Characterization of homologous DNA recombination activity in normal and immortal mammalian cells.

Homologous DNA recombination levels were measured in normal and spontaneously immortalized murine and human fibroblasts, and in a number of primate and murine established fibroblast cell lines. Immortal cell lines and tumor-derived clones homologously recombined extrachromosomal plasmid substrates at frequencies approximately 100-fold higher than did normal cells. To further explore the mechanism responsible for this phenotype, homologous recombination frequency was measured using nuclear extracts derived from normal and immortalized murine and human fibroblasts. Extracts prepared from immortal cells catalyzed high levels of homologous recombination, whereas very little recombination activity was detected in extracts prepared from normal fibroblasts. Similarly, only extracts derived from immortal cells contained strand-transferase activity as measured by the recently described pairing-on-membrane assay. Mixing experiments indicated that a recombination enhancing factor or factors present in immortal cells, rather than a recombination inhibitor in normal cells, was responsible for the enhanced homologous recombination activity observed using extracts derived from the former.     

Homologous recombination is elevated in some Werner-like syndromes but not during normal in vitro or in vivo senescence of mammalian cells

Werner syndrome (WS) is a recessive genetic condition associated with markedly reduced replicative lifespans of cells in culture, high chromosomal instability in vivo and in vitro, and premature appearance of many characteristics of normal aging, including an increased incidence of cancer. We have monitored plasmid homologous recombination frequencies in diploid fibroblasts from 6 Werner or Werner-like syndrome patients, following transfection with a plasmid substrate containing 2 overlapping fragments of the TN5 Neor gene. Plasmid DNA recovered from these cells was then assayed for homologous recombination by (a) transformation of recA- bacteria to Ampr (indicating total viable plasmid) or Neor (indicating viable recombinant plasmid), and (b) by limited-cycle polymerase chain reaction (PCR) to co-amplify a recombinant fragment containing the overlap region, and a control region of the same plasmid, without bacterial transformation. Bacterial assay data indicated that recombination rates in 3 of the 6 WS strains were significantly elevated above normal controls; 4 of 6 appeared elevated by PCR assay. The highest-recombination WS strain showed evidence of reduced degradation of transfected plasmid DNA. For this small sample of WS strains, clinical severity of WS was not well correlated with recombination rate as determined by either assay (Pearson r = 0.78, not significant, for PCR assay); elevated recombination may, however, define a subset of WS at greatest risk for cancer and/or atherosclerosis. PCR assay of a hyperoxia-resistant HeLa cell line, displaying substantially increased chromosome breakage, indicated increased recombination between direct-repeat fragments. Nevertheless, elevated recombination in WS strains is unlikely to be secondary to impaired replicative capacity characteristic of WS cells, or to defective repair of chromosome damage which is increased in WS, since recombination in non-WS strains was unaffected by passage level or repeated UV irradiation.     

Intermolecular plasmid recombination in fibroblasts from humans with DNA damage-processing defects

We have evaluated the ability of immortalized human fibroblasts to recombine transfected plasmid DNA. A number of cell lines from normal individuals and from patients with DNA damage-processing defects were examined. Two plasmid recombination substrates were derived from pSV2neo and contained nonoverlapping deletions in the aminoglycoside phosphotransferase II gene. Intermolecular recombination was assessed by two methods after cotransfection. In a short-term, extrachromosomal recombination assay, low molecular weight DNA was extracted from the human cells 48 h after transfection, and recombinant plasmids were detected by transformation into appropriate indicator bacteria. In a long-term stable recombination assay the fibroblasts were cotransfected and G418-resistant colonies allowed to form. By the former assay all but two cultures were recombination-proficient, whereas all were recombination-proficient by the latter assay. The efficiency of transfection of human cells with plasmids appears to be a major variable affecting recombination. Recombination can be stimulated by uv irradiation of plasmid DNA prior to transfection. Cells from patients with Fanconi anemia, ataxia telangiectasia, and xeroderma pigmentosum complementation groups A, C, D, E, and G are not defective at intermolecular plasmid recombination.     

Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control

A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability. Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-microns plasmid DNA and an intact actin gene, were constructed. Neither plasmid alone yielded transformants in the haploid Leu- strain AH22, but when cotransformed, a number of colonies were obtained. Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-microns plasmid. The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level. The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event. Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a high frequency of transformants resulting primarily from single-crossover events between the two plasmids. This was presumably because such events no longer generated an intact actin gene on a multicopy plasmid. Infrequently a transformant from a plasmid with an intact gene was recovered, but in these cases the plasmid was not present in multiple copies. These cells exhibited a slower growth rate, and Northern blot analysis revealed an elevated level of actin mRNA.     

Molecular rearrangements between two plasmids of the FII incompatibility group in different recombination—Deficient Escherichia Coli strains

The frequencies and types of plasmid molecular rearrangements generated in different recombinant mutants which carried two plasmids of the FII incompatibility group were studied. The wild-type cells generated molecular rearrangements mainly by interplasmidic recombination with a frequency of 2.4 x 10(-6) per cell per cell doubling. Cells in which RecF was the principal recombination pathway generated different types of molecular rearrangements that involved either both plasmids or one of the plasmids and the chromosome. The frequencies of molecular rearrangements for these cells were 50-fold greater than those of wild-type cells. The recA- cells, even when the RecE pathway was derepressed, generated rearrangements only between one of the plasmids and the chromosome, at very low frequencies (10(-9]. In wild-type cells and in RecF cells, interplasmidic recombination generated mainly cointegrates carrying DNA deletions. These cointegrates were stable in recA- or recA- RecE+ cells, but unstable in wild-type or RecF+ cells. In the latter, the cointegrates generated smaller plasmids with different molecular structures at relatively low frequencies.     

A potent DNA synthesis inhibitor expressed by the immortal cell line SUSM-1

We have previously reported the production of DNA synthesis inhibitor proteins by both quiescent and senescent human diploid fibroblasts. Young, proliferating fibroblasts do not produce such inhibitors, but are capable of responding to either the quiescent or senescent cell DNA synthesis inhibitors. Recently, we have analyzed the immortal cell line SUSM-1 (derived from normal liver fibroblasts following exposure to carcinogen) for inhibitory activity. We have found that SUSM-1 cells produce a factor capable of inhibiting DNA synthesis in young fibroblasts. Crude extracts prepared from SUSM-1 cells inhibit DNA synthesis in a dose-dependent manner at concentrations 10-fold lower than those of either senescent or quiescent fibroblast cell extracts. SUSM-1 cells are incapable of responding to the inhibitor they produce, as are three other immortal human cell lines tested. One immortal cell line, HeLa, does respond to the SUSM-1 inhibitor, though to a lesser degree than observed with normal young fibroblasts. One hypothesis is that the DNA synthesis inhibitor protein(s) of senescent cells plays a role in determining the finite in vitro life span of normal cells. The results reported here suggest that SUSM-1 cells may have escaped senescence through loss of a receptor or cofactor for the inhibitor protein(s).     

以杆状病毒为载体表达可溶性55kD人肿瘤坏死因子受体

从培养的HeLa细胞中提取总RNA,通过反转录PCR技术, 从该总RNA中扩增了约530 bp的shTNFR55基因的cDNA,将cDNA克隆至转移载体pAcGp67B的多角 体蛋白基因启动子的下游与转移载体构建成重组转移质粒pAcTNFR。pAcTNFR与杆状病毒AcNP V的DNA共转染昆虫细胞sf9,通过同源重组形成含有shTNFR55基因的重组病毒。经空斑分析 和DNA斑点杂交获得了纯化的重组病毒AcNPVTNFR。采用肿瘤坏死因子(TNF)敏感的L929细 胞检测表达产物的生物学活性。结果表明表达产物可以中和TNF对L929的细胞毒性。蛋白配 基印迹(Ligand blot)分析表明表达产物分子量约在20～25kD之间,有三条带。     

Recovery of recombinant bacterial plasmids from E. coli transformed with DNA from microinjected mouse cells

We have previously described the isolation of thymidine kinase positive (TK+), human beta-globin gene-containing colonies following co-microinjection of mouse TK- L cells with two recombinant pBR322 plasmids, one containing the TK gene of herpes simplex virus type I (plasmid pXl), and the second containing a human genomic DNA fragment within which is the human beta-globin gene (plasmid pRKl). DNA isolated from one such clone was used in bacterial transformation experiments with a selection for tetracycline-resistant colonies (that is, for cells containing pRKl). A total of forty-two tetracycline-resistant colonies were isolated, thirty of which contained circular pRK1 molecules identical to those originally injected. The remaining twelve colonies contained unique plasmids that were grouped into five different classes of recombinant molecules. All five of these unique recombinant classes appear to contain a common deletion endpoint occurring at a specific region of the pBR322 segment of pRKl. Four of the unique recombinant classes appear to have arisen from the deletion of a segment of a pRKl trimer or dimer molecule, while the fifth class appears to have resulted from recombination between pRKl and pXl followed by a deletion event within this recombinant. It is uncertain whether these deletions are occurring within the eukaryotic cell or upon subsequent transformation of the bacterial cell. If the latter, then the passage of the plasmid DNA through the eukaryotic cell alters a specific site of the pBR322 DNA in such a way that deletions can occur at a high frequency in this region when the plasmid DNA is introduced back into a bacterial cell. Thus, we have established a prokaryote-eukaryote-prokaryote DNA transfer and recovery system which should be useful in studies on DNA replication and the regulation of gene expression in higher eukaryotes.     

A cloned polyoma DNA fragment representing the 5'' half of the early gene region is oncogenic.

The two polyoma DNA fragments generated by cleavage with BamHI and EcoRI were cloned in pBR322, and their oncogenic potential was tested in vivo and in vitro. Only recombinant plasmid DNA containing a polyoma DNA fragment which extends clockwise from 58 to 0 map units and include approximately the 5'-proximal half of the early gene region produced tumors in newborn hamsters and transformed rat embryo cells in tissue culture. Southern blotting analysis indicated that the entire 2.2-kilobase polyoma BamHI-EcoRI fragment was intact in both a tumor cell line and a cell line transformed in culture which we examined. The presence of polyoma middle and small T antigen in these lines was demonstrated by immunoprecipitation and tryptic peptide mapping. DNA from a recombinant plasmid containing a polyoma genome deleted between 90 and 4 map units failed to induce tumors or transform cells.     

Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei.

We have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus.     

Correlation between complementation group for immortality and DNA synthesis inhibitors

Previous studies had demonstrated that a DNA synthesis inhibitor(s) was produced by senescent but not young human diploid fibroblasts (HDF). Analysis of immortal human cell lines led to the finding that SUSM-1, carcinogen-treated immortal human liver fibroblast cells, expressed a potent inhibitor of DNA synthesis that was active in proliferation-competent young HDF but did not affect the SUSM-1 cell line itself. To determine whether one mechanism of escape from senescence to the immortal phenotype involved the loss of response to such DNA synthesis inhibitors, we initiated the present study analyzing a larger number of immortal human cell lines representative of the four complementation groups for indefinite division identified to date. We have found a correlation between the assignment of a cell line to Complementation Group D and the production of DNA synthesis inhibitors coupled with inability to respond to the inhibitory factors. We have also observed a correlation between the ability of immortal cell lines to respond to such DNA synthesis inhibitory factors and assignment to Complementation Group B. These data suggest DNA synthesis inhibitors are involved in the limited lifespan of normal cells and that the immortalization process may involve alterations in the activity of or response to such inhibitors.     

FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

TCDD-induced homologous recombination: the role of the Ah receptor versus oxidative DNA damage

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicits numerous biological responses including carcinogenicity. The molecular mechanism by which TCDD exerts its tumorigenic effects is unclear, since it does not directly damage DNA. TCDD-initiated toxicity can be mediated by the aryl hydrocarbon receptor (AhR) pathway and/or via increased oxidative stress. DNA damage, including DNA oxidation, can induce DNA double-strand breaks, which can be repaired through homologous recombination. Excessive DNA double-strand breaks may promote aberrant DNA recombination, which can lead to detrimental genetic changes and ultimately to carcinogenesis. TCDD has been shown to induce homologous recombination but the molecular mechanism mediating these events are unknown. To investigate the role of the AhR and oxidative DNA damage in mediating TCDD-induced homologous recombination we used a Chinese hamster ovary (CHO) cell line containing a neo direct repeat recombination substrate (CHO 3-6). CHO 3-6 cells were exposed to TCDD (50, 500 or 1000 pM) in the presence or absence of an AhR antagonists (0.1 microM alpha-naphthoflavone (alpha-NF)) for 6 or 24 h and 2 weeks later homologous recombination frequencies were determined by counting the number of neo expressing, G418-resistant colonies per live cells plated. TCDD-initiated DNA oxidation was determined by measuring the formation of 8-hydroxy-2'-deoxyguanosine via HPLC and electrochemical detection. Exposure to 500 pM TCDD for 24 h significantly increased the frequency of homologous recombination. Southern blot analysis on G418-resistant colonies determined that TCDD induced both conservative gene conversion events and deletion events. DNA oxidation was not increased in cells exposed to TCDD for either 6 or 24 h. However, alpha-naphthoflavone exposure resulted in a significant decrease in TCDD-induced homologous recombination frequency. These results suggest that TCDD-initiated homologous recombination in CHO 3-6 cells is mediated by the AhR and not via increased oxidative stress.     

High levels of genetic recombination among cotransfected plasmid DNAs in poxvirus-infected mammalian cells.

The frequency of recombination between transfected plasmid DNAs was measured by using cultured cells infected with a variety of poxviruses. Plasmid derivatives of pBR322 containing XhoI linker insertion mutations in the tetracycline gene were used to assess recombination frequencies in rabbit cells infected with the leporipoxviruses Shope fibroma virus and myxoma virus and the orthopoxvirus vaccinia virus. Recombination frequencies were calculated by Southern blotting, which detects novel plasmid restriction fragments generated by genetic recombination, and by a plasmid rescue procedure in which the reconstruction of an intact tetracycline gene in the transfected rabbit cell was monitored by transformation back into Escherichia coli. The highest recombination frequencies were measured in cells infected with Shope fibroma virus and myxoma virus, and a minimum recombination frequency of at least one recombination event per 7 kilobases was calculated within 24 h posttransfection under these conditions. The deduced recombination frequency in vaccinia virus-infected cells was at least fivefold lower and was not detectable in mock-infected cells, suggesting that the induced recombination activity detected by these methods was under viral control. The results of kinetic studies, analysis with methylation-sensitive restriction enzymes, and the use of phosphonoacetic acid, a specific inhibitor of poxvirus DNA polymerase, indicated that recombination between transfecting DNAs occurred concomitantly with DNA replication but that the two processes could be partially uncoupled. We conclude that the dramatic expansion of recombination activities in the cytoplasm of poxvirus-infected cells is virus specific and offers a good model system with which to analyze the mechanism of recombination in a eucaryotic environment.     

Homologous recombination in variants of the B16 murine melanoma with reference to their metastatic potential

Genomic instability has been accepted as providing a phenotypic variety of malignant cells within a developing tumour. Defects in genetic recombination can often lead to phenotypic differences; therefore, it is possible that metastatic variant cell lines exhibit their particular phenotype as a result of an altered ability to catalyse homologous recombination. We have investigated recombination efficiency in B16 melanoma metastatic variants, using a plasmid, pDR, as a recombination substrate. The plasmid contains two truncated, nontandem but overlapping segments of the neomycin resistance gene (neo 1 and neo 2), separated by the functional gpt gene unit. Only a successful recombination of the two neo segments will generate a functionally intact neomycin gene. Extrachromosomal recombination here was a transient measure of the cells to recombine the neo fragments in an intra- or intermolecular manner. Extrachromosomal recombination frequencies were higher in the high metastasis variants (BL6, ML8) compared with the low metastatic F1 cells. On the other hand, the frequency of chromosomal recombination (after plasmid integration) was higher for the low metastasis (F1) cell line compared with the highly metastatic variants, BL6 and ML8. Since the recombination assay measures only successful recombination events, we have interpreted the observed higher incidence of chromosomal recombination in the low metastatic variant line as indicative of a more stable genome. Similarly, a higher inherent instability in the genome of the high metastasis variants would render these less efficient at producing and maintaining successful recombination events, and this was found to be true by Southern analysis. The results presented show that frequency of recombination may be adduced as evidence for implicating genomic instability in the generation of variant cell populations during metastatic spread. Such an interpretation is also compatible with the Nowell hypothesis for tumour progression. © 1996 Wiley-Liss, Inc.     

Homologous recombination in a Chinese hamster X-ray-sensitive mutant

We have tested the mutant Chinese hamster cell line xrs-5, which is sensitive to ionizing radiation, for the ability to carry out homologous recombination. In an in vivo assay to detect recombination between two transfected plasmids carrying non-complementing mutants in the neomycin resistance gene, xrs-5 showed a 6-fold reduction in recombination frequency when compared to the parental cell line K1. Extracts prepared from nuclei of the mutant were also tested for their ability to catalyze homologous recombination between the same two plasmids in vitro. Extracts from xrs-5 were found to mediate recombination in this assay at frequencies not significantly different from those obtained with extracts from the parental cell line.     

Transformation of rodent cells by a cloned DNA fragment of herpes simplex virus type 2.

Transformation of rodent cells with isolated restriction endonuclease fragments of herpes simplex virus type 2 DNA identified a region of the genome located between map positions 0.58 and 0.62. These sequences were cloned into pBR322, and the recombinant plasmid was used to transform primary rat embryo cells and NIH 3T3 cells. The transformants were selected for their ability to form dense foci on a monolayer or to form colonies in semisolid medium. In contrast to the parental rat or mouse cells, cell lines transformed with the cloned herpes simplex virus type 2 fragment grow to high saturation densities, replicate in medium containing 1% serum, form colonies in dilute methylcellulose, show reduced levels of fibronectin, and are tumorigenic in nude mice and in their syngeneic hosts. Southern blot hybridizations have detected sequences homologous to the viral fragment in high-molecular-weight DNA from the transformed cell lines that are not present in DNA from normal rodents. In all cases, the plasmid DNA was present in less than one copy per cell, and the patterns of viral sequences changed with passage of the cell line in vivo.