Determination of the single polyadenylation site of the human pro alpha 1(II) collagen gene.

Several cDNA clones for the human type II procollagen mRNA were isolated from a cartilage cDNA library. Six of the clones containing the longest inserts were subjected to restriction site mapping for alignment. All these six clones extended to the poly A tail. The longest clone, containing a 1470 bp insert, was named pHCAR3. Sequencing of pHCAR3 made it clear that neither of the two canonical AATAAA sequences of the human type II collagen gene is used as the polyadenylation signal. Two 60 bp stretches of high interspecies homology terminating in a hexanucleotide ATTAAA, located 23 nucleotides upstream of the poly A tail, apparently have an important role in determining the single polyadenylation signal for this gene. S1 protection experiments confirmed these observations.     

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.     

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein.     

GDF5基因真核表达载体的构建及其在恒河猴骨髓间充质干细胞中的表达

目的克隆人软骨组织生长分化因子5（GDF5）基因及构建GDF5基因真核表达载体,观察其在恒河猴骨髓间充质干细胞（MSCs）中的表达情况。方法采用反转录聚合酶链式反应（RT-PCR）从人胎儿软骨组织克隆hGDF5基因全长cDNA,插入pEGFP-C2载体,构建重组真核表达质粒pEGFP-C2-GDF5。重组质粒脂质体介导法转染MSCs细胞,荧光显微镜观察报告基因的表达,RT-PCR法检测目的基因表达。结果成功克隆人软骨组织GDF5基因和构建GDF5真核表达质粒pEGFP-C2-GDF5,克隆在载体上的基因长度为1505bp,包含全部cDNA编码序列1505bp,测序显示与Genbank上的序列一致。重组质粒转染恒河猴MSCs细胞得到表达,绿色荧光蛋白在转染24h后开始表达,72h达高峰,然后表达逐渐减弱。转染后72h可检测到GDF5mRNA表达。结论人GDF5基因在恒河猴MSCs细胞的成功表达为应用恒河猴模型开展基于细胞的基因疗法修复骨和软骨损伤研究奠定了必要基础。     

Transcriptional products of the human placental lactogen gene

Poly(A+)RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to preplacental lactogen (pre-hPL) and a minor band co-migrating with mature hPL represent approximately 15% of the total radioactively labeled proteins. Analysis of the poly(A+)RNA by agarose gel electrophoresis showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with 32P-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460, and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A+)RNA was also used to construct a cDNA library. Approximately 5% of the clones were found to hybridize to hPL DNA sequences, indicating that hPL mRNA is indeed very abundant in term placental tissue. One recombinant plasmid containing an insert of approximately 815 base pairs was isolated and characterized by restriction enzyme mapping and electron microscopy. Heteroduplexes constructed between the cDNA and the DNA isolated from an hPL genomic clone revealed four small intervening sequences which can account for the lengths observed for the hnRNA molecules.     

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。     

人促红细胞生成素基因组基因和cDNA的克隆及其在COS－7细胞中的表达

利用ＰＣＲ技术,从正常人胎肝染色体ＤＮＡ中克隆到长度为１５７２ｂｐ的人促红细胞生成素（ＥＰＯ）基因组基因片段,它包含除第一个外显子和第一个内含子外所有外显子及内含子。再人工合成１３ｂｐ外显子１的编码区,并与１５７２ｂｐ片段拼接,从而得到除第一个内含子的人促红细胞生成素基因组基因。将克隆得到的ＥＰＯ基因插入载体ｐＳＶ２－ｄｈｆｒ得到ｐＳＶ２－ＥＰＯ表达载体,转染ＣＯＳ－７细胞后获得高效表达。利用自行研制的小鼠抗人ＥＰＯ单抗及兔抗人ＥＰＯ多抗,对表达产物进行ＥＬＩＳＡ定量测定,细胞分泌ＥＰＯ量高达２５１±７Ｕ／ｍｌ．Ｋｒｙｓｔａｌ法测得体外生物活性２４１．５±６．５Ｕ／ｍｌ．用ＥＰＯ单抗免疫沉淀结合ＳＤＳ－ＰＡＧＥ对转染细胞的表达产物做了进一步鉴定,清晰地看到了ＥＰＯ条带。从高效表达ＥＰＯ的转染细胞中分离纯化ｍＲＮＡ,用ＲＴ－ＰＣＲ方法扩增并克隆到ＥＰＯ的ｃＤＮＡ,这为ＥＰＯ在其它系统中的表达及ＥＰＯ的功能与结构的研究打下了基础。     

Expression of the genes coding for the skeletal muscle and cardiac actions in the heart

Several types of evidence indicate that the gene coding for the skeletal muscle actin is expressed in the rat heart: 1) A recombinant plasmid containing an insert with a nucleotide sequence identical to that of the homologous region of skeletal muscle actin gene was isolated from a cDNA library prepared on rat cardiac mRNA template. 2) Using specific probes it was found that the hearts of newborn rats contain a significant amount of skeletal muscle actin mRNA. The quantity of this mRNA in the heart decreases during development. 3) The skeletal muscle actin gene is DNAase I sensitive in nuclei from rat heart tissue. A plasmid containing a cDNA insert homologous to a part of the cardiac actin mRNA was isolated and sequenced. It was found that in spite of the great similarity between the amino acid sequence of the skeletal muscle and cardiac actins, the nucleotide sequences of the two mRNAs are considerably divergent. There is only limited sequence homology between the 3' untranslated regions of the two mRNAs. However, there is an extensive sequence homology between the 3' untranslated regions of the rat and human cardiac mRNAs, suggesting a functional role for this region of the gene or mRNA.     

Cloning of the human oestrogen receptor cDNA

Poly A+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and those fractions enriched in oestrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt11 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kbase) and lambda OR8 (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains DNA sequences which cross-hybridize with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Using lambda OR8 as a hybridization probe revealed a single poly A+ RNA band of approx. 6.2 kbase in the ER containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-cell line HeLa. The same probe hybridizes to a chicken gene which is expressed in oviduct tissue as a 7.5 kbase poly A+ RNA.     

Human and mouse S-protein mRNA detected in Northern blot experiments and evidence for the gene encoding S-protein in mammals by Southern blot analysis

Summary Human S-protein is a serum glycoprotein that binds and inhibits the activated complement complex, mediates coagulation through interaction with antithrombin III and plasminogen activator inhibitor I, and also functions as a cell adhesion protein through interactions with extracellular matrix and cell plasma membranes. A full length cDNA clone for human S-protein was isolated from a lambda gt11 cDNA library of mRNA from the HepG2 hepatocellular carcinoma cell line using mixed oligonucleotide sequences predicted from the amino-terminal amino acid sequence of human S-protein. The cDNA clone in lambda was subcloned into pUC18 for Southern and Northern blot experiments. Hybridization with radiolabeled human S-protein cDNA revealed a single copy gene encoding S-protein in human and mouse genomic DNA. In addition, the S-protein gene was detected in monkey, rat, dog, cow and rabbit genomic DNA. A 1.7 Kb mRNA for S-protein was detected in RNA from human liver and from the PLC/PRF5 human hepatoma cell line. No S-protein mRNA was detected in mRNA from human lung, placenta, or leukocytes or in total RNA from cultured human embryonal rhabdomyosarcoma (RD cell line) or cultured human fibroblasts from embryonic lung (IMR90 cell line) and neonatal foreskin. A 1.6 Kb mRNA for S-protein was detected in mRNA from mouse liver and brain. No S-protein mRNA was detected in mRNA from mouse skeletal muscle, kidney, heart or testis.     

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.     

Cloning and expression of murine lymphotoxin cDNA

The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.