Biomedical subjects

L P Goscin

Publications and source records attributed to L P Goscin.

3 recordsLinked to original sources

PubMed1982

Isolation of the catalytic core of DNA polymerase alpha from rabbit bone marrow.

Modification of the purification procedures for rabbit bone marrow DNA polymerase [Byrnes, J.J., & Black, V.L. (1978) Biochemistry 17, 4226-4231] has increased the yield and stability of the enzyme thus allowing further purification. In particular, the higher molecular weight form, alpha 1, has been more abundant. Additional purification has been obtained upon phosphocellulose and chromatofocusing column chromatography. SDS slab gel electrophoretic analyses of the eluates demonstrate a 135,000 molecular weight polypeptide in nearly pure form which correlates with DNA polymerase activity. Approximately 200,000 nmol of thymidine monophosphate is incorporated into DNA (mg of protein) -1h -1 at 37 degrees C. Similar to DNA polymerase alpha from other sources this enzyme is an acidic protein, is very sensitive to aphidicolin, and has no detectable 3' to 5' nuculease activity.

Animals↗

PubMed1982

DNA polymerase delta: one polypeptide, two activities.

DNA polymerase delta from rabbit bone marrow has an associated 3'-5'-exonuclease. Previous studies demonstrated a Stokes radius of 45.5 A by gel filtration and a sedimentation coefficient of 6.5 S by zone sedimentation. Thus, a molecular weight of 122000 and a frictional coefficient of 1.39 were calculated [Byrnes, J. J., & Black, V. L. (1978) Biochemistry 17, 4226-4231]. Several problems obstructed further purification and definition of DNA polymerase delta. The small amount of protein obtained limited further purification as the nonspecific loss of enzyme in subsequent procedures was excessive. Furthermore, the amount of protein recovered was insufficient for conventional analysis. These difficulties have been overcome, and DNA polymerase delta has been purified to apparent homogeneity. Under conditions of nondenaturing microgel electrophoresis, DNA polymerase b aggregates to molecular weight species of 300000 and higher. In situ assays for DNA polymerase and exonuclease in these gels generate concordant activity profiles. Upon sodium dodecyl sulfate gel electrophoresis, delta is a single polypeptide of 122000 apparent molecular weight. The DNA polymerase incorporates between 250000 and 300000 nmol of thymidine deoxyribonucleoside monophosphate (dTMP) into poly(dA)/oligo(dT) (mg of protein)-1 h-2 at 37 degrees C; the exonuclease simultaneously hydrolyzes 13% of the newly synthesized DNA. Aphidicolin, considered to be a specific inhibitor of DNA polymerase alpha, inhibits both the DNA polymerase and 3'-5'-exonuclease activities of delta. DNA polymerase alpha from rabbit bone marrow does not share a common subunit with delta. Therefore, aphidicolin binding is not specific for alpha, and conclusions based upon the supposition that it is must be reconsidered.

Animals↗

PubMed1982

Enhancement of pneumococcal transfection by protamine sulfate.

Protamine sulfate enhanced transfection of Streptococcus pneumoniae by DNA of omega 3 phage by factors as large as 10(5)-fold, provided it was present at the time the cells were added to the DNA. For DNA concentrations well below 1 microgram/ml, the optimum amount of protamine sulfate was near 1 microgram/ml of cells. Higher DNA concentrations required more protamine for maximum effect, and in all cases transfection fell when protamine was in excess. Transformation was not enhanced by low protamine levels and was inhibited by higher levels. A recipient strain with low but finite endonuclease activity and normal transformability showed higher transfection than did the wild type at low DNA concentrations but less than did the wild type at high DNA concentrations. Protamine sulfate enhanced its transfection at low, but not high, DNA concentrations. The behavior of this strain and the enhancement of transfection by protamine sulfate of wild-type cells were each consistent with less cutting of the donor DNA at the cell surface, which is part of the normal entry process in naturally competent gram-positive bacteria. Less cutting would lead to entry of fewer but longer strands that would be more efficient in reconstruction of the 33-megadalton phage replicon. We suggest that in this system protamine enhances transfection by inhibition of the surface nuclease action that is part of the normal entry process.

Bacteriophages↗