A novel genetic tool for clonal analysis of fourth chromosome mutations

The fourth chromosome of Drosophila remains one of the most intractable regions of the fly genome to genetic analysis. The main difficulty posed to the genetic analyses of mutations on this chromosome arises from the fact that it does not undergo meiotic recombination, which makes recombination mapping impossible, and also prevents clonal analysis of mutations, a technique which relies on recombination to introduce the prerequisite recessive markers and FLP-recombinase recognition targets (FRT). Here we introduce a method that overcomes these limitations and allows for the generation of single Minute haplo-4 clones of any fourth chromosome mutant gene in tissues of developing and adult flies.     

USING HITCHHIKING GENES TO STUDY ADAPTATION AND DIVERGENCE DURING SPECIATION WITHIN THE DROSOPHILA MELANOGASTER SPECIES COMPLEX

Several studies of intraspecific and interspecific DNA sequence variation from Drosophila loci have revealed a pattern of low intraspecific variation from genomic regions of low recombination. The mechanisms consistently invoked to explain these patterns are the selective sweep of advantageous mutations together with genetic hitchhiking of linked loci. To examine the effect of selective sweeps on genetic divergence during speciation, we studied two loci in different genomic regions thought to be subject to selective sweeps. We obtained DNA sequences from 1.1kb pair portions of the fourth chromosome locus cubitus interruptus Dominant (ci^D) and from the asense locus near the telomere of the X chromosome. At ci^D, we found very low variation among multiple lines of Drosophila mauritiana and D. sechellia. This finding is consistent with an earlier report of very low variation in D. melanogaster and D. simulans at ci^D and supports the conclusion of selective sweeps and genetic hitchhiking on the nonrecombining fourth chromosome. The pattern of variation found at asense suggests that a selective sweep has occurred recently at the tip of the X chromosome in D. simulans, but not in D. melanogaster or D. mauritiana. The data from ci^D and asense are compared with data from three X chromosome loci (period, zeste, and yolk protein 2) that experience normal levels of recombination. By examining estimated genealogies and the rates at which different classes of mutations have accumulated, we conclude that selective sweeps are common occurrences on the fourth chromosome but less common near the tip of the X chromosome. An interesting pattern of low variation at ci^D among D. simulans, D. mauritiana, and D. sechellia suggests that a selective sweep may have occurred among these forms even after divergence into separate species had begun.     

High recombinagenic activities of three antiviral agents, adenine derivatives, in the Drosophila wing spot test

Three adenine derivatives (R,S)-9-(2,3-dihydroxypropyl)adenin (DHPA), D-eritadine (EA), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), prospective antiviral drugs, were subjected to genotoxicity analysis using the somatic mutation and recombinatino test in Drosophila melanogaster. All three compounds were found to be very potent inducers of mosaic spots on Drosophila wings in a dose-related fashion. Data obtained in inversion-free flies revealed that the compounds, in particualr DHPA and EA (nucleoside analogues), are highly effective in the induction of mitotic recombination. PMEA, a nucleotide, exhibited a rather different genotoxic profile from those of DHPA and EA, indicating a different mechanism of genetic action of this compound. Of somatic mutations, chromosome aberrations, rather than point mutations seem to play a major role in the genotoxicity of PMEA. In flies carrying an inversion chromosome, which eliminates most products of mitotic recombination, reduced spot frequencies were obtained, which, however, were still unexpectedly high for compounds with strong recombinagenic activities. Most probably, in additino to structural mutations of chromosomes, double mitotic crossing-over and non-reciprocal recombinatino events similar to unequal sister-strand recombination of gene conversion significantly contributed to spot induction in the inversion heterozygous flies. Concerning the mechanism of genotoxic action, we suggest that these adenine derivatives can be incorporated into DNa chains during replication. This would result, via breaks and DNa repair mechanisms, either in various recombination events or in chromosome aberrations.     

Genetic Screening for Meiotic Mutations of the Female Germline Mosaic Clones in Drosophila melanogaster

A method of screening for meiotic mutations based on genetic analysis of chromosome disjunction in germline mosaic clones of females homozygous for potential mutations is proposed. The clones are obtained at high frequency due to the use of the transgenic FLP/FRT system of mitotic recombination. This system permits obtaining homozygous clones in the first generation after mutagenesis, whereas the cultures are set up after selection for potential meiotic mutations. This significantly enhances, the efficiency of screening by the elimination of the limiting stage. Using this method, the following mutations were revealed in the 3L arm of Drosophila: ff6leading to disturbed centriole disjunction, which results in appearance of multi-tail spermatids and three-pole spindles during male meiosis; ff3leading to the formation of chromosome bridges in anaphase and telophase, chromosome nondisjunction, and premature chromatin condensation after metaphase; embryonic lethal ff29, with disturbed coordination between nuclear and centrosome cycles during syncytial cleavage; and a series of other mutations causing a wide spectrum of disturbances in male meiosis. Comparison of the proposed method with procedures of screening for yeast cell-cycle mutations showed that we succeeded in attaining the efficiency of screening in the Drosophilamodel close to that in the yeast model.     

Architecture of the X Chromosome,Expression of LIM Kinase 1, and Recombination in the agnostic Mutants of Drosophila: A Model for Human Williams Syndrome

As the Human Genome and Drosophila Genome Projects were completed, it became clear that functions of human disease-associated genes may be elucidated by studying the phenotypic expression of mutations affecting their structural or functional homologs in Drosophila.Genomic diseases were identified as a new class of human disorders. Their cause is recombination, which takes place at gene-flanking duplicons to generate chromosome aberrations such as deletions, duplications, inversions, and translocations. The resulting imbalance of the dosage of developmentally important genes arises at a frequency of 10^-3 (higher than the mutation rate of individual genes) and leads to syndromes with multiple manifestations, including cognitive defects. Genomic DNA fragments were cloned from the Drosophila melanogaster agnostic locus, whose mutations impair learning ability and memory. As a result, the locus was exactly localized in X-chromosome region 11AB containing the LIM kinase 1 (LIMK1) gene (CG1848), which is conserved among many species. Hemizygosity for the LIMK1 gene, which is caused by recombination at neighboring extended repeats, underlies cognitive disorders in human Williams syndrome. LIMK1 is a component of the integrin signaling cascade, which regulates the functions of the actin cytoskeleton, synaptogenesis, and morphogenesis in the developing brain. Immunofluorescence analysis revealed LIMK1 in all subdomains of the central complex and the visual system of Drosophila melanogaster.Like in the human genome, theD. melanogaster region is flanked by numerous repeats, which were detected by molecular genetic methods and analysis of ectopic chromosome pairing. The repeats determined a higher rate of spontaneous and induced recombination, including unequal crossing over, in theagnostic gene region. Hence, the agnostic locus was considered as the first D. melanogaster model suitable for studying the genetic defect associated with Williams syndrome in human.     

Homolog pairing and sister chromatid cohesion in heterochromatin in <Emphasis Type="Italic">Drosophila</Emphasis> male meiosis I

Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X–Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.     

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster

Summary The present report describes the recovery and genetic characterization of mutant alleles at zygotic loci on the third chromosome ofDrosophila melanogaster which alter the morphology of the larval cuticle. We derived 12600 single lines from ethyl methane sulfonate (EMS)-treatedst e orrucuca chromosomes and assayed them for embryonic lethal mutations by estimating hatch rates of egg collections. About 7100 of these lines yielded at least a quarter of unhatched eggs and were then scored for embryonic phenotypes. Through microscopic examination of unhatched eggs 1772 lines corresponding to 24% of all lethal hits were classified as embryonic lethal. In 198 lines (2.7% of all lethal hits), mutant embryos showed distinct abnormalities of the larval cuticle. These embryonic visible mutants define 45 loci by complementation analysis. For 32 loci, more than one mutant allele was recovered, with an average of 5.8 alleles per locus. Complementation of all other mutants was shown by 13 mutants. The genes were localized on the genetic map by recombination analysis, as well as cytologically by complementation analysis with deficiencies. They appear to be randomly distributed along the chromosome. Allele frequencies and comparisons with deficiency phenotypes indicate that the 45 loci represent most, if not all, zygotic loci on the third chromosome, where lack of function recognizably affects the morphology of the larval cuticle.     

Recombination, dominance and selection on amino acid polymorphism in the Drosophila genome: contrasting patterns on the X and fourth chromosomes

Surveys of nucleotide polymorphism and divergence indicate that the average selection coefficient on Drosophila proteins is weakly positive. Similar surveys in mitochondrial genomes and in the selfing plant Arabidopsis show that weak negative selection has operated. These differences have been attributed to the low recombination environment of mtDNA and Arabidopsis that has hindered adaptive evolution through the interference effects of linkage. We test this hypothesis with new sequence surveys of proteins lying in low recombination regions of the Drosophila genome. We surveyed >3800 bp across four proteins at the tip of the X chromosome and >3600 bp across four proteins on the fourth chromosome in 24 strains of D. melanogaster and 5 strains of D. simulans. This design seeks to study the interaction of selection and linkage by comparing silent and replacement variation in semihaploid (X chromosome) and diploid (fourth chromosome) environments lying in regions of low recombination. While the data do indicate very low rates of exchange, all four gametic phases were observed both at the tip of the X and across the fourth chromosome. Silent variation is very low at the tip of the X (thetaS = 0.0015) and on the fourth chromosome (thetaS = 0.0002), but the tip of the X shows a greater proportional loss of variation than the fourth shows relative to normal-recombination regions. In contrast, replacement polymorphism at the tip of the X is not reduced (thetaR = 0.00065, very close to the X chromosome average). MK and HKA tests both indicate a significant excess of amino acid polymorphism at the tip of the X relative to the fourth. Selection is significantly negative at the tip of the X (Nes = -1.53) and nonsignificantly positive on the fourth (Nes approximately 2.9), analogous to the difference between mtDNA (or Arabidopsis) and the Drosophila genome average. Our distal X data are distinct from regions of normal recombination where the X shows a deficiency of amino acid polymorphism relative to the autosomes, suggesting more efficient selection against recessive deleterious replacement mutations. We suggest that the excess amino acid polymorphism on the distal X relative to the fourth chromosome is due to (1) differences in the mutation rate for selected mutations on the distal X or (2) a greater relaxation of selection from stronger linkage-related interference effects on the distal X. This relaxation of selection is presumed to be greater in magnitude than the difference in efficiency of selection between X-linked vs. autosomal selection.     

Involvement of the PS03 gene of Saccharomyces cerevisiae in intrachromosomal mitotic recombination and gene amplification

Using a genetic system of haploid strains of Saccharomyces cerevisiae carrying a duplication of the his4 region on chromosome III, the pso3-1 mutation was shown to decrease the rate of spontaneous mitotic intrachromosomal recombination 2- to 13-fold. As previously found for the rad52-1 mutant, the pso3-1 mutant is specifically affected in mitotic gene conversion. Moreover, both mutations reduce the frequency of spontaneous recombination. However, the two mutations differ in the extent to which they affect recombination between either proximally or distally located markers on the two his4 heteroalleles. In addition, amplifications of the his4 region were detected in the pso3-1 mutant. We suggest that the appearance of these amplifications is a consequence of the inability of the pso3-1 mutant to perform mitotic gene conversion.     

A comparison of physical distribution of recombination in chromosome 1R in diploid rye and in hexaploid triticale

Summary Polymorphism for six C-bands on chromosome 1R was used to study the frequency and distribution of recombination along the chromosome in a diploid rye (Secale cereale L.) and in a hexaploid triticale (X Triticosecale Wittmack) derived from it. In rye, the total recombination frequency in five segments of chromosome 1R was 93.7%. Recombination was concentrated in the distal regions of both chromosome arms and was infrequent in the proximal regions. In hexaploid triticale the total recombination frequency in the same chromosome was reduced to 51.7%. In both backgrounds the distal half of the long arm showed similar recombination frequencies, 51.4% and 45.7% for rye and triticale, respectively. The remaining about two-thirds of the chromosome length showed 42.3% recombination in rye but only 6% recombination in triticale. The results demonstrate that the genetic background in which mapping is performed not only affects the total amount of recombination, but also its distribution along the chromosome length.     

Participation of translesion synthesis DNA polymerases in the maintenance of chromosome integrity in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We employed a genetic assay based on illegitimate hybridization of heterothallic Saccharomyces cerevisiae strains (the α-test) to analyze the consequences for genome stability of inactivating translesion synthesis (TLS) DNA polymerases. The α-test is the only assay that measures the frequency of different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material simultaneously. All these events are manifested as illegitimate hybridization and can be distinguished by genetic analysis of the hybrids and cytoductants. We studied the effect of Polζ, Polη, and Rev1 deficiency on the genome stability in the absence of genotoxic treatment and in UV-irradiated cells. We show that, in spite of the increased percent of accurately repaired primary lesions, chromosome fragility, rearrangements, and loss occur in the absence of Polζ and Polη Our findings contribute to further refinement of the current models of translesion synthesis and the organization of eukaryotic replication fork.     

On the increase of chromosome mutations under random mating.

After a short introduction on karyotypes and chromosome mutations, we review the ways by which a chromosome mutation can increase in a random mating population, despite the mutation's deleterious effect on the fertility of heterozygotes. Random drift, segregation distortion, viability advantage, and recombination modification are the mechanisms considered. When possible, the models are illustrated with examples of chromosome mutations involving autosomes in mammals, but the arguments apply, of course, to any genetic factor in any outbreeding species that causes a fertility decrease in heterozygotes.     

How did the guppy Y chromosome evolve?

The sex chromosome pairs of many species do not undergo genetic recombination, unlike the autosomes. It has been proposed that the suppressed recombination results from natural selection favouring close linkage between sex-determining genes and mutations on this chromosome with advantages in one sex, but disadvantages in the other (these are called sexually antagonistic mutations). No example of such selection leading to suppressed recombination has been described, but populations of the guppy display sexually antagonistic mutations (affecting male coloration), and would be expected to evolve suppressed recombination. In extant close relatives of the guppy, the Y chromosomes have suppressed recombination, and have lost all the genes present on the X (this is called genetic degeneration). However, the guppy Y occasionally recombines with its X, despite carrying sexually antagonistic mutations. We describe evidence that a new Y evolved recently in the guppy, from an X chromosome like that in these relatives, replacing the old, degenerated Y, and explaining why the guppy pair still recombine. The male coloration factors probably arose after the new Y evolved, and have already evolved expression that is confined to males, a different way to avoid the conflict between the sexes.     

A molecular map of the apomixis-control locus in <Emphasis Type="Italic">Paspalum procurrens</Emphasis> and its comparative analysis with other species of <Emphasis Type="Italic">Paspalum</Emphasis>

Since apomixis was first mapped in Paspalum, the absence of recombination that characterizes the related locus appeared to be the most difficult bottleneck to overcome for the dissection of the genetic determinants that control this trait. An approach to break the block of recombination was developed in this genus through an among-species comparative mapping strategy. A new apomictic species, P. procurrens (Q4094) was crossed with a sexual plant of P. simplex and their progeny was classified for reproductive mode with the aid of morphological, embryological and genetic analyses. On this progeny, a set of heterologous rice RFLP markers strictly co-segregating in coupling phase with apomixis was identified. These markers were all located on the telomeric region of the long arm of the chromosome 12 of rice. In spite of the lack of recombination exhibited by the apomixis-linked markers in P. procurrens, a comparative mapping analysis among P. simplex, P. malacophyllum, P. notatum and P. procurrens, allowed us to identify a small group of markers co-segregating with apomixis in all these species. These markers bracketed a chromosome region that likely contains all the genetic determinants of apomictic reproduction in Paspalum. The implications of this new inter-specific approach for overcoming the block of recombination to isolate the genetic determinants of apomixis and gain a better comprehension of genome structure of apomictic chromosome region are discussed.     

Meiotic checkpoints and the interchromosomal effect on crossing over in Drosophila females

During prophase of meiosis I, genetic recombination is initiated with a Spo11-dependent DNA double-strand break (DSB). Repair of these DSBs can generate crossovers, which become chiasmata and are important for the process of chromosome segregation. To ensure at least one chiasma per homologous pair of chromosomes, the number and distribution of crossovers is regulated. One system contributing to the distribution of crossovers is the pachytene checkpoint, which requires the conserved gene pch2 that encodes an AAA+ATPase family member. Pch2-dependent pachytene checkpoint function causes delays in pachytene progression when there are defects in processes required for crossover formation, such as mutations in DSB-repair genes and when there are defects in the structure of the meiotic chromosome axis. Thus, the pachytene checkpoint appears to monitor events leading up to the generation of crossovers. Interestingly, heterozygous chromosome rearrangements cause Pch2-dependent pachytene delays and as little as two breaks in the continuity of the paired chromosome axes are sufficient to evoke checkpoint activity. These chromosome rearrangements also cause an interchromosomal effect on recombination whereby crossing over is suppressed between the affected chromosomes but is increased between the normal chromosome pairs. We have shown that this phenomenon is also due to pachytene checkpoint activity.     

A paracentric inversion suppresses genetic recombination at the <Emphasis Type="Italic">FON3</Emphasis> locus with breakpoints corresponding to sequence gaps on rice chromosome 11L

Paracentric inversion is known to inhibit genetic recombination between normal and inverted chromosomal segments in heterozygous arrangements. Insect inversion polymorphisms have been studied to reveal adaptive processes for maintaining genetic variation. We report the first paracentric inversion in rice (Oryza sativa), which was discovered in our effort to clone the floral organ number gene FON3. Recombination at the FON3 locus on the long arm of chromosome 11 was severely suppressed over a distance of more than 36 cM. An extensive screening among 8,242 F₂ progeny failed to detect any recombinants. Cytological analysis revealed a loop-like structure on pachytene chromosomes, whereas FISH analysis showed the migration of a BAC clone from a distal location to a position closer to the centromere. Interestingly, the locations where the genetic recombination suppression began were coincided with the positions of two physical gaps on the chromosome 11, suggesting a correlation between the physical gaps, the inversion breakpoints. Transposons and retrotransposons, and tandemly arranged members of gene families were among the sequences immediately flanking the gaps. Taken together, we propose that the genetic suppression at the FON3 locus was caused by a paracentric inversion. The possible genetic mechanism causing such a spontaneous inversion was proposed.     

Construction of pBR322-ara hybrid plasmids by in vivo recombination

Summary In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analysis demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%–3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araCmutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed.     

Chromosome arm‐specific patterns of polymorphism associated with chromosomal inversions in the major African malaria vector,Anopheles funestus

Chromosomal inversions facilitate local adaptation of beneficial mutations and modulate genetic polymorphism, but the extent of their effects within the genome is still insufficiently understood. The genome of Anopheles funestus, a malaria mosquito endemic to sub‐Saharan Africa, contains an impressive number of paracentric polymorphic inversions, which are unevenly distributed among chromosomes and provide an excellent framework for investigating the genomic impacts of chromosomal rearrangements. Here, we present results of a fine‐scale analysis of genetic variation within the genome of two weakly differentiated populations of Anopheles funestus inhabiting contrasting moisture conditions in Cameroon. Using population genomic analyses, we found that genetic divergence between the two populations is centred on regions of the genome corresponding to three inversions, which are characterized by high values of F_ST, absolute sequence divergence and fixed differences. Importantly, in contrast to the 2L chromosome arm, which is collinear, nucleotide diversity is significantly reduced along the entire length of three autosome arms bearing multiple overlapping chromosomal rearrangements. These findings support the idea that interactions between reduced recombination and natural selection within inversions contribute to sculpt nucleotide polymorphism across chromosomes in An. funestus.     

The effect of chromosome geometry on genetic diversity

Although organisms with linear chromosomes must solve the problem of fully replicating their chromosome ends, this chromosome configuration has emerged repeatedly during bacterial evolution and is evident in three divergent bacterial phyla. The benefit usually ascribed to this topology is the ability to boost genetic variation through increased recombination. But because numerous processes can impact linkage disequilibrium, such an effect is difficult to assess by comparing across bacterial taxa that possess different chromosome topologies. To test directly the contribution of chromosome architecture to genetic diversity and recombination, we examined sequence variation in strains of Agrobacterium Biovar 1, which are unique among sequenced bacteria in having both a circular and a linear chromosome. Whereas the allelic diversity among strains is generated principally by mutations, intragenic recombination is higher within genes situated on the circular chromosome. In contrast, recombination between genes is, on average, higher on the linear chromosome, but it occurs at the same rate as that observed between genes mapping to the distal portion of the circular chromosome. Collectively, our findings indicate that chromosome topology does not contribute significantly to either allelic or genotypic diversity and that the evolution of linear chromosomes is not based on a facility to recombine.     

Occasional recombination of a selfish X‐chromosome may permit its persistence at high frequencies in the wild

The sex‐ratio X‐chromosome (SR) is a selfish chromosome that promotes its own transmission to the next generation by destroying Y‐bearing sperm in the testes of carrier males. In some natural populations of the fly Drosophila neotestacea, up to 30% of the X‐chromosomes are SR chromosomes. To investigate the molecular evolutionary history and consequences of SR, we sequenced SR and standard (ST) males at 11 X‐linked loci that span the ST X‐chromosome and at seven arbitrarily chosen autosomal loci from a sample of D. neotestacea males from throughout the species range. We found that the evolutionary relationship between ST and SR varies among individual markers, but genetic differentiation between SR and ST is chromosome‐wide and likely due to large chromosomal inversions that suppress recombination. However, SR does not consist of a single multilocus haplotype: we find evidence for gene flow between ST and SR at every locus assayed. Furthermore, we do not find long‐distance linkage disequilibrium within SR chromosomes, suggesting that recombination occurs in females homozygous for SR. Finally, polymorphism on SR is reduced compared to that on ST, and loci displaying signatures of selection on ST do not show similar patterns on SR. Thus, even if selection is less effective on SR, our results suggest that gene flow with ST and recombination between SR chromosomes may prevent the accumulation of deleterious mutations and allow its long‐term persistence at relatively high frequencies.