Chromosome polymorphisms close to the cm-ADE1 locus of Candida maltosa

The imperfect yeast Candida maltosa has an ill-defined genetic constitution; it is nominally diploid, but probably highly aneuploid, in nature. We report on polymorphisms specifically affecting those chromosomes which bear the cm-ADE1 gene. This gene encodes phosphoribosylaminoimidazole-succinocarboxamide synthetase, an enzyme in the adenine biosynthetic pathway. By electrophoretic karyotype analysis, three differently sized chromosomes were demonstrated to carry cm-ADE; the size (but not the number) of these chromosomes was also found to vary, both between strains and during the mitotic growth of a single strain. Four different alleles of cm-ADE1 have been cloned and sequenced from one prototrophic strain. DNA sequence divergence between these different alleles is as high as 8%, with the greatest divergence being found in the upstream region. Mitotic recombination events that led to changes in the karyotype were followed by using cm-ADE1 DNA as an hybridization probe. A recombination hot-spot in the neighbourhood of the gene appears to be responsible for the instability of the chromosomes on which it resides.     

The DNA of Arabidopsis thaliana

Summary Arabidopsis thaliana is a small flowering plant of the mustard family. It has a four to five week generation time, can be self- or cross-pollinated and bears as many as 10⁴ seeds per plant. Many visible and biochemical mutations exist and have been mapped by recombination to one of the five chromosomes that comprise the haploid karyotype. With the experiments reported here we demonstrate that Arabidopsis has an extraordinarily small haploid genome size (approximately 7×10⁷ nucleotide pairs) and a low level of cytosine methylation for an angiosperm. In addition, it appears to have little repetitive DNA in its nuclear DNA, in contrast to other higher plants.     

Structure and organization of rhodophyte and chromophyte plastid genomes: implications for the ancestry of plastids

Summary Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15–20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms⁺ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.     

Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe

In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1 ⁺ gene was present in the introduced leu1::ura4 ⁺ DNA fragment; but a decrease in length of homology leads to an increase in the ratio of nonhomologous to homologous recombination events. The introduced DNA fragments were integrated into different sites in the chromosomes by nonhomologous recombination. The results suggested that there are multiple modes of integration; most events simply involve both ends of the fragments, while in other cases, fragments were integrated in a more complicated manner, probably via circularization or multimerization. To analyze the mechanism of the major type of integration, DNA fragments containing the recombination junctions of three recombinants were amplified by inverted polymerase chain reaction (IPCR) and their nucleotide sequences were determined. There was no obvious homology between introduced DNA and chromosomal DNA at these recombination sites. Furthermore it was found that each terminal region of the introduced DNA was deleted, but that there were no or very small deletions in the target sites of chromosomal DNA. Two models are proposed to explain the mechanism of nonhomologous integration.     

Hyper-recombination in uvrD mutants of Escherichia coli K-12

Summary A mutant strain of E. coli which was isolated initially because of its strong hyper-recombination phenotype was shown to carry a lesion in uvrD. The presence of this mutation, designated uvrD210, increased the frequency of recombination between chromosomal duplications in F-prime repliconant cells and reduced linkage between closely linked markers in crosses with Hfr donors. A comparable hyper-rec phenotype was demonstrated in strains carrying other alleles of uvrD previously referred to as mutU4, uvr502 and recL152. The recombination activity of a uvrD210 strain was abolished by mutation of recA but the mutator activity associated with this allele proved to be independent of recA. It is suggested that uvrD mutations reduce the fidelity of DNA replication and that the accumulation of lesions in the newly synthesized strand provides additional sites for initiating recombination.     

Magnification in Drosophila: evidence for an inducible rDNA-specific recombination system

An experiment is described that provides evidence for an exchange mechanism to explain the increase in ribosomal gene number that occurs during bobbed magnification. We show that bobbed and bobbed-lethal alleles do not magnify in closed X chromosomes, but that a spontaneous ring opening restores normal magnification. The results provide strong evidence that the elementary magnifying event is unequal sister chromatid exchange, and can be interpreted in the framework of an inducible rDNA-specific recombination system as the basis of ribosomal gene magnification. Correspondence to: S.A. Endow at the above address     

Complementation pattern of lexB and recA mutations in Escherichia coli K12; mapping of tif-1, lexB and recA mutations

Summary Three lexB mutations, whose phenotypes have been previously characterized, are studied here in relation to a few recA mutations as to their complementation pattern and relative location.The restoration of resistance to UV-light and to X-rays in the hetero-allelic diploid bacteria was used as a test for dominance and complementation. The wild type allele was always dominant over the mutant allele. Only partial complementation was found between lexB and two recA alleles. There was no complementation between the recA alleles. All the data taken together strongly suggest that the complementations found are intragenic: lexB and recA mutations are in one gene.Mapping of lexB, recA and tif-1 mutations in relation to srl-1 and cysC by phage P1 transduction shows that lexB and the tif-1 mutations form a cluster proximal to srl-1 whereas recA mutations are located at the other extremity of the gene. Variability with temperature of cotransduction frequencies as well as their extended range of values prevent a meaningful calculation of the length of the recA gene.Our hypothesis is that the recA protein has two functional regions called A and B respectively defined at the genetical level by recA and lexB mutations and that it is, in vivo, an oligomeric protein forming a complex with the lexA protein. This complex is postulated to be multifunctional: recombination and control of exonuclease V are effected by the A region while the B region and lexA protein effect induced DNA repair and lysogenic induction.     

Molecular karyotyping and chromosome length polymorphism in Cochliobolus sativus

Fungi are known to have variable genomes that can generate new virulence types capable of attacking important crop plants. To assess chromosome length polymorphisms in the barley spot blotch pathogen (Cochliobolus sativus), we analyzed the karyotypes of 16 isolates using contour-clamped homogeneous electric field (CHEF) electrophoresis. The collection of isolates studied were from diverse regions of the world (USA, Canada, Japan, Brazil, Uruguay, and Poland) and included representatives comprising the three known C. sativus pathotypes of 0, 1, and 2. Under two different running conditions, the number of CHEF bands observed ranged from 8 to 13 with a size range of 0.85 to 3.80 mega-bases (Mb). Each of the 16 isolates showed a unique banding pattern, except for two North Dakota isolates ND90Pr and ND91-Bowman, which were very similar. Single-copy DNA probes, previously assigned to each of the 15 chromosomes identified in reference isolate ND93-1, were hybridized to Southern blots of CHEF-separated chromosomes and revealed highly polymorphic chromosomes among isolates. Chromosomal rearrangements (translocations, deletions, duplications) were found in several isolates. DNA markers previously found linked to VHv1, a gene in pathotype 2 isolates conferring virulence on barley cultivar Bowman, also were used as probes in hybridizations with the CHEF blots. The results showed that the chromosome carrying the virulence gene in pathotype 2 isolates is larger than its counterpart without the gene in other isolates. This suggests that the genomic region carrying the virulence locus VHv1 is unique to pathotype 2 isolates. This study provides useful information on genome structure and divergence, which is essential for advancing our understanding of the genetics and biology of C. sativus.     

The Tetracycline Resistance Genetet(M) Exhibits Mosaic Structure

Tetracycline resistance genes of the M class,tet(M), are typically found on mobile genetic elements as the conjugative transposons of gram-positive bacteria. By comparing the sequences of eight differenttet(M) genes (fromEnterococcus faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Ureaplasma urealyticum,andNeisseria), a mosaic structure was detected which could be traced to two distinct alleles. The two alleles displayed a divergence of 8% and a different G/C content. The block structure of these genes provides evidence for the contribution of homologous recombination to the evolution and the heterogeneity of thetet(M) locus. Unlike described cases of chromosomally located mosaic loci,tet(M) is a relatively recently acquired determinant in the species examined and it would appear that mosaic structure withintet(M) has evolved after acquisition of the gene by the mobile genetic elements upon which it is located.     

Genetic recombination in Bacillus subtilis 168: effect of recN, recF, recH and addAB mutations on DNA repair and recombination

A recN ^– (recN1) strain of Bacillus subtilis was constructed. The effects of this and recF, recH and addAB mutations on recombination proficiency were tested. Mutations in the recN, recF recH and addAB genes, when present in an otherwise Rec⁺ B. subtilis strain, did not affect genetic exchange. Strains carrying different combinations of mutations in these genes were constructed and examined for their sensitivity to 4-nitroquinoline1-oxide (4NQO) and recombination proficiency. The recH mutation did not affect the 4NQO sensitivity of recN and recF cells and it only marginally affected that of addA addB cells. However, it reduced genetic recombination in these cells 10²- to 10⁴-fold. The addA addB mutations increased the 4NQO sensitivity of recF and recN cells, but completely blocked genetic recombination of recF cells and marginally affected recombination in recN cells. The recN mutation did not affect the recombinational capacity of recF cells. These data indicate that the recN gene product is required for, DNA repair and recombination and that the recF, recH and addAB genes provide overlapping activities that compensate for the effects of single mutants proficiency. We proposed that the recF, recH, recB and addA gene products define four different epistatic groups.     

白念珠菌CaGDT1基因的功能研究

人类跨膜蛋白TMEM165与酿酒酵母Sc Gdt1均属于阳离子/钙离子交换器家族的成员,在本研究中,通过序列比对在白念珠菌中发现了Sc GDT1的同源基因Ca GDT1,表型互补实验显示Ca GDT1基因的表达能够抑制Sc GDT1基因缺失所造成的钙离子敏感性,证明Ca GDT1是Sc GDT1的同功基因。此外,通过同源重组原理敲除了Ca GDT1的2个等位基因。表型筛选结果表明gdt1/gdt1缺失株对钙离子、细胞壁和内质网3种胁迫均不敏感,而对酮康唑和特比萘芬2种抗真菌药物具有耐受性。     

Accurate homologous recombination is a prominent double-strand break repair pathway in mammalian chromosomes and is modulated by mismatch repair protein Msh2

We designed DNA substrates to study intrachromosomal recombination in mammalian chromosomes. Each substrate contains a thymidine kinase (tk) gene fused to a neomycin resistance (neo) gene. The fusion gene is disrupted by an oligonucleotide containing the 18-bp recognition site for endonuclease I-SceI. Substrates also contain a “donor” tk sequence that displays 1% or 19% sequence divergence relative to the tk portion of the fusion gene. Each donor serves as a potential recombination partner for the fusion gene. After stably transfecting substrates into mammalian cell lines, we investigated spontaneous recombination and double-strand break (DSB)-induced recombination following I-SceI expression. No recombination events between sequences with 19% divergence were recovered. Strikingly, even though no selection for accurate repair was imposed, accurate conservative homologous recombination was the predominant DSB repair event recovered from rodent and human cell lines transfected with the substrate containing sequences displaying 1% divergence. Our work is the first unequivocal demonstration that homologous recombination can serve as a major DSB repair pathway in mammalian chromosomes. We also found that Msh2 can modulate homologous recombination in that Msh2 deficiency promoted discontinuity and increased length of gene conversion tracts and brought about a severalfold increase in the overall frequency of DSB-induced recombination.     

LINKAGE TO THE MATING‐TYPE LOCUS ACROSS THE GENUS MICROBOTRYUM: INSIGHTS INTO NONRECOMBINING CHROMOSOMES

Parallels have been drawn between the evolution of nonrecombining regions in fungal mating‐type chromosomes and animal and plant sex chromosomes, particularly regarding the stages of recombination cessation forming evolutionary strata of allelic divergence. Currently, evidence and explanations for recombination cessation in fungi are sparse, and the presence of evolutionary strata has been examined in a minimal number of fungal taxa. Here, the basidiomycete genus Microbotryum was used to determine the history of recombination cessation for loci on the mating‐type chromosomes. Ancestry of linkage with mating type for 13 loci was assessed across 20 species by a phylogenetic method. No locus was found to exhibit trans‐specific polymorphism for alternate alleles as old as the mating pheromone receptor, indicating that ages of linkage to mating type varied among the loci. The ordering of loci in the ancestry of linkage to mating type does not agree with their previously proposed assignments to evolutionary strata. This study suggests that processes capable of influencing divergence between alternate alleles may act at loci in the nonrecombining regions (e.g., gene conversion) and encourages further work to dissect the evolutionary processes acting upon genomic regions that determine mating compatibility.     

Chromosomal location of 45S rDNA and dfr gene in Citrus sinensis

Dihydroflavonol 4-reductase (DFR) is a key enzyme in the anthocyanin biosynthesis. In this study, the localization of 45S rDNA and dfr gene (named as CsDFR-bo) of the blood orange (Citrus sinensis Osbeck cv. Ruby) on chromosomes was investigated by fluorescence in situ hybridization (FISH). A karyotype of C. sinensis was reconstructed based on the length of mitotic metaphase chromosomes. The 45S rDNAs were localized on chromosomes 2p and 7q. The detection ratios of 45S rDNA in the two chromosomes were 69.5 and 77.3 %, respectively. The CsDFR-bo was proved to be a single-copy gene and localized on chromosome 3p. The detection ratio of CsDFR-bo was 8.2 %.     

Inheritance of chromosomal length polymorphisms in the ascomycete Leptosphaeria maculans

Pulsed field gel electrophoresis experiments show that chromosomal length polymorphisms are produced during meiosis in the ascomycetous plant pathogen Leptosphaeria maculans. Homologues in tetrads of L. maculans were identified on the basis of their binding to chromosome-specific probes that included -tubulin, nitrate reductase, 18S ribosomal DNA and two Random Amplified Polymorphic DNA (RAPD) markers. Changes in size of homologues were followed during meiosis. Significant karyotype variation was evident due to the random assortment of parental homologues of different sizes. In most cases, the progeny had the same-sized homologues as the parents; however, in some instances novel-sized homologues were detected that varied in size from those of the parents by up to 50 kb. Our results are consistent with the hypothesis that these novel chromosomal length polymorphisms are produced by reciprocal recombination between parental homologous chromosomes of unequal sizes.     

Gene flow and selection interact to promote adaptive divergence in regions of low recombination

Adaptation to new environments often occurs in the face of gene flow. Under these conditions, gene flow and recombination can impede adaptation by breaking down linkage disequilibrium between locally adapted alleles. Theory predicts that this decay can be halted or slowed if adaptive alleles are tightly linked in regions of low recombination, potentially favouring divergence and adaptive evolution in these regions over others. Here, we compiled a global genomic data set of over 1,300 individual threespine stickleback from 52 populations and compared the tendency for adaptive alleles to occur in regions of low recombination between populations that diverged with or without gene flow. In support of theory, we found that putatively adaptive alleles (F_ST and d_XY outliers) tend to occur more often in regions of low recombination in populations where divergent selection and gene flow have jointly occurred. This result remained significant when we employed different genomic window sizes, controlled for the effects of mutation rate and gene density, controlled for overall genetic differentiation, varied the genetic map used to estimate recombination and used a continuous (rather than discrete) measure of geographic distance as proxy for gene flow/shared ancestry. We argue that our study provides the first statistical evidence that the interaction of gene flow and selection biases divergence toward regions of low recombination.     

Mos1 transposition as a tool to engineer the Caenorhabditis elegans genome by homologous recombination

Gene knockouts and knock-ins have emerged as powerful tools to study gene function in model organisms. The construction of such engineered alleles requires that homologous recombination between a transgenic fragment carrying the modifications desired in the genome and the locus to engineer occurs at high frequencies. Homologous recombination frequency is significantly increased in the vicinity of a DNA double-strand break. Based on this observation, a new generation of transgene-instructed genome engineering protocols was developed. Here, we present MosTIC (for “Mos1 excision-induced transgene-instructed gene conversion”), a new technique that provides a means to engineer the Caenorhabditis elegans genome. MosTIC is initiated by the mobilization of Mos1, a Drosophila transposon experimentally introduced in C. elegans. During MosTIC, a Mos1 insertion localized in the genomic region to engineer is mobilized after germline expression of the Mos transposase. Mos1 excision generates a DNA double-strand break, which is repaired by homologous recombination using a transgenic repair template. This results in the transfer of information from the transgene into the genome. Depending on the method used to trigger Mos1 excision, two alternative MosTIC protocols are available, which are presented here in detail. This technique can be used for a wide range of applications, such as structure-function analysis, protein localization and purification, genetic screens or generation of single copy transgenes at a defined locus in the genome.