Recombination rates of human microRNA

The fact that microRNAs play a role in almost all biological processes is well established, as is the importance of recombination in generating genome variability. However, the association between microRNAs and recombination remains largely unknown. In order to investigate the recombination patterns of microRNAs, we performed a comprehensive analysis of the recombination rate of human microRNAs. We observed that microRNAs that are expressed in several tissues tend to have lower recombination rates than tissue-specific microRNAs. Additionally, microRNAs that are associated with a number of diseases are also likely to have lower recombination rates. Furthermore, microRNAs with higher expression levels are found to have fewer recombination events. These findings reveal patterns in recombination rates of microRNAs that could help in understanding the function, evolution, and disease-related roles of microRNAs.     

Sex-Related Differences in Crossing over in Caenorhabditis Elegans

In the nematode Caenorhabditis elegans, hermaphrodite recombination has been characterized and is the basis of the genetic map used in this organism. In this study we have examined male recombination on linkage group I and have found it to be approximately one-third less than that observed in the hermaphrodite. This decrease was interval-dependent and nonuniform. We observed less recombination in the male in 5 out of 6 intervals examined, and no observable difference in one interval on the right end of LG I. Hermaphrodite recombination frequencies are the result of recombination in two germlines; oocyte and hermaphrodite spermatocytes. We have measured recombination in the oocyte and have found it to be approximately twofold lower than that calculated for hermaphrodite spermatocytes and not significantly different from the male spermatocyte frequency. Thus, recombination frequencies appear to be a function of gonad physiology rather than the sex of the germline. Evidence from experiments examining the effect of karyotype on recombination in males sexually transformed by the her-1 mutation into XO hermaphrodites (normally XX), suggests the sexual phenotype rather than genotype determines the recombination frequency characteristic of a particular sex. Hermaphrodite recombination is known to be affected by temperature, maternal age, and the rec-1 mutation. We have examined the effect of these parameters on recombination in the male and have found male recombination frequency increased with elevated temperatures and in the presence of Rec-1, and decreased with paternal age.     

RNA recombination in animal and plant viruses.

An increasing number of animal and plant viruses have been shown to undergo RNA-RNA recombination, which is defined as the exchange of genetic information between nonsegmented RNAs. Only some of these viruses have been shown to undergo recombination in experimental infection of tissue culture, animals, and plants. However, a survey of viral RNA structure and sequences suggests that many RNA viruses were derived form homologous or nonhomologous recombination between viruses or between viruses and cellular genes during natural viral evolution. The high frequency and widespread nature of RNA recombination indicate that this phenomenon plays a more significant role in the biology of RNA viruses than was previously recognized. Three types of RNA recombination are defined: homologous recombination; aberrant homologous recombination, which results in sequence duplication, insertion, or deletion during recombination; and nonhomologous (illegitimate) recombination, which does not involve sequence homology. RNA recombination has been shown to occur by a copy choice mechanism in some viruses. A model for this recombination mechanism is presented.     

Recombination occurs uniformly within the bronze gene, a meiotic recombination hotspot in the maize genome.

The bronze (bz) gene is a recombinational hotspot in the maize genome: its level of meiotic recombination per unit of physical length is > 100-fold higher than the genome's average and is the highest of any plant gene analyzed to date. Here, we examine whether recombination is also unevenly distributed within the bz gene. In yeast genes, recombination (conversion) is polarized, being higher at the end of the gene where recombination is presumably initiated. We have analyzed products of meiotic recombination between heteroallelic pairs of bz mutations in both the presence and absence of heterologies and have sequenced the recombination junction in 130 such Bz intragenic recombinants. We have found that in the absence of heterologies, recombination is proportional to physical distance across the bz gene. The simplest interpretation for this lack of polarity is that recombination is initiated randomly within the gene. Insertion mutations affect the frequency and distribution of intragenic recombination events at bz, creating hotspots and coldspots. Single base pair heterologies also affect recombination, with fewer recombination events than expected by chance occurring in regions of the bz gene with a high density of heterologies. We also provide evidence that meiotic recombination in maize is conservative, that is, it does not introduce changes, and that meiotic conversion tracts are continuous and similar in size to those in yeast.     

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammalian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct DNA segments to predetermined chromosomal locations by homologous recombination.     

Manipulating mitotic recombination in the zebrafish embryo through RecQ helicases

RecQ DNA helicases resolve Rad-51-mediated recombination and suppress aberrant homologous recombination. RecQ gene loss is associated with cancer susceptibility and increased mitotic recombination. We have developed an in vivo assay based on a zebrafish pigment mutant for suppression of RecQ activity, and demonstrate that zebrafish RecQ genes have conserved function in suppressing mitotic recombination.     

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.     

Topological analysis of Hin-catalysed DNA recombination in vivo and in vitro

In vitro studies have demonstrated that Hin-catalysed site-specific DNA inversion occurs within a tripartite invertasome complex assembled at a branch on a supercoiled DNA molecule. Multiple DNA exchanges within a recombination complex (processive recombination) have been found to occur with particular substrates or reaction conditions. To investigate the mechanistic properties of the Hin recombination reaction in vivo, we have analysed the topology of recombination products generated by Hin catalysis in growing cells. Recombination between wild-type recombination sites in vivo is primarily limited to one exchange. However, processive recombination leading to knotted DNA products is efficient on substrates containing recombination sites with non-identical core nucleotides. Multiple exchanges are limited by a short DNA segment between the Fis-bound enhancer and closest recombination site and by the strength of Fis-Hin interactions, implying that the enhancer normally remains associated with the recombining complex throughout a single exchange reaction, but that release of the enhancer leads to multiple exchanges. This work confirms salient mechanistic aspects of the reaction in vivo and provides strong evidence for the propensity of plectonemically branched DNA in prokaryotic cells. We also demonstrated that a single DNA exchange resulting in inversion in vitro is accompanied by a loss of four negative supercoils.     

Study of two different recombination events in maize cmsT-regenerated plants during reversion to fertility

Summary The change of phenotype from sterility to fertility for some cmsT callus tissue culture regenerated plants and their progenies has been correlated with changes in their mitochondrial genome. Those changes that have been analyzed here are the result of recombination events. Two different sets of repeated sequences have been found to be involved in those recombination events. The most common one is a recombination through a 127-bp repeat between various independently isolated revertants. The second one is a recombination through a 58-bp repeat. In every case the products of recombination containing the urf13 gene have been deleted.     

Mechanisms of nondisjunction in human spermatogenesis

A reduction in recombination in the pseudoautosomal region is associated with an increased frequency of aneuploid 24,XY human sperm. Similarly, individuals with paternally derived Klinefelter syndrome (47,XXY) also have a paucity of recombination in the chromosomes that have undergone nondisjunction. Meiotic studies using newly developed immunocytogenetic techniques have demonstrated errors of chromosome synapsis and significantly reduced recombination in infertile men with nonobstructive azoospermia. These men have an increased risk of aneuploidy in sperm that have been surgically removed from the testes. Thus, evidence is starting to accumulate that reduced recombination has a marked effect on the generation of aneuploid sperm.     

Distance-Independence of Mitotic Intrachromosomal Recombination in Saccharomyces Cerevisiae

Many genetic studies have shown that the frequency of homologous recombination depends largely on the distance in which recombination can occur. We have studied the effect of varying the length of duplicated sequences on the frequency of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. We find that the frequency of recombination resulting in the loss of one of the repeats and the intervening sequences reaches a plateau when the repeats are short. In addition, the frequency of recombination to correct a point mutation contained in one of these repeats is not proportional to the size of the duplication but rather depends dramatically on the location of the mutation within the repeated sequences. However, the frequency of mitotic interchromosomal reciprocal recombination is dependent on the distance separating the markers. The difference in the response of intrachromosomal and interchromosomal mitotic recombination to increasing lengths of homology may indicate there are different rate-limiting steps for recombination in these two cases. These findings have important implications for the maintenance and evolution of duplicated sequences.     

A neutral explanation for the correlation of diversity with recombination rates in humans

One of the most striking findings to emerge from the study of genomic patterns of variation is that regions with lower recombination rates tend to have lower levels of intraspecific diversity but not of interspecies divergence. This uncoupling of variation within and between species has been widely interpreted as evidence that natural selection shapes patterns of genetic variability genomewide. We revisited the relationship between diversity, divergence, and recombination in humans, using data from closely related species and better estimates of recombination rates than previously available. We show that regions that experience less recombination have reduced divergence to chimpanzee and to baboon, as well as lower levels of diversity. This observation suggests that mutation and recombination are associated processes in humans, so that the positive correlation between diversity and recombination may have a purely neutral explanation. Consistent with this hypothesis, diversity levels no longer increase significantly with recombination rates after correction for divergence to chimpanzee.     

Crossing over is rarely associated with mitotic intragenic recombination in Schizosaccharomyces pombe

Chromosomal rearrangements can result from crossing over during ectopic homologous recombination between dispersed repetitive DNA. We have previously shown that meiotic ectopic recombination between artificially dispersed ade6 heteroalleles in the fission yeast Schizosaccharomyces pombe frequently results in chromosomal rearrangements. The same recombination substrates have been studied in mitotic recombination. Ectopic recombination rates in haploids were approximately 1-4 x 10(-6) recombinants per cell generation, similar to allelic recombination rates in diploids. In contrast, ectopic recombination rates in heterozygous diploids were 2.5-70 times lower than allelic recombination or ectopic recombination in haploids. These results suggest that diploid-specific factors inhibit ectopic recombination. Very few crossovers occurred in ade6 mitotic recombination, either allelic or ectopic. Allelic intragenic recombination was associated with 2% crossing over, and ectopic recombination between multiple different pairing partners showed 1-7% crossing over. These results contrast sharply with the 35-65% crossovers associated with meiotic ade6 recombination and suggest either differential control of resolution of recombination intermediates or alternative pathways of recombination in mitosis and meiosis.     

The minimum amount of homology required for homologous recombination in mammalian cells.

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.     

Genetic Control of Chromosomal and Plasmid Recombination in STAPHYLOCOCCUS AUREUS

Recombination-deficient mutants of Staphylococcus aureus have been isolated and found to have properties similar to those of recombination-deficient Escherichia coli. In addition, one Rec(-) mutant was found to be defective in the restriction and modification of DNA. There is a marked reduction ( approximately 10(4)-fold) in recombination between penicillinase plasmids in the Rec(-) mutants suggesting that these elements do not encode an efficient recombination system. There is, however, a demonstrable residuum of interplasmid recombination; evidence is lacking on whether this residuum is a plasmid or host function. In the absence of the generalized host recombination system it has been possible to demonstrate that interplasmid recombination occurs during vegetative bacteriophage growth and is presumably mediated by a phage-determined recombination system.     

Site-specific recombination between ColE1 tcer and NTP16 nmr sites in vivo

The site-specific recombination system used by multicopy plasmids of the ColE1 family uses two identical plasmid-encoded recombination sites and four bacterial proteins to catalyze the recombination reaction. In the case of the Escherichia coli plasmid ColE1, the recombination site, cer, is a 280 by DNA sequence which is acted on by the products of the argR, pepA, xerC and xerD genes. We have constructed a model system to study this recombination system, using tandemly repeated recombination sites from the plasmids ColE1 and NTP16. These plasmids have allowed us precisely to define the region of strand exchange during site-specific recombination, and to derive a model for cer intramolecular site-specific recombination.     

Male and female recombination landscapes of diploid Arabidopsis arenosa

The number and placement of meiotic crossover events during meiosis have important implications for the fidelity of chromosome segregation as well as patterns of inheritance. Despite the functional importance of recombination, recombination landscapes vary widely among and within species, and this can have a strong impact on evolutionary processes. A good knowledge of recombination landscapes is important for model systems in evolutionary and ecological genetics, since it can improve interpretation of genomic patterns of differentiation and genome evolution, and provides an important starting point for understanding the causes and consequences of recombination rate variation. Arabidopsis arenosa is a powerful evolutionary genetic model for studying the molecular basis of adaptation and recombination rate evolution. Here, we generate genetic maps for 2 diploid A. arenosa individuals from distinct genetic lineages where we have prior knowledge that meiotic genes show evidence of selection. We complement the genetic maps with cytological approaches to map and quantify recombination rates, and test the idea that these populations might have distinct patterns of recombination. We explore how recombination differs at the level of populations, individuals, sexes and genomic regions. We show that the positioning of crossovers along a chromosome correlates with their number, presumably a consequence of crossover interference, and discuss how this effect can cause differences in recombination landscape among sexes or species. We identify several instances of female segregation distortion. We found that averaged genome-wide recombination rate is lower and sex differences subtler in A. arenosa than in Arabidopsis thaliana.     

Common sites for recombination and cleavage mediated by bacteriophage T4 DNA topoisomerase in vitro

We have previously shown that purified T4 DNA topoisomerase promotes illegitimate recombination between two lambda DNA molecules, or between lambda and plasmid DNA in vitro (Ikeda, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 922-926). Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous sequences of lambda DNA. In this paper, we have examined the sequences of the recombination junctions produced by the recombination between two lambda DNA molecules mediated by T4 DNA topoisomerase. We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of lambda recombination sites, indicating that homology is not essential for the recombination. Next, we have shown an association of the recombination sites with the topoisomerase cleavage sites, indicating that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in the illegitimate recombination. A consensus sequence for T4 topoisomerase cleavage sites, RNAY decreases NNNNRTNY, was deduced. The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4-base pair staggered fashion and produces 5'-protruding ends.     

Site-specific recombination between ColE1 tcer and NTP16 nmr sites in vivo

The site-specific recombination system used by multicopy plasmids of the ColE1 family uses two identical plasmid-encoded recombination sites and four bacterial proteins to catalyze the recombination reaction. In the case of the Escherichia coli plasmid ColE1, the recombination site, cer, is a 280 by DNA sequence which is acted on by the products of the argR, pepA, xerC and xerD genes. We have constructed a model system to study this recombination system, using tandemly repeated recombination sites from the plasmids ColE1 and NTP16. These plasmids have allowed us precisely to define the region of strand exchange during site-specific recombination, and to derive a model for cer intramolecular site-specific recombination.