共查询到20条相似文献,搜索用时 31 毫秒
1.
Transcription of Simian virus 40 DNA in a HeLa whole cell extract 总被引:22,自引:0,他引:22
H Handa R J Kaufman J Manley M Gefter P A Sharp 《The Journal of biological chemistry》1981,256(1):478-482
2.
3.
DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA. 总被引:12,自引:4,他引:8
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo. 相似文献
4.
5.
6.
7.
8.
9.
抗水稻条纹叶枯病毒核酶的设计,克隆及体外活性测定 总被引:10,自引:0,他引:10
为探索控制水稻条纹叶枯病毒(Ricestripevirus,RSV)设计合成了特异切割该病毒RNA保守区及编码病害特异性蛋白(DiseaseSpecificProtein,DSP)基因的核酶,核酶基因的长度均为40个碱基,用化学合成方法合成其正链及与其3'-末端互补的15个碱基引物,用TagDNA多聚酶合成其互补链。双链DNA直接插入克隆载体PGEM3zf(+)的Smal位点。序列测定表明,克隆得到的核酶序列与设计的核酶序列完全一致。经SP6RNA多聚酶体外转录得到核酶RNA。当核酶RNA与以同样方法转录得到的靶基因RNA混合反应,可得到预期结果相同的切割片段,表明两种核酶在体外均具有特异性切割活性。 相似文献
10.
11.
12.
Attenuation in the control of SV40 gene expression 总被引:57,自引:0,他引:57
13.
14.
A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression. 总被引:5,自引:4,他引:1
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence. 相似文献
15.
16.
17.
18.
19.
20.
Characterization of a site-specific restriction endonuclease from Streptomyces aureofaciens. 总被引:3,自引:0,他引:3
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A new type II sequence-specific restriction endonuclease, SauI, was isolated from Streptomyces aureofaciens IKA18/4. The purified enzyme was free of contaminating exonuclease and phosphatase activities. SauI cleaved lambda DNA at two sites, but did not cleave pBR322, simian virus 40, or phi X174 DNA. SauI recognized the septanucleotide sequence 5'-CCTNAGG-3' and cleaved at the position indicated by the arrow, producing a trinucleotide 5'-terminal extension. 相似文献