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Biomedical subjects

M Wigler

Publications and source records attributed to M Wigler.

106 records · Page 6Linked to original sources
PubMed1980

Isolation of the chicken thymidine kinase gene by plasmid rescue.

We have used the bacterial plasmid pBR322 as a vehicle to isolate genes coding for selectable markers from higher eukaryotes. In this way, we have obtained the chicken thymidine kinase (tk) gene as a 2.2-kilobase EcoRI/HindIII insert in BR322. The cloned gene transforms tk- animal cells with an efficiency equal to that of the cloned herpes simplex virus-1 tk gene.

Animals↗
PubMed1980

Characteristics of an SV40-plasmid recombinant and its movement into and out of the genome of a murine cell.

A bacterial plasmid carrying the early region of SV40 (pOT) has been stably established in high molecular weight (hmw) DNA of mouse L cells by selection for the herpes virus thymidine kinase (tk) gene. DNA blotting has demonstrated that most cell lines contain multiple discrete copies of pOT, generally with an intact SV40 early region. No free copies of pOT have been detected. Both pOT and tk sequences may be amplified up to 20-200 copies of the SV40 early region. In contrast to the uniform staining pattern normally observed in SV40-transformed lines, indirect immunofluorescence using antiserum to the SV40 T antigen has demonstrated that the expression of the early region is heterogeneous in these cell lines. This fraction expressing T is characteristic of a given cell line, and varies from 0 to 99% positive. Several pOT cell lines have been fused to simian cells, and replicating low molecular weight DNAs were isolated from the heterokaryons. Transformation of E. coli with this DNA demonstrates that pOT can be rescued from hmw DNA in L cells and reestablished as a plasmid in E. coli. Excision is generally precise when pOT is introduced to the murine cells as supercoiled molecule, and imprecise when pOT is introduced in linear form.

Animals↗
PubMed1980

Genetic and physical linkage of exogenous sequences in transformed cells.

The calcium phosphate precipitation method of Graham and van der Eb (1973) is an efficent means of introducing DNA into culturaed animal cells. Cells which incorporate one selectable marker are also likely to incorporate one selectable marker are also likely to incorporate sequences from the carrier DNA. Both selected and unselected markers are found integrated in the high molecular weight nuclear DNA of the host. In the present study, we demonstrate that exogenously acquired sequences are gentically linked, segregating and amplifying coordinately, and that their flanking sequences derive primarily from the carrier species rather than the host species. Based on these results, we propose that, upon transformation, the host cell ligates incorporated DNA into a large concatameric structure which may at times be as large as 2000 kilobases. From blotting data alone we cannot determine whether this structure is chromosomal or extrachromosomal in location.

Animals↗
PubMed1980

Transformation of mammalian cells with an amplifiable dominant-acting gene.

We have transferred a mutant hamster gene coding for an altered dihydrofolate reductase to wild-type cultured mouse cells by using total genomic DNA from methotrexate-resistant Chinese hamster ovary A29 cells as donor. By demonstrating the presence of hamster gene sequences in transformants we have provided direct evidence for gene transfer. Transformants selected for increased resistance to methotrexate contain increased amounts of the newly transferred gene. We have used this mutant dhfr gene to introduce the Escherichia coli antibiotic resistance plasmid pBR322 into animal cells. Amplification of the dhfr sequences results in amplification of the pBR322 sequences as well. The use of this gene may allow the introduction and amplification of virtually any genetic element in various new cellular environments.

Animals↗
PubMed1979

Introduction and expression of a rabbit beta-globin gene in mouse fibroblasts.

The cloned chromosomal rabbit beta-globin gene has been introduced into mouse fibroblasts by DNA-mediated gene transfer (transformation). In this report, we examine the expression of the rabbit gene in six independent transformants that contain from 1 to 20 copies of the cloned globin gene. Rabbit globin transcripts were detected in two of these transformants at steady-state concentrations of 5 and 2 copies per cell. The globin transcripts from one cell line are polyadenylylated and migrate as 9S RNA on methylmercury gels. These transcripts reflect correct processing of the two intervening sequences but lack 48 +/- 5 nucleotides present at the 5' terminus of rabbit erythrocyte globin mRNA.

Animals↗
PubMed1979

DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells.

In this report, we demonstrate the feasibility of transforming mouse cells deficient in adenine phosphoribosyltransferase (aprt; AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) to the aprt+ phenotype by means of DNA-mediated gene transfer. Transformation was effected by using unfractionated high molecular weight genomic DNA from Chinese hamster, human, and mouse cells and restriction endonuclease-digested DNA from rabbit liver. The transformation frequency observed was between 1 and 10 colonies per 10(6) cells per 20 microgram of donor DNA. Transformants displayed enzymatic activity that was donor derived as demonstrated by isoelectric focusing of cytoplasmic extracts. These transformants fall into two classes: those that are phenotypically stable when grown in the absence of selective pressure and those that are phenotypically unstable under the same conditions.

Adenine Phosphoribosyltransferase↗
PubMed1978

Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor.

Previous studies from our laboratories have demonstrated the feasibility of transferring the thymidine kinase (tk) gene from restriction endonuclease-generated fragments of herpes simplex virus (HSV) DNA to cultured mammalian cells. In this study, high molecular weight DNA from cells containing only one copy of the HSV gene coding for tk was successfully used to transform L+K-cells to the tk+ phenotype. The acquired phenotype was demonstrated to be donor-derived by analysis of the electrophoretic mobility of the tk activity, and the presence of HSV DNA sequences in the recipient cells was demonstrated. In companion experiments, we used high molecular weight DNA derived from tissues and cultured cells of a variety of species to transfer tk activity. The tk+ mouse cells transformed with human DNA were shown to express human type tk activity as determined by isoelectric focusing.

Animals↗
PubMed1978

The transfer and stable integration of the HSV thymidine kinase gene into mouse cells.

Treatment of mutant mouse cells (Ltk-) deficient in thymidine kinase with Bam I restriction endonuclease-cleaved HSV-1 DNA results in the appearance of numerous surviving colonies which stably express thte tk+ phenotype. Through a series of electrophoretic fractionations in concert with transfection assays, we isolated a 3.4 kb fragment which contains the thymidine kinase gene and which alone is competent in the biochemical transformation of Ltk- cells. In this report, we have examined the distribution of tk sequences in the DNA of several transformed clones following stable gene transfer. A series of complementary experiments involving reassociation kinetics in solution and annealings with tk DNA to restriction-cleaved cellular DNA following electrophoresis and transfer to filters allow us to make the following general conclusions concerning the fate of the tk gene in all clones examined: the tk gene is present in all cells at a frequency of one copy per chromosomal complement; the tk gene is stably integrated in the DNA of all transformants; and integration is not site-specific and occurs at different loci in the DNA of all transformants examined. The existence of a single active tk gene in tk+ transformants now facilitates an analysis of the sequence organization of tk- mutant cells and provides a useful model system for studies on the transfer of cellular genes.

Cell Line↗
PubMed1978

Induction of plasminogen activator in cultured cells by macrocyclic plant diterpene esters and other agents related to tumor promotion.

In vitro systems that are responsive to tumor-promoting agents may facilitate the identification of such agents and the analysis of their mode of action. We have previously reported that the potent tumor promoter phorbol-12-myristate-13-acetate induces the synthesis of the enzyme plasminogen activator in cultured chick embryo fibroblasts. We have, therefore, tested various compounds for their ability to induce plasminogen activator in chicken embryo fibroblasts. Among these, phorbol esters and other macrocyclic diterpene esters isolated from species of the families Euphorbiaceae and Thymelaeaceae were potent inducers of plasminogen activator. These compounds maximally induced enzyme to the same levels, although they differed in their relative molar potencies. Structural requirements for in vitro activity paralleled the requirements for activity in vivo. These results indicate that induction of plasminogen activator is a useful marker for the biologically active macrocyclic diterpene esters. On the other hand, tumor-promoting agents such as anthralin, cantharidin, Tween 60, and tobacco leaf extract failed to induce plasminogen activator.

Cells, Cultured↗
PubMed1977

Effects of antiinflammatory agents on mouse skin tumor promotion, epidermal DNA synthesis, phorbol ester-induced cellular proliferation, and production of plasminogen activator.

The antinflammatory ateroids fluocinoine acetonide, fluocinonide, and fluclorolone acetonide were found to be very effectiveinhibitory agents of mouse skin tumor promotion. These steroids also drastically inhibited epidermal DNA synthesis and epidermal cellular proliferation induced by a phorbal ester tumor promoter. In addition, these compounds were potent inhibitors, of plasminogen activator production in tumor cell cultures. The clinically used non-steroidal antiinflammatory agents oxyphenbutazone, indomethacin, and Seclazone also inhibite tumor promotion but were much less effective. Although these agents are useful against inflammatory disorders in general when given p.o., in our studies they had little effect on inflammation and epidermal cellular proliferation induced by a phorbol ester tumor promoter when given topically. The afore mentioned nonsteroidal antiinflammatory agents also had little effect on epidermal DNA synthesis. Oxyphenbutazone and indomethacin were less potent inhibitors of plasminogen activator production in tumor cells than were the antiinflammatory steroids, and Seclazone produced a negligible inhibition. There is, therefore, a general correlation in the potencies of a series of steroidal antiinflammatory agents for inhibition of tumor promotion and their ability to inhibit plasminogen activator production by tumor cell cultures and epidermal DNA synthesis.

Animals↗
PubMedDate not supplied

Structure of the Ki-ras gene of the human lung carcinoma cell line Calu-1.

The homologue of the viral Kirsten ras (v-Ki-ras) gene found in the human lung carcinoma cell line, Calu-1, has an intron-exon structure similar to that of the human homologue of the viral Harvey ras (v-Ha-ras) gene. A second, potential fourth coding exon is present in the human Ki-ras gene and similar sequences are found in the Kirsten murine sarcoma virus. Cysteine is encoded at the twelfth amino acid position, suggesting that the Calu-1 Ki-ras gene has undergone a mutational activation at the same position as the human Ha-ras gene of the bladder carcinoma cell line, T24. A comparison of their predicted amino acid sequences suggests that ras proteins have a 'constant' region and a 'variable' region. Here we propose a common modular structure for ras gene products in which the variable region forms a physiologically important combining site.

Amino Acid Sequence↗