Recombination Can Initiate and Terminate at a Large Number of Sites within the Rosy Locus of Drosophila Melanogaster

This report presents the results of a recombination experiment designed to question the existence of special sites for the initiation or termination of a recombination heteroduplex within the region of the rosy locus. Intragenic recombination events were monitored between two physically separated rosy mutant alleles ry301 and ry2 utilizing DNA restriction site polymorphisms as genetic markers. Both ry301 and ry2 are known from previous studies to be associated with gene conversion frequencies an order of magnitude lower than single site mutations. The mutations are associated with large, well defined insertions located as internal sites within the locus in prior intragenic mapping studies. On the molecular map, they represent large insertions approximately 2.7 kb apart in the second and third exons, respectively, of the XDH coding region. The present study monitors intragenic recombination in a mutant heterozygous genotype in which DNA homology is disrupted by these large discontinuities, greater than the region of DNA homology and flanking both sides of the locus. If initiation/or termination requires separate sites at either end of the locus, then intragenic recombination within the rosy locus of the heterozygote should be eliminated. Contrary to expectation, significant recombination between these sites is seen.     

Molecular Mapping of the ROSY Locus in DROSOPHILA MELANOGASTER

The DNA from the chromosomal region of the Drosophila rosy locus has been examined in 83 rosy mutant strains. Several spontaneous and radiation-induced alleles were associated with insertions and deletions, respectively. The lesions are clustered in a 4-kb region. Some of the alleles identified on the DNA map have been located on the genetic map by fine-structure recombination experiments. The genetic and molecular maps are collinear, and the alignment identifies the DNA location of the rosy control region. A rosy RNA of 4.5 kb has been identified; its 5' end lies in or near the control region.     

Recombination and selection in the maintenance of the adaptive value of inversions

A huge amount of data seem to confirm the adaptive value of inversions in Drosophila. The inhibition of recombination in heterokaryotypes mediated by inversions seems fundamental in maintaining their adaptive role. This study shows that recombination is highly suppressed in Drosophila subobscura because of chromosomal inversions, not only inside the inversions but also outside them. It seems that the region outside the inversion where recombination is inhibited is asymmetrical and independent of the inversion length. Despite the difficulty of crossovers taking place near inversion breakpoints, the only two recombination events detected inside inversions were located close to the breakpoint. Thus, selection could be largely responsible for the recombination reduction maintaining sets of adaptive alleles inside the inverted region. Heterokaryotype descendants were always in higher frequency than inbred or outbred homokaryotypes, regardless of the geographical origin of the chromosome, suggesting that chromosomes carrying the same arrangement, although with a different set of alleles for neutral markers, could be submitted to the same selection processes.     

The Effect of DNA Sequence Polymorphisms on Intragenic Recombination in the Rosy Locus of Drosophila Melanogaster

The effect of simple DNA sequence polymorphisms on intragenic recombination in the rosy locus of Drosophila melanogaster was assayed. Two crosses were performed involving nearly identical molecular distances between selective ry null mutations (3778 nucleotides and 3972 nucleotides). In one heterozygote (ry606/ry531), in addition to the nucleotide substitution ry- mutations, there were 11 simple nucleotide polymorphisms between the selective markers as well as additional flanking simple nucleotide polymorphisms within the rosy locus. In the other heterozygote (ry606/ry609), there were no additional polymorphisms because the two rosy nucleotide substitution mutations were induced on the same rosy isoallele (ry+6). From ry606/ry531 heterozygous females, 27 intragenic crossovers and five marker conversions were seen among 4.53 x 10(5) progeny. From ry606/ry609 heterozygous females, 23 intragenic crossovers and eight marker conversions were seen among 4.18 x 10(5) progeny. The intragenic crossover frequencies per kilobase of DNA were very similar, 1.6 x 10(-5) for ry606/ry531 and 1.4 x 10(-5) for ry606/ry609. Thus, simple DNA sequence polymorphisms neither inhibit nor promote intragenic recombination in D. melanogaster.     

Studies of the species barrier between Drosophila subobscura and D. madeirensis V: the importance of sex-linked inversion in preserving species identity

The X chromosome is known to exert a disproportionately large effect on characters related to post-zygotic reproductive isolation. There is also growing evidence about the important role of the chromosomal regions with reduced recombination (such as inversions) in maintaining the identity of closely related species. Using molecular markers, we examine the effect of different regions of the X chromosome on determination of hybrid traits (viability, testes size, sperm motility and morphological anomalies) in hybrid males between Drosophila madeirensis and Drosophila subobscura. The preponderant effect of a region localized inside the A2 inversion in the X chromosome in all hybrid traits is identified. Other marked regions exert a weaker influence or only influence some of the hybrid trait. Our results confirm the crucial role of sex-linked chromosomal inversion in preserving the identity of species with incomplete reproductive isolation. The specific genomic make-up of parental lines used to perform crosses has a great effect on hybrid fitness.     

Two new balancer chromosomes on mouse chromosome 4 to facilitate functional annotation of human chromosome 1p

To facilitate genetic screens to identify and maintain recessive mutations that map to the short arm of human chromosome 1, we have utilized chromosome engineering to generate two mouse strains that carry large inversions on the distal region of mouse chromosome 4. The inversion intervals are 16 and 22 cM in size together they cover approximately half of chromosome 4. Since recombination between the wild-type and inversion chromosomes does not occur within these inversion intervals, mutant alleles of genes mapping to this region can be identified and maintained. Therefore, these inversion chromosomes work as balancer chromosomes. These inversions have the additional advantage that they are tagged with genes encoding the visible coat color markers tyrosinase and agouti, and therefore the dosage of the inversion chromosome (+/+, Inv/+, Inv/Inv) can be visually recognized. These inversion strains will be extremely useful for mutagenesis screens that focus on functional annotation of human chromosome 1p.     

Cytogenetic Analysis of the Chromosomal Region Immediately Adjacent to the Rosy Locus in DROSOPHILA MELANOGASTER

This report describes the genetic analysis of a region of the third chromosome of Drosophila melanogaster extending from 87D2–4 to 87E12–F1, an interval of 23 or 24 polytene chromosome bands. This region includes the rosy (ry, 3–52.0) locus, carrying the structural information for xanthine dehydrogenase (XDH). We have, in recent years, focused attention on the genetic regulation of the rosy locus and, therefore, wished to ascertain in detail the immediate genetic environment of this locus. Specifically, we question if rosy is a solitary genetic unit or part of a larger complex genetic unit encompassing adjacent genes. Our data also provide opportunity to examine further the relationship between euchromatic gene distribution and polytene chromosome structure.——The results of our genetic dissection of the rosy microregion substantiate the conclusion drawn earlier (Schalet, Kernaghan and Chovnick 1964) that the rosy locus is the only gene in this region concerned with XDH activity and that all adjacent genetic units are functionally, as well as spatially, distinct from the rosy gene. Within the rosy micro-region, we observed a close correspondence between the number of complementation groups (21) and the number of polytene chromosome bands (23 or 24). Consideration of this latter observation in conjunction with those of similar studies of other chhromosomal regions supports the hypothesis that each polytene chromosome band corresponds to a single genetic unit.     

Genetic Limits of the Xanthine Dehydrogenase Structural Element within the Rosy Locus in DROSOPHILA MELANOGASTER

Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3:52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH). This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.     

Normal synaptonemal complex and abnormal recombination nodules in two alleles of the Drosophila meiotic mutant mei-W68

The meiotic phenotypes of two mutant alleles of the mei-W68 gene, 1 and L1, were studied by genetics and by serial-section electron microscopy. Despite no or reduced exchange, both mutant alleles have normal synaptonemal complex. However, neither has any early recombination nodules; instead, both exhibit high numbers of very long (up to 2 microm) structures here named "noodles." These are hypothesized to be formed by the unchecked extension of identical but much shorter structures ephemerally seen in wild type, which may be precursors of early recombination nodules. Although the mei-W68(L1) allele is identical to the mei-W68(1) allele in both the absence of early recombination nodules and a high frequency of noodles (i.e., it is amorphic for the noodle phene), it is hypomorphic in its effects on exchange and late recombination nodules. The differential effects of this allele on early and late recombination nodules are consistent with the hypothesis that Drosophila females have two separate recombination pathways-one for simple gene conversion, the other for exchange.     

The genotoxicity of chromium(VI) oxide in the wing spot test of Drosophila melanogaster is over 90% due to mitotic recombination.

Chromium(VI) oxide and chromium(III) chloride were tested in the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster according to standard procedures. The hexavalent compound was highly genotoxic in both chronic and acute treatments whereas the trivalent one was clearly negative. Further analysis of wings carrying an inversion chromosome which eliminates all recombination events showed that over 90% of the spots induced by chromium(VI) oxide are due to mitotic recombination.     

"Mode for mutation" in the natural population of Drosophila melanogaster from Umani is caused by distribution of a hobo-induced inversion in the regulatory region of the yellow gene

A mutation outburst of the yellow gene occurred in a Drosophila melanogaster population from the town of Uman' from 1982 to 1991 and was associated with the instability of several alleles. Molecular genetic analysis revealed a deletion variant of the hobo transposable element in the same site of the regulatory region of yellow in the mutant alleles and their derivatives. The outburst of the yellow-2 mutations was attributed to the spreading of the X chromosome, which contained an inversion of the yellow regulatory region, through the population. Reinversion resulted in the wild-type phenotype. Crossing lines carrying the inversion with laboratory line C(1)DX, ywf induced instability of the yellow alleles, which was associated with duplication or multiplication of a fragment of the yellow gene. Most derivative lines eventually became stable. The loss of instability was not associated with phenotypic changes; molecular genetic changes included a loss of the duplicated sequences or a deletion of the inverted regulatory region of the yellow gene.     

Use of a Recombination Reporter Insert To Define Meiotic Recombination Domains on Chromosome III of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by DNA double-strand breaks (DSBs). DSBs usually occur in intergenic regions that display nuclease hypersensitivity in digests of chromatin. DSBs are distributed nonuniformly across chromosomes; on chromosome III, DSBs are concentrated in two "hot" regions, one in each chromosome arm. DSBs occur rarely in regions within about 40 kb of each telomere and in an 80-kb region in the center of the chromosome, just to the right of the centromere. We used recombination reporter inserts containing arg4 mutant alleles to show that the "cold" properties of the central DSB-deficient region are imposed on DNA inserted in the region. Cold region inserts display DSB and recombination frequencies that are substantially less than those seen with similar inserts in flanking hot regions. This occurs without apparent change in chromatin structure, as the same pattern and level of DNase I hypersensitivity is seen in chromatin of hot and cold region inserts. These data are consistent with the suggestion that features of higher-order chromosome structure or chromosome dynamics act in a target sequence-independent manner to control where recombination events initiate during meiosis.     

Spontaneous Chromosome Breakage at Male Meiosis Associated with Male Recombination in DROSOPHILA MELANOGASTER

An inbred line (OK1) of Drosophila melanogaster , recently derived from a natural population in Oklahoma, has been found by Woodruff and Thompson to exhibit a low frequency of spontaneous male recombination when outcrossed to marker stocks. There is also a reciprocal-cross effect, such that recombination is found only if OK1 males are used in the initial cross. When OK1 females are used, however, male recombination is again found if their male progeny are used for a subsequent cross.-In the present cytological analysis, chromosome behavior at male meiosis was studied in reciprocal crosses between the OK1 line and both a marker gene stock and an inversion stock. If the recombination events were "conventional" and premeiotic (gonial) in origin, no chromosome aberrations would be expected during meiosis. If they were "conventional" and meiotic, some dicentric bridges with free fragments would be expected in the inversion heterozygote, but none should be present in the marker gene cross.-The results demonstrated that the occurrence of recombination in males is most likely a meiotic event, though the occurrence of some limited premeiotic recombination can not be disproven. Meiosis was found to be perfectly normal in all crosses lacking male recombination. In all of the inversion stock and noninversion marker stock crosses that showed male recombination, however, anaphase bridges were found at both first and second meiotic divisions. These were often accompanied by more than the single fragment expected from a conventional inversion bridge and fragment situation. In extreme cases, almost complete pulverization of one or more autosomes was found.-All metaphase I stages were perfectly normal, suggesting that no comparable breakage occurs in premeiotic gonial mitoses. The form of chromosome damage is similar in many ways to that produced by some DNA synthesis inhibitors, or by some viral or mycoplasma infections. This possibility is discussed, and some of the evolutionary implications of the system are briefly considered.     

Approaches to Half-Tetrad Analysis in Bacteria: Recombination between Repeated, Inverse-Order Chromosomal Sequences

In standard bacterial recombination assays, a linear fragment of DNA is transferred to a recipient cell and, at most, a single selected recombinant type is recovered from each merozygote. This contrasts with fungal systems, for which tetrads allow recovery of all meiotic products, including both ultimate recombinant products of an apparent single act of recombination. We have developed a bacterial recombination system in which two recombining sequences are placed in inverse order at widely separated sites in the circular chromosome of Salmonella typhimurium. Recombination can reassort markers between these repeated sequences (double recombination and apparent gene conversion), or can exchange flanking sequences, leading to inversion of the chromosome segment between the recombining sequences. Since two recombinant products remain in the chromosome of a recombinant with an inversion, one can, in principle, approach the capability of tetrad analysis. Using this system, the following observations have been made. (a) When long sequences (40 kb) recombine, conversion frequently accompanies exchange of flanking sequences. (b) When short sequences (5 kb) recombine, conversion rarely accompanies exchange of flanks. (c) Both recA and recB mutations eliminate inversion formation. (d) The frequency of exchanges between short repeats is more sensitive to the distance separating the recombining sequences in the chromosome. The results are presented with the assumption that inversions occur by simple interaction of two sequences in the same circular chromosome. In an appendix we discuss mechanistically more complex possibilities, some of which could also apply to standard fungal systems.     

The interchromosomal effect of inversions in maize

Summary The interchromosomal effect of inversions in maize along the short arm of chromosome 9 yields results which are distinctly different from those which are reported with Drosophila melanogaster. Recombination was increased in the c ₁-sh₁ region of chromosome 9 while the sh ₁-wx region was unaffected. This increased frequency of recombination appears to be due to an increase in single exchange events as multiple events were unchanged. Increases in recombination were accompanied by either an increase in chromosome interference or normal interference levels. The magnitude of increase in recombination was much smaller than that seen in interchromosomal effects in Drosophila and is consistent with other observations made in maize. When two inversions were present in the same nucleus simultaneously, the effect on recombination was of the same magnitude as the effect of a single inversion. All inversions tested, regardless of size or position with respect to the centromere showed the same magnitude of increase.     

Recombination Load in a Chromosomal Inversion Polymorphism of Drosophila subobscura

Chromosomal inversions suppress recombination in heterokaryotypes and may help to maintain positive epistatic interactions among groups of alleles at loci contained in the inversion. Here I evaluate the protective effect of inversions on recombination when different chromosomal segments, or even the whole chromosome O of Drosophila subobscura, can be effectively prevented from undergoing recombination in several naturally occurring heterokaryotypes. The fitness of flies made homozygous for recombinant chromosomes was generally lower when compared to their nonrecombinant counterparts, thus suggesting that segregating gene arrangements in this species hold together favorable combinations of alleles that interact epistatically.     

Analysis of crossing-over in a family with translocation 9;10 involving a chromosome 9 with a pericentric inversion

Summary The presence of two markers on chromosome 9, both a balanced reciprocal translocation and an inversion, allows morphologic demonstration of recombination between the normal and rearranged homologues. In the family under discussion 50% of the progeny studied (two of four) received a translocated 9 without the inversion from a parent with a translocated and inverted 9, indicating crossing-over between members of the chromosome 9 pair. Thus the morphology of the chromosomes allows a recombinat event which is normally invisible to be seen cytologically. Theoretically after crossing-over the balanced reciprocal translocation heterozygote results from adjacent-1 segregation and unbalanced derivative chromosome combinations from alternate segregation. Therefore it cannot be assumed that the balanced progeny necessarily result from alternate segregation and the unbalanced from adjacent-1. The prenatal diagnostic studies presented in this report also show that chromosome analysis of other family members is required when the recombination between homologues produces differences in chromosome morphology between parent and fetus.     

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.