DNA修复基因RAD24的分子克隆和序列分析

利用缺口修复（ｇａｐｒｅｐａｉｒ）方法克隆啤酒酵母（Ｓ．ｃｅｒｅｖｉｓｉａｅ）野生型ＲＡＤ２４基因,并将其亚克隆到Ｍ１３ｍｐ１８和Ｍ１３ｍｐ１９,用双脱氧末端终止法对该基因的两条链均进行了序列测定,ＤＮＡＳｔｒｉｄｅｒ程序分析显示该基因编码２６８个氨基酸的蛋白质,基因缺失试验表明,该基因为细胞生存所必需。     

鸡马立克氏病病毒B抗原片段在大肠杆菌中表达

鸡马立克氏病病毒ＢａｍＨＩ基因文库Ｉ３质粒中含有编码Ｂ抗原膜外Ｄｏｍａｉｎ的ＤＮＡ序列。经ＳｃａＩ和ＳｐｈＩ双酶酶解Ｉ３质粒,分离获得７６４ｂｐＤＮＡ片段,并克隆进Ｍ１３ｍｐ１９中。ＤＮＡ序列测定分析表明克隆片段为ＭＤＶ－Ｂ抗原基因的４９４－１２５８ｂｐ部分序列。进一步分离ＮｃｏＩ－ＨｉｎｄＩＩＩ部分基因片（５３０ｂｐ）,克隆于ＰＬＰｒｏｍｏｔｅｒ控制下的含有修饰型ｃＩｔｓ８５７基因的表达载体中,     

7号淀粉酶链霉菌M1033木糖异构酶基因的启动子活性检测,转录起始点…

分别从重组质粒ＰＵＢ１及其Ｍ１３亚克隆Ｓ１中将７号淀粉酶链霉菌（Ｓｔｒｃｐｔｏｍｙｃｅｓ ｄｉａｓｔａｔｉｃｕｓ Ｎｏ．７）Ｍ１０３３（以下简称Ｓ。ｄｉ．Ｍ１０３３）木糖异构酶基因的－１９２－＋５８１ｂｐ片段克隆入链霉菌启动子探测质粒ＰＩＪ４０８３中,转化变铅青链霉菌（Ｓ。ｌｉｖｉｄａｎｓ）ＴＫ２４。通过对其邻苯二酚加双氧酶活性的检测表明,该片段具有启动子活性。应用Ｍ１３亚克隆Ｓ１和合成引物Ｐ６延     

7号淀粉酶链霉菌M1033木糖异构酶基因的启动子活性检测、转录起始点的确定

分别从重组质粒ｐＵＢ１及其Ｍ（１３）亚克隆Ｓ１中将７号淀粉酶链霉菌（ＳｔｒｅｐｔｏｍｙｃｅｓｄｉａｓｔａｔｉｃｕｓＮｏ．７）Ｍ１０３３（以下简称Ｓ．ｄｉ．Ｍ１０３３）木糖异构酶基因的－１９２－＋５８１ｂｐ片段克隆入链霉菌启动子探测质粒ｐＩＪ４０８３中,转化变铅青链霉菌（Ｓ．ｌｉｖｉｄａｎｓ）ＴＫ２４。通过对其邻苯二酚加双氧酶活性的检测表明,该片段具有启动子活性。应用Ｍ１３亚克隆Ｓ１和合成引物Ｐ６延伸制备放射性标记的单链ＤＮＡ探针;通过Ｓ．ｄｉ．Ｍ１０３３的总ＲＮＡ的Ｓ１核酸酶保护实验,确定了其转录的起始位点,并由此探讨了与木糖异构酶基因表达有关的一些因素。     

u—PA／t—PA嵌合型纤溶酶原激活剂（ut—PA）的构建,表达,纯化和性…

将尿激酶原ｃＤＮＡ和组织型纤溶酶原激活剂Ａ链ｃＤＮＡ克隆到Ｍ１３ｍｐ１８中,经过二次寡核苷酸诱导的大片段定点删除了一次寡核苷酸诱导的多位点突变,得到了ｊ－ＰＡ／ｔ－ＰＡ融合基因。     

人脑源性神经营养因子cDNA在COS7细胞中的表达及活性研究

本文从质粒Ｍ１３ｍｐ１８－ｈＢＤＮＦ中酶切回收人脑源性神经营养因子（ｈＢＤＮＦ）全长基因,构建真核表达载体ｐＣＭＶ４－ｈＢＤＮＦ。利用脂质体的方法转染ＣＯＳ７细胞,对转染后的ＣＯＳ７细胞提取ＲＮＡ进行狭缝杂交分析和免疫细胞化学反应,分别从转录及翻译水平上检测ＢＤＮＦ基因在ＣＯＳ７细胞中的表达。实验还证实在ＣＯＳ７细胞中表达的ｈＢＤＮＦ蛋白可分泌至胞外并可促进中脑黑质细胞的发育和生长,具有良好的生物学活性。     

人脑源性神经营养因子cDNA在COS7细胞中的表达及活性…

本文从质粒Ｍ１３ｍｐ１８－ｈＢＤＮＦ中酶切回收入及源性神经营养因子（ｈＢＤＮＦ）全长基因,构建真核表达载体ｐＣＭＶ４－ｈＢＤＮＦ。利用脂质体的方法转染ＣＯＳ７细胞,对转染后的ＣＯＳ７细胞提取ＲＮＡ进行狭缝杂交分析和免疫细胞化学反应,分别从转录及翻译水平上检测ＢＤＮＦ基因在ＣＯＳ７细胞中的表达。实验还证实在ＣＯＳ７细胞中表达的ｈＢＤＦ蛋白可分泌至胞外并可促进中脑黑质细胞的发育和生长,具有良好的生物学     

HFRSV内影像型抗独特型人单抗重链可变区基因克隆

利用反转录－ＰＣＲ方法,从分泌肾综合症出血热病毒（ＨＦＲＳＶ）内影像型抗独特型人单抗杂交瘤细胞系（Ｃ８）中,成功地克隆了抗ＨＦＲＳＶ１ｄ人单抗重链可变区基因,并将此基因重组入Ｍ１３噬菌体ＤＮＡ中,测定了此重链可变区基因全序列。经计算机分析,基因全长共３５１ｂｐ、编码１１７个氨基酸,氨基酸序列同源性分析发现所推得的该单抗ＣＤＲ２区与ＨＦＲＳＶＧ２蛋白Ｃ端有同源区,此区可能具有较强的抗原性。     

u-PA/t-PA嵌合型纤溶酶原激活剂(ut-PA)的构建、表达、纯化和性质研究

将尿激酶原（ｐｒｏ－ＵＫ）ｃＤＮＡ和组织型纤溶酶原激活剂（ｔ－ＰＡ）Ａ链ｃＤＮＡ克隆到Ｍ１３ｍｐ１８中,经过二次寡核甘酸诱导的大片段定点删除和一次寡核苷酸诱导的多位点突变,得到ｕ－ＰＡ（Ｌｅｕ１４４－Ｇｌｙ４０８）／ｔ－ＰＡ（Ｓｅｒ１－Ｔｈｒ２６３）（ｕｔ－ＰＡ）融合基因．将ｕｔ－ＰＡ融合基因克隆到表达载体ｐＣＭ－βｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞,筛选稳定表达株．收集无血清表达上清,经苯甲脒柱纯化得到ｕｔ－ＰＡ纯品,ＳＤＳ－ＰＡＧＥ和纤维蛋白自显影显示ｕｔ－ＰＡ有两种分子量形式,分子量分别为６８ｋＤ和６１ｋＤ．纤维蛋白亲和性试验表明,ＬＵＫ（低分子量尿激酶）对纤维蛋白没有亲和性,而含有ＬＵＫ的ｕｔ－ＰＡ则对纤维蛋白表现出很强的亲和性,但ｕｔ－ＰＡ的亲和性略低于亲本ｔ－ＰＡ．     

一种中国发生的真菌传小麦花叶病毒RNA－13＇末端核苷酸序列分析

以采自河南的真菌传小麦花叶病毒（ＦＷＭＶ－Ｃ）为材料,抽提病毒ＲＮＡ,合成互补ＤＮＡ（ｃＤＮＡ）。对杂交筛选所得ｃＤＮＡ克隆进行亚克隆及序列分析,结果表明,亚克隆ｐＧＳＩ含有一个长度为８９１个核苷酸的不完整开放阅读框架（ＯＲＦ）和长度为２５８个核苷酸的３＇末端非编码区（ＮＴＲ）,并带有Ｐｏｌｙ（Ａ）尾序。此段序列与大麦黄花叶病毒（ＢａＹＭＶ）及法国报道的一种小麦梭条花叶病毒（ＷＳＳＭＶ－Ｆ）的ＲＮＡ－１３＇末端分别具有６７．６％和６９．９％的同源性。由所测序列编码区（１－８９１ｎｔ）可推导产生２９６个氢基酸,并与ＷＳＳＭＶ－Ｆ及ＢａＹＭＶ外壳蛋白氨基酸序列分别具有７５．９％和７１％的同源性。此结果表明,ＦＷＭＶ－Ｃ为另外一种不同于ＷＳＳＭＶ－Ｆ的大麦黄花叶病毒组（Ｂａｙｍｏｖｉｒｕｓ）病毒,所测基因组片段应为ＲＮＡ－１３＇末端序列,其中可能包括了病毒全长外壳蛋白编码区域。     

Activity expressed from cloned Anacystis nidulans large and small subunit ribulose bisphosphate carboxylase genes

Summary The ribulose bisphosphate carboxylase/oxygenase (EC4.1.1.39) (RubisCO) large and small subunit genes from Anacystis nidulans have been cloned as a single fragment into M 13mp10 and pEMBL8 and expressed in Escherichia coli. From M 13mp10 a low yield of enzyme with high specific activity was obtained. The molecular weight of the active enzyme was 260 000 Da and of the inactive enzyme approximately 730 000 Da. The small and large subunits cloned separately did not express activity. The RubisCO gene cloned into pEMBL8 expressed activity up to 22 times that from the M 13 cloned RubisCO DNA. The RubisCO protein produced by the pEMBL cloned gene had a normal MW (550 000). Immunoprecipitation and polyacrylamide gel electrophoresis showed the presence of both large and small subunits.     

Gene expression in vectors constructed on the basis of the genome of filamentous phages

Expression of the human leucocyte interferon alpha 2 gene has been studied, cloned into filamentous phages (M13mp8, M13mp9) and plasmid vehicles comprising the regulatory regions and signal sequence of the filamentous phage main coat protein gene (plasmids pFPCP2 and pFPCP8).     

血小板生成素改构体基因的克隆和序列分析

采用PCR技术,根据文献报道的鼠TPO成熟肽基因序列,设计并合成两对引物,以鼠TPO cDNA为模板,扩增获得mTPO N端153个氨基酸的478bp cDNA片段及鼠TPO全长1032bp cDNA片段,mTPO153片段与合成的碱性成纤维生长因子序列中Lys119-Lys135as的51bp肝素结合位点DNA片段连接,克隆到M13mp18及M13mp19载体中进行双向测序;同时将扩增的鼠TPO全长cDNA片段克隆到M13mp18及M13mp19载体中进行双向测序。证明获得鼠血小板生成素与肝素结合位点基因及鼠TPO全长基因,继之以pMAL-C2X为表达载体构建表达质粒,并经PCR及酶切鉴定。     

A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2.

In this paper we describe the synthesis and cloning of a short segment of DNA complementary to the region immediately adjacent to the EcoRI insertion site in the single-stranded bacteriophage vector M13mp2. This segment is useful as a "universal" primer for DNA sequencing by the dideoxynucleotide chain termination method; the template can be any DNA species cloned in M13mp2 or its derivatives. The primer has been cloned into the tetracycline resistance gene of plasmid pBR322 as one strand of a 26 bp EcoRI/BamHI fragment. This fragment may be readily prepared from an EcoRI + BamHI restriction digest of the parent plasmid (designated pSP14) by a simple size fractionation.     

恶性疟原虫子孢子CS抗原融合基因的克隆

利用381-A型DNA合成仪,参照恶性疟原虫子孢子CS抗原决定簇相应氨基酸的核苷酸序列,设计了CS-Ⅰ和CS-Ⅱ两个片段。以噬菌体M13mp18质粒作为载体,将两片段进行磷酸化、退火、连接和克隆,经过原位杂交,序列分析,获得了(NANP)_(10)重复串联基因的重组质粒M13mp18-CS。再以酶切,从重组质粒中回收(NANP)_(10)次的片段,将其片段基因与CT-B基因融合,最后将融合基因CT-B-CS插入载体PMC055中,成功地得到含有(NANP)_(10)与CT-B基因融合的重组质粒pMC055-CS。     

A system for shotgun DNA sequencing.

A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.     

Genetic analysis of attTn7, the transposon Tn7 attachment site in Escherichia coli, using a novel M13-based transduction assay

The large (14 kb; kb = 10(3) bases) bacterial transposon, Tn7 (encoding resistance to trimethoprim and streptomycin/spectinomycin), has unusual properties. Like other elements, Tn7 transposes with low efficiency and low target-site specificity, but Tn7 also transposes, with high frequency in a unique orientation, to a preferred "attachment" site, called attTn7, in the Escherichia coli chromosome and similarly into plasmids containing attTn7. We developed a novel bacteriophage M13-based assay system to measure the transposition frequency of Tn7 to M13mp phage vectors containing attTn7 on a cloned 1 kb fragment of chromosomal DNA. Phage harvested from a Tn7 donor strain were used to infect recipient bacteria with selection for trimethoprim resistance. Transposition frequency, expressed as the number of trimethoprim-resistant colonies per plaque-forming unit, was found to be approximately 10(-4) to M13mp::attTn7, in contrast to 10(-10) to M13mp recombinants with approximately 1 kb insertions of other, "generic brand", DNA. By deletion analysis of M13mp::attTn7, we show that attTn7 is contained within a 64 base-pair region; sequences adjacent to the actual insertion site and encoding the carboxy terminus of the glmS gene are required. This assay also provided evidence for transposition immunity conferred by the right end of Tn7.     

Quantitation of type II procollagen mRNA levels during chick limb cartilage differentiation

A single-stranded DNA probe complementary to chicken type II procollagen mRNA has been used to quantitate levels of that mRNA present in chicken limb mesenchyme during cartilage differentiation. Excess labeled probe prepared from a cDNA template cloned in M13mp9 was hybridized to completion to increasing amounts of total RNA and assayed by protection from S1 nuclease digestion. Estimates of the absolute levels of type II procollagen RNA were determined using the M13mp9 template containing the coding strand as a standard. RNA complementary to the probe increased from 20 copies per diploid genome in stage 24 limb to approximately 2000 copies per diploid genome in stage 24 limb mesenchyme which had differentiated to cartilage in culture. Similar levels were found in cartilage from stage 31 limb. Sternal cartilage from 17-day embryos contained approximately 10,000 copies per diploid genome suggesting that the level of expression of this gene is different in limb growth cartilage compared with sternal cartilage. Low but detectable levels of RNA complementary to the probe were observed in limb at stages 20-24. Since a large fraction of the type II procollagen RNA in these early limbs is associated with polysomes, the type II procollagen gene appears to be expressed at a low level prior to phenotypic differentiation and prior to the accumulation of immunologically detectable levels of type II collagen.