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A retrovirus shuttle vector is described that contains the dominant selectable neo gene which confers resistance to kanamycin in bacteria and to the drug G418 in animal cells. The bacterial supF gene and the origins of DNA replication from polyomavirus and the ColE1 replicon also have been included in this vector. Infection of normal rodent cells results in single-copy proviral integration, whereas infection of mouse (MOP) cells expressing polyoma large T antigen results in extrachromosomal replication of the DNA form of the virus. The copy number of the extrachromosomal circles in MOP cells varies from 0 to 100 copies per cell. G418-resistant MOP cells lose their drug-resistant phenotype after passage under nonselective conditions, suggesting that maintenance of the extrachromosomal circles is unstable. The extrachromosomal form of the virus can be recovered as plasmids in Escherichia coli. Two-thirds of the circles analyzed were found to be structurally intact. The others have undergone rearrangements including deletions and insertions. The bacterial supF gene was found to be intact in the majority of recovered plasmids. The data presented here suggest that these retroviruses should be useful as gene transfer vectors for animal cells in culture or in vivo.
Mol Cell Biol 1985 Feb
PMID:Characterization of a retrovirus shuttle vector capable of either proviral integration or extrachromosomal replication in mouse cells. 298 88

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.
Mol Cell Biol 1985 Mar
PMID:gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes. 298 55

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.
Mol Cell Biol 1985 Apr
PMID:Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids. 298 70

A recombinant murine retrovirus was constructed which contains, within its genome, a truncated version of the gene encoding the murine H-2Ld major histocompatibility antigen. The H-2Ld gene, which was inserted 3' of the env splice acceptor site in the recombinant retrovirus MSV-neo, lacked the 5' promoter and TATA sequences and the 3' transcription termination and polyadenylate addition sites of the normal H-2Ld gene. Transfection of the MSV-neo/H-2Ld plasmid (pLTV-11) into Y-2 cells resulted in the production of the transmissible recombinant retrovirus LTV-11. Cells infected with LTV-11 virus were resistant to the eucaryotic antibiotic G418 and expressed H-2Ld on the cell surface. These infected cells contained a viral RNA species which possessed both the H-2Ld and the neomycin resistance gene sequences but did not contain significant levels of the smaller H-2Ld-specific mRNA. The H-2Ld antigen expressed on the surface of infected cells functioned as a target for cytolytic T cells specific for the H-2Ld antigen.
Mol Cell Biol 1985 Jun
PMID:H-2Ld antigen encoded by a recombinant retrovirus genome is expressed on the surface of infected cells. 299 61

Antibiotic resistance to G418 has been transferred into Chinese hamster cell lines via a plasmid vector. The same plasmid, which also contained the Leu2 gene, has been used to transform Leu2- yeast (strain MC16) to leucine prototrophy. Subsequent fusion between transformed yeast and untransformed hamster cells demonstrated that plasmid DNA could be transferred and its genes expressed within the mammalian cell genome. The fusion of transformed hamster cells with untransformed MC16 yeast cells demonstrated that DNA integrated within the mammalian cell genome could be transferred to correct the Leu2 deficiency and also confer G418 resistance on some yeast colonies.
Somat Cell Mol Genet 1986 Mar
PMID:Transfer of antibiotic resistance genes between yeast and mammalian cells under conditions favoring cell fusion. 300 48

We have studied the extrachromosomal maintenance and the transcription regulation of two glucocorticoid-inducible genes on bovine papilloma virus (BPV) vectors in c127 mouse fibroblasts. These genetic elements were the rat tryptophan oxygenase (TOase) gene promoter, which is active in vivo only in hepatocytes, and the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR). From both genes, fusions of the 5'-flanking region of the transcription unit to the bacterial gene for chloramphenicol acetyltransferase (CATase) were constructed. These fusion genes were inserted either into pCGBPV9, a BPV vector encoding G418 resistance, into pBPV-BV1, a vector containing "stabilizing" segments of the human beta-globin gene, or into a BPV construct, whose bacterial plasmid sequences could be removed before transfection. Five constructs of the two latter groups, selectable in c127 cells only as foci, were normally maintained in the extrachromosomal state. In contrast, three out of five constructs based on pCGBPV9 and selectable for resistance against G418 were maintained in a high molecular weight form, most probably of intrachromosomal concatemeric nature, while the remaining two G418-resistant constructs appeared alternatively in this or the extrachromosomal monomeric form. In contrast to its absence of expression in fibroblasts in vivo, the TOase gene element present on BPV vectors was found to be active in fibroblasts in these transfection experiments. As judged by CATase activities and for TOase also by mapping of the transcription start sites, transcription of both genes was under hormonal regulation. All BPV vectors proved to be useful tools in the study of these regulated genes, and in only one out of ten constructs was regulation atypical, possibly due to effects from flanking vector sequences.
J Mol Biol 1986 Feb 20
PMID:Physical state, expression and regulation of two glucocorticoid-controlled genes on bovine papilloma virus vectors. 301 94

The Moloney leukemia virus (M-MuLV) genome was introduced into undifferentiated teratocarcinoma cells by transfection of a plasmid with the virus genome linked to pSV2neo, which carries a bacterial drug resistance gene, neo, or by cotransfection with pSV2neo. In the resulting cells, the M-MuLV genome remained hypomethylated, but its expression was suppressed in cells in an undifferentiated state. The pattern of DNA methylation of the viral genome remained unchanged when the cells were induced to differentiate into epithelial tissues. However, spontaneous M-MuLV expression was detected with differentiation of the cells. To determine to what extent the viral long terminal repeat (LTR) was responsible for this suppression in undifferentiated cells, I constructed plasmids in which neo was placed under the control of the promoter sequence of the dihydrofolate reductase gene or the M-MuLV LTR, and compared the biological activities of the plasmids in Ltk- cells and in undifferentiated teratocarcinoma cells. In Ltk- cells, these plasmids were highly efficient in making the cells resistant to selection by G418. However, in undifferentiated teratocarcinoma cells, the M-MuLV LTR promoted neo gene expression at only 10% of the expected efficiency, as compared with the expression of the neo gene under the control of the simian virus to or dihydrofolate reductase promoter. Thus, the mechanisms of gene regulation are not the same in undifferentiated and differentiated teratocarcinoma cells.
Mol Cell Biol 1985 Sep
PMID:Suppression of the hypomethylated Moloney leukemia virus genome in undifferentiated teratocarcinoma cells and inefficiency of transformation by a bacterial gene under control of the long terminal repeat. 301 27

The intracellular structure of several vectors based on BPV-1 DNA has been analyzed following transfection into mouse C127 cells by the calcium phosphate method or, for the first time, by microinjection directly into the nucleus. It is shown that the method of introduction markedly affects the fate of a BPV-1 based vector. In general, microinjection appears to do little damage to DNA and is more likely to result in a vector replicating extrachromosomally as a monomeric structure of the same size as the input DNA. The method of selection for transformed cells, e.g., focus formation versus resistance to the neomycin analog G418, can also affect the intracellular state of the BPV-1 vector DNA. The nature of the recipient mammalian cell also influences whether a vector can replicate extrachromosomally or whether it integrates. BPV-1 based vectors, which replicated predominantly as multicopy intact extrachromosomal forms in mouse C127 cells, were always found to have integrated at low copy number in mouse LtAp20 cells.
Somat Cell Mol Genet 1986 Jul
PMID:Comparison of methods for introducing vectors based on bovine papillomavirus-1 DNA into mammalian cells. 301 16

Transformed monkey cell lines (CMT and BMT) that inducible express simian virus 40 (SV40) T antigen from the metallothionein promoter have been isolated and characterized. Immunoprecipitation of pulse-labeled T antigen demonstrates a 5- to 12-fold increase in the rate of synthesis on addition of heavy-metal inducers to the culture medium. Radioimmunoassay of cell extracts indicates the accumulation of three- to fourfold more total T antigen after 2 days of induction by comparison with uninduced controls. A direct correlation was found between the level of T-antigen synthesis and the extent of SV40 DNA replication in inducible cells. Inducible BMT cells expressing a low basal level of T antigen were efficiently transformed by a vector carrying the neomycin resistance marker and an SV40 origin of replication. These vector sequences were maintained in an episomal form in most G418-resistant cell lines examined and persisted even in the absence of biochemical selection. Extensive rearrangements were observed only if the vector contained bacterial plasmid sequences. Expression of a protein product under the control of the SV40 late promoter in such vectors was increased after heavy-metal-dependent amplification of the template. These results demonstrate the ability of BMT cells to maintain a cloned eucaryotic gene in an amplifiable episomal state.
Mol Cell Biol 1985 Nov
PMID:New host cell system for regulated simian virus 40 DNA replication. 301 9

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.
Mol Cell Biol 1985 Nov
PMID:Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum. 301 10


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