野生大豆未成熟种子总mRNA的分离及其cDNA的分子克隆

用氯化锂沉淀法从野生大豆(G.soja)未成熟种子中制备总RNA,经oligo(dT)-纤维素柱亲和层析,获得总mRNA,在兔网织红细胞体系中表现出一定翻译活性。以总mRNA为模板,oligo(dT)_(12)(?)为引物,反转录酶催化合成第一链cDNA,RNase H-DNA聚合酶Ⅰ协同合成第二链cDNA。双链cDNA的长度大约为200—5000 bp,且不存在发夹结构。将双链cDNA修补后钝端连接到pUC 19质粒的Sma 1位点,转化E.coli JM107,获得800多个白色重组子克隆。快速电泳检测及酶切分析表明,多数重组子带有插入片段,其中3个重组子的插入片段长度大致为1700 bp、2600 bp和1400 bp。     

黄地老虎颗粒体病毒cDNA文库的构建

以感染黄地老虎颗粒体病毒（Agrotis segetum ganulosis virus,AsGV)黄地老虎幼虫为材料提取总RNA,分离mRNA,并反转录合成cDNA ,构建了包括黄地老虎（Agrotis segetum,As)幼虫和黄地老虎颗粒体病毒的cDNA 文库。用Eco RI和HindⅢ限制性内切酶酶切AsGV基因组DNA,制备地高辛探针,与上述总RNA,mRNA,cDNA 杂交,从文库中筛选出AsGV的阳性克隆1081个,经cDNA测序,cDNA 编码序列与基因组编码序列相符。并根据基因组阅读框序列合成引物,PCR扩增出59个阅读框的编码基因,也完全与基因组序列相符。     

RT－PCR克隆里氏木霉甘露聚糖酶cDNA

为了省去繁杂的mRNA提取纯化过程，简便，快速，重现性好的克隆里氏木霉RutC-30β-甘露聚糖酶cDNA，本研究依据其cDNA序列设计了一对引物F1和R1，然后用总RNA直接进行RT-PCR，其产物经过EcoRI,BamHI酶切后插入P^GEM-32克隆载体，再转化到JM109感受态细胞中，克隆转化子，酶切测序。结果获得了β-甘露聚糖酶编码的成熟肽cDNA，其序列与GenBank报道完全一样，另外，对有利于用RT-PCR克隆cDNA的高诱导产生β-甘露聚糖酶的里氏木霉RutC-30菌体培养条件进行了研究。     

黄地老虎颗粒体病毒cDNA文库的构建

以感染黄地老虎颗粒体病毒(Agrotis segetum ganulosis virus,AsGV)的黄地老虎幼虫为材料提取总RNA、分离mRNA,并反转录合成cDNA,构建了包括黄地老虎(Agrotis segetura,As)幼虫和黄地老虎颗粒体病毒的cDNA文库.用EcoR Ⅰ和HindⅢ限制性内切酶酶切AsGV基因组DNA,制备地高辛探针,与上述总RNA、mR-NA、cDNA杂交,从文库中筛选出AsGV的阳性克隆1081个,经cDNA测序,cDNA编码序列与基因组编码序列相符.并根据基因组阅读框序列合成引物,PCR扩增出59个阅读框的编码基因,也完全与基因组序列相符.     

应用限制性显示PCR技术构建细菌poly(A)化mRNA的cDNA文库

针对细菌mRNA poly(A)化位点的高度多态性,利用oligo(dT)与poly(A)特异结合的特性,以oligo(dT)一纤维素纯化mRNA,并以oligo(dT)18为引物逆转录合成cDNA,用限制性内切酶消化cDNA,所得的限制性内切酶片段与通用接头相连,通过10个选择性引物组合进行选择性PCR,使各片段得以扩增并分布于10个亚组中,并进行克隆,成功地克隆了100多个基因片段,已对其中40个进行了测序分析,探讨了限制性显示PCR技术在细菌poly(A)化mRNA cDNA库构建中的应用价值。     

应用逆转录聚合酶链反应法扩增类胰岛素生长因子Ⅱ基因

利用酸性异硫氰酸胍-酚-氯仿一步法从人胚胎组织中提取总RNA,经Oligo(dT)纤维柱分离纯化出mRNA。用逆转录与聚合酶链反应相结合的RT-PCR法,扩增出人类胰岛素生长因子Ⅱ(IGFⅡ)的cDNA片段,在限制性内切酶Sma Ⅰ存在的连接体系中,将扩增出的cDNA片段克隆进PUC12的Sma Ⅰ位点处。经限制性内切酶EcoR Ⅰ、Sal Ⅰ、Eco47Ⅲ酶切鉴定其方向。以重组质粒的双链DNA为模板,用末端终止法测定其全部核苷酸顺序,证实其核苷酸编码的IGFⅡ在氨基酸顺序上与文献报道的相同。     

大麦条纹花叶病毒新疆株(BSMV-XJ)RNA_2的cDNA克隆及5′端序列分析

分离了大麦条纹花叶病毒(BSMV)新疆株基因组的3个RNA组份。以RNA2为模板,3′端互补寡核苷酸为引物,合成了第一条cDNA链和第二条cDNA链,将ds-cDNA重组在pUC9质粒中,转化大肠杆菌细胞,获得含RNA3′端的克隆,并证明所选克隆的cDNA含有新疆株几近全长的RNA2组份。对于插入片段为3.3kbp的112号克隆进行了酶谱分析,得到了与国外典型株类似的结果;用双脱氧终止法分析了相当于RNA 2 5′端250bp的cDNA酶切片段,表明与国外典型株有十分相似的一级结构。     

限制性内切酶介导的重叠延伸在人环氧合酶-1(COX-1)基因克隆中的应用

建立一种用于克隆全长基因的、限制性内切酶介导的重叠延伸法 .对全长基因进行分段扩增 ,并利用适当的限制性内切酶对基因序列内相应的限制性位点进行酶切 ,从而使分段扩增片段得以重叠并互为模板 ,在DNA聚合酶的作用下延伸获得全长基因 .将环氧合酶 1 (COX 1 )基因的外显子 9巧妙地拼接到了缺失外显子 9的COX 1cDNA片段中 ,获得了COX 1基因的全长cDNA .该方法分 3步进行 .首先 ,通过RT PCR分别扩增跨外显子 9的cDNA片段和缺失外显子 9的cDNA片段 ,并克隆到pMD1 8 T载体上 ;其次 ,PCR扩增外显子 9片段 ,限制性内切酶StuI酶切缺失外显子9cDNA片段的重组质粒 ,二者以一定的比例混合 ,互为模板 ,在pfuDNA聚合酶的作用下进行延伸 ,从而产生一个双链的DNA分子 .最后 ,以延伸产物为模板 ,用COX 1cDNA两端的引物进行PCR扩增 ,产生包含外显子 9的COX 1基因的全长cDNA .这种限制性内切酶介导的重叠延伸方法 ,对于克隆mRNA剪接水平上受调控的基因尤为有用 ,同时也为基因的重组和修饰提供一个新的思路     

人成纤维细胞成纤维蛋白质cDNA片段的分离及亚克隆

我们用噬菌斑原位杂交的方法从人成纤维细胞cDNA基因库(λNMT-pCD-cDNA重组体)中分离到一株成纤维蛋白质cDNA克隆。经限制性内切酶酶切电泳及吸印转移法分析证明这是一个成纤维蛋白质cDNA片段克隆,并进一步将此cDNA片段克隆到pBR322质粒上。     

Genetic engineering of peptide hormones

The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated. The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs. Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin. The gene of human calcitonin was created by combination of chemical and enzymatic synthesis. This synthetic gene was further cloned in pBR322. The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E. coli were tested.     

对虾白斑综合症病毒重组cDNA克隆的构建与分析

取经人工注射感染了对虾白班综合症病毒40-45h的凡纳对虾鳃组织,分离mRNA,以mRNA为模板合成双链cDNA,并克隆于PUC质粒的Not I/Sal I位点,构建了1000余株对虾感染后期鳃细胞的重组cDNA克隆,重组质粒经PCR鉴定插入片段,DNA斑点杂交分析目的片段,测定了20株对虾白斑综合症病毒的重组cDNA克隆的末端DNA序列,并对其进行了包含存在的开放阅读框架,启动区上游序列、编码产物的特性等分析。结果显示,PCR产物在0.3-1.6kb之间;大于1kb的克隆中有31.8%的克隆为白斑综合症病毒的重组cDNA克隆。已测序的不包含同源序列的13株克隆中含有14个开放阅读框,其中11个上游可检出启动子基序,4个可检出启动子调制元件,ORF转译产物的特性基序分析显示,有2个ORFs可检出锌指基序,3个ORFs可检出亮氮酸拉链基序,2个ORFs可检出NTP结合基序,未检定核定位信号基序。     

猪胰脏细胞cDNA文库的构建

 本文以新鲜猪胰脏为材料,采用异硫氰酸胍法和寡聚(dT)-纤维素亲和层析法,提取了Poly(A)~+RNA,经麦胚无细胞体系鉴定其体外翻译活性,~3H-Leu参入量为空白对照的5倍。参照Gubler和Hoffman等人的方法,以此poly(A~+)RNA为模板,合成总cDNA,并采用多聚物加尾法,与pUC19质粒重组,转化入感受态E.coliJM107,进行分子克隆,其转化率为3.6×10~4克隆子/μgcDNA。并对重组质粒DNA进行了酶切鉴定。     

Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae.

Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae. Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E. coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions. A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E. coli EF-Tu, respectively.     

Maize Glutamine Synthetase Cdnas: Isolation by Direct Genetic Selection in Escherichia Coli

Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli delta glnA mutant growing on medium lacking glutamine. The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter. E. coli delta glnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 ampr gene, and challenged to grow on agar medium lacking glutamine. Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences. Two cDNAs identical except for different 5' and 3' termini have been sequenced. The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes. In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF. This mini-ORF was shown not to be essential for the functional rescue of the E. coli delta glnA mutant. Expression of the cDNAs in E. coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids. Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.     

ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'

BACKGROUND: Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples. MATERIALS AND METHODS: Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples. RESULTS: After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for ILG1. CONCLUSIONS: In the course of our studies using cDNA RDA, we have isolated and identified ILG1, a likely active site-specific recombinase and new member of the bacteriophage P4 family of integrases. This family of integrases is implicated in the horizontal DNA transfer of pathogenic genes between bacterial species, such as those found in pathogenic strains of E. coli, Shigella, Yersinia, and Vibrio cholera. Using ILG1 as a marker of our laboratory E. coli strain TOP10F', our evidence suggests that contaminating bacterial DNA in our subtraction experiment is due to this laboratory bacterial strain, which colonized exposed surfaces of the laboratory worker. Thus, identification of differentially expressed genes between normal and diseased states could be dramatically improved by using extra precaution to prevent bacterial contamination of samples.     

Expression in Escherichia coli cells of mutant human interferon alpha2

cDNA library was obtained from mRNA isolated from human leukocytes induced by Newcastle disease virus. Clones containing cDNA for alpha 2-interferons were identified by colony hybridization with two synthetic hexadecanucleotides. One of the positive clones contained a NH2-terminal part of cDNA of human interferon identical to cDNA for IFN-alpha 2. The only difference between these two clones was the Ser-8 leads to Asn-8 substitution in deduced sequenced of mature interferons. This mutant interferon, named alpha 2, was expressed in E. coli and its properties were compared with those of interferon alpha 2.