Frequency Of Intragenic Recombination Research Articles

Development of a high-throughput method for examining the intra-genic recombination frequency in Wx locus in rice using an image analysis

Development of a high-throughput method for examining the intra-genic recombination frequency in <i>Wx</i> locus in rice using an image analysis

Evidence of recombination in intrapatient populations of hepatitis C virus.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and a potential cause of substantial morbidity and mortality in the future. HCV is characterized by a high level of genetic heterogeneity. Although homologous recombination has been demonstrated in many members of the family Flaviviridae, to which HCV belongs, there are only a few studies reporting recombination on natural populations of HCV, suggesting that these events are rare in vivo. Furthermore, these few studies have focused on recombination between different HCV genotypes/subtypes but there are no reports on the extent of intra-genotype or intra-subtype recombination between viral strains infecting the same patient. Given the important implications of recombination for RNA virus evolution, our aim in this study has been to assess the existence and eventually the frequency of intragenic recombination on HCV. For this, we retrospectively have analyzed two regions of the HCV genome (NS5A and E1-E2) in samples from two different groups: (i) patients infected only with HCV (either treated with interferon plus ribavirin or treatment naïve), and (ii) HCV-HIV co-infected patients (with and without treatment against HIV). The complete data set comprised 17712 sequences from 136 serum samples derived from 111 patients. Recombination analyses were performed using 6 different methods implemented in the program RDP3. Recombination events were considered when detected by at least 3 of the 6 methods used and were identified in 10.7% of the amplified samples, distributed throughout all the groups described and the two genomic regions studied. The resulting recombination events were further verified by detailed phylogenetic analyses. The complete experimental procedure was applied to an artificial mixture of relatively closely viral populations and the ensuing analyses failed to reveal artifactual recombination. From these results we conclude that recombination should be considered as a potentially relevant mechanism generating genetic variation in HCV and with important implications for the treatment of this infection.

Spo5/Mug12, a Putative Meiosis-Specific RNA-Binding Protein, Is Essential for Meiotic Progression and Forms Mei2 Dot-Like Nuclear Foci

We report here a functional analysis of spo5(+)(mug12(+)) of Schizosaccharomyces pombe, which encodes a putative RNA-binding protein. The disruption of spo5(+) caused abnormal sporulation, generating inviable spores due to failed forespore membrane formation and the absence of a spore wall, as determined by electron microscopy. Spo5 regulates the progression of meiosis I because spo5 mutant cells display normal premeiotic DNA synthesis and the timely initiation of meiosis I but they show a delay in the peaking of cells with two nuclei, abnormal tyrosine 15 dephosphorylation of Cdc2, incomplete degradation of Cdc13, retarded formation and repair of double strand breaks, and a reduced frequency of intragenic recombination. Immunostaining showed that Spo5-green fluorescent protein (GFP) appeared in the cytoplasm at the horsetail phase, peaked around the metaphase I to anaphase I transition, and suddenly disappeared after anaphase II. Images of Spo5-GFP in living cells revealed that Spo5 forms a dot in the nucleus at prophase I that colocalized with the Mei2 dot. Unlike the Mei2 dot, however, the Spo5 dot was observed even in sme2Delta cells. Taken together, we conclude that Spo5 is a novel regulator of meiosis I and that it may function in the vicinity of the Mei2 dot.

Molecular Diagnostics of Genetic Diseases: Experience from Studies of DMD, APC, TSC1, and OPG Genes. Part 1.

Over the past decade geneticists have searched for rapid and efficient means of identification of genetic diseases. Nucleic acid techniques, such as restriction fragment length polymorphisms, polymerase chain reaction, and sequence analysis of amplified DNA are making increasing inroads as tools in diagnostic laboratories. Our laboratory is involved in the molecular diagnostics of several genetic diseases including Duchenne muscular dystrophy, familial adenomatous polyposis coli, tuberous sclerosis, and osteoporosis. Each disease varies in type of molecular lesions and different approaches are applied to achieve the best mutation detection rate. Duchenne/Becker muscular dystrophy (DMD/ BMD) is a lethal X-linked recessive disease caused by mutations within the dystrophin gene (DMD gene). The DMD gene is the largest known human gene, spanning about 2,500kb and consisting of 79 exons. In approximately 60% of DMD/BMD patients, deletions of one or more exons are detected. Carrier detection still causes problems for many cases, because of the enormous size of the DMD gene and the high intragenic recombination frequency. Familial adenomatous polyposis coli (FAP) is a dominantly inherited autosomal disorder caused by germ line mutation in the adenomatous polyposis coli (APC) gene and is characterized by early onset of multiple adenomatous polyps, which leads to the development of colorectal carcinoma. The development of colon carcinoma is associated with loss of heterozygosity (LOH) in the APC gene and accumulation of mutations in a number of tumor suppressor genes. Detection of mutation carriers in FAP families before pathological symptoms occur is very important and makes possible clinical treatment. Molecular changes in the APC gene were found in 30% of the studied patients with familial adenomatous polyposis coli, with a similar frequency for the common delta 1309 mutation as in other populations. In a Polish population of FAP patients, most mutations were localized in a region of the APC gene encompassing codons 1040-1309.

Selection, convergence, and intragenic recombination in HLA diversity.

To account for high degrees of human leukocyte antigen (HLA) diversity, a method is proposed for detecting intragenic recombination or gene conversion separately from parallel substitutions or convergent evolution. An application of the method to HLA protein sequences suggests that intragenic recombination played important roles in HLA-B and DPB1, some in HLA-A and DRB1, and least in HLA-C and DQB1 diversity. However, the extent of diversity of these molecules does not necessarily correlate with the frequency of intragenic recombination, supporting the view that (balancing) selection is a primary agent of HLA diversity and often leads to convergent evolution. Computer simulation is carried out to examine two models of balancing selection under the coupled effect with mutation, intragenic recombination, and random drift in a diploid population. It is emphasized that break points by intragenic recombination need be specified to account for HLA diversity. Implications of HLA diversity in human evolution are briefly discussed.

Intragenic recombination in the CSR1 locus of Arabidopsis.

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 +/- 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.

Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells

The mutation pat1-114 has been used to synchronize meiosis in the fission yeast Schizosaccharomyces pombe. We have investigated several aspects of such synchronized meiotic cultures. In both pat1-114 and pat1+ diploids, meiotic landmark events are initiated at the same time after meiosis induction, but synchrony is much more pronounced in the pat1-114-driven meiosis. Commitment to recombination and to meiosis have been timed at 2 h after meiotic induction. Due to a seven-fold reduction of intragenic recombination frequency in the ade6 region of pat1-114 diploids, physical analysis of recombination has not been possible. We have distinguished three factors that influence intragenic recombination frequencies: temperature, azygotic versus zygotic meiosis, and the nature of the pat1 allele. Differences and similarities in the timing of meiotic landmarks in S. cerevisiae and S. pombe are discussed.

Accurate assessment of intragenic recombination frequency within the Duchenne muscular dystrophy gene

Polymorphic loci that lie at the two extremities of the Duchenne/Becker muscular dystrophy ( DMD BMD ) gene have been used to estimate intragenic recombination rates. Multipoint linkage analysis of the CEPH panel of families suggests a total intragenic recombination frequency of nearly 0.12 (confidence intervals 0.041–0.226) over the genomic length of approximately 2 Mb.

Direct selection of mutants influencing gene conversion in the yeast Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, a suppressor-active mutation at the anticodon site of the tRNASerUCA gene sup3 leads to opal (UGA)-specific suppression. Second-site mutations (rX) in sup3 inactivate the suppressor. The sup3-UGA, rX double mutants are genetically unstable in meiotic selfings, due to the intergenic transfer of information between sup3 and the unlinked genes sup9 and sup12 (Hofer et al. 1979; Munz and Leupold 1981; Munz et al. 1982). These three genes have considerable sequence homology over about 200 base pairs (Hottinger et al. 1982). Mutants showing a decrease or an increase of the meiotic instability at sup3 have been selected. One mutation (rec3-8) increases both the genetic instability and the frequency of intragenic recombination in sup3 by one order of magnitude. It has no effect on the stability of the nonsense alleles arg1-230 (UAA), ade6-704 and ural1-61 (UGA) or on the frequency of crossing-over between sup3 and the closely linked gene cdc8. The existence of a common genetic control over intragenic recombination and genetic instability at sup3 provides a direct way of selecting for rec mutants in homothallic haploid strains of S. pombe carrying a suppressor-inactive allele of sup3. It also supports the hypothesis that the instability of mutant alleles of this gene is due to chromosome mispairing at meiosis allowing sup3 to pair with sup9 or sup12 and then to undergo recombination by gene conversion restoring the suppressor-active allele sup3-UGA from the suppressor-inactive allele sup3-UGA, rX.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure of cdc8 homozygous diploids of Saccharomyces cerevisiae to nonpermissive temperature leads to an increase in mitotic conversion frequencies

In a diploid strain homozygous for the cdc8-1 mutation, a block in DNA synthesis caused by restrictive temperature resulted in a significant increase in the frequency of intragenic recombination at the HOM2 locus. Under restrictive conditions, incorporation of radioactivity into DNA was reduced to 2% of the control and alkaline sucrose gradient centrifugation revealed that only short DNA fragments were synthesized. There was no considerable fragmentation of template DNA during incubation of cdc8-1 strains under restrictive conditions.

Variation of gene conversion and intragenic recombination frequencies in the genome of Ascobolus immersus

Eighty mutants in 17 ascospore character genes were studied for their conversion patterns. The correlation between conversion pattern and mutagenic origin, previously found in genes b1 and b2 was extended to all the genes studied. Aberrant 4:4 asci were found in most genes irrespective of their conversion frequency. From gene to gene, the conversion frequency showed an almost 100 times variation. The frequency of intragenic recombination also showed sharp variation from gene to gene. The mean conversion frequency and the maximal intragenic recombination frequency were shown to be highly correlated in 5 genes for which these 2 values are known. This correlation was extended to 12 other genes in other Ascomycetes: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora, and Sordaria. From this study it is concluded that, 1) the probability of hybrid DNA formation undergoes considerable changes according to the region of the genome; 2) the intragenic recombination frequency primarily reflects the frequency of hybrid DNA formation rather than the physical length of the gene; 3) for a given physical distance on the DNA, a similar fraction of the gene conversion events lead to recombination in the 5 Ascomycetes.

8-Hydroxyquinoline inhibition of DNA synthesis and intragenic recombination during yeast meiosis

Complete inhibition of sporulation was observed in two strains of Saccharomyces cerevisiae to which 8-hydroxyquinoline was added at a final concentration of 5 microgram/ml during the initial 4 to 6 h of sporulation. The cells were most sensitive to the inhibitor during 4 to 6 interval beginning at approximately 2 h (T2). Its addition during that interval resulted in 70 to 80% lethality in strain 4579 and about 40% in API at T24. When present from T0 onward, 5 microgram/ml of 8-hydroxyquinoline severely inhibited premeiotic DNA replication and reduced the frequency of intragenic recombination at the ade 2 and leu 2 loci by 70 and 100%, respectively, relative to control cultures which did not have the inhibitor present. During the period when the cells were most sensitive, the incorporation of 14C-leucine into protein and 14C-adenine into RNA was not inhibited nor was the polysome content affected. At 150 microgram/ml of inhibitor, incorporation of labeled precursors into RNA and protein were inhibited and the percentage of active ribosomes was reduced by 35% within 45 min, but neither transcription or translation appeared to be completely inhibited at this concentration of the inhibitor.

GENETIC RECOMBINATION IN SEXUAL CROSSES OF PHYCOMYCES

Sexual crosses between strains of Phycomyces blakesleeanus , involving three auxotrophic and one color marker and yielding a high proportion of zygospore germination, are described. Samples of 20-40 germ spores from 311 individual fertile germ sporangia originating from five two-factor and three three-factor crosses were characterized. The results show: (1) absence of any contribution of apogamic nuclei to the progeny, (2) confirmation of Burgeff's conjecture that the germ spores of any germ sporangium in most cases derive from one meiosis. In a cross involving two allelic markers the analysis of 175 pooled germ sporangia suggests an intragenic recombination frequency of 0.6%. All other factor combinations tested are unlinked. The bulk of the germ spores are homokaryotic. However, a small portion (4%) are heterokaryotic with respect to mating type.

Another two genes controlling mitotic intragenic recombination and recovery from UV damage in Aspergillus nidulans IV. Genetic analysis of mitotic intragenic recombinants from uvs+/ uvs+, uvsD/ uvsD and uvsE/ uvsE diploids

This paper presents the results of a genetic analysis of a number of spontaneous mitotic p-aminobenzoic acid-independent recombinants from uvs +/ uvs +, uvsD 53/ uvsD 53 and uvsE82/ uvsE82 diploids that are heteroallelic at the pabaA locus. Intragenic recombination in each of the three strains is largely nonreciprocal and may usually result from gene conversion. Gene conversion may be strongly correlated with crossing-over between outside markers in the uvsE82/ uvsE82 diploid. Conversion shows no polarity in the uvs +/ uvs +diploid; in the uvsE82/ uvsE82 diploid there is almost no conversion of the distal pabaA allele; the proximal allele probably converts more often in uvsE82/ uvsE82 but this is obscured by the high back-mutation rate of the proximal allele. Whether there is any polarity of gene conversion in the uvsD 53 / uvsD 53 diploid cannot be inferred from the results. More than half of the recombinants from the uvsD 53 / uvsD 53 diploid have an apparently complex origin and may have arisen by double exchanges or by second-order recombination. No such complex recombinants have arisen from the uvsE82/ uvsE82 diploid and their frequency among recombinants from the uvs +/ uvs + strain is 16%. The frequency of appearance of these complex recombinants in our strains seems to be correlated with the frequency of intragenic recombination in the particular strains, as influenced by the uvs allele they bear.

The effects of dark holding and photoreactivation on ultraviolet light-induced mitotic recombination and survival in yeast.

UV induces lesions in DNA which lead to the death of cells, mutation and, in yeast, intragenic and intergenic mitotic recombination. We have investigated the interaction of two post-treatments, dark holding and photoreactivation, on the frequencies of these events. It was found that dark holding reduces cell death and intergenic recombinants, but causes an increase in intragenic recombination frequency. Photoreactivation reduces cell death and intragenic recombination, but has no effect on intergenic recombination. After dark holding, photoreactivation has no further effect on cell survival or intergenic recombination, but may reduce the frequency of intragenic recombinants. After photoreactivation, dark holding still causes an increase in cell survival and the frequency of intragenic recombination, and reduces the frequency of intergenic recombination.

EFFECT OF GENE INDUCTION ON FREQUENCY OF INTRAGENIC RECOMBINATION OF CHROMOSOME AND F-MEROGENOTE IN ESCHERICHIA COLI K-12

EFFECT OF GENE INDUCTION ON FREQUENCY OF INTRAGENIC RECOMBINATION OF CHROMOSOME AND F-MEROGENOTE IN <i>ESCHERICHIA COLI</i> K-12