The REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability,recombination and sporulation: cell-type and life cycle stage-specific expression of the rec46-1 mutation

Summary The recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MAT n+1 chromosome VII disomic strain LBW (Esposito et al. 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MAT n+1 chromosome VII disomic strains. The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types. MATaIMaT ree46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MAT rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intergenic recombination resulting in prototrophy. When compared to MAT rec46-1 n+1 disomics however, MATa/MAT rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants. MATaIMAT rec46-1/rec46-1 diploids are sporulation-deficient. The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).     

乙型肝炎病毒全基因克隆及序列分析发现一D/C基因型重组株

目的 构建含有adr、adw、ayw3个血清型的HBV全基因组质粒，并进行测序及序列分析。方法 从HBsAg携带者的血清中扩增出S区克隆，筛选出adr、adw、ayw血清型的血清，然后扩增HBV的全基因组，对扩增产物进行克隆测序。利用DNAStar的MegAlign，Phylogenetic tree对HBV全基因序列以及分段序列进行比较和进化树分析。结果 构建了含有adw、adr、ayw 3种血清型的HBV全基因组质粒，代码分别为：H1、H2、H3，对3株完成序列测定，按照S区核苷酸顺序划分基因型分别为B、C、D，全序列分型显示H1、H2为B、C基因型，与按S区的分型一致，但H3为C基因型，与按S区的分型不同，进一步的序列分析表明H3为一株异常的基因型，按照S区和preS2区分型为D基因型，但是全基因组／C／P／X区的进化树分析，H3株均位于C基因型，提示该异常的基因型可能是由于D／C基因型间基因重组引起，对重组位点进行了分析：3181nt-10nt、799-834nt为可能的重组位点所在区。结论 H3病毒株的发现进一步证明了HBV基因型间的基因重组，但澄清该问题需要在HBV感染的高流行区进一步的积累HBV异常基因型的全序列，以及发现新的基因型资料。     

Recombination between parental mitochondrial DNA following protoplast fusion can occur in a region which normally does not undergo intragenomic recombination in parental plants

Summary A novel restriction fragment, which was not present in either parent's mitochondrial DNA, was cloned from the mitochondrial genome of a somatic hybrid Petunia line. This fragment resulted from interspecific recombination between homologous mitochondrial DNA regions of the parental plants, Petunia line 3704 and line 3688. Hybridization with a cloned Petunia atp9 gene revealed that the regions involved in recombination carry atp9 coding sequences. Intragenomic recombination within the parental mitochondrial genomes was not detected between the atp9 regions which recombined following protoplast fusion.     

单链抗体（ScFv）表达载体的构建及抗HBs ScFv的表达

通过ＤＮＡ重组技术和ＰＣＲ技术，将Ｆａｂ表达载体改建成了ＳｃＦｖ表达载体。该载体可以表达表达方式产生噬菌体抗体，用于抗体库技术，也可以分泌型表达产生有活性的ＳｃＦｖ。所表达的抗ＨＢｓ ＳｃＦｖ具有与其亲本Ｆａｂ相同的抗体活性。载体上具有聚组氨酸编码序列，使表达的ＳｃＦｖ可经金属螯合层析法进行简便有效的纯化。     

Single-strand scissions of nuclear yeast DNA occur without meiotic recombination

Summary In meiotic cells of Saccharomyces cerevisiae, reduction in molecular weights of DNA in alkaline sucrose gradients is observed concomittantly with premeiotic DNA replication and with commitment to recombination. Following the completion of the latter processes, higher molecular weights are obtained. These single-stranded breaks are found in both old and newly synthesised strands. Similar scissions in DNA are also found in a temperature-sensitive mutant (cdc40/cdc40), which does not undergo commitment to recombination at the restrictive temperature, and in vegetative wild type cells that were previously exposed to sporulation medium. The suggestion that these scissions are the physical manifestation of commitment to recombination is therefore rejected.     

Nonrandomly-associated forward mutation and mitotic recombination yield yeast diploids homozygous for recessive mutations

We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L--aminoadipate as a nitrogen source (-Aa⁺) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. -Aa^- diploid cells yield -Aa⁺ mutants at a rate of 7.8±3.6×10^-9. As in haploid strains, approximately 97% (30/31) of -Aa⁺ mutants are spontaneous lys2-x recessive mutations. -Aa⁺ mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.2±0.4×10^-6. Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.3±0.6×10^-3. This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene conversion of LYS2 to lys2-x; 85% of lys2-x/lys2-x diploids appear to have arisen by mitotic recombination in the CENII-LYS2 interval. lys2-1/lys2-1 mitotic segregants of a control LYS2/lys2-1 diploid consist similarly of 18% of lys2-1/lys2-1 diploids that appear to have arisen by gene conversion of LYS2 to lys2-1 and 82% of lys2-1/lys2-1 diploids that appear to have arisen by mitotic recombination in the CENII-LYS2 interval. The methods described can be used to simultaneously monitor the effects of yeast gene mutations and carcinogens on the principal parameters affecting the genomic stability of diploid mitotic cells: mutation, gene conversion, intergenic recombination, and chromosomal loss or rearrangement.The research of the authors was supported by the Director, Office of Energy Research, Biological Research Division of the U.S. Department of Energy under Contract No. DE-ACO3-76SF00098, grants to M. S. E. and C. V. B. from the National Institutes of Health and the National Aeronautics and Space Administration, and a National Science Foundation postdoctoral research fellowship award to R. M. R.     

TAT-凋亡素基因重组质粒的构建、蛋白的表达纯化及体内活性实验

目的 克隆构建TAT-凋亡素质粒并提取融合蛋白，为进一步研究该蛋白功能奠定基础。方法 PCR合成TAT-凋亡素基因，与pTYB2质粒连接后转入Rosetta菌，经IPTG诱导表达，几丁质亲和层析一步纯化目的蛋白，用昆明小鼠H22动物模型检测活性。结果 克隆载体经过PCR筛选、测序鉴定，其大小和核苷酸序列正确，诱导后融合蛋白出现在上清，纯化出的TAT-凋亡素蛋白具有明显的抗肿瘤活性。结论 本实验所构建的重组质粒pTYB2/TAT-apoptin经诱导表达出了可溶性目的蛋白TAT-凋亡素，纯化后具有明显的生物活性，为研究TAT-凋亡素蛋白的进一步研究奠定了基础。     

表达反义单核细胞趋化蛋白-1的重组逆转录病毒对家兔动脉平滑肌单核细胞趋化蛋白-1基因表达的影响

为研究反义单核细胞真挚化蛋白－１转基因表达对单核细胞进入动脉壁的作用,首先,构建了表达反义单核细胞趋化蛋白－１基因的逆转录病道理毒重组体,并观察它在培养的细胞中的表达。将家多单核细胞趋化蛋白－１ｃＤＮＡ反向插入到ｐＬＮＣＸ,构成ＬＮＣＸ－ａｎｔｉ－ＭＣＰ－１重组病毒质粒。再将重组质粒转染Ψ－２细胞,继以Ψ－２细胞产生的病毒上清感染ＰＡ３１７细胞,取得Ｇ４１８ＰＡ３１７抗细胞克隆。上述细胞经扩增培养     

The influence of mutation rad57-1 on the fidelity of DNA double-strand gap repair in Saccharomyces cerevisiae

The role of the RAD57 gene in double-strand gap (DSG) repair has been examined. The repair of a linearized plasmid, bearing a DSG, has been analyzed in a rad57-1 mutant of Saccharomyces cerevisiae. For effective rejoining of the ends of plasmid DNA in the rad57 mutant the sequence of chromosomal DNA homologous to the DSG region is required. However, DSG repair (restoration of plasmid circularity) in rad57 cells is not accompanied by the recovery of DSGs. The DSG repair, which depends on an homologous chromosomal DNA sequence, requires the cohesive ends of DSGs. The non-cohesive-ended DSGs are repaired in rad57 cells by a pathway independent of the homologous recombination between chromosomal and plasmid DNA. We presume that the rad57-1 mutation is connected with the inhibition of DNA repair synthesis, required for filling the DSG. This situation produces a condition of “homology-dependent ligation”, the alternative minor mechanism of recombinational DSG repair, that takes place in mutant cells. A molecular model for “homology-dependent ligation” in rad57 cells is proposed. Received: 26 March / 29 October 1998     

2012年重庆单中心腹泻患儿感染诺如病毒及其基因型别和重组的研究

目的 了解重庆地区门诊腹泻患儿感染诺如病毒（NV）流行株的变迁及基因重组情况。方法 采集2012年1~12月就诊于重庆医科大学附属儿童医院的疑似病毒性腹泻患儿的粪便标本。采用JVl2/JVl3、GⅠ SKF /GⅠ SKR(COG2F/GⅡ SKR)两对引物,对NV基因组的部分RNA依赖的RNA聚合酶区和衣壳蛋白的N/S区分别进行RT-PCR核酸检测,所有阳性产物进行回收纯化、测序,用DNAstar和MEGA 5.05软件对序列分别进行比对和构建进化树。并将疑似重组株的标本再用JVl2/GⅠ SKR(JVl2/GⅡ SKR)进行PCR扩增,用SimPlot软件对序列进行重组鉴定。结果 384例腹泻患儿粪便标本进入本文分析,男248例,女136例,年龄（13.1±14.4）个月。①NV阳性84/384例（21.9%）,<60月龄组NV阳性构成比为96.4%（81/84）。NV在6、8和9月份检出率较高,3和4月份检出率最低。②84例NV阳性标本capsid的N/S区基因片段测序显示,GⅡ.4 2006b型37例,GⅡ.4 New Orleans 2009型1例,GⅡ.4 Sydney 2012型27例,GⅡ.3型12例,GⅡ.6型3例, GⅡ.13型3例,GⅡ.5型1 例;1~7月份 GⅡ.4 2006b型为主要流行株,8~12月GⅡ.4 Sydney 2012型为主要流行株;③共检出44株重组株,分别是GⅡ.e/GⅡ.4 Sydney 2012型27株、GⅡ.7/GⅡ.6型1株、GⅡ.22/GⅡ.5型1株、GⅡ.12/GⅡ.3型12株,GⅡ.16/GⅡ.13型3株。结论 重庆地区NV重组现象非常明显,2012年8~12月NV优势株逐渐由GⅡ.4 2006b型转为GⅡ.e/GⅡ.4 Sydney 2012型的重组株,并检出GⅡ.22/GⅡ.5型和GⅡ.16/GⅡ.13型2种新型重组株。