Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11-Rad50-Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.     

Different recombination site specificity of two developmentally regulated genome rearrangements

In the absence of a combined nitrogen source, such as ammonia, approximately every tenth vegetative cell along filaments of the cyanobacterium Anabaena develops into a heterocyst, a terminally differentiated cell that is morphologically and biochemically specialized for nitrogen fixation. At least two specific DNA rearrangements involving the nitrogen-fixation (nif) genes occur during heterocyst differentiation, one within the nifD gene and the other near the nifS gene. The two rearrangements have several properties in common. Both occur quantitatively in all heterocyst genomes, both occur at approximately the same developmental time, late in the process of heterocyst differentiation, and both result from site-specific recombination between short repeated DNA sequences. We report here the nucleotide sequences found at the site of recombination near the nifS gene. These sequences differ from those found previously for the nifD rearrangement, suggesting that the two rearrangements are catalysed by different enzymes and may be regulated independently. We also show that the nifS gene is transcribed only from rearranged genomes.     

山新杨蔗糖合酶基因PCR介导的重组现象研究

[目的]山新杨(Poputlus davidiana ×P.bolleana)是林木基因工程育种基础和应用研究的良好材料;以山新杨蔗糖合酶基因(PdbSUS)为例研究聚合酶链式反应(polymerase chain reaction,PCR)过程介导的重组现象,在此基础上区分每一个PdbSUS基因的两种单倍型,为后续研...     

Multiple intron gain and loss events occurred during the evolution of Cenp-A gene

Centromere protein A(CENP-A) is a histone H3 like protein,and it plays a very important role in chromosomal segregation during mitosis and meiosis.The analyses on the exon-intron organization of the Cenp-A gene in representative genomes revealed that multiple intron gain and loss events have occurred during the evolution of Cenp-A gene in opisthokonta(common ancestor of fungi and animals).Moreover,our results revealed that at least two positions were conserved in the intron gain and loss events during the evolution of the Cenp-A gene.     

Spontaneous shuffling of domains between introns of phage T4

The three self-splicing introns in phage T4 (in the td, sunY and nrdB genes) (Fig. 1a) each have the conserved group I catalytic RNA core structure (Fig. 1b), out of which is looped an open reading frame. Although the core sequences are very similar (approximately 60% identity), the open reading frames seem to be unrelated. Single crossover recombination events between homologous core sequences in the closely linked td and nrdB introns have led to 'exon shuffling. Here we describe spontaneous double crossovers between the unlinked td and sun Y introns that result in shuffling of an intron structure element, P7.1 (refs 3 and 4). The intron domain-switch variants were isolated as genetic suppressors of a splicing-defective P7.1 deletion in the td intron. This unprecedented example of suppression through inter-intron sequence substitution indicates that the introns are in a state of genetic flux and implies the functional interchangeability of the two analogous but nonidentical P7.1 elements. The implications of such recombination events are discussed in the light of the evolution of the introns themselves as well as that of their host genomes.     

Recombination: Multiply infected spleen cells in HIV patients

The genome of the human immunodeficiency virus is highly prone to recombination, although it is not obvious whether recombinants arise infrequently or whether they are constantly being spawned but escape identification because of the massive and rapid turnover of virus particles. Here we use fluorescence in situ hybridization to estimate the number of proviruses harboured by individual splenocytes from two HIV patients, and determine the extent of recombination by sequencing amplified DNA from these cells. We find an average of three or four proviruses per cell and evidence for huge numbers of recombinants and extensive genetic variation. Although this creates problems for phylogenetic analyses, which ignore recombination effects, the intracellular variation may help to broaden immune recognition.     

狂犬病毒糖蛋白衍生物作为靶脑载体初探

血脑屏障（blood-brain barrier, BBB）阻碍了具有治疗潜力的大分子化合物从外周组织进入脑内。为了寻找一种高效、快速通过BBB的靶向性载体,本实验通过罗丹明B标记的狂犬病毒糖蛋白衍生肽（RDP）注射入昆明小鼠体内,与15min、5h取大脑、脊髓及肝、肾等外周组织,冷冻切片观察其在体内的分布,并通过构建pET28a-RDP-luciferase重组质粒,结果发现融合蛋白能快速的穿过血脑屏障分布于中枢神经系统,为治疗中枢神经系统的药物开发提供新的思路。     

Gene targeting in normal and amplified cell lines

Targeted recombination in mammalian cells is rare compared with non-homologous integration. In Saccharomyces cerevisiae the reverse is true. Differences in targeting efficiency could arise because a target of unique DNA is 200 times more dilute in mammalian genomes than it is in yeast. We tested this possibility by measuring gene targeting in normal CHO cells with two copies of the dihydrofolate reductase (DHFR) gene and in amplified CHOC 400 cells, which carry 800 copies. If the concentration of the target gene is critical, amplified cells should show an enhanced frequency of targeted recombination relative to non-homologous integration. Using a positive/negative selection protocol, we demonstrated that the efficiency of targeting into DHFR genes is indistinguishable in normal and amplified CHO cells. As targeting does not depend on the number of targets, the search for homology is not a rate-limiting step in the mammalian pathway of gene targeting. Thus, the difference in genome size is not the basis for the different outcomes of targeting experiments in S. cerevisiae and mammals.     

Adeno-associated virus-mediated integration and expression of human P-globin gene with the core fragment of locus control region in erythroid cells

To improve the integration stability and expression of the transferred human p-globin gene, the two recombinant adeno-associated virus (AAV) vectors containing the human [3-globin gene with a single or multiple DNase I hypersensitive site (HS) core fragment of the LCR were constructed. These recombinants were respectively introduced into MEL cells via AAV-mediated gene transfer to investigate their integration and expression. The results suggested that following AAV vector-mediated gene transfer, the human [3-globin gene with the multiple HS core fragment of the LCR could steadily integrate into MEL cells and confer an expression level comparable with endogenous mouse a-globin gene.     

AcNPV增强子hr5增强HBsAg基因表达的研究

用形成包涵体（ＯＯＣ＋）并能利用人工合成启动序列和多角体ＸＩＶ启动子表达外源基因的转移载体质粒ｐＳＸＩＶＶＩ＋Ｘ３将多角体基因、乙型肝炎病毒表面抗原（ＨＢＳＡｇ）基因和苜蓿丫纹夜蛾核型多角体病毒（ＡｃＮＰＶ）的增强子ｈｒ５部分序列同时插入无包涵体的粉纹夜蛾核型多角体病毒ＴｎＮＰＶ－ＳＶＩ－Ｇ基因组中，得到两株高效表达ＨＢｓＡｇ基因又形成包涵体的重组病毒ＴｎＮＰＶ－ｓｈｒ３５－ＯＣＣ＋和ＴｎＮＰＶ－ｓｈｒ２６－ＯＣＣ＋．对重组病毒的酶切鉴定、ＤＮＡ斑点杂交和Ｓｏｕｔｈｅｒｎｂｌｏｔ分析证实，外源基因及其相应的启动子和增强子序列已正确插入病毒基因组中．插入顺序中，ｈｒ５增强子是插入ＨＢｓＡｇ基因下游，多角体基因与ＨＢｓＡｇ基因方向相反．１２５Ⅰ－固相放射免疫检测和Ｗｅｓｔｅｒｎｂｌｏｔ结果表明，ＨＢｓＡｇ基因在昆虫离体细胞中得到高效表达并保留了抗原活性．ＴｎＮＰＶ－ｓｈｒ２６－ＯＣＣ＋和ＴｎＮＰＶ－ｓｈｒ３５－ＯＣＣ＋表达的ＨＢｓＡ吕蛋白与没有插入增强子序列的重组病毒ＴｎＮＰＶ—ＨＢｓ８５－ＯＣＣ＋的比较，分别提高了４０％和４６％．     

Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells

A full understanding of the function of genes that control developmental events can be obtained only by a combination of molecular and mutational analysis. One putative developmental gene is the mouse engrailed-like gene En-2, which was isolated by virtue of its extensive homology to Drosophila engrailed, which contributes to the control of segmentation in the developing insect. Our hybridization analysis in situ has revealed that expression of En-2 is restricted to a specific domain of the developing central nervous system from 8 days of development on, indicating a role for the gene in establishing spatial domains in the brain. Unfortunately no En-2 mutations are available to elucidate further its function in development. To this end, we report here the isolation of three pluripotent embryonic stem cell lines in which one copy of the homoeobox-containing gene, En-2, has been altered by homologous recombination.     

The map-based sequence of the rice genome

Rice, one of the world's most important food plants, has important syntenic relationships with the other cereal species and is a model plant for the grasses. Here we present a map-based, finished quality sequence that covers 95% of the 389 Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 non-transposable-element-related protein-coding genes were identified, of which 71% had a putative homologue in Arabidopsis. In a reciprocal analysis, 90% of the Arabidopsis proteins had a putative homologue in the predicted rice proteome. Twenty-nine per cent of the 37,544 predicted genes appear in clustered gene families. The number and classes of transposable elements found in the rice genome are consistent with the expansion of syntenic regions in the maize and sorghum genomes. We find evidence for widespread and recurrent gene transfer from the organelles to the nuclear chromosomes. The map-based sequence has proven useful for the identification of genes underlying agronomic traits. The additional single-nucleotide polymorphisms and simple sequence repeats identified in our study should accelerate improvements in rice production.     

Filamentous phage integration requires the host recombinases XerC and XerD

Many bacteriophages and animal viruses integrate their genomes into the chromosomal DNA of their hosts as a method of promoting vertical transmission. Phages that integrate in a site-specific fashion encode an integrase enzyme that catalyses recombination between the phage and host genomes. CTX phi is a filamentous bacteriophage that contains the genes encoding cholera toxin, the principal virulence factor of the diarrhoea-causing Gram-negative bacterium Vibrio cholerae. CTX phi integrates into the V. cholerae genome in a site-specific manner; however, the approximately 6.9-kilobase (kb) CTX phi genome does not encode any protein with significant homology to known recombinases. Here we report that XerC and XerD, two chromosome-encoded recombinases that ordinarily function to resolve chromosome dimers at the dif recombination site, are essential for CTX phi integration into the V. cholerae genome. The CTX phi integration site was found to overlap with the dif site of the larger of the two V. cholerae chromosomes. Examination of sequences of the integration sites of other filamentous phages indicates that the XerCD recombinases also mediate the integration of these phage genomes at dif-like sites in various bacterial species.     

Overview of HBV whole genome data in public repositories and the Chinese HBV reference sequences

The number of Hepatitis B virus （HBV） whole genomic sequences in public nucleotide databases （GenBank, EMBL, and DDBJ） had reached 866 by January 1, 2007. Coming from 46 countries and regions, these sequences were categorized as eight genotypes （A-H）. With the statistical and phylogenetic analysis on all available complete genomic data of HBV, we here present an overview of HBV sequences in public databases. From all registered 229 HBV genomes in Chinese regions as well as 59 sequencing data from our research group, we report the establishment of reference sequences of HBV strains prevailing in China. These analyses provide clues for the effects of HBV genotypes in host clinical progressions, geographic distribution of the infection, and the viral evolutionary history. Moreover, the viral sequence reference would be helpful in the identification of various HBV mutations. Based on the analysis of various public databases, we suggest that the Chinese HBV database with the clinical information should be constructed.     

水稻基因整合平台系统供体质粒pURKMT的构建

提高水稻产量,改良稻米品质是育种学家广泛研究的课题．随着现代生物技术的发展,水稻已成为植物基因工程的重要研究对象．许多实验室已成功地建立了一系列供外源基因转化水稻的系统．但是这些转化系统主要应用Ｔｉ质粒衍生的载体,通过Ｔ－ＤＮＡ左右两端的序列将目的基...     

南宁地区肝细胞性肝癌中突变型p53蛋白表达

用ＡＢＣ免疫组织化学方法，对南宁地区居民中１３例肝细胞性肝癌（ＨＣＣ）的活检组织进行研究。发现其中８例有突变型ｐ＾５３蛋白呈强阳性表达，占ＨＣＣ病例数６４．３％。南宁地区某些县，如扶绥县是我国著名的ＨＣＣ高发区，不但ＨＢＶ感染率高，而且是全国少有的ＡＦＢ１高污染区，后者的作用不能忽视。与江苏启东和南部非洲ＨＣＣ高发区一样，估计也会出现ｐ＾５３基因突变热点。     

Fine-scale recombination rate differences between sexes, populations and individuals

Meiotic recombinations contribute to genetic diversity by yielding new combinations of alleles. Recently, high-resolution recombination maps were inferred from high-density single-nucleotide polymorphism (SNP) data using linkage disequilibrium (LD) patterns that capture historical recombination events. The use of these maps has been demonstrated by the identification of recombination hotspots and associated motifs, and the discovery that the PRDM9 gene affects the proportion of recombinations occurring at hotspots. However, these maps provide no information about individual or sex differences. Moreover, locus-specific demographic factors like natural selection can bias LD-based estimates of recombination rate. Existing genetic maps based on family data avoid these shortcomings, but their resolution is limited by relatively few meioses and a low density of markers. Here we used genome-wide SNP data from 15,257 parent-offspring pairs to construct the first recombination maps based on directly observed recombinations with a resolution that is effective down to 10 kilobases (kb). Comparing male and female maps reveals that about 15% of hotspots in one sex are specific to that sex. Although male recombinations result in more shuffling of exons within genes, female recombinations generate more new combinations of nearby genes. We discover novel associations between recombination characteristics of individuals and variants in the PRDM9 gene and we identify new recombination hotspots. Comparisons of our maps with two LD-based maps inferred from data of HapMap populations of Utah residents with ancestry from northern and western Europe (CEU) and Yoruba in Ibadan, Nigeria (YRI) reveal population differences previously masked by noise and map differences at regions previously described as targets of natural selection.