牛疱疹病毒Ⅳ型(DN-599株)DNA的分子克隆

从感染的MDBK(牛肾)细胞中直接抽提出牛疱疹病毒IV型(BHV-4)DNA。分别经限制性内切酶EcoRⅠ,HindⅢ或BamHI消化后,用鸟枪法和选择法将病毒DNA片段克隆到质粒pBR322和pUC9的相应位点中,构建了三组病毒DNA基因库。阳性克隆是用光生物素标记的病毒DNA和牛胸腺细胞DNA与各重组质粒DNA杂交而筛选的。总共克隆了病毒基因组的94％,其中用EcoRI克隆了83.4％,BamHI克隆了63.4％,HindIlI克隆了45％。     

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。     

番茄1-氨基环丙烷羧酸(ACC)合成酶基因的反义RNA-核酶嵌合DNA序列的构建

根据番茄ACC合成酶基因（LE—ACC2）DNA序列,以番茄(LycopersiconesculentumMill)果实的总DNA为模板,利用PCR技术扩增得到预期大小的该基因编码区内部分DNA序列,插入到质粒载体pGEM—3zf(＋)的BamHⅠ和HindⅢ位点之间后转化E．coliDH—5α,可选出重组子pRE,经酶切,PCR及DNA序列分析证明克隆成功;将pRE上的目的DNA序列以反义方式构建到我室已合成并克隆的含核酶DNA序列的重组质粒pRⅠ的BamHⅠ和HindⅢ之间,构成含有反义RNA-核酶嵌合DNA序列的重组质粒pREⅠ,经酶切及序列分析,结果与预期一致.     

萘降解质粒ND1.860和NAH7的DNA同源性研究

通过DNA:DNA杂交技术,用NAH7质粒的全部EcoR Ⅰ片段或ECOR Ⅰ A片段作为~(32)P标记的DNA探针,研究了萘降解质粒ND1.860和NAH7之间的DNA同源性。在ND1.860的9个HindⅢ片段中,5个与NAH7有同源性,其中3个与NAH7的编码萘降解途径的EcoR Ⅰ A片段有同源性。     

棒杆菌启动子片段的酶谱分析及定位

用大肠杆菌启动子探测质粒pSDS I (Ap^r,Tc^s)从钝齿棒杆菌(Corynebacterium crenatum)6282染色体的HindⅢ酶切片段中,克隆到两个具有启动功能的DNA片段,分别将两个重组质粒命名为pSDB5和pSDB21。含有这两个质粒的菌株均可以在含300μg/ml Tc的平板上生长。通过酶切分析,pSDB5的插入片段为1.6kb,pSDB21的插入片段为3.4kb,并分别作出了它们的限制性酶切图谱。对pSDB21利用其EcoR Ⅰ和BglⅡ位点,通过亚克隆删除了与启动功能无关片段,构建成pSDB210和pSDB211,从而使启动子定位于约0.1kb的BglⅡ/HindⅢ外源片段上。分子杂交实验证明所得到的这两个具有启动功能的DNA片段确实来源于钝齿棒杆菌6282的染色体DNA。     

油桐尺蠖核多角体病毒多角体蛋白基因定位与克隆

以[~(32)P]-dATP标记含AcNPV DNA的EcoRI-I片段的重组质粒为探针,在35℃条件下对油桐尺蠖核多角体病毒(BsNPV)多角体蛋白基因进行了定位,将其分别定位在BamH Ⅰ-A,Bgl Ⅰ-A,Bgl Ⅱ-F,EocR Ⅰ-R,Hind Ⅲ-A,Kpn Ⅰ-Ⅰ,Pst Ⅰ-D,Xba Ⅰ-A(或B),Xho Ⅰ-F和G片段上,并以M13mp18为载体,克隆了Kpn Ⅰ-Ⅰ片段。     

鹅源腺病毒基因组DNA物理图谱的构建

将鹅源腺病毒Y81G4株全基因组DNA的HindⅢ酶切片段分别插入质粒pUC18, 成功构建了全基因组DNA文库.在此基础上,将重组质粒携带的插入片段切出、回收并分别用地高辛标记后作为探针,与经限制酶BamHI、EcoRI、PstI、Eco RV消化的病毒基因组DNA进行Southern Blotting,杂交结果经比较综合后获得了该病毒基因组DNA的HindⅢ、Ba mH I、EcoR I、PstI、EcoR V限制性内切酶的物理图谱.利用已发表的含有鸡EDS76病毒AA-2 株基因组DNA右末端的重组质粒pBE42作为探针,与本病毒两末端重组质粒进行Southern Blo tting,根据同源性杂交结果确定了本病毒基因组DNA物理图谱与EDSVAA-2株相应的方向. 本病毒基础因组DNA物理图谱的精确构建,为进行基因组结构分析,筛选复制非必需区,构建禽腺病毒载体打下了基础.     

苏芸金杆菌MS07A的Cry Ⅰ基因在大肠杆菌中的克隆及表达

分离了苏芸金杆菌库斯塔克变种MS07A(Bacillus thuringiensis var.kurstaki MS07A)的质粒,经HindⅢ酶解、凝胶电泳和Southern转移后,用DIGdUTP标记的质粒pES1的EcoR Ⅰ—F片段作探针进行DNA分子杂交,发现5.3、6.6和7kb左右的DNA片段含Cry Ⅰ基因。用Glass milk合并回收这些片段,克隆到pUC18的HindⅢ位点上并转化大肠杆菌JM109。通过菌落原位杂交、重组质粒的限制性消解等分析方法,选出带有Cry Ⅰ基因并能在大肠杆菌中表达其毒蛋白的转化子。初步生物测定结果表明。转化子TM48和TM76对松毛虫和菜青虫有毒杀活性。     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒(CAV)环形基因组DNA(全长2.3kb)的EcoRI位点和BamHI位点的两侧选择适当序列合成两对引物,用PCR技术,从斑点杂交检测到病毒核酸的CAV感染的MDCC-RP1细胞基因组DNA中,分别扩增出包含EcoRI和BamHI分割开的病毒基因组两部分(1.5kb和0.8kb)约1.5kb和约1.25kb的两个片段.再将其中相应序列拼接克隆进pUC18载体,获得包含CAV全基因组序列DNA片段的克隆质粒pCAV2.4.酶切分析表明,该质粒具有预期的BamHI位点、PstI位点、HindⅢ位点,而预期的EcoRI位点消失.重组质粒插入DNA片段的两端序列分析表明,质粒pCAV2.4是包含CAV全基因组序列的重组质粒,插入DNA片段序列中的EcoRI位点序列发生了一个碱基突变.     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

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。     

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.     

人鼻咽组织中Epstein-Barr病毒基因组存在状态的分析

Burkitt淋巴瘤和未分化型鼻咽癌的细胞中存在着Epstein-Barr病毒(EBV)的基因组,在前者虽经长期体外培养,病毒基因组仍持续存在,并部分地表达。这些现象提示,至少有一些EBV基因组是整合在寄主细胞的DNA中。Kieff等用染色体原位杂交检测Burkitt淋巴瘤Namalwa细胞株和EBV转化的人脐带血B淋巴细胞IB_4株,发现病毒DNA是整合在前者的1号染色体上,在后者则整合     

中国安徽庐江鸭乙型肝炎病毒全基因组克隆酶谱分析和部分病毒基因正负单链探针的构建

用鸭乙型肝炎病毒(DHBV)阳性的安徽庐江鸭血清感染DHBV阴性的北京雏鸭,扩增病毒,将提取的DHBV-DNA插入pUC18质粒,转化E.coli JM 105。酶切重组质粒及South-ern转膜杂交结果证实,质粒pLJ76的插入片段为DHBV全基因组。用EcoR Ⅰ等11种限制性内切酶对pLJ76进行酶谱分析,并与美国,西德的已知DHBV基因组比较。定向克隆该株病毒不同基因编码区片段,构建正负单链探针,将斑点杂交和单链电泳检出的M13阳性重组子与已知序列的DHBV基因组作比较,提示获得了该株病毒基因组的S、Pre-S、P和X/C等蛋白编码区的正、负单链克隆株。     

Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus

Deletions of polyoma virus DNA around the region that codes for the C-terminus of the viral middle T-antigen were created using a transforming fragment (BamH I/EcoR I) of viral DNA cloned in the plasmid vector pAT153. These species were recloned and assayed for their ability to transform Rat-1 cells in culture. Our results showed that whereas the DNA sequence between the presumed translational termination codon for the viral middle T-antigen and the single viral EcoR I site could be removed with no apparent effect on transformation, the removal of the termination codon itself or any amino acid coding sequences of this protein caused a drastic decrease in the transforming ability of the DNA. Transfection of Rat-1 cells with plasmids that contained viral DNA with deletions which corresponded to the last fourteen or more amino acids of the middle T-antigen never gave rise to cellular transformation.     

pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts.

A multipurpose plasmid, pUR 250, has been constructed. It contains seven unique cloning sites (Hind III, Xba I, Sal I, Acc I, Hinc II, BamH I and EcoR I) in a small region of its lac Z gene part. Insertion of foreign DNA into the plasmid can be easily detected. Plasmid DNA isolated from a clone by a rapid method can be used to determine the DNA sequence of the insert from both ends directly without isolation of labeled fragment.     

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。     

鼠伤寒沙门氏菌Ⅰ相鞭毛蛋白基因fliC~I的克隆和鉴定

以提纯的鼠伤寒沙门氏菌8705染色体DNA为材料,经EcoR Ⅰ消化,过SepharcylS-400柱,得到大于400bp的酶切片段;然后随机克隆到质粒pGEM-3Zf(—)中,转化大肠杆菌LC2a(hag~-,recA~-);在氨苄青霉素平板上共得到6013个转化子,从中筛选出1个有动力的克隆,小量制备质粒DNA,经酶切电泳鉴定,该克隆的外源片段大小为15.3kb,将其命名为pGI4015。动力、动力抑制试验和Southern blot分子杂交试验证明pGI4015中载有鼠伤寒沙门氏菌Ⅰ相鞭毛蛋白基因fliC~I;利用其BamH Ⅰ和Sal Ⅰ位点删除与鞭毛蛋白表达无关的序列,构建亚克隆质粒pGI4015BS,使得fliC~I定位于更小的区域——3.8kb的BamH Ⅰ/Sal Ⅰ插入片段上。     

Expression of bacterial endonucleases in Saccharomyces cerevisiae mitochondria

Expression vectors were created in which the 5' end of the Saccharomyces cerevisiae CDC9 gene, which encodes a mitochondrial targeting peptide, was cloned in-frame with the coding regions of the EcoR I, Hind III, and Pst I endonuclease genes. Expression of the EcoR I and Hind III fusion proteins inhibited growth of yeast on glycerol-containing media and resulted in the nearly quantitative restriction digestion of their mitochondrial DNA. In contrast, expression of Pst I, which does not recognize any sites within yeast mitochondrial DNA, had no effect on growth in glycerol-containing media, and did not affect the integrity of the mitochondrial genome.