Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
DNA polymerase activity assays: new techniques and applications in tumor and viral diseases, notably HIV-infection.
Diss Abstr Int [C]; 53(2):289 1992. Unique Identifier : AIDSLINE ICDB/93686581 Neumuller M; Uppsala Universitet, Sweden
Abstract:
DNA polymerase (DNA-pol) activity was detected in serum samples of patients (pts) with leukemia, vitamin B12 deficiency or cytomegalovirus infection, using a standard assay technique based on detection of [3H]dTMP incorporated into DNA. DNA-pol activity was only detected in serum samples with high levels of thymidine kinase activity, previously shown to appear during severe conditions of several diseases. Biochemical and immunological characterization indicated that the DNA-pol was of alpha-type. The apparent mol wt of the native serum DNA-pol was approx 240 kD. 125I-dUTP was synthesized enzymatically from 125I-dU and purified by liquid chromatography. The 125I-dUTP was found to be a dTTP-analogous substrate for several prokaryotic and eukaryotic DNA-pols, taking advantage of the high sensitivity obtained by use of 125I-dUTP as tracer substrate. The gamma radiation of 125I was advantageous for the development of a reverse transcriptase (RT) activity assay based on the use of template bound to polycarbonate beads or agarose. The immobilized template was also used for combined affinity purification and assay of RT from serum and cell extracts prior to assay. This procedure reduced the disturbing effects of these specimens and greatly increased the substrate turnover and the linearity of the RT assays. The new assay techniques were used in studies of RT-inhibiting antibodies (RTI-Abs) in serum samples from HIV-infected subjects. RTI-Abs could be detected without purification of the IgG from serum prior to assay. The high prevalence (98.8%) of RTI-Ab in sera positive for HIV Ab in Western blot assay was in the same order as the prevalence of HIV gp41 Abs (94.7%). The levels of RTI-Abs in serum samples from HIV-infected subjects in Stage IV were found to be significantly lower than the levels in Stage II and III, which confirmed previous reports that RTI-Abs relate to the stage of disease. The serum levels of RTI-Abs were found to be higher in a group of pts not progressing to AIDS during follow-up than the RTI-Ab levels in a group of pts who did progress. The specificity of the assay was 100% with serum samples from HIV-negative blood donors. (Abstract shortened by UMI)
Keywords: Blotting, Western Cytomegalovirus Infections/*ENZYMOLOGY DNA/METABOLISM DNA Polymerase II/*METABOLISM Human HIV Antibodies/METABOLISM HIV Envelope Protein gp41/METABOLISM HIV Infections/*ENZYMOLOGY Leukemia/*ENZYMOLOGY RNA-Directed DNA Polymerase/METABOLISM Sensitivity and Specificity Thymidine Kinase/METABOLISM Vitamin B 12 Deficiency/ENZYMOLOGY THESIS 930228
M9320859
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Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
