Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase.

We have labeled the primer binding domain of HIV1-RT with 5'-32P-labeled (dT)15 primer using ultraviolet light energy. The specificity of the primer cross-linking to HIV1-RT was demonstrated by competition experiments. Both synthetic and natural primers, e.g., p(dA)15, p(dC)15, and tRNA(Lys), inhibit p(dT)15 binding and cross-linking to the enzyme. The observed binding and cross-linking of the primer to the enzyme were further shown to be functionally significant by the observation that tRNA(Lys) inhibits the polymerase activity on poly(rA).(dT)15 template-primer as well as the cross-linking of p(dT)15 to the enzyme to a similar extent. At an enzyme to p(dT)15 ratio of 1:3, about 15% of the enzyme can be cross-linked to the primer. To identify the domain cross-linked to (dT)15, tryptic peptides were generated and purified by a combination of HPLC on a C-18 reverse-phase column and DEAE-Sephadex chromatography. A single peptide cross-linked to p(dT)15 was identified. This peptide corresponded to amino acid residues 288-307 in the primary sequence of HIV1-RT as judged by amino acid composition and sequence analyses. Further, Leu(289)-Thr(290) and Leu(295)-Thr(296) of HIV1-RT appear to be the probable sites of cross-linking to the primer p(dT)15.

Chromosome aberrations and dominant lethals in Culex fatigans due to mercuric chloride.

The mosquito, Culex fatigans, was used for determining the possible mutagenic potential of mercuric chloride, using chromosome aberrations and frequency of dominant lethals as the parameters. As many as 27.75 +/- 0.85% aberrations against 2.75 +/- 0.35% (P less than 0.001) in the controls were observed in the chromosomes of the animals treated with 0.001 microgram/ml of mercuric chloride. Similarly the frequency of dominant lethals was statistically significant in the treated lot. The results indicate that this compound is genotoxic.