Mismatch-specific post-meiotic segregation frequency in yeast suggests a heteroduplex recombination intermediate

Post-meiotic segregation of alleles, which is seen, for example, in the 5:3 distribution of alleles in the products of a single meiosis in fungi, has been thought to be due to the non-repair of heteroduplex regions formed during genetic recombination. In current models of genetic recombination, heteroduplex DNA is formed either as the primary intermediate generated by two interacting non-sister chromatids or as a short region flanking a double-stranded gap. The frequency of post-meiotic segregation differs for different alleles, and this is presumed to reflect the varying efficiencies with which different types of mismatches in the heteroduplex are repaired. To gain some insight into this process, we have now determined the nucleotide sequences of various yeast alleles with different post-meiotic segregation frequencies and compared the mismatches predicted to occur in heteroduplexes of these alleles with wild-type DNA with those repaired with varying efficiency in bacterial systems. A striking correlation is observed, with the mismatches predicted for high post-meiotic segregation frequency alleles being similar to mismatches repaired with low efficiency in bacteria. These results support the view that postmeiotic segregation frequency reflects heteroduplex repair efficiency and the contention that meiotic gene conversion is the result of the successful repair of heteroduplex mismatches.     

黑腹果蝇杂合体中ADH杂二聚体的形成

研究了醇脱氢酶基因 (Adh)诱变体与正常基因相互作用后的部分显性现象 .所有 8个由乙基亚硝基尿素 (ENU)和 1个 X-射线诱变体仅为单碱基置换体 ,其余 3个 X-射线诱变体则为 9～2 1个碱基的缺失体 .这 1 2个诱变体 (除 1个外 )都能产生可测的突变肽 ,其中 7个不能与正常肽形成二聚体 ,杂合体酶活性约为正常纯合体的 1 /2 ;另 4个形成二聚体 .形成二聚体突变基因产物中所有氨基酸突变均发生在肽链 1 82～ 1 94氨基酸区域 ,可见该区域对于二聚化不是必需的 ,该序列可能是重要的催化表面功能区     

Conformational mutations in human mitochondrial DNA

Variation in the human mitochondrial DNA (mtDNA) sequence has been extensively analysed using restriction fragment length polymorphisms (RFLPs). MtDNA RFLPs have previously been attributed to nucleotide changes within restriction endonuclease recognition sites or to small insertion-deletion mutations. We now report that RFLPs detected by polyacrylamide gel electrophoresis can also result from single nucleotide substitutions which alter the mobility of small- to medium-sized restriction fragments that incorporate the sequence. We have defined the mutation responsible at two loci and have identified several possible additional loci. When screening human mtDNAs with multiple restriction endonucleases, such mutations can be misidentified as insertion-deletion mutations or counted as multiple polymorphic restriction sites. This can lead to errors in constructing restriction maps and estimating sequence diversity.     

Tight linkage between a splicing mutation and a specific DNA haplotype in phenylketonuria

The first phenylketonuria mutation identified in the human phenylalanine hydroxylase gene is a single base substitution (GT----AT) in the canonical 5'-splice donor site of intron 12. Direct hybridization analysis using specific oligonucleotide probes demonstrates that the mutation is tightly associated with a specific restriction fragment-length polymorphism haplotype among mutant alleles. The splicing mutation is the most prevalent phenylketonuria allele among Caucasians, and the results suggest the possibility of detecting carriers of the genetic trait who have no family history of phenylketonuria.     

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes

Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.     

DNA restriction fragments associated with alpha 1-antitrypsin indicate a single origin for deficiency allele PI Z

The alpha 1-protease inhibitor, or alpha-antitrypsin (AAT), a major plasma inhibitor of leukocyte elastase and bacterial proteases, is encoded at the PI locus on chromosome 14 (14q24.3-q32.1). A deficiency of AAT in individuals homozygous for the PI Z allele occurs in about 1 in 2,000-8,000 caucasians and is associated with an increased risk of early adult onset emphysema and liver disease in childhood. We have now used DNA polymorphisms associated with the AAT gene to investigate the origin of the PI Z allele. Using two genomic probes extending into the 5' and 3' flanking regions, respectively, we have identified eight polymorphic restriction sites. Extensive linkage disequilibrium occurs throughout the probed region with the PI Z allele, but not with normal PI M alleles. The Z allele occurs mainly with one haplotype, indicating a single, relatively recent, origin in caucasians.     

The role of heteroduplex correction in gene conversion in Saccharomyces cerevisiae

Two different models have been proposed to explain the relative frequencies of the non-mendelian allelic segregations which are detected by tetrad analysis after meiosis in fungi. The first model maintains that 6:2 type tetrads result from correction of heteroduplexes containing mismatched sites and 5:3 type tetrads result from failure to correct mismatched sites. The second model suggests that 6:2 segregations result from the filling-in of double-strand gaps using information obtained from both strands of a homologous duplex. In this model 5:3 type tetrads result if the allele is included in the heteroduplex regions flanking the gap and the resulting mismatched nucleotides are not corrected. We have studied the correction of heteroduplex plasmid DNA in pms1 mutant strains of Saccharomyces cerevisiae, which are known to exhibit higher frequencies of 5:3 type tetrads and lower frequencies of 6:2 tetrads than wild-type strains. Our results suggest that the pms1 mutation causes a defect in mismatch correction, supporting the hypothesis that meiotic gene conversion in wild-type yeast cells often results from the correction of heteroduplex DNA.     

Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation.

Physical mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the generalizability of the recognition motif, and the cleavage yield. Oligonucleotide-directed triple helix formation is a generalizable motif for specific binding to sequences longer than 12 base pairs within DNA of high complexity. Studies with plasmid DNA show that triple helix formation can limit the operational specificity of restriction enzymes to endonuclease recognition sequences that overlap oligonucleotide-binding sites. Triple helix formation, followed by methylase protection, triple helix-disruption, and restriction endonuclease digestion produces near quantitative cleavage at the single overlapping triple helix-endonuclease site. As a demonstration that this technique may be applicable to the orchestrated cleavage of large genomic DNA, we report the near quantitative single-site enzymatic cleavage of the Saccharomyces cerevisiae genome mediated by triple helix formation. The 340-kilobase yeast chromosome III was cut uniquely at an overlapping homopurine-EcoRI target site 27 base pairs long to produce two expected cleavage products of 110 and 230 kilobases. No cleavage of any other chromosome was detected. The potential generalizability of this technique, which is capable of near quantitative cleavage at a single site in at least 14 megabase pairs of DNA, could enable selected regions of chromosomal DNA to be isolated without extensive screening of genomic libraries.     

草鱼基因组随机扩增多态性引物及多态性位点的筛选

为了建立草鱼基因组DNA多态性分析的指标体系,应用RAPD技术,从180条10碱基随机引物中筛选出了31条能扩增出多态性DNA片段的引物。用这31条多态性引物共扩增出了327条重复性好、带型清晰、分辨率高的谱带。扩增产物的片段大小范围在400—2000bp之间。单一引物扩增条带为5—14条。用这些多态性引物在草鱼基因组DNA中检测到了93个多态性位点,并对这些多态性位点上的等位基因频率进行了统计分析。这31条多态性引物和所检测到的93个多态性位点初步为草鱼基因组的多态性分析提供了可靠的分析指标体系。     

Functional inactivation of genes by dominant negative mutations

Molecular biologists are increasingly faced with the problem of assigning a function to genes that have been cloned. A new approach to this problem involves the manipulation of the cloned gene to create what are known as 'dominant negative' mutations. These encode mutant polypeptides that when overexpressed disrupt the activity of the wild-type gene. There are many precedents for this kind of behaviour in the literature--some oncogenes might be examples of naturally occurring dominant negative mutations.     

Optical mapping of a rice BAC clone using restriction endonuclease and imaging with fluorescent microscopy at single molecule level

A method of constructing restriction map by optical mapping and single molecule fluorescent microscopy is described. DNA molecules were aligned and adsorbed on a glass coverslip surface by a modified “molecular combing” technique, and then the surface-immobilized DNAs were cleaved in situ with a restriction endonuclease. Individual DNA molecules digested by the endonuclease EcoRⅠ were observable with fluorescent microscopy. Using optical mapping, a physical map of a rice bacterial artificial chromosome clone was constructed. This method will facilitate genomic mapping and tracing the dynamic process in real time at a single molecule level with fluorescence microscopy.     

HLA DQ位点多样性模拟分析及其PCR—RFLP分型

选择２７个识别序列为４ｎｔ的限制性内切酶,通过计算机对最新获得的１９个ＤＱＡ１和３５个ＤＱＢ１等位基因第２外显子中的一段序列进行模拟酶切分析,根据酶切格局数目及各格局所代表的等位基因数的均衡性,确定酶的优先次序,在此基础上进行ＰＣＲ－ＲＦＬＰ模拟分析,确定最佳的酶组合。结果表明,分别采用４个和６个酶的组合,可有效鉴定ＤＱＡ１和ＤＱＢ１等位基因。同时通过比较分析ＤＱＢ１各等位基因核苷酶位点的变异系数     

Mutations in the p53 gene occur in diverse human tumour types

The p53 gene has been a constant source of fascination since its discovery nearly a decade ago. Originally considered to be an oncogene, several convergent lines of research have indicated that the wild-type gene product actually functions as a tumour suppressor gene. For example, expression of the neoplastic phenotype is inhibited, rather than promoted, when rat cells are transfected with the murine wild-type p53 gene together with mutant p53 genes and/or other oncogenes. Moreover, in human tumours, the short arm of chromosome 17 is often deleted. In colorectal cancers, the smallest common region of deletion is centred at 17p13.1; this region harbours the p53 gene, and in two tumours examined in detail, the remaining (non-deleted) p53 alleles were found to contain mutations. This result was provocative because allelic deletion coupled with mutation of the remaining allele is a theoretical hallmark of tumour-suppressor genes. In the present report, we have attempted to determine the generality of this observation; that is, whether tumours with allelic deletions of chromosome 17p contain mutant p53 genes in the allele that is retained. Our results suggest that (1) most tumours with such allelic deletions contain p53 point mutations resulting in amino-acid substitutions, (2) such mutations are not confined to tumours with allelic deletion, but also occur in at least some tumours that have retained both parental 17p alleles, and (3) p53 gene mutations are clustered in four 'hot-spots' which exactly coincide with the four most highly conserved regions of the gene. These results suggest that p53 mutations play a role in the development of many common human malignancies.     

Evidence for Base Substitutions and Repair of DNA Mismatch Damage Induced by Low Energy N^＋ Ion Beam Implantation in E. coli

Ever since the low energy N^ ion beam has been accepted, the mutations of ionizing radia-tion are attributable mainly to avoidance of DNA damages repair. Evidences based on in vivo proof results are limited. Using the E. coli wild type and mutator strains, the mutant frequencies suggest that base substitutions in rpoB gene are induced by the N^ implantation. A highly con-served region is selected to get the direct evidence for base substitutions by sequence of the high fi-delity PCR amplification products in mutants. Most of the mutants (90.9%, 40/44) have at least one base substitution in the amplification region. The evidences for CG to TA (55 %, 22/40), AT to GC (20%, 8/40) and TA to CG (5%, 2/40) transitions are identified. The transversions are AT to TA ( 15 %, 6/40) and GC to CG (5 %, 2/40). It is suggested that DNA cytosine methylase might play an important role in mismatch repair of DNA damage induced by N^ implantation by analysis of the mutant frequencies of mutator strains.     

Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.     

Molecular and phenotypic variation in the achaete-scute region of Drosophila melanogaster

Variation in quantitative characters underlies much adaptive evolution and provides the basis for selective improvement of domestic species, yet the genetic nature of quantitative variation is poorly understood. Many loci affecting quantitative traits have been identified by the segregation of mutant alleles with major qualitative effects. These alleles may represent an extreme of a continuum of allelic effects, and most quantitative variation could result from the segregation of alleles with subtle effects at loci identified by alleles with major effects. The achaete-scute complex in Drosophila melanogaster plays a central part in bristle development and has been characterized at the molecular level. The hypothesis that naturally occurring quantitative variation in bristle number could be associated with wild-type alleles of achaete-scute was tested by correlating phenotypic variation in bristle number with molecular variation in restriction maps in this region among chromosomes extracted from natural populations. DNA insertion variation in the achaete-scute region was found to be strongly associated with variation in bristle number.     

鸭瘟病毒基因组核酸的提取方法比较

将鸭瘟病毒分离株SD-01在鸡胚成纤维细胞上增殖.收集病变细胞及培养液,采用两种方法提取病毒基因组核酸.第一种方法,首先用差速离心的方法纯化病毒粒子,然后用酚/氯仿抽提的方法提取病毒基因组.第二种方法,先用高渗缓冲液将感染DPV的细胞完全裂解,然后加入微球菌核酸酶消化细胞DNA,最后用酚/氯仿抽提.限制性内切酶EcoR I消化,结果表明,第一种方法提取的样品中含有细胞DNA,酶切后为弥散模糊的拖带,而方法二可以最大限度的消除细胞DNA污染,得到纯净的病毒基因组DNA,酶切后为清晰的梯状条带.     

Genome-wide genetic analysis of polyploidy in yeast

Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution. Despite its prevalence, little is known about the physiological alterations that accompany polyploidy. We previously described 'ploidy-specific lethality', where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids. Here we report a genome-wide screen to identify ploidy-specific lethal functions. Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality. Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function. We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability. We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole. We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores. Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.     

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.