HIV-1 RT is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA(Lys,3) to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating RT are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-1 RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.
The collagen prolyl 4-hydroxylases (P4Hs, EC ) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are alpha(2)beta(2) tetramers, in which protein disulfide isomerase (PDI) serves as the beta subunit. Two conserved alpha subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single beta subunit PDI-2, both in a recombinant expression system and in vivo, namely a PHY-1/PHY-2/(PDI-2)(2) mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans but phy-2-/- and phy-1-/- (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln(121)-Ala(271) and Asp(1)-Leu(122) in PHY-1 and PHY-2, respectively, are critical for its assembly.
Five grams of seafood products were inoculated with one to 500 viable or 10(9) heat-killed cells of Listeria monocytogenes. The presence of the pathogen was detected by the polymerase chain reaction (PCR) with primers specific for fragments of the listeriolysin O (hly) gene (two sets) and for the invasion-associated protein (iap) gene (one set). For DNA preparation, boiling, either alone or in combination with lysozyme and proteinase K treatment, was not always sufficient to lyse L. monocytogenes, while treatment with Triton X-100 produced consistently good DNA suitable for amplification. To avoid false-negative and false-positive results, 48 h incubations were necessary and a subculturing step after an initial 24 h incubation greatly improved the results. The primers that amplified regions of the listeriolysin O gene gave clearer and stronger products than primers for the invasion-associated protein gene. Using this method we were able to detect one to five L. monocytogenes cells in 5 g of product in a total of 55 h.
Protein-disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b', and a'. It is a ubiquitous protein folding catalyst that in addition functions as the beta-subunit in vertebrate collagen prolyl 4-hydroxylase (C-P4H) alpha(2)beta(2) tetramers. We report here that point mutations in the primary peptide substrate binding site in the b' domain of PDI did not inhibit C-P4H assembly. Based on sequence conservation, additional putative binding sites were identified in the a and a' domains. Mutations in these sites significantly reduced C-P4H tetramer assembly, with the a domain mutations generally having the greater effect. When the a or a' domain mutations were combined with the b' domain mutation I272W tetramer assembly was further reduced, and more than 95% of the assembly was abolished when mutations in the three domains were combined. The data indicate that binding sites in three PDI domains, a, b', and a', contribute to efficient C-P4H tetramer assembly. The relative contributions of these sites were found to differ between Caenorhabditis elegans C-P4H alphabeta dimer and human alpha(2)beta(2) tetramer formation.