Isolation and characterization of the porcine growth hormone gene

A cosmid clone containing the entire porcine growth hormone (PGH) gene has been isolated using a full-length PGH cDNA as the hybridization probe. The gene within the cosmid was subcloned into plasmids and completely sequenced. The coding, promoter, and both 5'- and 3'-noncoding sequences of the PGH gene were found to be highly conserved when compared to the previously sequenced genes coding for rat, human and bovine growth hormones, and also to the human placental lactogen gene. The high degree of conservation between the 5'- and 3'-noncoding regions of the genes from these different species indicates that growth hormone genes may be evolving by some unusual mechanism. The PGH gene was found to contain the unusual variant GC donor splice site.     

Molecular cloning of DNA complementary to bovine growth hormone mRNA

We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH). Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E. coli x 1776. A recombinant plasmid containing bGH cDNA ws identified by hybridization to cloned rat growth hormone cDNA. It contains the entire coding and 3'-untranslated regions and 31 bases in the 5'-untranslated region. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal peptide and confirmed the published amino acid sequence of the secreted hormone at all but 2 residues. Codon usage is nonrandom, with 81.7% of the codons ending in G or C. The nucleotide sequence of bGH mRNA is 83.9% homologous with rat GH mRNA and 76.5% homologous with human GH mRNA, while the respective amino acid sequence homologies are 83.5% and 66.8%.     

猪生长激素cDNA的分子克隆

用改进的氯化锂沉淀沉法和寡聚(dT)一纤维素亲和层析法由猪垂体制得总mRNA。以此总mRNA为模板,合成cDNA。钝端连接重组到质粒pUC19的SmaI位点,转化E.coli JM107,筛选出阳性克隆。用限制性内切酶酶切签定及5’端部分核苷酸序列分析证明:克隆了全长猪生长激素cDNA,其长度约为896bp。     

Cloning and sequencing of beta-mannosidase gene from Aspergillus aculeatus no. F-50

The manB gene, coding for a unique beta-mannosidase (MANB) of Aspergillus aculeatus, was cloned from genomic and cDNA libraries, and sequenced. The gene consists of 2,811 bp encoding a polypeptide of 937 amino acid residues with a molecular mass of 104,214 Da. The A. aculeatus MANB shared amino acid sequence identity with MANB of human (24%), goat (24%), bovine (24%), and Caenorhabditis elegans (22%). When the A. aculeatus MANB was compared with other related enzymes, a Glu residue corresponding to the active site identified by the Escherichia coli beta-galactosidase and the human beta-guclonidase was conserved. This is the first fungal gene that encodes MANB.     

Effective method for obtaining long nucleotide chains on partially complementary templates. Processed bovine gamma-interferon gene obtained from human gamma-interferon gene

The method of obtaining the bovine gamma-interferon gene by means of simultaneous multidirected mutagenesis of the human gamma-interferon gene is presented. The first strand of the bovine gamma-interferon gene was obtained by ligation of synthetic oligonucleotides, using the cDNA of human gamma-interferon, cloned in the single-stranded phage M13mp19 as a template. The second strand was synthesized using a large fragment of E. coli DNA-polymerase I. The double-stranded gene was then treated by restriction nucleases and cloned in a pUC-18 derived vector. The primary structure was confirmed by sequencing.     

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor.

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.     

Characterization of the bovine prothrombin gene

The bovine prothrombin gene was characterized by Southern blot analysis of bovine genomic DNA using bovine prothrombin cDNA fragments as hybridization probes. These analyses suggested that the bovine genome contains a single prothrombin gene that is at least 10 kilobase pairs (kbp) in size. To characterize the gene more thoroughly, two bovine genomic phage libraries were screened by using prothrombin cDNAs as hybridization probes. Heteroduplex analysis of the cloned genomic DNA and cDNA showed that the prothrombin gene is 14.9 kbp in size and contains at least 14 exons interrupted by 13 introns. The exons vary in size from 28 to 317 base pairs (bp), while the introns vary in size from less than 100 to 6940 bp. Regions of self-complementarity were observed within some of the introns, suggesting the presence of inverted repeat sequences. The bovine prothrombin gene shows similarities in structure to both the human prothrombin gene and the human factor IX gene.     

兔单个植入前克隆胚胎cDNA文库的构建

人与小鼠和牛在正常胚胎植入前发育过程中基因活化的研究已经取得了长足的进展 ,但是还没有对同期克隆胚胎相关研究的报道 .利用单个家兔植入前移核重构胚胎成功地构建了MⅡ卵母细胞及发育至 4 、8 细胞期的胚胎和囊胚的特异性cDNA文库 .并用 β肌动蛋白和LAPTM4α证实这类文库是可靠的 .以 8 细胞期移核重构胚cDNA文库为例 ,随机挑取克隆进行测序分析 :其中 2 3的基因EST片段可以在GenBank或EST库中找到同源序列 ,约 1 3的EST片段属于未知的新片段 ,表明这是一类重要的新兴基因资源库 (期特异性EST库 ) .这种利用单胚胎构建cDNA文库的方法 ,解决了胚胎研究材料受限的问题 ,在时间上更加精确 ,更符合胚胎发育的规律 ,也能够更加准确地反映出一些克隆胚胎的异常表型 ,是研究早期胚胎发育基因表达以及克隆胚胎再程序化基因表达的一种有效手段 .     

人生长激素基因的cDNA克隆,原核高效表达及纯化

用ＲＴ－ＰＣＲ方法,从含有全长人生长激素（ＨＧＨ）基因的ＣＨＯ细胞中获得ｈｇｈ的ｃＤＮＡ,将其克隆入ｐＥＴ－１１ｂ载体中,在大肠杆菌中获得高效表达,表达量占菌体蛋白总量的２０％。经色谱纯化后,纯度大于９５％。     

A Candida albicans homolog of a human cyclophilin gene encodes a peptidyl-prolyl cis-trans isomerase

A Candida albicans cDNA and its genomic counterpart were isolated from lambda phage libraries using a human T-cell cyclophilin (Cyp) cDNA as a hybridization probe. The clones contain a 486-bp open reading frame predicting a 162-amino acid, approx. 18 kDa protein which is similar in size to, and which shares 68 and 81% homology with, human T-cell Cyp and cytosolic Saccharomyces cerevisiae Cyp, respectively. Northern blots show the presence of a single mRNA species of about 800 bp. However, genomic Southern blots suggest the presence of at least one other Cyp-related gene in C. albicans. The cDNA was engineered for expression in Escherichia coli, and the resulting recombinant protein, like mammalian Cyps, exhibited a peptidyl-prolyl cis-trans isomerase (PPIase) activity which was sensitive to inhibition by cyclosporin A in vitro. These results indicate that the gene which we have cloned encodes a C. albicans Cyp. We designate this gene CYP1 (cyclophilin). Interestingly, the predicted C. albicans protein contains only two cysteine residues which do not align with any of the four cysteines conserved among mammalian Cyps. This suggests that the PPIase catalytic mechanism may not involve an enzyme-bound hemithioorthoamide, as previously reported for porcine Cyp.     

Application of recombinant DNA technologies to studies on chicken growth hormone

Messenger RNA isolated from chicken pituitaries was used to construct a chicken pituitary cDNA library. A chicken growth hormone cDNA clone was isolated using 32P-labeled mammalian growth hormone cDNA probes. The amino acid sequence (derived from the DNA sequence) of the mature form of chicken growth hormone shows 77% homology with that of bovine growth hormone. The chicken growth hormone cDNA clone was used to generate a vector capable of producing chicken growth hormone in Escherichia coli. The recombinant E. coli-derived chicken growth hormone was similar to pituitary chicken growth hormone in several biochemical and immunological properties. The recombinant-derived hormone has been used to establish a sensitive radioimmunoassay for growth hormone determinations made from chicken sera. The chicken growth hormone gene has also been introduced into a retroviral vector capable of establishing productive infections of chicken cells both in in vitro and in vivo. The resulting infections are accompanied by the production of radioimmunoassay-detectable growth hormone. The concentrations of growth hormone in sera of Leghorn chickens infected with the recombinant retrovirus are three- to tenfold higher than in control animals.     

重组人Endostatin／MBP融合蛋白片段的构建及体外抑制内皮细胞增殖活性

为了寻找人endostain基因的核心作用自然,先用PCR方法从人的肝脏cDNA库克隆出人endostain基因。然后,利用限制性内匹酶酶切得到5个不同的endostatin基因片段,并净它们构建到肠杆菌表达得到rh Endo-statin/MPB6个不同片段的融合蛋白,用亲和层析技术分离纯化,并分别作用于 外培养的牛肾上腺毛细血管内皮细胞（BCE）,检测它们对内皮细胞增殖的影响,rh Endostain/MBP不同片段的融合蛋白对经bFGF刺激引起的BCE细胞的快速增殖有不同程序的抑制作用。Endostain作用的活性片段位于Endostain蛋白N端的第55－96氨基酸位置内。