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.     

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.     

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.     

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.     

Homologous Recombination between Episomal Plasmids and Chromosomes in Yeast

We have observed genetic recombination between ura3( -) mutations (among them extensive deletions) carried on "episomal" (i.e., 2micro DNA-containing) plasmids and other ura3( -) alleles present at the normal chromosomal URA3 locus. The recombination frequency found was comparable to the level observed for classical mitotic recombination but was relatively insensitive to sunlamp radiation, which strongly stimulates mitotic recombination. Three equally frequent classes could be distinguished among the recombinants. Two of these are the apparent result of gene conversions (or double crossovers) which leave the URA3(+) allele on the chromosome (class I) or on the plasmid (class II). The third class is apparently due to a single crossover that results in the integration of the plasmid into a chromosome. Plasmid-chromosome recombination can be useful in fine structure genetic mapping, since recombination between a chromosomal point mutation and a plasmid-borne deletion mutation only 25 base pairs distant was easily detected.     

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.     

Genetic control of recombination processes in Aspergillus nidulans. I. Effect of uvs-mutations on the frequency of spontaneous and ultraviolet ray-induced intergenic recombination]

Effect of 3 uvs mutations (uvs 12, 19 and 25) on recombination processes in Aspergillus nidulans is studied. All the mutations are found either to affect the fertility of carp bodies and germination ability of askospores, or result in complete inability of heterokaryons to form cleistocarpia. Two mutations change the frequency of spontaneous meitotic crossing-over at pro-paba region of the chromosome I and do not affect the rate of mitotic recombination at w-centromeric region of the chromosome II: uvs 12 mutation increases, and uvs 19 mutation decreases the frequency of meiotic recombination. One mutation (uvs 25) decreases the rate of spontaneous mitotic crossing-over. All uvs mutations decrease the frequency of VU light induced mitotic recombination at w-centromeric region of the chromosome II. The data obtained, together with earlier reported characteristics of uvs mutants, suggest that recombination mechanisms in yeast participate in reparation processes more actively than in prokariotes. Different effects of the same uvs mutations on spontaneous frequency of meiotic and mitotic crossing-over draw to the conclusion that genetic control and molecular mechanisms of these processes in A. nidulans are not identical.     

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.     

New class of Bacillus subtilis glutamine-requiring mutants

By using genetic analysis, the mutations of eight glutamine-requiring mutants isolated from Bacillus subtilis 168 were all shown to be linked to the thyA marker. A three-factor transduction analysis performed with one of the gln mutations indicated that the gene order in this region of the B. subtilis chromosome was gltA-thyA-gln. On the basis of recombination index values, two closely linked groups were identified. The mutations belonging to one group were assigned to the structural gene for glutamine synthetase, and those belonging to the other group might impair a regulatory locus. The residual glutamine synthetase activities and the cross-reacting materials of the mutants from both recombination groups supported these conclusions.     

Meiotic Mutants That Cause a Polar Decrease in Recombination on the X Chromosome in Caenorhabditis Elegans

Recessive mutations in three autosomal genes, him-1, him-5 and him-8, cause high levels of X chromosome nondisjunction in hermaphrodites of Caenorhabditis elegans, with no comparable effect on autosomal disjunction. Each of the mutants has reduced levels of X chromosome recombination, correlating with the increase in nondisjunction. However, normal or elevated levels of recombination occur at the end of the X chromosome hypothesized to contain the pairing region (the left end), with recombination levels decreasing in regions approaching the right end. Thus, both the number and the distribution of X chromosome exchange events are altered in these mutants. As a result, the genetic map of the X chromosome in the him mutants exhibits a clustering of genes due to reduced recombination, a feature characteristic of the genetic map of the autosomes in non-mutant animals. We hypothesize that these him genes are needed for some processive event that initiates near the left end of the X chromosome.     

The influence of recombination on human genetic diversity

In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution.     

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.     

Genetic Evidence for Two T Complex Tail Interaction (Tct) Loci in T Haplotypes

The t complex on chromosome 17 of the house mouse is an exceptional model for studying the genetic control of transmission ratio, gametogenesis, and embryogenesis. Partial haplotypes derived through rare recombination between a t haplotype and its wild-type homolog have been essential in the genetic analysis of these various properties of the t complex. A new partial t haplotype, which was derived from the complete tw71 haplotype and which is called tw71Jr1, was shown to have unexpected effects on tail length and unique recombination breakpoints. This haplotype, either when homozygous or when heterozygous with the progenitor tw71 haplotype, produced short-tailed rather than normal-tailed mice on certain genetic backgrounds. Genetic analysis of this exceptional haplotype showed that the recombination breakpoints were different from those leading to any other partial t haplotype. Based on this haplotype, a model is proposed that accounts for genetic interactions between the brachyury locus (T), the t complex tail interaction (tct) locus, and their wild-type homolog(s) that determine tail length. An important part of this model is the hypothesis that the tct locus, which enhances the tail-shortening effect of T mutations, is in fact at least two, genetically separable genes with different genetic activities. Genetic analysis of parental and recombinant haplotypes also suggests that intrachromosomal recombination involving an inverted duplicated segment can account for the variable orientation of loci within an inverted duplication on wild-type homologs of the t haplotype.     

Genetic analysis of the RecE pathway of genetic recombination in Escherichia coli K-12.

The RecE pathway of genetic recombination in Escherichia coli K-12 was defined to be the pathway that is utilized in deoxyribonucleic acid exonuclease V (ExoV)-defective cells which express constitutively recE+, the structural gene for deoxyribonucleic acid exonuclease VIII. Dependence on ExoVIII was shown by the occurrence in a recB21 sbcA23 strain of recombination deficiency mutations in recE, the structural gene for ExoVIII. Point mutations in recE were found as well as deletion mutations in which the entire Rac prophage, carrying recE, was lost. In addition, strain construction and mutagenesis revealed the dependence of the RecE pathway on recA+ and on recF+. Dependence on a fourth gene was shown by a mutation (rec-77) which does not map near the other genes. The problem of distinguishing the RecE pathway from that previously called RecF is discussed.     

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.     

The temporal dynamics of processes underlying Y chromosome degeneration

Y chromosomes originate from ordinary autosomes and degenerate by accumulating deleterious mutations. This accumulation results from a lack of recombination on the Y and is driven by interference among deleterious mutations (Muller's ratchet and background selection) and the fixation of beneficial alleles (genetic hitchhiking). Here I show that the relative importance of these processes is expected to vary over the course of Y chromosome evolution due to changes in the number of active genes. The dominant mode of degeneration on a newly formed gene-rich Y chromosome is expected to be Muller's ratchet and/or background selection due to the large numbers of deleterious mutations arising in active genes. However, the relative importance of these modes of degeneration declines rapidly as active genes are lost. In contrast, the rate of degeneration due to hitchhiking is predicted to be highest on Y chromosomes containing an intermediate number of active genes. The temporal dynamics of these processes imply that a gradual restriction of recombination, as inferred in mammals, will increase the importance of genetic hitchhiking relative to Muller's ratchet and background selection.     

Nucleotide variation and recombination along the fourth chromosome in Drosophila simulans

The fourth chromosome of Drosophila melanogaster and its sister species are believed to be nonrecombining and have been a model system for testing predictions of the effects of selection on linked, neutral variation. We recently examined nucleotide variation along the chromosome of D. melanogaster and revealed that a low average level of recombination could be associated with considerably high levels of nucleotide variation. In this report, we further investigate the variation along the fourth chromosome of D. simulans. We sequenced 12 gene regions evenly distributed along the fourth chromosome for a worldwide collection of 11 isofemale lines and 5 gene regions in a local population of 10 isofemale lines from South America. In contrast to predictions for regions of very low recombination, these data reveal that the variation levels in many gene regions, including an intron region of the ci gene, vary considerably along the fourth chromosome. Nucleotide diversity ranged from 0.0010 to 0.0074 in 9 gene regions interspersed with several regions of greatly reduced variation. Tests of recombination indicate that the recombination level is not as low as previously thought, likely an order of magnitude higher than that in D. melanogaster. Finally, estimates of the recombination parameters are shown to support a crossover-plus-conversion model.     

Genetic screening of meiotic mutations in mosaic clones of the Drosophila melanogaster female germline]

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: ff6 leading to disturbed centriole disjunction, which results in appearance of multi-tail spermatids and three-pole spindles during male meiosis; ff3 leading 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 Drosophila model close to that in the yeast model.     

Suppression of recJ mutations of Escherichia coli by mutations in translation initiation factor IF3.

We have isolated genetic suppressors of mutations in the recJ gene of Escherichia coli in a locus we term srjA. These srjA mutations cause partial to complete alleviation of the recombination and UV repair defects conferred by recJ153 and recJ154 mutations in a recBC sbcA genetic background. The srjA gene was mapped to 37.5 min on the E. coli chromosome. This chromosomal region from the srjA5 strain was cloned into a plasmid vector and was shown to confer recJ suppression in a dominant fashion. Mutational analysis of this plasmid mapped srjA to the infC gene encoding translation initiation factor 3 (IF3). Sequence analysis revealed that all three srjA alleles cause amino acid substitutions of IF3. Suppression of recJ was shown to be allele specific: recJ153 and recJ154 mutations were suppressible, but recJ77 and the insertion allele recJ284::Tn10 were not. In addition, growth medium-conditional lethality was observed for strains carrying srjA mutations with the nonsuppressible recJ alleles. When introduced into recJ+ strains, srjA mutations conferred hyperrecombinational and hyper-UVr phenotypes. An interesting implication of these genetic properties of srjA suppression is that IF3 may regulate the expression of recJ and perhaps other recombination genes and hence may regulate the recombinational capacity of the cell.     

Genetic Analysis of Fusion Recombinants in Bacillus Subtilis: Function of the Rece Gene

Bacillus subtilis protoplast fusion allows the study of the genetic recombination of an entire procaryotic genome. Protoplasts from bacterial strains marked genetically by chromosomal mutations were fused using polyethylene glycol and the regenerated cells analyzed. Recombinants represent 19.3% of heterozygotic cells; they are haploids. Individual characterization of clones show a unique particular phenotype in each colony suggesting that recombination takes place immediately after fusion, probably before the first cellular division. Recombination occurs in the whole chromosome; in one-third of the cases both reciprocal recombinants could be shown in the colony. The genetic interval that includes the chromosome replication origin shows the highest recombination level. Our results suggest that the RecE protein accounts for most of the fused protoplast recombination; however, some "replication origin-specific" recombination events were independent of the recE gene product.