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Biomedical subjects

S H Wilson

Publications and source records attributed to S H Wilson.

At least 235 records · Page 13Linked to original sources
PubMed1977

Polynucleotide recognition by DNA alpha-polymerase.

In a survey of template-primer preference of a mouse myeloma DNA alpha-polymerase, the fastest rate of DNA synthesis was with poly(dT) as template and (rA)24 as primer. Such a preference for poly(dT).oligo(rA) was not observed with other DNA polymerases of mouse origin. DNA synthesis in this system resulted in formation of oligo(dA) chains, not template-length poly(dA); thus, the average enzyme molecule bound to a poly(dT).(rA)24 complex and initiated a new oligo(dA) chain many times during the incubation. Binding experiments revealed that the alpha-polymerase had high affinity for poly(dT). Although the alpha-polymerase did not bind to poly(dl) and failed to replicate it inreactions with a base pair complementary primer, poly(dl) was replicated after a (dT) block had been grafted to its 3'-end and the oligo(rA) primer had been added. In similar experiments, the (dT) block was found to be much more effective than other 3'-terminal blocks in promoting replication of denatured calf thymus DNA. The results indicate that specific base sequences may regulate initiation of DNA syntehsis by this alpha-polymerase.

Cell Line↗
PubMed1976

Studies on DNA alpha-polymerase of mouse myeloma: partial purification and comparison of three molecular forms of the enzyme.

Activity of DNA alpha-polymerase in extracts from MOPC-104E was not associated with a single protein molecule, but with several molecular species that differed in isoelectric point. The three most abundant of these enzyme species were first separated from other DNA polymerases and then resolved from each other by repeated chromatography on diethylaminoethylcellulose columns. Next, with the use of glycerol gradient centrifugation and DNA-cellulose column chromatography, the three species were further purified to a state representing more than 5000-fold purification over the crude extract. These three highly purified enzyme species exhibited very similar catalytic properties, and the main activity of each species sedimented at the same rate (6-7S) in glycerol gradients containing 0.5 M KCl. Analysis of the polypeptide content of each species revealed that polypeptides of about 150 000 and 60 000 daltons cofractionated with the DNA polymerase activity. The multiple alpha-polymerase species did not appear to result from in vitro proteolytic cleavage, since multiple species were observed in extracts prepared under several different types of conditions, including the presence of the protease inhibitors, phenylmethanesulfonyl fluoride, or trasylol. The three species were recovered in about the same relative amounts from both the nuclear and cytoplasmic fractions of MOPC-104E, and it appeared that multiple species of alpha-polymerase were also present in extracts from fetal bovine liver.

Animals↗
PubMed1976

Template specific inhibitor of mammalian DNA polymerases.

A study of the inhibition of mouse cellular DNA polymerases by poly-nucleotides and their vinyl analogs is presented. Poly(dT)-directed poly(dA) synthesis by representatives of all three classes of cellular DNA polymerase could be completely inhibited by poly(9-vinyladenine), although higher concentrations were required in the case of the gamma class enzyme. Studies on the mechanism of the inhibition using the alpha class DNA polymerase and different templates showed that the enzyme activity was inhibited in all cases where base-pairing between the vinyl polymer and the template occurred; poly(9-vinyladenine) did not interfere with the replication of templates to which it does not bind. The inhibition occurred shortly after addition of poly(9-vinyladenine) to ongoing reactions, yet the enzyme was not displaced from the template - primer complex.

Animals↗
PubMed1976

Differential response of type C and intracisternal type A particle markers in cells treated with iododeoxyuridine and dexamethasone.

Mouse neuroblastoma cells containing intracisternal type A particles were treated with iododeoxyuridine and dexamethasone to induce the release of type C oncornavirus particles. For 5 days after treatment, antigenic markers and DNA polymerase activities specific to particles of each of the two types were assayed in the cells and in pellets obtained by high-speed centrifugation of the culture fluid. There was a marked release of C-particle antigen (p30) and DNA polymerase activity in extracellular particulate form, reaching a maximum on day 3 after treatment and falling thereafter. In contrast, no extracellular A-particle antigen was detected, and A-particle-specific DNA polymerase activity in the medium pellets did not increase from the original very low level. Electron microscopy confirmed the presence of free type C virus particles, but not intracisternal type A particles, in the culture fluid. Although intracellular levels of C-particle antigen rose 20- to 30-fold per milligram of cell protein, intracellular A-particle antigen and DNA polymerase activity did not vary more than two-fold. The relative rate of A-particle synthesis in the treated cells, as judged by incorporation of radioactive amino acids into the major structural protein (P73), was also unchanged over the period of observation. Thus, the induction of type C virus particle formation in cultured neuroblastoma cells had no detectable effect on the quantity, synthesis rate, or location of intracisternal type A particles.

Animals↗
PubMed1975

Differential sensitivity of low molecular weight DNA polymerase to sulfhydryl-blocking reagents.

Activity of a 2.5 S mouse myeloma DNA polymerase (termed DNA polymerase II) measured with either poly(rA) or poly(dA) as template did not require sulfhydryl-reducing reagents, but was sensitive to inhibition by p-hydroxymercuribenzoate and the sulfhydryl-alkylating reagent, N-ethylmaleimide; however, the activity was much more sensitive to inhibition by p-hydroxymercuribenzoate than by the sulfhydryl-alkylating reagent. The p-hydroxymercuribenzoate inhibition appeared to involve the mercurial portion of the p-hydroxymercuribenzoate molecule because HgCl2 was an equally effective inhibitor, while p-hydroxybenzoate had little effect upon enzyme activity. The p-hydroxymercuribenzoate inhibition was reversed by an equal concentration of the sulfhydryl-reducing reagent, dithiothreitol.

Alkylation↗
PubMed1975

On the DNA polymerase III of mouse myeloma: partial purification and characterization.

A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...

Animals↗
PubMed1974

Multiple forms of DNA polymerase in mouse myeloma.

FIVE DISTINCT FORMS OF DNA POLYMERASE (DEOXYNUCLEOSIDETRIPHOSPHATE: DNA deoxynucleotidyltransferase, EC 2.7.7.7) were separated from extracts of mouse myeloma MOPC-104E using a fractionation procedure based upon sequential ion-exchange column chromatography. The enzymes were characterized according to sedimentation behavior, subcellular localization, chromatographic behavior on hydroxyapatite columns, and reaction properties. The results indicate that myeloma contains two enzymes that appear to correspond to well characterized DNA polymerases found in many other mammalian tissues, a 6S DNA polymerase localized in the cytoplasmic supernatant fraction, and a lower molecular weight (2-3S) DNA polymerase. Also present were a second 6S DNA polymerase localized exclusively in the nuclear fraction and a 6-8S DNA polymerase localized in the cytoplasmic membrane fraction. The enzyme in the cytoplasmic membrane fraction, which accounted for the predominant activity in the myeloma, was active with poly(rA).(dT)(12-18) as template-primer, but not with activated calf thymus DNA. The detection of this distinct 6-8S membrane-bound DNA polymerase is of particular interest.

Animals↗
PubMed1974

Oncornavirus expression in human x mouse hybrid cells segregating mouse chromosomes.

Human x mouse hybrid clones obtained by fusing transformed human (VA2) cells with embryonic mouse brain cells were tested for their ability to spontaneously express type C virus particles. It had been previously shown that these hybrid cells preferentially retained human chromosomes while mouse chromosomes were lost. The culture fluid from one cell line was found to contain type C particle markers in abundance, and typical budding C particles were observed in the cells by electron microscopy. In contrast, no particle markers were detected in the culture fluid from parental cells and several other hybrid cell lines. Subclones of the virus-positive cell line continued to lose mouse chromosomes and were found to vary more than 100-fold in their culture fluid DNA polymerase activity. The hybrid cell viruses, termed HMV1, banded in a sucrose gradient between 1.14 and 1.16 g/ml, possessed viral group-specific antigens, and exhibited B-tropic host range for replication in mouse embryo cells, but did not replicate in human cells when directly applied. The virus did not transform mouse cells but was able to rescue the defective murine sarcoma virus from sarcoma-positive, helper-virus-negative cells. Activity of the DNA polymerase associated with HMV1 was similar to the activity of Rauscher murine leukemia virus (MuLV) DNA polymerase in its preference for poly(rA) over poly(dA) as a template, use of endogenous template, detergent requirement, and inhibition by antiserum directed against MuLV.DNA polymerase. The results suggest that human x mouse hybrid cells segregating mouse chromosomes can spontaneously express endogenous type C viruses and that such hybrid cell lines may be used for the isolation of latent mammalian oncornaviruses and analysis of viral gene regulation.

Animals↗