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Biomedical subjects

R Axel

Publications and source records attributed to R Axel.

At least 109 records · Page 6Linked to original sources
PubMed1980

Altering genotype and phenotype by DNA-mediated gene transfer.

Transformation, or DNA-mediated gene transfer, permits the introduction of new genetic information into a cell and frequently results in a change in phenotype. The transforming DNA is ultimately integrated into a recipient cell chromosome. No unique chromosomal locations are apparent, different lines contain the transforming DNA on different chromosomes. Expression of transformed genes frequently results in the synthesis of new polypeptide products which restore appropriate mutant cells to the wild-type phenotype. Thus transformation provides an in vivo assay for the functional role of DNA sequence organization about specific genes. Transforming genes coding for selectable functions, such as adenine phosphoribosyltransferase or thymidine kinase, have now been isolated by utilizing transformation in concert with molecular cloning. Finally, transformation may provide a general approach to the analysis of complex heritable phenotypes by permitting the distinction between phenotypic changes without concomitant changes in DNA and functional genetic rearrangements.

Adenine Phosphoribosyltransferase↗
PubMed1980

Isolation of transforming DNA: cloning the hamster aprt gene.

We have isolated the hamster gene coding for the enzyme adenine phosphoribosyl transferase (aprt) using gene transfer and molecular cloning of transforming DNA. Mouse aprt- cells were transformed to the aprt+ phenotype with the product of ligation of Hind III-cleaved hamster genomic DNA and pBR322 DNA. In this manner, the aprt gene was linked to a marked plasmid sequence and segregated from other hamster sequences. A lambda-recombinant phage containing pBR322 DNA sequences was isolated from a library of aprt+ transformed cell DNA. The phage DNA transfers hamster aprt+ activity at a frequency expected of a pure gene. Furthermore, sequences homologous to this clone are present in all hamster aprt+ transformants examined. This experimental design should in theory permit the isolation of any gene coding for selectable or identifiable functions for which DNA-mediated gene transfer can be effected.

Adenine Phosphoribosyltransferase↗
PubMed1980

Introduction of a viral thymidine kinase gene and the human beta-globin gene into developmentally multipotential mouse teratocarcinoma cells.

Teratocarcinoma (TCC) stem cells provide unique prospects for the introduction of specific genes into mice, by virtue of their dual capacity for propagation in vitro and for normal differentiation in embryos. In this study, we have demonstrated that foreign genes amenable to selection in culture can be transferred into the stem cells and expressed. These cells maintain expression of the gene for long periods during differentiation in tumors in vivo in the absence of selective pressure. The cells also integrate an unlinked nonselectable gene at high frequency. Addition of the cloned herpes simplex virus (HSV) thymidine kinase (tk; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene to cultures of tk(-)TCC cells yielded tk(+) colonies at a frequency of one colony per 4 mug of plasmid DNA. This transformation efficiency, although appreciably lower than for mouse L tk(-) cells, permits the isolation of many transformants. The HSV provenance of the transformed phenotype was verified by the characteristic electrophoretic mobility of the tk protein and by neutralization of the tk activity with specific antiserum. Moreover, blot hybridization tests revealed at least one intact copy of the viral tk gene integrated into the DNA of transformed cells. When injected into syngeneic mice, the cells formed solid tumors with various differentiating tissues. From blot hybridization comparisons with their cell lines of origin, seven of nine tumors examined had maintained the HSV tk gene without significant loss or rearrangement. Viral tk enzyme activity could also be demonstrated in at least some of the tumors. Cotransfer of the cloned human beta-globin gene along with the unlinked HSV tk gene was successful in 2 of 10 tk(+) transformants. Thus, defined genes can be stably introduced into TCC cells in culture and maintained in vivo in a form in which they are transcribed and translated to produce a functional protein.

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

Intragenic DNA spacers interrupt the ovalbumin gene.

We have performed restriction endonuclease mapping to examine the linear organization of the ovalbumin gene in chromosomal DNA. Treatment of genomic DNA with restriction endonucleases that do not cleave the ovalbumin mRNA sequence results in the generation of multiple DNA fragments capable of annealing with ovalbumin-specific probes in molecular hybridization reactions. These data strongly suggest that the linear order of DNA sequences coding for ovalbumin is interrupted by at least two intragenic DNA spacers absent from the corresponding RNA. At least one of these spacer sequences interrupts the coding sequence; therefore the chromosomal ovalbumin gene is not colinear with its translational product. We can discern no difference in the sequence organization about this gene in producer and nonproducer somatic cells, suggesting that the presence of intragenic spacers does not reflect that transcriptional activity of this gene. Furthermore, profiles obtained for gametes are identical to those observed for somatic cells, indicating that significant translocation during development is not responsible for the generation of this split sequence. Divergence of the intragenic spacer, however, is observed between individual chickens; thus multiple alleles may exist for this gene that are identifiable by differences in the organization of spacer with no apparent phenotypic evidence for their presence.

Animals↗