Replisome pausing in mutagenesis

E. coli cells containing a temperature-sensitivednaE mutation, in the α-subunit of holoenzyme DNA polymerase III, do not survive at the restrictive temperature. Such cells may survive in the presence of thepcbA1 mutation, an allele of thegyrB gene. Such survival is dependent on an active DNA polymerase I. Evidence indicates that DNA polymerase I interacts directly in the replisome (REP·A). Despite normal survival for cells using thepcbA replication pathway after some type of DNA damage, we have noted a failure of damage-induced mutagenesis. Here we present evidence supporting a model of replisome pausing in cells dependent upon thepcbA replication pathway. The model argues that the (REP·A) complex pauses longer at the site of the lesion, allowing excision repair to occur completely. In the normal replication pathway (REP·E) bypass of the lesion occurs, fixing the mutation.     

Kinetics of deactivation for thermostable DNA polymerase enzymes

Summary The kinetics of thermal deactivation for thermostable DNA polymerase enzymes were investigated by using the experimental data published elsewhere (Nielson et al. 1996. Strategies. 9, 7–8). The order of deactivation (a) and the deactivation rate constants (k _d) were determined for different Taq DNA polymerase enzymes and were found to be of first order.     

The natural history, thermal physiology, and ecological impacts of intertidal mesopredators, Oedoparena spp. (Diptera: Dryomyzidae)

Abstract. The ecological roles of small (1–1000 mg) predators in benthic marine systems are poorly understood. We investigated the natural history and predatory impact of one group of such mesopredators—larvae of dipteran flies in the genus Oedoparena —which prey on intertidal barnacles. We 1) quantified patterns of larval Oedoparena distribution and abundance in the Northwest Straits of Washington State, USA, 2) determined larval physiological tolerance limits in the laboratory, and 3) conducted a manipulative field experiment to assess the role of microhabitat temperature on predation rates in Oedoparena . Members of Oedoparena in Washington are univoltine, with peak larval abundance in late spring and early summer. Infestation frequencies in the barnacles Balanus glandula and Chthamalus dalli were as high as 22% and 35%, respectively. In laboratory studies, larvae of O . glauca were able to tolerate temperatures up to 37°C; however, this temperature is often exceeded in high intertidal habitats. In a field manipulation using experimental shades, we demonstrate that the alleviation of physiological stress greatly increased the abundance of larvae of Oedoparena spp. As a result of increased larval densities under shades, adult B. glandula mortality increased from 5% to nearly 30%, and C. dalli mortality increased from less than 20% to over 60%. Because high intertidal barnacles serve as food and habitat for a diverse array of species, Oedoparena spp. have the potential to play a major role in structuring high intertidal communities, particularly in cooler microhabitats.     

Phylogenetic comparative analysis of RNA structure on Macintosh computers

A Macintosh Hypertalk program (Hypercard ‘stack’)for use in phylogenetic comparative analysis of RNA structureis described. The program identifies covariations and compensatorychanges in RNA sequence alignments, for use in the constructionof secondary structure models or the identification of tertiaryinteractions. The results of an analysis are presented eitheras a list of positions in the alignment which covary, or asa 2-dimensional matrix in which potential helices in the secondarystructure appear as diagonal patterns. Received on January 7, 1991; accepted on March 19, 1991     

Activation of AMPK by Bitter Melon Triterpenoids Involves CaMKKβ

We recently showed that bitter melon-derived triterpenoids (BMTs) activate AMPK and increase GLUT4 translocation to the plasma membrane in vitro, and improve glucose disposal in insulin resistant models in vivo. Here we interrogated the mechanism by which these novel compounds activate AMPK, a leading anti-diabetic drug target. BMTs did not activate AMPK directly in an allosteric manner as AMP or the Abbott compound (A-769662) does, nor did they activate AMPK by inhibiting cellular respiration like many commonly used anti-diabetic medications. BMTs increased AMPK activity in both L6 myotubes and LKB1-deficient HeLa cells by 20–35%. Incubation with the CaMKKβ inhibitor, STO-609, completely attenuated this effect suggesting a key role for CaMKKβ in this activation. Incubation of L6 myotubes with the calcium chelator EGTA-AM did not alter this activation suggesting that the BMT-dependent activation was Ca²⁺-independent. We therefore propose that CaMKKβ is a key upstream kinase for BMT-induced activation of AMPK.