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Biomedical subjects

B Vogelstein

Publications and source records attributed to B Vogelstein.

At least 289 records · Page 16Linked to original sources
PubMed1983

A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.

Animals↗
PubMed1983

Mutation affecting the 12th amino acid of the c-Ha-ras oncogene product occurs infrequently in human cancer.

A point mutation alters the 12th amino acid of the c-Ha-ras oncogene product p21 in a human bladder cancer cell line. This is, at present, the only mutation known to result in a human transforming gene. This mutation may therefore represent a possible target for mutagenesis leading to carcinogenesis in humans. By means of restriction enzyme analysis, 29 human cancers, including 20 primary tumor tissues, derived from organs commonly exposed to environmental carcinogens, were tested for the presence of this mutation. None of ten primary bladder carcinomas exhibited the mutation; nor did nine colon carcinomas or ten carcinomas of the lung. Thus the point mutation affecting the 12th amino acid of the c-Ha-ras gene product, while a valuable model for carcinogenesis, does not appear to play a role in the development of most human epithelial cancers of the bladder, colon, or lung.

Adenocarcinoma↗
PubMed1983

Hypomethylation of ras oncogenes in primary human cancers.

We have examined the methylation status of two cellular oncogenes, c-Ha-ras and c-Ki-ras, in primary human carcinomas and the adjacent analogous normal tissues from which the tumors derived. The c-Ha-ras gene was hypomethylated in six of eight carcinomas, including five colonic adenocarcinomas and one small cell lung carcinoma, when compared to adjacent normal tissues. The c-Ki-ras gene was hypomethylated to a lesser extent in two colonic adenocarcinomas. This is the first demonstration of alterations in methylation of cellular oncogenes in human cancer.

Adenocarcinoma↗
PubMed1983

Hypomethylation distinguishes genes of some human cancers from their normal counterparts.

It has been suggested that cancer represents an alteration in DNA, heritable by progeny cells, that leads to abnormally regulated expression of normal cellular genes; DNA alterations such as mutations, rearrangements and changes in methylation have been proposed to have such a role. Because of increasing evidence that DNA methylation is important in gene expression (for review see refs 7, 9-11), several investigators have studied DNA methylation in animal tumours, transformed cells and leukaemia cells in culture. The results of these studies have varied; depending on the techniques and systems used, an increase, decrease, or no change in the degree of methylation has been reported. To our knowledge, however, primary human tumour tissues have not been used in such studies. We have now examined DNA methylation in human cancer with three considerations in mind: (1) the methylation pattern of specific genes, rather than total levels of methylation, was determined; (2) human cancers and adjacent analogous normal tissues, unconditioned by culture media, were analysed; and (3) the cancers were taken from patients who had received neither radiation nor chemotherapy. In four of five patients studied, representing two histological types of cancer, substantial hypomethylation was found in genes of cancer cells compared with their normal counterparts. This hypomethylation was progressive in a metastasis from one of the patients.

Aged↗
PubMed1982

The ovalbumin gene is associated with the nuclear matrix of chicken oviduct cells.

The DNA in a eucaryotic nucleus is arranged into a series of supercoiled loops that are anchored at their bases to the nuclear matrix. Using nuclease digestion, one can progressively cleave DNA from the loops, thereby isolating residual DNA that is progressively closer to the nuclear matrix anchorage sites. We have determined that the ovalbumin gene is preferentially associated with the nuclear matrix of chicken oviduct cells, but is not preferentially associated with the nuclear matrix of chicken liver cells. As a control, the beta-globin gene, which is not transcribed in oviduct cells, was found not to be preferentially associated with the oviduct nuclear matrix. The observation that the transcriptionally active ovalbumin gene is preferentially associated with the nuclear matrix may have significant implications for gene expression and the organization of nuclear DNA into supercoiled-loop domains.

Animals↗
PubMed1982

Nonrandom distribution of repeated DNA sequences with respect to supercoiled loops and the nuclear matrix.

The DNA in a eukaryotic nucleus is arranged into a series of supercoiled loops that are anchored at their bases to the nuclear matrix. We have analyzed the DNA sequences that are closest to the matrix attachment points for their relative content of specific repeated sequences. Sequences were enriched (mouse satellite, human Alu family) or depleted (mouse EcoRI repeat, monkey alpha component), depending on the specific sequence and species examined. These results can be understood in terms of a nonrandom arrangement of DNA sequences with respect to nuclear DNA loops.

Animals↗
PubMed1982

Specific cellular stimulation in the primary immune response: a quantized model.

A general theory for the initial phase of T cell independent immune response is derived from elementary physical-chemical considerations and from the premise that response entails a quantized linkage of cell surface receptors. The theory leads to the construction of explicit antigen dose--response and antigen dose--suppression curves, to the calculation of intrinsic affinities for receptors, and to the deduction that receptors are divalent in character. The theory may be applicable to other cell surface phenomena wherein requirements for stimulation and suppression are equivalent to those found in the immune system.

Antibody Formation↗
PubMed1982

Specific cellular stimulation in the primary immune response: experimental test of a quantized model.

Dose-response and dose-suppression curves have been measured for the primary immune response in mice, in vivo and in vitro, by using size-fractionated linear polymers of acrylamide substituted with hapten. The results are in general agreement with a simple theory based on the premise that the specific primary immunological response is quantized at some fundamental and limiting step, requiring a minimum number of linked antigen receptors for response.

Acrylic Resins↗
PubMed1981

Association of newly replicated DNA with the nuclear matrix of Physarum polycephalum.

We have studied the role of the nuclear matrix in DNA replication in a naturally synchronized eucaryote, Physarum polycephalum. When P. polycephalum. When P. polycephalum macroplasmodia were pulse labeled with 3H-thymidine, the DNA remaining tightly associated with the matrix was highly enriched in newly synthesized DNA. This enrichment was found both in nuclei that had just initiated DNA replication as well as in nuclei isolated later during S phase. Pulse chase experiments showed that the association of newly replicated DNA with the matrix is transient, since most of the newly replicated DNA could be chased from the matrix by incubating pulse labeled macroplasmodia in media containing unlabeled thymidine. Studies measuring the size distribution of the matrix DNA supported the hypothesis that replication forks are attached to the nuclear matrix. Reconstitution controls indicated that these results were unlikely to be due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. These results with P. polycephalum in combination with previous studies in non-synchronized rodent cells, suggest that the association of newly replicated DNA with the nuclear matrix may be a general feature of eucaryotic DNA replication.

Cell Cycle↗
PubMed1980

Localization of SV40 genes within supercoiled loop domains.

Recent studies indicate that eukaryotic DNA is organized into supercoiled loop domains. These loops appear to be anchored at their bases to an insoluble nuclear skeleton or matrix. Most of the DNA in the loops can be released from the matrix by nuclease digestion; the residual DNA remaining with the nuclear matrix represents sequences at the base of the loops, and possibly other sequences which are intimately associated with the nuclear matrix for other reasons. Using a quantitative application of the Southern blotting technique, we have found this residual DNA from SV40 infected 3T3 cells to be enriched in SV40 sequences, indicating that they reside near matrix-DNA attachment points. An enrichment of 3-7 fold relative to total cellular DNA, was found in each of three different lines of SV40 infected 3T3 cells. Control experiments with globin genes showed no such enrichment in this residual matrix DNA. This sequence specificity suggests that the spatial organization of DNA sequences within loops may be related to the functionality of these sequences within the cell.

Animals↗
PubMed1980

Divergence of primate ribosomal RNA genes as assayed by restriction enzyme analysis.

Primate ribosomal RNA (rRNA) genes have been compared by restriction endonuclease mapping. In all species examined, the restriction map of the reiterated ribosomal DNA is simple (within the limits of detection by hybridization with rRNA) and is consistent with a high degree of homogeneity among the repeats. Within a species, all members have similar rDNA restriction patterns. However, different species of primates have distinctly different rDNA restriction maps; even chimpanzee and man can be discerned by their rDNA restriction patterns. Possible mechanisms for maintenance of homogeneity of the rDNA repeats within a species, while allowing divergence among closely related species, are discussed.

Animals↗
PubMed1979

Preparative and analytical purification of DNA from agarose.

Two procedures were developed for removing DNA from agarose after electrophoretic separation of DNA fragments according to size. Both involve dissolving the DNA-containing agarose in NaI. The preparative technique uses binding of DNA to glass in the presence of NaI. The method is rapid and convenient, and DNA of all molecular weight ranges can be recovered in high yield and without degradation. The DNA is free of agarose and remains susceptible to digestion by restriction enzymes. The analytical technique uses selective precipitation of DNA with acetone and has been adapted to molecular hybridization scans of sequences in agarose gels. The sequence-monitoring system is quantitative, directly measuring the proportion of the probe complementary to a given DNA fragment and vice versa. It is especially suitable for analyzing restriction enzyme digests of DNA in mapping experiments.

Acetone↗