Recognition sites and coordinate control of recombination in Schizophyllum

The genetic control of recombination in two chromosomal regions was studied in dikaryons of Schizophyllum commune with joint control of recombination in the A and B factors. The role of the controlled regions in the determination of their recombination frequencies was studied by substituting one or the other or both by homologous segments. The B factor was shown to affect both its own and the A factors' recombination frequency. A model of genetic control of recombination in several regions is proposed. The model is based on identical recognition sites within the jointly controlled segments. It is consistent with the fact that jointly-controlled segments can show either negative or positive correlation between their recombination frequencies.     

Mutations arising during transformation in the blue-green alga Anacystis nidulans

Summary Genetic transformation experiments have been performed in Anacystis nidulans using donor material from two sources, namely chemically extracted DNA and extracellular nucleic acids. A high proportion of the transformants became mutant at sites which were wild type in both parental strains. Linkage was less extensive in transformation mediated by chemically extracted DNA, and this increased frequency of recombination was associated with enhanced mutation frequencies. The frequencies of recombination and mutation were varied to the same extent by changing the DNA concentration, and both processes were prevented by pretreatment of donor DNA with DNase. Mutational events are, therefore, closely associated with recombination in A. nidulans.     

Genetic variation in piñon pine, Pinus edulis, associated with summer precipitation

Three previous reports of microgeographical variation of glycerate dehydrogenase (Gly) frequencies in piñon, Pinus edulis, established the hypothesis that Gly frequencies contribute to adaptation to heterogeneous environments, specifically to variation in soil moisture. In each of these studies, the frequency of the Gly‐3 allele or of Gly‐33 homozygotes was higher on dry sites than on nearby moist sites. Here we attempt to extend these observations by testing the hypothesis that Gly frequencies respond to soil moisture variation on a range‐wide scale. Gly frequencies were surveyed in 11 natural populations, and the frequency of the Gly‐3 allele varied from 0.27 to 0.65 among the sample sites. Elevation varied from 1650 to 3100 m, and summer precipitation, defined as precipitation from April to August, varied from 13.7 to 26.4 cm. The soil types at the collection sites were schist, quaternary volcanic or a mixture of shale and sandstone. Logistic regression revealed that Gly frequencies did not respond to either elevation or soil type, but were related to summer precipitation (P < 0.01). The correlation between summer precipitation and the frequency of the Gly‐3 allele was r = ?0.92 (P < 0.001). Thus, the patterns of differentiation on microgeographical scales are consistent with greater differentiation on a range‐wide scale.     

A diversity of serine phage integrases mediate site-specific recombination in mammalian cells

This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, φFC1, and φRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage att sites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage att site with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attB and attP sites at a frequency of ∼ 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attB and attP sites of A118. The integrases from phages Bxb1, φFC1, and φRV1 carried out such recombination at their attB and attP sites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attP site on a plasmid and pseudo attB sites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo att sites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.     

Worldwide allele frequencies of the human apolipoprotein E gene: Climate,local adaptations,and evolutionary history

The ?4 allele of the apolipoprotein E (APOE) gene is associated with increased cholesterol levels and heart disease. Population allele frequencies of APOE have previously been shown to vary, with ?4 frequencies generally increasing with latitude. We hypothesize that this trend resulted from natural selection protecting against low‐cholesterol levels. In high‐latitude cold environments and low‐latitude hot environments, metabolic rate is elevated, which could require higher cholesterol levels. To explore this hypothesis, we compiled APOE allele frequencies, latitude, temperature, and elevation from populations around the world. ?4 allele frequencies show a curvilinear relationship with absolute latitude, with lowest frequencies found in the mid‐latitudes where temperatures generally require less expenditure on cooling/thermogenesis. Controlling for population structure in a subset of populations did not appreciably change this pattern of association, consistent with selection pressures that vary by latitude shaping ?4 allele frequencies. Temperature records also predict APOE frequency in a curvilinear fashion, with lowest ?4 frequencies at moderate temperatures. The model fit between historical temperatures and ?4 is less than between latitude and ?4, but strengthened after correcting for estimated temperature differences during the Paleolithic. Contrary to our hypothesis, we find that elevation did not improve predictive power, and an integrated measure of the cholesterol effect of multiple APOE alleles was less related to latitude than was ?4 alone. Our results lend mixed support for a link between past temperature and human APOE allele distribution and point to the need to develop better models of past climate in future analyses. Am J Phys Anthropol 143:13–20, 2010. © 2010 Wiley‐Liss, Inc.     

Frequency and character of alternative somatic recombination fates of paralogous genes during T-DNA integration

A synthetic RBCSB gene cluster was transformed into Arabidopsis in order to simultaneously evaluate the frequency and character of somatic illegitimate recombination, homologous recombination, and targeted gene replacement events associated with T-DNA-mediated transformation. The most frequent type of recombination event observed was illegitimate integration of the T-DNA without activation of the silent ΔRBCS1B: LUC transgene. Sixteen luc⁺ (firefly luciferase positive) T1 plants were isolated. Six of these were due to illegitimate recombination events resulting in a gene trapping effect. Nine resulted from homologous recombination between paralogous RBCSB sequences associated with T-DNA integration. The frequency of somatic homologous recombination associated with T-DNA integration was almost 200 times higher than previously reported rates of meiotic homologous recombination with the same genes. The distribution of (somatic homologous) recombination resolution sites generally fits a fractional interval length model. However, a small region adjacent to an indel showed a significant over-representation of resolution sites, suggesting that DNA mismatch recognition may also play an important role in the positioning of somatic resolution sites. The frequency of somatic resolution within exon-2 was significantly different from that previously observed during meiotic recombination. Electronic Supplementary Material Supplementary material is available for this article at     

Variability of allelic recombination frequencies

Summary Estimates of allelic recombination frequencies are shown to have coefficients of variation of between 20 and 40%. In Coprinus this is true of both high and low recombination frequencies and is also true when the alleles involved show marker effect. This variability is not confined to Coprinus but is a general feature of both meiotic and mitotic allelic recombination. Experimental errors do not make a major contribution to the observed variation althought it has the nature of a sampling variation. It is suggested that the variation arises from the diversity of ways in which the initial errors introduced by hybrid DNA formation can be resolved during the excision-repair stages of recombination. If the enzymes responsible for these processes are present in low concentrations then much latitude can be anticipated in the way the same errors are dealt with by separate, though isogenic, diploid or dikaryotic organisms. Each separate cross is thus interpreted as providing an estimate of the recombination frequency which is but a sample from a varied population of possible estimates of the same recombination frequency. Each pair of alleles exhibits a recombination frequency which, within the statistical boundaries of the general variation, is sufficiently reproducible to be described as a characteristic of them. Combinations of allelic recombination frequencies derived from pair-wise crosses fall into patterns that are sufficiently consistent for allele maps to be drawn; and, providing a sufficient number of replicate crosses have been analysed, the allele map can be shown to be statistically soundly based. Two marker effect situations are examined. One causes reduction of recombination frequency and is probably intrinsic to the mutant site itself, the other causes enhancement of recombination frequency and is due to a factor or factors distinct from the allelic mutant site in the strain in which it was first identified. When intercrossed the two effects counteract one another.     

Transformation and recombination in rad mutants of Saccharomyces cerevisiae

Summary Disruption/deletion mutations in genes of the RAD52 epistasis group of Saccharomyces cerevisiae were examined for their effects on recombination between single-and double-stranded circular DNA substrates and chromosomal genes in a transformation assay. In rad50 mutants there was a small reduction in recombination with single-stranded DNA at the leu2-3, 112 allele; in addition there was an almost complete elimination of recombination at trpl-1 for both single- and double-stranded DNA. Reintroduction of a wild-type RAD50 gene on a replicating plasmid carrying CEN4 restored recombinational competence at trpl-1, indicating that rad50 is defective in gene replacement of this allele. In rad52 mutants a reduction of 30%-50% in recombination involving either single- or double-stranded circular DNA was observed in each experiment when compared to the wild type. This reduction of recombination in rad52 mutants was similar for recombination at the ura352 mutant locus where only integration events have been observed, and at the trpl-1 mutant locus, where recombination occurs predominantly by gene replacement. Neither the rad54 nor the rad57 mutations had a significant effect on recombination with single- or double-stranded DNA substrates.     

Genetic variation in two populations of the rough‐skinned newt (Taricha granulosa) assessed using novel tetranucleotide microsatellite loci

Seven novel tetranucleotide microsatellite loci were identified from a partial genomic DNA library, enriched for GATA‐motif microsatellites, from the rough‐skinned newt (Taricha granulosa). All loci were polymorphic, and one displayed a high frequency null allele. A related species, T. rivularis, displays strong site fidelity and detectable population structure over small spatial scales, so we assessed genetic variation in two samples of T. granulosa separated by 16 km. Distributions of allele frequencies differ significantly between our two sites, but small values of F_ST and Rho_ST suggest that the populations are linked by a large amount of gene flow.     

Evidence for adaptation from standing genetic variation on an antimicrobial peptide gene in the mussel Mytilus edulis

Genome scans of population differentiation identify candidate loci for adaptation but provide little information on how selection has influenced the genetic structure of these loci. Following a genome scan, we investigated the nature of the selection responsible for the outlying differentiation observed between populations of the marine mussel Mytilus edulis at a leucine/arginine polymorphism (L31R) in the antimicrobial peptide MGD2. We analysed DNA sequence polymorphisms, allele frequencies and population differentiation of polymorphisms closely linked to L31R, and pairwise and third‐order linkage disequilibria. An outlying level of population differentiation was observed at L31R only, while no departure from panmixia was observed at linked loci surrounding L31R, as in most of the genome. Selection therefore seems to affect L31R directly. Three hypotheses can explain the lack of differentiation in the chromosomal region close to L31R: (i) hitchhiking has occurred but migration and recombination subsequently erased the signal, (ii) selection was weak enough and recombination strong enough to limit the hitchhiking effect to a very small chromosomal region or (iii) selection acted on a pre‐existing polymorphism (i.e. standing variation) at linkage equilibrium with its background. Linkage equilibrium was observed between L31R and linked polymorphisms in every population analysed, as expected under the three hypotheses. However, linkage disequilibrium was observed in some populations between pairs of loci located upstream and downstream to L31R, generating a complex pattern of third‐order linkage disequilibria which is best explained by the hypothesis of selection on a pre‐existing polymorphism. We hypothesise that selection could be either balanced, maintaining alleles at different frequencies depending on the pathogen community encountered locally by mussels, or intermittent, resulting in sporadic fluctuations in allele frequency.     

Somatic mutagenesis in human lymphocytes depending on genotypes for detoxification and oxidative response loci

Associations of polymorphism of seven detoxification genes and three genes of oxidative response with the frequency of chromosome aberrations in human peripheral blood lymphocytes were studied. The genotyping data were correlated with the frequencies of spontaneous and γ-induced (1 Gy in vitro) chromosome aberrations estimated for a group of healthy donors (97 males under 25 years of age) by analyzing 500–1000 metaphase cells per individual. The spontaneous level of chromosome-type aberrations was reduced in homozygotes for the GSTM1 locus deletion, and especially in double homozygotes for deletions of the GSTM1 and GSTT1 genes. The frequency of γ-induced chromosome-type aberrations was reduced in G/G homozygotes for the minor allele of the poorly studied CYP1A1 T606G site: 0.094 ± 0.006 against 0.112 ± 0.002 for T allele carriers (P = 0.004). Linkage of the T606G site with well known and functionally important sites of the CYP1A1 gene (A4889G, T3801C) was analyzed.     

Genetic and Phenotypic Analysis of <Emphasis Type="Italic">lax1-6</Emphasis>, a Mutant Allele of <Emphasis Type="Italic">LAX PANICLE1</Emphasis> in Rice

Proper function of the LAX1 gene is required for the development of axillary meristem in rice. Here, we report genetic and phenotypic characters of a novel recessive mutant allele of rice LAX1 gene, lax1-6, which showed abnormal panicle phenotypes with few numbers of elongated primary rachis branches. Beside typical lax mutant phenotype, abnormalities of lax1-6 mutant allele were observed with defect lemma and palea primordial in floral organs. The lax1-6 mutant locus was linked between SSR markers RM7594 and RM5389 on chromosome 1 with 1.02% and 1.0% recombination frequencies, respectively. Molecular analysis revealed that the lax1-6 mutant allele was caused by a transversion mutation of nucleotide T to G substitution that resulted in an amino acid substitution from serine (S) to alanine (A) at the 117^th position from amino terminus of a basic helix-loop-helix protein coded by LAX1 gene. Furthermore, we found that the Oryza sativa indica type cv. IRRI347 contained 24 nucleotide deletion in the upstream sequence in the LAX1 gene, but this deletion did not influence panicle morphology, which demonstrated that the deletion is a polymorphism in rice. All together, the lax1-6 mutant is a newly identified allele of LAX1 gene displaying the abnormal axillary meristems and inflorescences in rice.     

Allozyme variation among natural populations of Holopedium gibberum (Crustacea; Cladocera)

• 1 Holopedium gibberum, from twenty lakes in Rhode Island and Maine, were examined for allozyme variation at five loci to determine the pattern and degree of generic variation among sites and the genetic structure within individual lakes.
• 2 There were significant differences in allele frequencies among sites. Most populations were fixed for a particular allele at each locus. Only five lakes had polymorphic populations.
• 3 Polymorphic populations showed significant deviation from expected Hardy-Weinberg genotype frequencies. In each case, there was an excess of homozygotes.
• 4 Two lakes were examined for intra-lake allele frequency differences. In one lake there were no differences. The other lake exhibited significant allele frequency differences between stations at the north and south ends of the lake.
• 5 Populations were examined for the frequency distribution of composite genotypes over three loci. Most populations were dominated by one or two genotypes.
• 6 The results suggest sporadic sexual recruitment and a high degree of genetic isolation among these populations of H. gibberum. In these respects they resemble the permanent pond populations of Daphnia magna examined by Hebert (1974a).

     

Inversion and allozyme polymorphism show contrasting patterns of microgeographical population structure in a natural population of Drosophila buzzatii from Argentina

Second chromosome inversions and genotypic frequencies at seven allozyme loci were determined in a natural population of the cactophilic species Drosophila buzzatii that uses as breeding sites the necrotic cladodes of the prickly pear Opuntia quimilo and the rotting stems of cardón, Trichocereus terschekii. Different processes govern the evolutionary fate of inversion and allozyme polymorphisms. A pattern of heterotic balance for inversions seems to be acting uniformly in each breeding site and could depend on different regimes of density‐dependent selection within cactus hosts. Patterns of variation of allozymes revealed significant heterogeneity in allele frequencies for Esterase‐1 (Est‐1) among O. quimilo rots and Aldehyde oxidase (Aldox) and Xanthine dehydrogenase (Xdh) among T. terschekii substrates and showed gene‐cactus effects only for Esterase‐2 (Est‐2). Consistent and significant excesses of homozygotes were detected at both the within‐rot and in the total population levels that could be accounted for by diversifying selection among individual breeding sites.     

MHC class I allele frequencies in pigtail macaques of diverse origin

Pigtail macaques (Macaca nemestrina) are an increasingly common primate model for the study of human AIDS. Major Histocompatibility complex (MHC) class I-restricted CD8⁺ T cell responses are a critical part of the adaptive immune response to HIV-1 in humans and simian immunodeficiency virus (SIV) in macaques; however, MHC class I alleles have not yet been comprehensively characterized in pigtail macaques. The frequencies of ten previously defined alleles (four Mane-A and six Mane-B) were investigated in detail in 109 pigtail macaques using reference strand-mediated conformational analysis (RSCA). The macaques were derived from three separate breeding colonies in the USA, Indonesia and Australia, and allele frequencies were analysed within and between these groups. Mane-A*10, an allele that restricts the immunodominant SIV Gag epitope KP9, was the most common allele, present in 32.1% of the animals overall, with similar frequencies across the three cohorts. Additionally, RSCA identified a new allele (Mane-A*17) common to three Indonesian pigtail macaques responding to the same Gag CD8⁺ T cell epitope. This broad characterization of common MHC class I alleles in more than 100 pigtail macaques further develops this animal model for the study of virus-specific CD8⁺ T cell responses.     

DEVELOPMENT OF THE HYBRID SWARM BETWEEN PECOS PUPFISH (CYPRINODONTIDAE: CYPRINODON PECOSENSIS) AND SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS): A PERSPECTIVE FROM ALLOZYMES AND mtDNA

A comparison of allozyme and mtDNA frequencies was used for insight into a situation in the Pecos River, Texas where contact between the endemic pupfish (Cyprinodon pecosensis) and an introduced congener (C. variegatus) has resulted in rapid, geographically extensive genetic introgression. Temporal changes in mean frequencies of diagnostic allozyme markers indicate that the clinal pattern of introduced genetic material (Echelle and Connor 1989) is slowly decreasing in amplitude. Significant rank concordance in diagnostic allele frequencies among sites and across sampling years indicates directional influences upon temporal allele frequency change. These observations are consistent with the theory of gene flow in neutral clines. Levels of introgression indicated by each of four allozyme loci and mtDNA were roughly equivalent. The early history of the hybrid swarm is explained by genetic swamping, possibly mediated by selection for C. variegatus or C. variegatus × C. pecosensis, at a time when the normally abundant endemic species had been catastrophically depleted. High frequencies of an introduced GPI-A allele in all samples of intergrades suggests that the introduced genome originated with a single founding event.     

Homologous recombination and allele replacement in transformants of Fusarium fujikuroi

The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss was due to gene conversion in some cases and to recombination between repeated sequences in others. Received: 5 November 1999 / Accepted: 16 March 2000     

Mitotic gene conversion, reciprocal recombination and gene replacement at the benA, beta-tubulin, locus of Aspergillus nidulans

Summary We have developed a procedure for determining the rates of mitotic recombination of an interrupted duplication created by integration of transforming plasmid sequences at the benA, beta-tubulin, locus of Aspergillus nidulans. Transformation of a strain carrying a benomyl-resistant benA allele with plasmid AIpGM4, which carries the wild-type benA allele and the pyr4 (orotidine-5-phosphate decarboxylase) gene of Neurospora crassa, creates an interrupted duplication with plasmid sequences flanked by two benA alleles, one wild type and one benomyl resdistant. Such transformants will not grow in the presence of high levels of benomyl. Mitotic recombination causes the loss of the wild-type benA allele or conversion of the wild-type to the mutant allele resulting in nuclei carrying only the benomylresistant allele. Conidia containing such nuclei can be selected on media with high benomyl allowing easy quantitation of mitotic recombination. We found that the rate of recombination giving rise to benomyl-resistant conidia was 4.6×10^-4. Reciprocal recombination leading to benomyl-resistant conidia lacking plasmid sequences occurred at a rate of 2.0×10^-4 and gene conversion leading to benomylresistant conidia occurred at a rate of 2.6×10^-4. We selected for reciprocal recombination leading to loss of pyr4 sequences on 5-fluoro-orotic acid and used this selection for two-step gene replacement of a mutant benA allele with the wild-type allele.     

Population structure of the intertidal copepod Tigriopus californicus as revealed by field manipulation of allele frequencies

Summary Allele frequencies in natural T. californicus populations were perturbed by introduction of copepods from neighboring differentiated populations. In five experiments, the Gpt ^F allele was introduced into single recipient pools at a frequency of approximately 20%. In each case, the introduced allele declined to low frequencies (<3%) in less than one month, apparently due to dilution by residents of other pools on the same outcrop. In a larger scale experiment, the Pgi ^F was introduced into four pools on a single small rock outcrop; all pools on the outcrop were subsequently monitored. While the allele frequency fell from approximately 40% to 10% during the first six weeks after the transplant, no further change in frequency was observed for the duration of the experiment (16 months). Within six weeks some spread of the allele to non-recipient pools on the same outcrop was observed; by eight months, allele frequencies in all pools on the outcrop were similar. Hence, despite the extensive turnover of subpopulations as single pools evaporate or are washed out, genetic homogeneity and stability of entire outcrops are maintained via extensive inter-pool gene flow; this contrasts sharply with the highly restricted levels of inter-outcrop gene flow.     

RAPID,GEOGRAPHICALLY EXTENSIVE GENETIC INTROGRESSION AFTER SECONDARY CONTACT BETWEEN TWO PUPFISH SPECIES (CYPRINODON,CYPRINODONTIDAE)

Apparently between 1980 and 1984, Cyprinodon variegatus was introduced into an area of the Pecos River in Texas, where it hybridized with an endemic species, C. pecosensis. Protein electrophoresis indicated that, by 1985, panmictic admixtures of these two pupfishes occupied approximately 430 river-kilometers of the Pecos River, roughly one-half of the historic range of the endemic species. The average frequency of introduced alleles at four diagnostic loci ranged from 0.18 to 0.84 at the 15 sites sampled from the Pecos River in Texas. Clinal patterns in allele frequencies suggest that C. variegatus was introduced into a mid-reach of the river and that this was followed by both upstream and downstream dispersal of the introduced alleles. All pairwise combinations of loci showed significant linkage disequilibrium. The level of disequilibrium indicates chromosomal linkage for one gene-pair, Gpi-A and Est-1. The change in pupfish allele frequencies in the Pecos River represents an extreme example of rapid natural selection in a seminatural situation.