Co-operation between the p53 protein tumor antigen and platelet-poor plasma in the induction of cellular DNA synthesis

Plasmids containing DNA sequences coding for p53 were microinjected into quiescent Swiss 3T3 cells. Three constructs were used, carrying either the whole gene sequence, a full-length cDNA, or a hybrid between the gene and the cDNA. All of them stimulated DNA synthesis when cells were incubated with platelet-poor plasma (PPP) following injection. The p53 gene stimulated DNA synthesis to a lesser extent, also in the absence of PPP. Several negative results were obtained with different plasmids, including deletion mutants in the p53 coding region. However, a deletion mutant in which the p53 reading frame ended in the middle of the coding part of the p53 gene still stimulated DNA synthesis in co-operation with PPP. The stimulation of DNA synthesis induced by p53 cDNA was more synchronous and more limited than that induced by serum. The present data suggest that p53 may act as a competence factor in cell cycle progression.     

人Ⅱ型囊泡单胺转运体基因的克隆

目的 为获得人Ⅱ型囊泡单胞转运体（vesicular monomine transporter-2VMAT2)以治疗帕金森病,克隆添加Kozak序列的VMAT2cDNA,用于真核细胞表达。方法 从人胎脑中提取总RNA,逆转录成cDNA,设计一对引物用PCR方法扩增目的cDNA片段并将其重级一起pGEM-Easy-T载体中,酶切鉴定插入片段后,进行全序列测定。结果 RT-PCR扩增到1637bp的带有Kozak序列的cDNA片段。结论 克隆人VMAT2cDNA片断,可用于真核细胞表达。     

Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5'-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5'-end non-coding region and a poly(A) signal AATAAA at the 3'-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3' region after poly(A) signal.     

人Neuritin在原核表达系统的构建及表达纯化

Neuritin是一种新发现能促进神经突起和轴突分支的蛋白,为了更清楚的研究它的生物学功能,我们在已克隆Neuritin cDNA的基础上, PCR扩增出Neuritin ORF,与原核表达载体pET32a重组后,成功的构建了Neuritin原核表达质粒pET32a-Neuritin。重组质粒转化BL21大肠杆菌, IPTG诱导后,表达产物用SDS-PAGE 和Western blot证实系Neuritin,用镍离子亲和层析的方法获得了纯化的Neuritin蛋白。     

Induction of transformation and DNA synthesis after microinjection of raf proteins.

Full-length and N-terminal deletion mutants of human c-raf-1 cDNA were cloned into Escherichia coli expression plasmids. Bacterially expressed c-raf proteins were purified by anion-exchange, gel filtration, and affinity chromatography. Microinjection of mutant c-raf proteins into G0-arrested NIH 3T3 cells induced DNA synthesis and morphological transformation, whereas microinjection of full-length c-raf had no effect. The amino terminus of the raf protein has an important negative regulatory influence; alteration of this region resulted in increased kinase activity and oncogenicity.     

含RGD受体结合位点口蹄疫病毒Asia1/JS/China/2005株的拯救及初步鉴定

摘要：【目的】构建含有RGD受体结合位点口蹄疫病毒（FMDV）Asia1/JS/China/2005株的全长感染性cDNA克隆。【方法】采用定点突变方法,构建Asia1型FMDV含有预期突变的全长cDNA克隆pFMDV-RGD。pFMDV-RGD重组质粒经NotI线化后,与表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,进行FMDV-RGD病毒拯救。【结果】序列测定结果表明成功构建了FMDV含有RGD受体位点的Asia1/JS/China/2005全长cDNA克隆。共转染实验获得拯救病毒,对拯救的病毒分别进行序列测定、间接免疫荧光、电子显微镜观察和乳鼠致病性分析,表明成功拯救了含有RGD受体结合位点的Asia1/JS/China/2005株FMDV。【结论】该实验为进一步研究含有RGD和RDD受体结合位点2个拯救病毒生物学特性的差异奠定了基础。     

The construction, identification and characterisation of plasmids containing human alpha-lactalbumin cDNA sequences.

We describe the cloning of double-stranded cDNA synthesized from lactating human mammary gland total poly(A)-containing RNA, into the EcoRI site of the plasmid pAT153. Nine recombinants were shown to contain alpha-lactalbumin cDNA sequences as determined by positive hybridisation translation of complementary RNA. Restriction enzyme maps were determined for six of these. Alignment of the restriction map with the known amino acid sequence of human alpha-lactalbumin provided evidence that two plasmids, designated phO-53 and phB-35, contained the complete coding sequence of the primary translation product (pre-alpha-lactalbumin). Hybridisation studies using purified human, monkey and guinea-pig alpha-lactalbumin cDNA demonstrated that greater nucleotide sequence divergence has occurred within the rodents than the primates, and that rodent alpha-lactalbumin mRNAs retain regions of homology with primate alpha-lactalbumin mRNAs.     

A novel low molecular weight ribonucleic acid (RNA) related to transforming growth factor alpha messenger RNA

Human transforming growth factor alpha (TGF alpha) is coded for by an mRNA of about 4800 nucleotides. The cDNA sequence demonstrates that the 50 amino acid TGF alpha is embedded in a larger 160 amino acid precursor protein. We report here that in addition to the 4800 nucleotide TGF alpha mRNA, there is a novel second RNA species of about 350 nucleotides that hybridizes to a human TGF alpha cDNA probe. This small RNA species has been found in the RNA of several human tumor cells including HT1080, A549, A431, A2058, and A673. We have demonstrated an inverse relationship between the amounts of the 4800 nucleotide TGF alpha mRNA and the 350 nucleotide novel RNA in these human cells. Restriction enzyme cleavage of a human TGF alpha cDNA probe into three separate domains consisting of a processed coding region and 5'- and 3'-preprocessed coding and untranslated regions showed that only the 3'-untranslated region hybridized to the 350 nucleotide RNA. Using sense and anti-sense single-stranded 3'-untranslated region probes, we determined that the 350 nucleotide RNA band may be composed of multiple species of RNA which are related to the anti-sense DNA strand that is opposite to the strand that codes for the 4800 nucleotide TGF alpha mRNA.     

Replication of RNA by the DNA-dependent RNA polymerase of phage T7

The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately. The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence. Replication of X RNA involves synthesis of complementary strands. Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure. Replication of X RNA by T7 RNA polymerase is both template and enzyme specific. No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA. The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases. Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.     

Random silent mutagenesis in the initial triplets of the coding region: a technique for adapting human glutathione reductase-encoding cDNA to expression in Escherichia coli

The introduction of random silent mutations into the 5'-coding region of a human cDNA as the basis for successful expression in Escherichia coli is demonstrated in four steps. (1) Plasmid pUB200 containing the pRpL promoters of phage lambda was found not to serve as an expression vector for a unchanged human glutathione reductase (hGR)-encoding cDNA. (2) When this cDNA was expressed in a two-cistron context using high-copy-number plasmids, recombinant protein was detected in low yield (0.03% of the total cell protein). (3) Silent mutations were introduced into the triplets coding for the N-terminal amino acids. When screening E. coli colonies transformed with expression plasmids containing cDNA mutants, we identified adapted clones that produced hGR in up to 70-fold higher yield than the clone containing the unchanged cDNA. Sequence analyses of adapted cDNA species revealed lower G + C contents in the modified regions, suggesting altered mRNA structures. (4) When the adapted cDNA sequences were recloned in the vector which had failed to express unchanged hGR cDNA in step 1, synthesis of recombinant protein was as high as in step 3. This means that the yield of expression for adapted cDNA was at least 1000-fold higher than for unchanged cDNA. In conclusion, random silent mutations introduced into the translation initiation region of cDNA might be a useful technique for designing sequence features which favour gene expression.     

Human 2-5A synthetase: characterization of a novel cDNA and corresponding gene structure.

The enzyme 2-5A synthetase is induced in cultured cells in response to interferon (IFN) treatment. A lambda gt10 cDNA library of mRNA from IFN-induced Daudi lymphoblastoid cells was screened with oligonucleotide probes. Several overlapping cDNAs were isolated and shown to be derived from the human synthetase gene using filter selection and oocyte microinjection assays. The nucleotide sequence of one of these, cDNA 8-2, extended the 2-5A synthetase sequence already described 72 bp in the 5' direction but was found to differ significantly in coding sequence at the 3' end. The longest cDNA isolated (6-2) was approximately 1.4 kb. By Northern hybridization analysis single mRNAs of 1.7 kb were detected in Daudi and T98G (glioblastoma) cells. However, in HeLa cells, four mRNAs ranging in size from 1.5 to 3.5 kb were found, one of which differed at the 3' end. Analysis of both phage and cosmid genomic clones and comparison with genomic DNA indicate that there is a single gene for 2-5A synthetase, comprising at least six exons and five introns, which can undergo a novel form of alternative RNA processing depending on cell type.     

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.     

马尾松毛虫质型多角体病毒多角体蛋白基因的cDNA克隆及序列分析

通过对马尾松毛虫质型多角体病毒的增殖、纯化,获得一株单一类型的质型多角体病毒。提纯的病毒粒子经SDS－酚抽提,琼脂糖凝胶电泳分离基因组dsRNDA,回收纯化第十片段S10。S10经DMSO变性,逆转录合成cDNA第一链,PCR扩增后,克隆在pGEM-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长763bp,起始密码AUG位于3－5残基,终止密码UGA位于747－749残基。推测DpCPV多角体蛋白基因编码248个氨基酸的多肽,分子量28kD。和家蚕质型多角体病毒（BmCPV）多角体蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为89.3%和97.6%。