows clustering towards the analysed end. The apparently complex recombination events observed can all be interpreted as a succession of elementary duplications-deletions of inter- as well as intra-chromosomal origin, suggesting a model in which sister chromatid as well as conversion-like exchanges are involved in these mutation processes.     

Two new mouse chromosome 11 balancers

Segmental inversions causing recombination suppression are an essential feature of balancer chromosomes. Meiotic crossing over between homologous chromosomes within an inversion interval will lead to nonviable gametes, while gametes generated from recombination events elsewhere on the chromosome will be unaffected. This apparent recombination suppression has been widely exploited in genetic studies in Drosophila to maintain and analyze stocks carrying recessive lethal mutations. Balancers are particularly useful in mutagenesis screens since they help to establish the approximate genomic location of alleles of genes causing phenotypes. Using the Cre-loxP recombination system, we have constructed two mouse balancer chromosomes carrying 8- and 30-cM inversions between Wnt3 and D11Mit69 and between Trp53 and EgfR loci, respectively. The Wnt3-D11Mit69 inversion mutates the Wnt3 locus and is therefore homozygous lethal. The Trp53-EgfR inversion is homozygous viable, since the EgfR locus is intact and mutations in p53 are homozygous viable. A dominantly acting K14-agouti minigene tags both rearrangements, which enables these balancer chromosomes to be visibly tracked in mouse stocks. With the addition of these balancers to the previously reported Trp53-Wnt3 balancer, most of mouse chromosome 11 is now available in balancer stocks.