中国东部栗疫病菌群体的遗传分化

本实验采用RFLP技术,对中国东部栗疫病菌(Cryphonectria parasitica)进行了群体遗传结构的研究。313个参试菌株来自10个省(市)的16个群体(子群体),样本分布在北纬24°N—41°N。各菌株的DNA分别用限制性内切酶Pst Ⅰ和EcoR Ⅰ酶切,先后以10个低拷贝DNA探针和1个DNA指纹图谱探针进行了杂交和检测。结果表明,两个探针(pCB29和pMS29.1)的杂交图谱呈单态性;探针pCB19的杂交图谱显示,菌株DNA以PstⅠ酶切的为单态性,以EcoR Ⅰ酶切的则呈多态性;其他7个低拷贝探针的杂交图谱都呈多态性(Pst Ⅰ酶切)、指纹图谱探针的检测结果显示,辽宁凤城群体的菌株与中国东部其他群体的菌株相比,具有更多的限制性杂交片段,菌株间的遗传变异性也更大。     

中国东部栗疫病菌群体的遗传分化

本实验采用ＲＦＬＰ技术,对中国东部栗疫病菌进行了群体遗传结构的研究,３１３个参试菌株来自１０个省（市）的１６个群体（子群体）,样本人布在北纬２４°Ｎ－４１°Ｎ。各菌株的ＤＮＡ分别用限制性内切酶Ｐｓｔ Ⅰ和ＥｃｏＲⅠ酶切,先后以１０个低拷贝ＤＮＡ探针和１个ＤＮＡ指纹图谱探针进行了杂交和检测。结果表明,两个探针的杂交图谱呈单态性,其他７个低拷贝探针的杂交图谱都呈多态性、指纹图谱探针的检测结果显示,辽宁     

中国昆明(KM)小鼠线粒体DNA限制性内切酶图谱的研究

线粒体DNA的限制性内切酶图谱在不同的种系及亚种间存在多态性。我们分别用五种限制性内切酶BamHⅠ、EcoRⅡ、HidⅢ、PstⅠ、MspⅠ对60只中国KM小鼠(雌雄各半)的线粒体DNA进行了酶切电泳分析,结果未发现多态性,且这五种限制性内切酶图谱均与BALB/c小鼠相同。这表明中国KM小鼠与绝大多数实验小鼠一样,均起源于欧州的野鼠M.m.domesticu。未见到KM小鼠经其它亚种野鼠母系遗传污染的迹象。     

犬Ⅰ型腺病毒DNA的酶切分析及分子克隆

犬Ⅰ型腺病毒(CAV-1)弱毒用限制性内切酶EcoR Ⅰ,BamH Ⅰ,Pst Ⅰ,Sph Ⅰ和Hind Ⅲ消化分析后其图谱与强毒株相比没有差异。将弱毒DNA用Pst Ⅰ完全消化后以鸟枪法克隆到载体质粒pBluescrip'SK中,经用光生物素标记的CAV-1 DNA杂交筛选以及Pst Ⅰ分析重组质粒证明已将分子量为5.5,3.5,2.85,1.2,0.32和0.28Kb的CAV-1DNA片段克隆到质粒中。克隆到的这些片段将可考虑进一步研究作为探针检测犬及狐狸等野生动物的腺病毒感染。     

树qumtDNA限制性内切酶图谱及其与小家鼠褐家鼠的新缘关系

本实验用ApaⅠ,AvaⅠ,BamHⅠ,BclⅠ,BglⅠ,ClaⅠ,EcoRⅠ,EcoRⅤ,HpaⅠ,PstⅠ,PvuⅡ,ScaⅠ,XbaⅠ等13种限制性内切酶分析树qu(Chiromyscus chiropus) 的mtDNA限制性片段长度多态性,并用双酶解法构建了其中8种酶的限制性内切酶图谱。根据限制性片段差异法和分子钟,计算并讨论树qu和小家鼠(Mus musculus)、褐家鼠(Rattus norvegicus)的mtDNA遗传距离和亲缘关系。结果表明树qu与褐家鼠的关系较接近,两者的分歧时间在距今1500-2000万年前,即处于中新世早中期。     

大熊猫线粒体DNA的九种限制酶图谱

本文用9种限制性内切酶（BamHⅠ,BglⅠ,BglⅡ,EcoRⅠ,EcoRⅤ,PstⅠ,PvuⅡ,SalⅠ,XhoⅠ）分析大熊猫的线粒体DNA（mtDNA）。构建其中5种酶（BamHⅠ,EcoRⅠ,EcoRⅤ,PstⅠ,PvuⅡ）的mtDNA物理图谱。大熊猫mtDNA的分子大小约为16.4 Kb,酶切位点是随机分布。我们的结果为进一步研究大熊猫mtDNA进化提供了基础资料。     

滑鼠蛇肝线粒体DNA的限制酶图谱

用六种限制性内切酶BamHⅠ、EcoRⅠ、PstⅠ、BglⅠ、BglⅡ和SalⅠ对滑鼠蛇肝脏线粒体DNA(mtDNA)进行酶解。发现BglⅡ、PstⅠ、BamHⅠ、BglⅠ和EcoRⅠ在滑鼠蛇肝mtDNA上分别有1、2、3、3和4个切点。SalⅠ不能切割滑鼠蛇肝mtDNA。根据滑鼠蛇肝mtDNA的单酶、双酶完全酶解及部分酶解片段的数目和分子量,建立了滑鼠蛇肝mtDNA的限制酶图谱。     

油菜叶绿体基因组雄性不育特异DNA片段的定位

实验选用油菜湘矮不育系及同核保持系叶绿体DNA为材料,用4种限制性内切酶EcoRⅠ、BamHⅠ、PstⅠ和SmaⅠ完全酶解。在EcoRⅠ酶解的保持系ctDNA图式中,观察到唯一的差异片段E3.2kb。将此片段连接于载体pUC9进行克隆,经克隆杂交及电泳分析鉴定,获得重组子。以rRNA基因为探针,与EcoRⅠ酶解的保持系ctDNA杂交,其中一条阳性带显示在差异片段E3.2。进一步以E3.2片段为探针,与经过限制酶BamHⅠ、EcoRⅠ、salⅠ、BgⅢ、HindⅢ和PstⅠ酶解后的rRNA基因反杂交。根据rRNA基因图谱,这一与不育性有关的E3.2kb片段被定位于距16S rRNA基因5'端+2.0至+5.4kb的前导序列中。此结果表明,这段可能与花粉育性形成有关的片段定位于叶绿体基因组反向重复区。     

结核分枝杆菌中插入序列的研究

用人型复合分枝杆菌的特异插入序列IS6110和IS1081制 备探针,对5种限制性内切酶消化的结核分枝杆菌DNA进行杂交。结果表明,经PvuⅡ酶消化 的结核分枝杆菌DNA,用IS6110制备的探针进行杂交呈现高度多态性,说明IS6110对于人型 复合分枝杆菌分型研究和结核病流行病学研究具有很大价值。用IS6110制备的317bp探针对4 6株人结核分枝杆菌分离株多态性分析研究证实,这些菌株呈现高度DNA多态性,而且所含拷 贝数也极为不同,一般含7～18个拷贝。     

AFLP分子标记技术的改进

对AFLP技术的限制性内切酶组合进行了改进。以两个低频酶组合代替传统的高频酶与低频酶组合进行分子标记筛选,结果表明,采用低频酶组合(EcoRⅠ PstⅠ)产生的条带比传统酶切组合(EcoRⅠ MseⅠ和PstⅠ MseⅠ)要少且分布不均匀,多集中在150~800bp之间,条带信号强度要大,多态性明显得到提高,分子标记筛选成功率也得到了提高,而且其成本更低的特点有利于AFLP技术在我国的进一步推广普及。     

鲤鱼mtDNA酶切图谱的构建

采用密度梯度离心法及ＲＮａｓｅ消化法制备并纯化了鲤（ＧｙｐｒｉｎｕｓｃａｒｐｉｏＬｉｎｎａｅｕｓ）肝脏线粒体ＤＮＡ（ｍｔＤＮＡ）,用１０种限制性内切酶对ｍｔＤＮＡ进行了分析,鲤鱼ｍｔＤＮＡ分子量约１０．１２×１０￣６,约１６．４９ｋｂ．ＳａｌⅠ、ＰｓｔⅠ、ＢａｍＨⅠ、ＸｂａⅠ、ＢｇｌⅠ、ＰｖｕⅡ、ＸｈｏⅠ、ＥｃｏＲⅠ、ＤｒａⅠ和ＨｉｎｄⅢ分别为１、１、３、３、３、４、１、４、４、和６个切点。根据单酶解及双酶解结果,构建了鲤ｍｔＤＮＡ１０种具酶３０个切点的限制性酶切图谱。     

HLA-DR4-associated haplotypes are genotypically diverse within HLA

Biochemical diversity among products of class II HLA genes has been observed in individuals who appear to be HLA-D and DR-identical by cellular and serologic typing. We used techniques of restriction enzyme fragment analysis by Southern blotting to analyze this diversity at the level of cellular DNA. A panel of 17 HLA-DR4 homozygous cell lines (HCL) were investigated by using cDNA probes homologous to DQ beta, DQ alpha, and DR beta genes. Each probe was hybridized to cellular DNA digested with a series of different restriction endonucleases. Polymorphisms were observed with the use of the enzymes Pst I, Hind III, and Bam HI: Hybridization of cellular DNA digested with Hind III and Pst I with the DQ beta probe revealed specific polymorphisms, as did hybridization of the Pst I digest with the DQ alpha cDNA probe and the Bam HI digest with the DR beta probe. The observed differences fall into two categories: first, considerable diversity was seen between HLA-DR4 HCL that represent different HLA-D-defined haplotypes; second, diversity was also observed among HCL of the same DR4-associated HLA-D cluster. In contrast to the DQ cDNA probes, hybridization with the DR beta probe revealed relatively limited polymorphism by using a panel of different restriction endonucleases. Thus, although there is a general pattern of polymorphic restriction enzyme fragments homologous to DQ probes within an HLA-D cluster, the pattern seen for any particular cell line was not sufficiently distinct to assign an HLA-D or DR specificity.     

金秋梨和新高梨的分子遗传学对比分析

金秋梨和新高梨的形态性状、生物学性状以及酯酶同工酶、过氧化物酶同工酶、细胞色素氧化酶同工酶酶谱均产生了变化。利用异源DNA探针—小麦rDNA克隆pTA71,与四种核酸内切酶PstⅠ、BamHⅠ、EcoRⅠ及BglⅡ酶切消化的金秋梨和新高梨叶片总DNA杂交,结果表明,pTA71-PstⅠ和pTA71-BamHⅠ二种探针—酶组合可在金秋梨和新高梨中检测到RFLP差异。     

The use of fractionation in a polyethylene glycol-dextran two-phase system for the testing and purification of restriction endonucleases

A simple procedure was developed for testing and purification of restriction endonucleases Msp I, Pst I, Bam HI, Pvu I, Pvu II that includes biomass destruction, fractionation of cell-free extracts in the aqueous two-phase (polyethylene glycol-dextran) system and chromatography oh phosphocellulose. Optimal conditions for the fractionation of Msp I, Pst I, Bam HI, Pvu II, EcoR I, EcoR II, BspR I, Alu I were chosen. For separation of Pvu I and Pvu II gel filtration through biogel A-0.5 m was additionally introduced.     

pBR322DNA与8-甲氧补骨脂素光化学反应部位的碱基顺序特异性

用限制性核酸内切酶酶切试验研究了质粒pBR322 DNA经8-MOP及近紫外线作用后损伤部位的碱基顺序特异性。实验研究发现PUVA损伤的DNA在HindⅢ及RsaⅠ识别位置上酶切反应受到严重抑制,而在SphⅠ,EcoRⅠ,PvuⅡ,BamHI,PstⅠ识到位置上抑制轻微。通过对不同识别位置上碱基顺序及其光化学反应敏感性的分析,推断出DNA的TpA顺序可能是最易接受8-MOP光化学反应的部位。     

Isolation of DNA fragment containing phoS gene of Escherichia coli K-12

The DNA fragment containing the phoS gene, a regulatory gene for alkaline phosphatase, has been isolated from Escherichia coli K-12 chromosomal DNA by cutting off the DNA with Hind III restriction enzyme and by cloning the gene with plasmid vector pTP 4 which was constructed in this study. The isolated fragment was of about 12.3 kbp and seemed to contain the phoT, glmS, and bgl genes. The 12.3 kbp Hind III fragment was subjected to restriction enzymes EcoR I, BamH I, Sal I, and Pst I, and was found to possess two EcoR I, no BamH I, a Sal I, and four Pst I sites. Partial deletion using these restriction enzymes suggested that the about 6 kbp Hind III-Pst I fragment contained the phoS and phoT genes. Further analysis with other restriction enzymes revealed that the 6 kbp Hind III-Pst I fragment contained a BstE II, two Mlu I and four Hpa I sites. The deletion of these restriction sites using single-strand-specific nuclease S1 suggested that the BstE II and one of Mlu I sites were in the phoT gene, and the BstE II and two Mlu I sites were not in the phoS gene.