栖热菌属热稳定DNA聚合酶

ＤＮＡ聚合酶是能够以ＤＮＡ或ＲＮＡ为模板 ,在寡核苷酸或蛋白质引物的引导下催化合成ＤＮＡ的一类酶 ,已经分离纯化的有上百种 ,其中有半数已经被克隆和测序。它们之间在ＤＮＡ序列、氨基酸序列、二、三级结构方面有较高的同源性 ,且与ＲＮＡ聚合酶和反转录酶之间也有不同程度的同源性[1～ 4] 。1　ＤＮＡ聚合酶的分类ＤＮＡ聚合酶有许多不同的类群 ,如很小的 (3 9ｋＤａ) ,来自哺乳动物的修复性单亚基ＤＮＡ聚合酶 ,巨大的 (可高达 90 0ｋＤａ)、多亚基(可达 2 0多个亚基 )的复制性ＤＮＡ聚合酶 (如Ｅｃｏ聚合酶Ⅲ )。有些ＤＮＡ聚合酶则…     

伊氏锥虫在人胚肺成纤维细胞和在新生鼠肾成纤维细胞型细胞中的体外培养

在HEPES缓冲,含15—20％胎牛血清,100单位青霉素/ml,100微克链霉素/ml的RPMI-1640培养基中,用人胚肺成纤维细胞(KMB)和新生鼠肾成纤维细胞型细胞(ZUKF)作支持细胞,成功地使伊氏锥虫(Trypanosoma evansi)正常动基体株(kinetoplastic strain)和异常动基体株(dyskinetoplastic strain)在37℃含5％ CO_2的二氧化碳培养箱中连续培养达60天以上,并保持其对小鼠的高度感染力和致病力。这是迄今为止首次利用来源于人的细胞作支持细胞连续培养家畜非洲锥虫血液型成功的报告。实验结果表明伊氏锥虫体外培养成功与否对提供支持细胞的宿主是否有感染性无必然的联系。此外,利用Baltz氏等建立的无支持细胞培养系统,我们亦成功地使上述两伊氏锥虫株在体外连续繁殖,并保持对小鼠的高度感染力和致病力。     

鸡贫血病毒SJ1株DNA的分子克隆

通过ＰＣＲ方法扩增,用ｐＵＣ１１９和ｐＢｌｕｅｓｃｒｉｐｔＳＫ质粒载体分段克隆了山东省分离的ＳＪ１株的全病毒ＤＮＡ。限制性内切酶酶切分析结果显示其与国际标准株的酶谱相似     

DNA芯片技术

ＤＮＡ芯片技术利用固定化在固体表面的高密度ＤＮＡ分子微点阵来进行生化分析。从生物体中提取的样品ＤＮＡ或ＲＮＡ作为靶分子,荧光或其它方法标记后,与微点阵上互补的探刈杂交,从而产生异源双链。因为在微点阵上,某一特定位置年的核苷酸序列是已知的,所以通过对微点阵每一位点的荧光强度进行检测,便可能样品进行定性及定量分析,检测技术包括激光共聚焦扫描和电耦合设备（ＣＣＤ）成像等等。ＤＮＡ芯片根据控针成分的不同大     

日本血吸虫中国大陆株TPI基团的克隆及其表达产物特性

磷酸丙糖异构酶（ＴＰＩ）是血吸虫病疫苗重要的候选抗原基因之一。参考日本血吸虫已发表的ＴＰＩ ｃＤＮＡ序列,以日本血吸虫中国大陆株成虫ｍＲＮＡ为模板,用ＲＴ－ＰＣＲ法快速克隆出一大小约８００ｂｐ的ＤＮＡ片段。ＤＮＡ序列分析证实,所扩增到的ＤＮＡ片段即为日本血吸虫中国大陆株磷酸丙糖异构酶（ＳｊＴＰＩｃ）基因。将该基因重组到表达型质粒ｐＧＥＸ－４Ｔ中,表达的ＧＳＴ融合蛋白分子量约５４ｋＤ。用谷胱甘肽琼脂     

钴~(60)γ照射及腹注醋酸氢化可的松的小鼠对伊氏锥虫多态现象的影响

以不同剂量的醋酸氢化可的松分别处理感染伊氏锥虫无动基体株和水牛株的白化小鼠,结果表明50毫克/公斤的剂量对两个虫株短型的比例都有明显的影响,其中无动基体株的短型从感染后第3天的0.73％上升到第5天的4.4％,对照组为0.67％—0.73％;相反,水牛株短型的比例则从感染后第3天的6.3％下降到第5天的0.73％,对照组为0.73％—0.2％。200毫克/公斤的剂量对无动基体株的变异型有显著的影响,从感染后第3天的0.2％上升到第5天的1.9％。两虫株在经过钴~(60)r照射后再用醋酸氢化可的松处理的小鼠中亦表现出相似结果。实验结果表明宿主的免疫功能与伊氏锥虫的多态现象无实质的关系,而动基体的存在与否对适量的醋酸氢化可的松有较明显的反应。     

DNA操作技术领域里的两种新工具酶

ＤＮＡ操作技术领域里的两种新工具酶张显亮（中国科学院国家基因研究中心，上海２００２３３）关键词Ⅰ－内切酶超多位点内切酶（ｈｙｐｅｒｆｒｅｑｕｅｎｔｅｎｄｏｎｕｃｌｅａｓｅ）序列专一的ＤＮＡ双链内切酶是ＤＮＡ重组领域必不可少的工具之一。应用最普遍的是来...     

ACS和ACO基因克隆及植物转化

根据已知序列设计PCR引物,分别扩增氨基环丙烷羧酸合酶（ACS）和氨基环丙烷羧酸氧化酶（ACO）基因并克隆到中间载体,经限制性内切酶酶切图谱分析、部分序列分析以及Southern印迹鉴定后,将两个基因单个或相互串联后反向亚克隆至植物表达载体pBI121。经农杆菌LBA4404转化番茄子叶,在抗性培养基上得到8株ACS基因反义转化的生根小苗以ACS基因的酶切小片段作为探针,经Southern印迹分析,证明获得了两株阳性转化株。     

用简并寡核苷酸探针筛选对虾白斑综合征病毒DNA多聚酸基因片段

根据一些病毒的ＤＮＡ多聚酶氨基酸序列中特有的保守序列ＶＹＧＤＴＤ设计的简并寡核苷酸,经地高辛标记后与对虾白斑综合征病毒基因库克隆杂交,筛选出一段长度为７０７ｂｐ的ＥｃｏＲＩ基因片段,该片段在一个开放阅读框内。并含ＤＮＡ多聚酶Ｂ家族特有的保守序列ＹＧＤＴＤＳ。经与基因库比较,其氨基酸序列与藻类ＤＮＡ病毒科（Ｐｈｙｃｏｄｎａｖｉｒｉｄａｅ）的几株藻类病毒的ＤＮＡ多聚酶片段有部分相似,因此推测该核苷酸片     

CAR－bacillus菌亚单位核糖核酸RNA限制性内切酶图谱的研究

利用生物体中编码亚单位核糖核酸ＲＮＡ（ｓｍａｌｌｓｕｂｕｎｉｔｒｉｂｏｓｏｍａｌ　ＲＮＡ,ＳＳＵｒＲＮＡ）的ＤＮＡ序列为引物,经ＰＣＲ法扩增到ＣＡＲ－ｂａｃｉｌｌｕｓ的大约１．５ｋｂ的ＳＳＵｒＲＮＡ序列,采用ＨｉｎｄⅡ、ＨｉｎｆⅠ、ＥｃｏＴ１４,ＨａｅⅢ和ｘｈｏⅠ等５种限制性内切酶进行酶切电泳分析,同时比较了来源于小鼠的ＣＢＭ株及来源于大鼠的ＣＢＲ株,未发现两者有差异。     

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approximately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restriction analysis of cloned DNA fragments and by hybridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abundance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites occurred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of approximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence containing single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replication.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Molecular and morphological studies of Brazilian Trypanosoma evansi stocks: the total absence of kDNA in trypanosomes from both laboratory stocks and naturally infected domestic and wild mammals

The kinetoplast DNA (kDNA) minicircle molecules of 14 Brazilian stocks of Trypanosoma evansi were studied by morphological approaches (Giemsa and 4'-6'-diamidino-2-phenylindole staining and transmission electron microscopy) and molecular approaches (probing with an oligonucleotide complementary to the minicircle origin of replication and polymerase chain reaction amplification of a minicircle sequence). All methods indicated the absence of both a typical kinetoplast and kDNA minicircles, even in a very small number of parasites of a single stock or in small numbers of copies of molecules per cell. We did not detect any altered kDNA molecules. There were no kDNA molecules in either old or new stocks of T. evansi maintained by successive passages in mice. Similarly, no kDNA minicircles were detected in trypanosomes in blood smears from naturally infected domestic and wild animals. Thus, the total absence of kDNA in Brazilian stocks of T. evansi from both domestic and wild mammals is probably the natural state of Brazilian T. evansi.     

The action of ultraviolet light on the patterns of banding induced by restriction endonucleases in human chromosomes

The irradiation of metaphase spreads of human cells with ultraviolet (UV) light blocked the chromosome banding induced by Alu I, Mbo I, Dde I, Hinf I, Hae III, and Rsa I restriction endonucleases. At 13 J/m² there was moderate inhibition of the nuclease action, which was detected as an increase in the stain intensity of chromosomes (Alu I, Mbo I, Dde I, Rsa I) or as a change in the banding pattern (Hinf I, Hae III). At 70–300 J/m² the UV-induced blockage was complete; the chromosomes showed no banding, and stain intensity was similar to that of control slides incubated with buffer. — BrdU substitution and the irradiation of BrdU-substituted chromosomes with 313 nm light at 1800–15000 J/m² did not block the action of restriction nucleases. On the other hand, UV irradiation of BrdU-substituted chromosomes inhibited the action of restriction enzymes at the same fluences that blocked the nuclease action in unsubstituted chromosomes. The data indicate that DNA-protein crosslinkage is the factor inhibiting DNA extraction and chromosome banding.     

A possible structure for calf satellite DNA I.

Calf satellite DNA I (p = 1.715) has been hydrolysed by a number or restriction endonucleases. It consists of a repeating unit of 1460 nucleotide pairs within which the sites of Eco R II Mbo I, Sac I, Alu I, Ava II and Hha I were localised in comparison with those of Eco R I and Hind II. The distribution of the Hpa II, Sac I, Hha I, Hinf I and Mbo II sites within calf satellite DNA I, as well as that of several restriction endonuclease sites within calf satellite DNA III (p = 1.705) allowed me to define subsatellite fractions. Furthermore, some of the sites of the CpG containing restriction enzymes Hpa II and Hha I are lacking. The possible implications of these results are discussed.     

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata: II. Sequence evolution of the minicircles

Minicircle sequence evolution has been studied by comparison of the minicircles from Crithidia fasciculata and C. luciliae (C. fasciculata, var. luciliae) by restriction enzyme analysis and hybridization experiments. In contrast to the maxicircle sequence, the minicircle sequence of these trypanosomes evolves very rapidly. No conservation of restriction fragments has been observed and cross-hybridization of minicircles, minicircle fragments, and total kDNA is relatively weak. We conclude that no fragment larger than 550 bp is perfectly conserved between all minicircles of the two trypanosomes. Alterations in the minicircle fragment patterns of our stocks of trypanosomes were even apparent in a cultivation period of 1.5 to 2 years. The alterations suggest a random drift of the sequence which supports a noncodogenic function for the minicircles. Double restriction enzyme digestion experiments show that primary fragments are homogeneous with respect to cleavage by the second enzyme. This suggests that sequence rearrangements, rather than point mutations are the basis of the minicircle sequence heterogeneity.     

Identification of Trichinella isolates by polymerase chain reaction--restriction fragment length polymorphism of the mitochondrial cytochrome c-oxidase subunit I gene.

We developed a polymerase chain reaction based approach using restriction fragment length polymorphisms of the mitochondrial cytochrome c-oxidase subunit I to identify nine genotypes (Trichinella spiralis, Trichinella britovi-European strains, Trichinella britovi-Japanese strains, Trichinella nativa, Trichinella nelsoni, Trichinella T5, Trichinella T6, Trichinella T8 and Trichinella pseudospiralis) in the genus Trichinella. Partial mitochondrial cytochrome c-oxidase subunit I genes of nine genotypes were amplified by polymerase chain reaction, sequenced, and digested with three restriction endonucleases (Mse I, Alu I and Bsp1248 I). This polymerase chain reaction based restriction fragment length polymorphism method allowed the identification of Trichinella genotypes. Trichinella spiralis, Trichinella britovi-Japanese strains, Trichinella nelsoni, T5 and Trichinella pseudospiralis were distinguishable by digestion with Mse I. Trichinella britovi-European strains and Trichinella T8 were distinguishable by digestion using Alu I, and Trichinella nativa and Trichinella T6 were distinguishable by double-digestion with Mse I and Bsp1286 I. The results obtained with this polymerase chain reaction based restriction fragment length polymorphism assay confirmed those previously reported by others and support the separation of the Japanese isolates as a new genotype, namely Trichinella T9.     

Specific cleavage of kinetoplast minicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes

Multiple sequence classes of kinetoplast minicircle DNA from Leishmania tarentolae were cleaved by mung bean nuclease in the presence of formamide, yielding unit length linear molecules which retained the anomalous electrophoretic mobility in acrylamide characteristic of minicircle DNA. No specific cleavage site sequence common to all minicircle sequence classes was apparent, although the main region of nuclease cleavage was localized approximately 350 bp from the unique SmaI restriction site of the conserved region found in all minicircle sequence classes. Covalent closure of the minicircle substrate was not a requirement for cleavage, as linearized network-derived or cloned minicircles were also cleaved by mung bean nuclease at similar locations. The partial sequences of several new minicircle sequence classes released from the network by mung bean nuclease are also reported.