An origin unwinding activity regulates initiation of DNA replication during mammalian cell cycle

An in vitro assay was developed to study the positive factors that regulate the onset of DNA replication during the mammalian cell cycle. Extracts prepared from cells at defined positions in the cell cycle were used to examine the replication of SV40 DNA in a cell free system. Extracts prepared from S phase cells were ten times more efficient at initiating replication at the SV40 origin than were extracts from G1 cells, whereas elongation rates were similar in G1 and S reactions. At a discrete point in the cell cycle, just before the cell's entry into S, an activity appeared that was required, in conjunction with SV40 T antigen, for site specific initiation at the SV40 origin. This factor had a role in unwinding DNA at the replication origin.     

表达hTERT及SV40T永生化奶牛乳腺上皮细胞系的建立

构建pcDNA3.1-hTERT、pcDNA3.1-SV40 T载体,线性化后,共转染荷斯坦奶牛乳腺上皮细胞,研究人端粒酶逆转录酶(hTERT)和猿猴病毒40大T抗原(SV40 T)对荷斯坦奶牛乳腺上皮细胞体外培养的作用,并对细胞进行RT-PCR检测分析及免疫荧光鉴定。结果表明,hTERT和SV40 T在奶牛乳腺上皮细胞中表达可以有效地延长细胞的体外培养时间,增加细胞传代次数,获得的细胞系可以正常表达角蛋白。说明在体外培养的乳腺细胞中共表达hTERT和SV40 T可以有效延长细胞寿命且不影响乳腺细胞特性。     

Isolation of mutants of an animal virus in bacteria

Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions.     

Human diploid cell transformation by DNA extracted from the tumor virus SV40

The DNA isolated from simian virus 40 (SV40) can transform human fibroblast cells in tissue culture. Clonal lines of DNA-transformed human cells have been obtained that have properties characteristic of cells transformed by whole virus. They all contain SV40 T-antigen, and infectious virus can be recovered by cocultivation. This is the first demonstration of a permanent genetic alteration produced in human cells by purified DNA.     

MIF-like activity in simian virus 40-transformed 3T3 fibroblast cultures

Simian virus 40-transformed fibroblasts (SV3T3), as compared with their untransformed counterparts (3T3), elaborate a macromolecular product that inhibits macrophage migration and causes macrophages to aggregate and lose one type of cell coat material. I'he SV3T3 cells also lack this surface material relative to 3T3 cells. There may be a relationz between migration inhibition factor (MIF), the cell coat, and cell migration.     

Immunologic manipulation of metastases due to herpesvirus transformed cells

Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and simian virus 40 (SV40) fail to induce immunity in weanling Syrian hamsters to transplant of hamster cells transformed by HSV-2. However, the development of metastatic tumors is markedly enhanced by prior immunization with HSV-1. Immunization with SV40, ultraviolet-irradiated tumor cells, or ultraviolet-irradiated normal hamster embryo cells inhibits the development of metastases. The HSV-hamster system appears a good one for the study of development, prevention, and control of metastases by mammalian cells transformed by a common human virus.     

Tet-on系统诱导猿猴病毒40T在CHO细胞中的表达

四环素诱导表达系统（Tet-off／Tet-on系统）是比较成熟的真核生物基因诱导表达系统之一,具有高效、无毒、严密开／关功能的特点。猿猴病毒40T（SV40T）是一种病毒癌蛋白,其与肿瘤抑制蛋白p53和Rb结合,并使之失活,从而消除它们抑制细胞生长的功能,使细胞分裂加速,形成肿瘤。利用Tet-on系统首先稳定筛选获得了表达Tet-on系统调节元件rtTA的阳性细胞CHO-pTet-on,再通过稳定筛选又成功得到导入其反应元件的双阳性细胞CHO-pTet-on-pTRE2-SV40T-Hyg,经强力霉素诱导表达了目的基因SV40T,建立了Tet-on基因诱导表达系统的细胞诱导表达研究平台。     

Persistence of abnormal chloride conductance regulation in transformed cystic fibrosis epithelia

An airway epithelial cell line (CF/T43) was developed by infecting cultured airway epithelial cells from patients with cystic fibrosis (CF) with the pZIPneoSV(X)1/SV40T retrovirus and selecting for G418 resistance and ion transport properties. The distinctive chloride secretory phenotypes of the CF cell line CF/T43 and a normal cell line (NL/T4) were not perturbed by SV40T-induced cell transformation. Epithelial cell lines generated from CF cells with the SV40T gene can be used to test candidate CF genes and to evaluate the molecular mechanisms responsible for the CF phenotype.     

脑部特异表达SV40Tag转基因动物模型的建立

将猿猴病毒抗原蛋白(SV40Tag)片段克隆进脑部特异表达载体pMM279中,通过显微注射法制作转基因小鼠。采用PCR和Southern blotting检测目的基因整合,并通过RT-PCR检测目的基因的表达。结果表明:共出生29只仔鼠,经PCR检测出3只阳性,Southern blotting检测出2只阳性。RT-PCR检测到SV40Tag仅在前脑部皮层和海马表达。成功获得的脑部特异表达SV40Tag转基因小鼠模型,为SV40Tag的致病机制及脑肿瘤的治疗等研究提供工具。     

Active T-cell receptor genes have intron deoxyribonuclease hypersensitive sites

The T-cell receptor beta-chain gene has a nuclease hypersensitive site in several kinds of T cells, which does not appear in B cells expressing immunoglobulins. Conversely, the kappa immunoglobulin gene shows a known hypersensitive site at its enhancer element in B cells, as expected, but this site is absent in T cells. As is the case with immunoglobulin genes, the T-cell receptor site lies within the gene, in the intron separating joining and constant region segments. These nuclease hypersensitive DNA configurations in the introns of active T-cell receptor and immunoglobulin genes may arise from control elements that share ancestry but have diverged to the extent that each normally acts only in lymphoid cells which use the proximal gene product.     

Positive control of transformed phenotype in hybrids between SV40-transformed and normal human cells

Somatic cell hybrids have been obtained between SV40-transformed Lesch-Nyhan fibroblasts, which are deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and display glucose-6-phosphate dehydrogenase A (G6PD-A) activity, and late-passage HGPRT-positive W138 human embryo fibroblasts, which display G6PD-B activity. The human-human hybrid clones, which display G6PD-A and G6PD-B and heteropolymers of the two enzyme forms, have the same growth characteristic as the SV40-transformed parental cells and behave as continuous cell lines. The SV40 tumor antigen, the gene for which has been assigned to human chromosome 7, is present in all clones examined.     

erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells

A wide variety of human tumors contain an amplified or overexpressed erbB-2 gene, which encodes a growth factor receptor-like protein. When erbB-2 complementary DNA was expressed in NIH/3T3 cells under the control of the SV40 promoter, the gene lacked transforming activity despite expression of detectable levels of the erbB-2 protein. A further five- to tenfold increase in its expression under influence of the long terminal repeat of Moloney murine leukemia virus was associated with activation of erbB-2 as a potent oncogene. The high levels of the erbB-2 product associated with malignant transformation of NIH/3T3 cells were observed in human mammary tumor cells that overexpressed this gene. These findings demonstrate a new mechanism for acquisition of oncogenic properties by genes encoding growth factor receptor-like proteins and provide a functional basis for the role of their overexpression in the development of human malignancies.