The Effects of Insertions on Mammalian Intrachromosomal Recombination

We examined the effects of insertion mutations on intrachromosomal recombination. A series of mouse L cell lines carrying mutant herpes simplex virus thymidine kinase (tk) heteroalleles was generated; these lines differed in the nature of their insertion mutations. In direct repeat lines with different large insertions in each gene, there was a 20-fold drop in gene conversion rate and only a five-fold drop in crossover rate relative to the analogous rates in lines with small insertions in each gene. Surprisingly, in direct repeat lines carrying the same large insertion in each gene, there was a larger drop in both types of recombination. When intrachromosomal recombination between inverted repeat tk genes with different large insertions was examined, we found that the rate of gene conversion dropped five-fold relative to small insertions, while the rate of crossing over was unaffected. The differential effects on conversion and crossing over imply that gene conversion is more sensitive to insertion mutation size. Finally, the fraction of gene conversions associated with a crossover increased from 2% for inverted repeats with small insertions to 18% for inverted repeats with large insertions. One interpretation of this finding is that during intrachromosomal recombination in mouse cells long conversion tracts are more often associated with crossing over.     

Elevated levels of expression associated with regions of the Drosophila genome that lack crossing over

The recombinational environment influences patterns of molecular evolution through the effects of Hill-Robertson interference. Here, we examine genome-wide patterns of gene expression with respect to recombinational environment in Drosophila melanogaster. We find that regions of the genome lacking crossing over exhibit elevated levels of expression, and this is most pronounced for genes on the entirely non-crossing over fourth chromosome. We find no evidence for differences in the patterns of gene expression between regions of high, intermediate and low crossover frequencies. These results suggest that, in the absence of crossing over, selection to maintain control of expression may be compromised, perhaps due to the accumulation of deleterious mutations in regulatory regions. Alternatively, higher gene expression may be evolving to compensate for defective protein products or reduced translational efficiency.     

Mitotic recombination in yeast

Mitotic crossing over in diploid cells, heterozygous for a recessive mutation, results in homozygosis and phenotypic expression of the mutant allele. In addition to crossing over, gene conversion also takes place during mitotic growth. As in meiosis, gene conversion and crossing over are associated.     

The relationship between homology length and crossing over during the repair of a broken chromosome

Homologous recombination can result in the transfer of genetic information from one DNA molecule to another (gene conversion). These events are often accompanied by a reciprocal exchange between the interacting molecules (termed "crossing over"). This association suggests that the two types of events could be mechanistically related. We have analyzed the repair, by homologous recombination, of a broken chromosome in yeast. We show that gene conversion can be uncoupled from crossing over when the length of homology of the interacting substrates is below a certain threshold. In addition, a minimal length of homology on each broken chromosomal arm is needed for crossing over. We also show that the coupling between gene conversion and crossing over is affected by the mismatch repair system; mutations in the MSH2 or MSH6 genes cause an increase in the crossing over observed for short alleles. Our results provide a mechanism to explain how chromosomal recombinational repair can take place without altering the stability of the genome.     

Effects of Mutagen-Sensitive Mus Mutations on Spontaneous Mitotic Recombination in Aspergillus

Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to musS. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations from mus+ controls in both tests. Two mutations, musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels only for intragenic recombination. Effects were relatively small, but averages between hypo- and hyperrec mus differed 15-20-fold. In musL diploids, most of the rare color segregants resulted from mitotic malsegregation rather than intergenic crossing over. This indicates that the musL gene product is required for recombination and that DNA lesions lead to chromosome loss when it is deficient. In addition, analysis of the genotypes of intragenic (ad+) recombinants showed that the musL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygous for ad+). Similar analysis revealed differences between the effects of two hyperrec mutations; musN apparently caused high levels solely of mitotic crossing over, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over, while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans.     

Fission yeast Mus81.Eme1 Holliday junction resolvase is required for meiotic crossing over but not for gene conversion

Most models of homologous recombination invoke cleavage of Holliday junctions to explain crossing over. The Mus81.Eme1 endonuclease from fission yeast and humans cleaves Holliday junctions and other branched DNA structures, leaving its physiological substrate uncertain. We report here that Schizosaccharomyces pombe mus81 mutants have normal or elevated frequencies of gene conversion but 20- to 100-fold reduced frequencies of crossing over. Thus, gene conversion and crossing over can be genetically separated, and Mus81 is required for crossing over, supporting the hypothesis that the fission yeast Mus81.Eme1 protein complex resolves Holliday junctions in meiotic cells.     

Roads as barriers to animal movement in fragmented landscapes

Roads can act as barriers to animal movement through mortality during crossing attempts or behavioral avoidance. This barrier effect has negative demographic and genetic consequences that can ultimately result in local or regional extinction. Here we use radio-telemetry data on three terrestrial vertebrates (eastern massasauga Sistrurus catenatus , eastern box turtle Terrapene carolina and ornate box turtle Terrapene ornata ) to test whether roads acted as barriers to movement. Specifically, we test whether individuals avoided crossing roads by comparing the number of observed crossings with the number of road crossings predicted by randomizations of individual movement paths. All species crossed roads significantly less often than predicted by chance, indicating strong road avoidance. Results of this study showing behavioral avoidance and previous studies on road mortality indicate that roads are strong barriers to these species. High mortality during crossing attempts would select for road avoidance, reducing the number of individuals killed on roads over time but leading to genetically partitioned subpopulations due to a lack of gene flow. In species that are long-lived and late-maturing, negative genetic effects might not be observable over short time-scales, thus placing populations at high risk of extinction because of a failure to detect an incrementally worsening problem. Formulating successful management strategies for many species in decline will require integrating data on road mortality, animal behavior and population genetics in order to understand more clearly the barrier effect of roads.     

Intra-chromosomal gene conversion induced by a DNA double-strand break in Saccharomyces cerevisiae

We have stimulated mitotic and meiotic gene conversion between non-tandem direct repeats of ADE4 by a defined double-strand break imparted in vivo to one of two copies of the gene. The experimental design permitted us to distinguish unambiguously between reciprocal intra-chromosomal crossing over and non-reciprocal break-join events that could accompany the induced conversions. We observed that (1) less than 10% of the induced conversion events are accompanied by intra-chromosomal crossing over in both mitosis and meiosis; (2) non-reciprocal break-join is not stimulated by the double-strand breaks; (3) a double-strand break in meiosis is repaired off intra-chromosomal homology (if available) with approximately sevenfold preference over repair off the homologous chromosome. Our observations, analyzed in the light of previous investigations of spontaneous inter and intra-chromosomal crossing over and gene conversion, lead to the view that chromosomal configuration constrains intra-chromosomal crossing over accompanying conversion between closely spaced repeated genes during resolution of the conversion intermediate.     

Cutting edge: expansion of the KIR locus by unequal crossing over

The killer Ig-like receptor (KIR) genes have high sequence similarity and are organized in a head-to-tail fashion. These properties may enhance misalignment of homologous chromosomes during synapsis preceding meiotic recombination, resulting in unequal crossing over. We have identified an extended KIR haplotype that contains a novel hybrid gene and two copies of each of two previously described KIR genes. A parsimonious mechanism for the derivation of this haplotype invokes unequal crossing over between two known ancestral KIR haplotypes. These data raise the possibility that unequal crossing over may be responsible in part for the expansion/contraction of KIR haplotypes as well as other homologous gene families that map in tandem.     

转录后基因沉默(PTGS)及其在作物遗传改良中的应用

转录后基因沉默(PTGS)或RNA干扰(RNAi)技术的发展为创造植物遗传变异体提供了新途径。PTGS于1998年被明确为双链RNA(dsRNA)诱导的序列特异性基因沉默,短短几年内有关研究取得了突破性进展。结合利用PTGS技术进行的淀粉合成关键酶基因沉默研究,概述了PTGS的作用机理和特点、dsRNA表达载体设计、沉默效应的遗传稳定性及在作物改良应用方面的研究进展。研究表明,基因沉默效应可在子代间稳定遗传并可通过杂交进行重组,显示了其在农作物改良方面的应用潜力。     

mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster

The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.     

Linkage disequilibrium patterns across a recombination gradient in African Drosophila melanogaster

Previous multilocus surveys of nucleotide polymorphism have documented a genome-wide excess of intralocus linkage disequilibrium (LD) in Drosophila melanogaster and D. simulans relative to expectations based on estimated mutation and recombination rates and observed levels of diversity. These studies examined patterns of variation from predominantly non-African populations that are thought to have recently expanded their ranges from central Africa. Here, we analyze polymorphism data from a Zimbabwean population of D. melanogaster, which is likely to be closer to the standard population model assumptions of a large population with constant size. Unlike previous studies, we find that levels of LD are roughly compatible with expectations based on estimated rates of crossing over. Further, a detailed examination of genes in different recombination environments suggests that markers near the telomere of the X chromosome show considerably less linkage disequilibrium than predicted by rates of crossing over, suggesting appreciable levels of exchange due to gene conversion. Assuming that these populations are near mutation-drift equilibrium, our results are most consistent with a model that posits heterogeneity in levels of exchange due to gene conversion across the X chromosome, with gene conversion being a minor determinant of LD levels in regions of high crossing over. Alternatively, if levels of exchange due to gene conversion are not negligible in regions of high crossing over, our results suggest a marked departure from mutation-drift equilibrium (i.e., toward an excess of LD) in this Zimbabwean population. Our results also have implications for the dynamics of weakly selected mutations in regions of reduced crossing over.     

Use of fluorescent protein to analyse recombination at three loci in Neurospora crassa

We have inserted a histone H1-GFP fusion gene adjacent to three loci on different chromosomes of Neurospora crassa and made mating pairs in which a wild type version of GFP is crossed to one with a mutation in the 5' end of GFP. The loci are his-3, am and his-5, chosen because recombination mechanisms appear to differ between his-3 and am, and because crossing over adjacent to his-5, like his-3, is regulated by rec-2. At his-3, the frequencies of crossing over between GFP and the centromere and of conversion of 5'GFP to GFP(+) are comparable to those obtained by classical recombination assays, as is the effect of rec-2 on these frequencies, suggesting that our system does not alter the process of recombination. At each locus we have obtained sufficient data, on both gene conversion and crossing over, to be able to assess the effect of deletion of any gene involved in recombination. In addition, crosses between a GFP(+) strain and one with normal sequence at all three loci have been used to measure the interval to the centromere and to show that GFP experiences gene conversion with this system. Since any gene expressed in meiosis is silenced in Neurospora if hemizygous, any of our GFP(+) strains can be used as a quick screen to determine if a gene deleted by the Neurospora Genome Project is involved in crossing over or gene conversion.     

The Evolution of Restricted Recombination and the Accumulation of Repeated DNA Sequences

We suggest hypotheses to account for two major features of chromosomal organization in higher eukaryotes. The first of these is the general restriction of crossing over in the neighborhood of centromeres and telomeres. We propose that this is a consequence of selection for reduced rates of unequal exchange between repeated DNA sequences for which the copy number is subject to stabilizing selection: microtubule binding sites, in the case of centromeres, and the short repeated sequences needed for terminal replication of a linear DNA molecule, in the case of telomeres. An association between proximal crossing over and nondisjunction would also favor the restriction of crossing over near the centromere. The second feature is the association between highly repeated DNA sequences of no obvious functional significance and regions of restricted crossing over. We show that highly repeated sequences are likely to persist longest (over evolutionary time) when crossing over is infrequent. This is because unequal exchange among repeated sequences generates single copy sequences, and a population that becomes fixed for a single copy sequence by drift remains in this state indefinitely (in the absence of gene amplification processes). Increased rates of exchange thus speed up the process of stochastic loss of repeated sequences.     

Spontaneous Mitotic Recombination in Yeast: The Hyper-Recombinational Rem1 Mutations Are Alleles of the Rad3 Gene

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.     

A yeast strain for simultaneous detection of induced mitotic crossing over,mitotic gene conversion and reverse mutation

A diploid yeast strain is described which can be used to study induction of mitotic crossing over, mitotic gene conversion and reverse mutation.Mitotic crossing over can be detected visually as pink and red twin sectored colonies which are due to the formation of homozygous cells of the genotype ade240/ade240 (deep red) and ade-2-119/ade2-119 (pink) from the originally heteroallelic condition ade2-40/ade2-119 which forms white colonies.Mitotic gene conversion is monitored by the appearance of tryptophan non-requiring colonies on selective media. The alleles involved are tryp5-12 and trp5-27 derived from the widely used strain D4.Mutation induction can be followed by the appearance of isoleucine non-requiring colonies on selective media. D7 is homoallelic ilv1-92/ilv1-92. The isoleucine requirement caused by ilv1-92 can be alleviated by true reverse mutation and allele non-specific suppressor mutation.The effects of ethyl methanesulfonate (EMS), nitrous acid, ultraviolet light and hycanthone methanesulfonate were studied with D7 stationary phase cells. Mitotic crossing over as monitored by red/pink twin sectored colonies was almost equally frequent among normal and convertant cells. This showed again that mitotic recombination is not due to the presence fo a few cells committed to meiosis in an otherwise mitotic cell population.The dose-response curves for induction of mitotic gene conversion and reversion of the isoleucine requirement were exponential. In contrast to this, the dose-response curve for induction of twin sectored red and pink colonies reached a plateau at doses giving about 30% cell killing. This could partly be due to lethal segregation in the progeny of treated cells.None of the agents tested would induce only one type of mitotic recombination, gene conversion or crossing over. There was, however, some mutagen specificity in the induction of isoleucine prototrophs.     

Simulating Evolution by Gene Duplication

By considering the recent finding that unequal crossing over and other molecular interactions are contributing to the evolution of multigene families, a model of the origin of repetitive genes was studied by Monte Carlo simulations. Starting from a single gene copy, how genetic systems evolve was examined under unequal crossing over, random drift and natural selection. Both beneficial and deteriorating mutations were incorporated, and the latter were assumed to occur ten times more frequently than the former. Positive natural selection favors those chromosomes with more beneficial mutations in redundant copies than others in the population, but accumulation of deteriorating mutations (pseudogenes) have no effect on fitness so long as there remains a functional gene. The results imply the following: Positive natural selection is needed in order to acquire gene families with new functions. Without it, too many pseudogenes accumulate before attaining a functional gene family. There is a large fluctuation in the outcome even if parameters are the same. When unequal crossing over occurs more frequently, the system evolves more rapidly. It was also shown, under realistic values of parameters, that the genetic load for acquiring a new gene is not as large as J.B.S. Haldane suggested, but not so small as in a model in which a system for selection started from already redundant genes.     

Transgenic trait deployment using designed nucleases

The demand for crops requiring increasingly complex combinations of transgenes poses unique challenges for transgenic trait deployment. Future value‐adding traits such as those associated with crop performance are expected to involve multiple transgenes. Random integration of transgenes not only results in unpredictable expression and potential unwanted side effects but stacking multiple, randomly integrated, independently segregating transgenes creates breeding challenges during introgression and product development. Designed nucleases enable the creation of targeted DNA double‐strand breaks at specified genomic locations whereby repair can result in targeted transgene integration leading to precise alterations in DNA sequences for plant genome editing, including the targeting of a transgene to a genomic locus that supports high‐level and stable transgene expression without interfering with resident gene function. In addition, targeted DNA integration via designed nucleases allows for the addition of transgenes into previously integrated transgenic loci to create stacked products. The currently reported frequencies of independently generated transgenic events obtained with site‐specific transgene integration without the aid of selection for targeting are very low. A modular, positive selection‐based gene targeting strategy has been developed involving cassette exchange of selectable marker genes which allows for targeted events to be preferentially selected, over multiple cycles of sequential transformation. This, combined with the demonstration of intragenomic recombination following crossing of transgenic events that contain stably integrated donor and target DNA constructs with nuclease‐expressing plants, points towards the future of trait stacking that is less dependent on high‐efficiency transformation.     

MLH1 mutations differentially affect meiotic functions in Saccharomyces cerevisiae

To test whether missense mutations in the cancer susceptibility gene MLH1 adversely affect meiosis, we examined 14 yeast MLH1 mutations for effects on meiotic DNA transactions and gamete viability in the yeast Saccharomyces cerevisiae. Mutations analogous to those associated with hereditary nonpolyposis colorectal cancer (HNPCC) or those that reduce Mlh1p interactions with ATP or DNA all impair replicative mismatch repair as measured by increased mutation rates. However, their effects on meiotic heteroduplex repair, crossing over, chromosome segregation, and gametogenesis vary from complete loss of meiotic functions to no meiotic defect, and mutants defective in one meiotic process are not necessarily defective in others. DNA binding and ATP binding but not ATP hydrolysis are required for meiotic crossing over. The results reveal clear separation of different Mlh1p functions in mitosis and meiosis, and they suggest that some, but not all, MLH1 mutations may be a source of human infertility.     

A role for DNA polymerase delta in gene conversion and crossing over during meiosis in Saccharomyces cerevisiae

A screen for mutants of budding yeast defective in meiotic gene conversion identified a novel allele of the POL3 gene. POL3 encodes the catalytic subunit of DNA polymerase delta, an essential DNA polymerase involved in genomic DNA replication. The new allele, pol3-ct, specifies a protein missing the last four amino acids. pol3-ct shows little or no defect in DNA replication, but displays a reduction in the length of meiotic gene conversion tracts and a decrease in crossing over. We propose a model in which DNA synthesis determines the length of strand exchange intermediates and influences their resolution toward crossing over.