A gene for late-onset fundus flavimaculatus with macular dystrophy maps to chromosome 1p13.

Fundus flavimaculatus with macular dystrophy is an autosomal recessive disease responsible for a progressive loss of visual acuity in adulthood, with pigmentary changes of the macula, perimacular flecks, and atrophy of the retinal pigmentary epithelium. Since this condition shares several clinical features with Stargardt disease, which has been mapped to chromosome 1p21-p13, we tested the disease for linkage to chromosome 1p. We report here the mapping of the disease locus to chromosome 1p13-p21, in the genetic interval defined by loci D1S435 and D1S415, in four multiplex families (maximum lod score 4.79 at recombination fraction 0 for probe AFM217zb2 at locus D1S435). Thus, despite differences in the age at onset, clinical course, and severity, fundus flavimaculatus with macular dystrophy and Stargardt disease are probably allelic disorders. This result supports the view that allelic mutations produce a continuum of macular dystrophies, with onset in early childhood to late adulthood.     

Interplasmid illegitimate recombination in Bacillus subtilis cells

The illegitimate recombination between S. aureus plasmids pE194 (or pGG20-the hybrid between pE194 and E. coli plasmid pBR322) and pBD17 (plasmid pUB110 without Hpa-II-C-fragment) in B. subtilis was studied. Plasmid cointegrates were generated with the frequency of 1-3.10(-8). Among the 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all the parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions has revealed that in 8 cases recombination occurred between short homologous regions (9-15 b.p.). One of the recombinants resulted from nonhomologous recombination. The similarity between nucleotide sequences of recombination sites of two types of contegrates and those used for pE194 integration into the B. subtilis chromosome (Bashkirov et al. 1987) was demonstrated. Possible mechanisms of illegitimate recombination are discussed.     

A staphylococcal plasmid that replicates and expresses ampicillin, gentamicin and amikacin resistance in Escherichia coli

Plasmid pPG1 from Staphylococcus aureus coding for ampicillin (Apr), gentamicin (Gmr) and amikacin (Akr) resistance was transformed into Escherichia coli. Transformation efficiency was about 2 x 10(3) transformants/micrograms of plasmid DNA. The plasmids present in the E. coli transformants were identical to pPG1 according to their restriction patterns. The copy number of pPG1 was estimated to be at least 20-times less in E. coli than in S. aureus. The minimal inhibitory concentrations (MICs) for Ap and Gm were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus. pPG1 was maintained in the E. coli transformants for at least 80 generations at 37 degrees C without antibiotic selection pressure.     

产SEB金黄色葡萄球菌α-溶血毒素基因缺失菌株的构建

构建产肠毒素B(Staphylococcal enterotoxin B ,SEB)的金黄色葡萄球菌α-溶血毒素（α-hemolysin, α-HL)缺失菌株。首先构建用于α-HL基因敲除的同源重组质粒pMHL-α,经金黄色葡萄球菌RN4220修饰后再通过原生质体转入金黄色葡萄球菌SM-01。含重组质粒pMHL-α的金黄色葡萄球菌SM-01在42℃诱导条件下培养多代,最终筛选出α-溶血毒素基因缺失菌株。经序列分析和血平板溶血实验结果证明最终获得产SEB金黄色葡萄球菌α-HL缺失菌株。为野生型金黄色葡萄球菌的体内遗传操作及构建产超抗原药物金黄色葡萄球菌基因工程菌株提供了一定的理论基础和方法。     

The Evolution of Recombination: Removing the Limits to Natural Selection

One of the oldest hypotheses for the advantage of recombination is that recombination allows beneficial mutations that arise in different individuals to be placed together on the same chromosome. Unless recombination occurs, one of the beneficial alleles is doomed to extinction, slowing the rate at which adaptive mutations are incorporated within a population. We model the effects of a modifier of recombination on the fixation probability of beneficial mutations when beneficial alleles are segregating at other loci. We find that modifier alleles that increase recombination do increase the fixation probability of beneficial mutants and subsequently hitchhike along as the mutants rise in frequency. The strength of selection favoring a modifier that increases recombination is proportional to λ(2)Sδr/r when linkage is tight and λ(2)S(3)δ r/N when linkage is loose, where λ is the beneficial mutation rate per genome per generation throughout a population of size N, S is the average mutant effect, r is the average recombination rate, and δr is the amount that recombination is modified. We conclude that selection for recombination will be substantial only if there is tight linkage within the genome or if many loci are subject to directional selection as during periods of rapid evolutionary change.     

Construction, transfer and properties of a novel temperature-sensitive integrable plasmid for genomic analysis of Staphyiococcus aureus

As an alternative approach to genetic transfer and analysis, a novel integrable plasmid system was developed that should prove useful for mapping and cloning various genes in Staphylococcus aureus and other Gram-positive bacteria. The use of a restriction-deficient recipient strain and an improved protocol for protoplast plasmid transformation facilitated direct cloning of a recombinant plasmid (pPQ126) in S. aureus NCTC 8325-4. Plasmid pPQ126 (13.6 kb) is a novel, temperature-sensitive integrable plasmid containing genes encoding resistance to erythromycin and chloramphenicol (from plasmid pTV1ts), and resistance to gentamicin (from transposon Tn4001). When introduced into an appropriate recipient strain at the permissive temperature (30 degrees C), pPQ126 replicates autonomously. Integration of pPQ126 is directed into homologous chromosomal target sequences (chromosomal insertions of Tn551 or Tn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39 degrees C (nonpermissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination-deficient host, and does not occur in the absence of host chromosomal homology. Integrated pPQ126 excises from the chromosome under permissive conditions (30 degrees C), and excision results in derivatives of pPQ126 that harbour DNA of chromosomal origin.     

Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae

In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements.     

Evidence for clonal evolution among highly polymorphic genes in methicillin-resistant Staphylococcus aureus

The evolution of Staphylococcus aureus has been described as predominantly clonal, based on evidence from seven housekeeping genes. We aimed to test if this was also true for more polymorphic genes. In a collection of 60 isolates including major European epidemic methicillin-resistant S. aureus (MRSA) and sporadic MRSA strains, we compared the partial gene sequences of seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL), six core adhesion genes (present in all strains) (clfA, clfB, fnbA, map, sdrC, and spa), and four accessory adhesion genes (not present in all strains) (ebpS, fnbB, sdrD, and sdrE). Nucleotide diversity of adhesion genes was 2- to 10-fold higher than genes used for multilocus sequence typing. All genes showed evidence for purifying selection with a weakly reduced level among accessory adhesion genes. Among these highly variable genes, there was no evidence for a difference in molecular evolution between epidemic and sporadic strains. Gene trees constructed from concatenated sequences of housekeeping, core adhesion, and accessory adhesion genes were highly congruent, indicating clonality, despite some evidence for homologous exchange. Further evidence for clonality was found with an overall positive correlation of allelic and nucleotidic divergence for both seven housekeeping genes and six core adhesion genes. However, for small allelic differences that fit the demarcations of clonal complexes (CCs) there was no such correlation, suggesting that recombination occurred. Therefore, despite an overall clonal population structure, recombination between related isolates within CCs might have contributed to S. aureus evolution.     

Allelic and Ectopic Interactions in Recombination-Defective Yeast Strains

Ectopic recombination in the yeast Saccharomyces cerevisiae has been investigated by examining the effects of mutations known to alter allelic recombination frequencies. A haploid yeast strain disomic for chromosome III was constructed in which allelic recombination can be monitored using leu2 heteroalleles on chromosome III and ectopic recombination can be monitored using ura3 heteroalleles on chromosomes V and II. This strain contains the spo13-1 mutation which permits haploid strains to successfully complete meiosis and which rescues many recombination-defective mutants from the associated meiotic lethality. Mutations in the genes RAD50, SPO11 and HOP1 were introduced individually into this disomic strain using transformation procedures. Mitotic and meiotic comparisons of each mutant strain with the wild-type parental strain has shown that the mutation in question has comparable effects on ectopic and allelic recombination. Similar results have been obtained using diploid strains constructed by mating MATa and MAT alpha haploid derivatives of each of the disomic strains. These data demonstrate that ectopic and allelic recombination are affected by the same gene products and suggest that the two types of recombination are mechanistically similar. In addition, the comparison of disomic and diploid strains indicates that the presence of a chromosome pairing partner during meiosis does not affect the frequency of ectopic recombination events involving nonhomologous chromosomes.     

New range of vectors with a stringent 5-fluoroorotic acid-based counterselection system for generating mutants by allelic replacement in Staphylococcus aureus

We have developed a range of vectors for allelic replacements in Staphylococcus aureus to facilitate genetic work in this opportunistic pathogen. The central feature of the vector range is a selection/counterselection system that takes advantage of the 5-fluoroorotic acid (FOA) resistance and pyrimidine prototrophy caused by the loss and gain, respectively, of the pyrF and pyrE genes. This system allows for stringent counterselection of the vectors during the second homologous recombination of a classic allelic replacement. The basic vector pRLY2, which contains the pyrFE genes from Bacillus subtilis, was combined with chloramphenicol, erythromycin, and tetracycline resistance genes and four different versions of nonreplicative or conditionally replicative origins of replication. The choice between these 12 different pRLY vectors allows for high versatility and ensures that the vectors can be used in virtually any genetic background. Finally, as proof of concept, we present six deletions or modifications of components in the S. aureus degradosome as well as the operon containing the cshB DEAD box helicase.     

The effect of chromosome geometry on genetic diversity

Although organisms with linear chromosomes must solve the problem of fully replicating their chromosome ends, this chromosome configuration has emerged repeatedly during bacterial evolution and is evident in three divergent bacterial phyla. The benefit usually ascribed to this topology is the ability to boost genetic variation through increased recombination. But because numerous processes can impact linkage disequilibrium, such an effect is difficult to assess by comparing across bacterial taxa that possess different chromosome topologies. To test directly the contribution of chromosome architecture to genetic diversity and recombination, we examined sequence variation in strains of Agrobacterium Biovar 1, which are unique among sequenced bacteria in having both a circular and a linear chromosome. Whereas the allelic diversity among strains is generated principally by mutations, intragenic recombination is higher within genes situated on the circular chromosome. In contrast, recombination between genes is, on average, higher on the linear chromosome, but it occurs at the same rate as that observed between genes mapping to the distal portion of the circular chromosome. Collectively, our findings indicate that chromosome topology does not contribute significantly to either allelic or genotypic diversity and that the evolution of linear chromosomes is not based on a facility to recombine.     

The impact of recombination on short-term selection gain in plant breeding experiments

Recombination is a requirement for response to selection, but researchers still debate whether increasing recombination beyond normal levels will result in significant gains in short-term selection. We tested this hypothesis, in the context of plant breeding, through a series of simulation experiments comparing short-term selection response (≤20 cycles) between populations with normal levels of recombination and similar populations with unconstrained recombination (i.e., free recombination). We considered additive and epistatic models and examined a wide range of values for key design variables: selection cycles, QTL number, heritability, linkage phase, selection intensity and population size. With few exceptions, going from normal to unconstrained levels of recombination produced only modest gains in response to selection (≈11 % on average). We then asked how breeders might capture some of this theoretical gain by increasing recombination through either (1) extra rounds of mating or (2) selection of highly recombinant individuals via use of molecular markers/maps. All methods tested captured less than half of the potential gain, but our analysis indicates that the most effective method is to select for increased recombination and the trait simultaneously. This recommendation is based on evidence of a favorable interaction between trait selection and the impact of recombination on selection gains. Finally, we examined the relative contributions of the two components of meiotic recombination, chromosome assortment and crossing over, to short-term selection gain. Depending primarily on the presence of trait selection pressure, chromosome assortment alone accounted for 40–75 % of gain in response to short-term selection.     

Staphylococcus aureus keratinocyte invasion is dependent upon multiple high-affinity fibronectin-binding repeats within FnBPA

Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs) to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs) which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α(5)β(1) integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity) FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA.     

New method for generating deletions and gene replacements in Escherichia coli.

We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44 degrees C. Subsequent growth of these cointegrates at 30 degrees C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.     

Construction of single-copy integration vectors for Staphylococcus aureus.

Single-copy integration vectors suitable for cloning in Staphylococcus aureus have been constructed. Their construction was based on the site-specific recombination system of staphylococcal phage, L54a. The vectors are capable of autonomous replication in Escherichia coli, but they are not endowed with a replication function in S. aureus. As a consequence, establishment of these vectors in S. aureus can only be achieved by the integration system of the phage. Once integrated into the chromosome, the vectors, or their derivatives, were stably inherited even without selective pressure. Because such a vector exists in an integrated form in S. aureus, the gene dosage of the DNA cloned in the vector matches that of the chromosome.     

Position Effects in Ectopic and Allelic Mitotic Recombination in Saccharomyces Cerevisiae

We have examined the role that genomic location plays in mitotic intragenic recombination. Mutant alleles of the LEU2 gene were inserted at five locations in the yeast genome. Diploid and haploid strains containing various combinations of these inserts were used to examine both allelic recombination (between sequences at the same position on parental homologs) and ectopic recombination (between sequences at nonallelic locations). Chromosomal location had little effect on mitotic allelic recombination. The rate of recombination to LEU2 at five different loci varied less than threefold. This finding contrasts with previous observations of strong position effects in meiosis; frequencies of meiotic recombination at the same five loci differ by about a factor of forty. Mitotic recombination between dispersed copies of leu2 displayed strong position effects. Copies of leu2 located approximately 20 kb apart on the same chromosome recombined at rates 6-13-fold higher than those observed for allelic copies of leu2. leu2 sequences located on nonhomologous chromosomes or at distant loci on the same chromosome recombined at rates similar to those observed for allelic copies. We suggest that, during mitosis, parental homologs interact with each other no more frequently than do nonhomologous chromosomes.     

Analysis of close stable homolog juxtaposition during meiosis in mutants of Saccharomyces cerevisiae

A unique aspect of meiosis is the segregation of homologous chromosomes at the meiosis I division. The pairing of homologous chromosomes is a critical aspect of meiotic prophase I that aids proper disjunction at anaphase I. We have used a site-specific recombination assay in Saccharomyces cerevisiae to examine allelic interaction levels during meiosis in a series of mutants defective in recombination, chromatin structure, or intracellular movement. Red1, a component of the chromosome axis, and Mnd1, a chromosome-binding protein that facilitates interhomolog interaction, are critical for achieving high levels of allelic interaction. Homologous recombination factors (Sae2, Rdh54, Rad54, Rad55, Rad51, Sgs1) aid in varying degrees in promoting allelic interactions, while the Srs2 helicase appears to play no appreciable role. Ris1 (a SWI2/SNF2 related protein) and Dot1 (a histone methyltransferase) appear to play minor roles. Surprisingly, factors involved in microtubule-mediated intracellular movement (Tub3, Dhc1, and Mlp2) appear to play no appreciable role in homolog juxtaposition, unlike their counterparts in fission yeast. Taken together, these results support the notion that meiotic recombination plays a major role in the high levels of homolog interaction observed during budding yeast meiosis.     

Recombination, balancing selection and phylogenies in MHC and self-incompatibility genes.

Using a coalescent model of multiallelic balancing selection with recombination, the genealogical process as a function of recombinational distance from a site under selection is investigated. We find that the shape of the phylogenetic tree is independent of the distance to the site under selection. Only the timescale changes from the value predicted by Takahata's allelic genealogy at the site under selection, converging with increasing recombination to the timescale of the neutral coalescent. However, if nucleotide sequences are simulated over a recombining region containing a site under balancing selection, a phylogenetic tree constructed while ignoring such recombination is strongly affected. This is true even for small rates of recombination. Published studies of multiallelic balancing selection, i.e., the major histocompatibility complex (MHC) of vertebrates, gametophytic and sporophytic self-incompatibility of plants, and incompatibility of fungi, all observe allelic genealogies with unexpected shapes. We conclude that small absolute levels of recombination are compatible with these observed distortions of the shape of the allelic genealogy, suggesting a possible cause of these observations. Furthermore, we illustrate that the variance in the coalescent with recombination process makes it difficult to locate sites under selection and to estimate the selection coefficient from levels of variability.     

Selection and inertia in the evolution of holocentric chromosomes in sedges (Carex, Cyperaceae)

? Changes in chromosome number as a result of fission and fusion in holocentrics have direct and immediate effects on the recombination rate. We investigate the support for the classic hypothesis that environmental stability selects for increased recombination rates. ? We employed a phylogenetic and cytogenetic data set from one of the most diverse angiosperm genera in the world, which has the largest nonpolyploid chromosome radiation (Carex, Cyperaceae; 2n = 12-124; 2100 spp.). We evaluated alternative Ornstein-Uhlenbeck models of chromosome number adaptation to the environment in an information-theoretic framework. ? We found moderate support for a positive influence of lateral inflorescence unit size on chromosome number, which may be selected in a stable environment in which resources for reproductive investment are larger. We found weak support for a positive influence on chromosome number of water-saturated soils and among-month temperature constancy, which would be expected to be negatively select for pioneering species. Chromosome number showed a strong phylogenetic signal. ? We argue that our finding of small but significant effects of life history and ecology is compatible with our original hypothesis regarding selection of optima in recombination rates: low recombination rate is optimal when inmediate fitness is required. By contrast, high recombination rate is optimal when stable environments allow for evolutionary innovation.     

The impact of recombination on dN/dS within recently emerged bacterial clones

The development of next-generation sequencing platforms is set to reveal an unprecedented level of detail on short-term molecular evolutionary processes in bacteria. Here we re-analyse genome-wide single nucleotide polymorphism (SNP) datasets for recently emerged clones of methicillin resistant Staphylococcus aureus (MRSA) and Clostridium difficile. We note a highly significant enrichment of synonymous SNPs in those genes which have been affected by recombination, i.e. those genes on mobile elements designated "non-core" (in the case of S. aureus), or those core genes which have been affected by homologous replacements (S. aureus and C. difficile). This observation suggests that the previously documented decrease in dN/dS over time in bacteria applies not only to genomes of differing levels of divergence overall, but also to horizontally acquired genes of differing levels of divergence within a single genome. We also consider the role of increased drift acting on recently emerged, highly specialised clones, and the impact of recombination on selection at linked sites. This work has implications for a wide range of genomic analyses.