Specificity of SOS mutagenesis in native M13lacI phage.

Base substitutions account for 90% of all forward mutations sequenced in unmodified M13lacI DNA grown in both UV-irradiated and nonirradiated hosts. The principal effect of SOS induction was an increase in the contribution of transversions, in particular A.T----T.A events.     

A mutant of Escherichia coli K12 deficient in the 5''-3'' exonucleolytic activity of DNA polymerase I. II. Purification and properties of the mutant enzyme

Summary The enzymatic properties of purified DNA polymerase I from a strain of Escherichia coli K12 with a mutation in the polA gene have been studied. The polymerizing activity of the mutant enzyme is similar to that of the enzyme from isogenic wild-type cells, when the activity is measured on exonuclease III treated calf-thymus DNA. Also the 3–5 exonucleolytic activity is not significantly different for both enzyme preparations. The 5–3 exonucleolytic activity of DNA polymerase I isolated from the mutant strain, however, is much lower than that of wild-type DNA polymerase I. The products formed by the action of the wild-type and the mutant enzyme on nicked circular double-stranded DNA of phage X174 (RFII DNA) were analysed by sucrose-gradient sedimentation and electron-microscopy. When RFII DNA was incubated with wild-type enzyme 80% of the molecules were converted into linear molecules. All linear molecules were shorter than one phage genome. Only 25% of the molecules were branched. After incubation of RFII DNA with the mutant enzyme 62% of the molecules have become linear. More than 90% of these linear molecules were branched and the majority of them was longer than one phage genome.     

Mechanism of mutation by thymine starvation in Escherichia coli: clues from mutagenic specificity.

To probe the mechanisms of mutagenesis induced by thymine starvation, we examined the mutational specificity of this treatment in strains of Escherichia coli that are wild type (Ung+) or deficient in uracil-DNA-glycosylase (Ung-). An analysis of Ung+ his-4 (ochre) revertants revealed that the majority of induced DNA base substitution events were A:T----G:C transitions. However, characterization of lacI nonsense mutations induced by thymine starvation demonstrated that G:C----A:T transitions and all four possible transversions also occurred. In addition, thymineless episodes led to reversion of the trpE9777 frameshift allele. Although the defect in uracil-DNA-glycosylase did not appear to affect the frequency of total mutations induced in lacI by thymine deprivation, the frequency of nonsense mutations was reduced by 30%, and the spectrum of nonsense mutations was altered. Furthermore, the reversion of trpE9777 was decreased by 90% in the Ung- strain. These findings demonstrate that in E. coli, thymine starvation can induce frameshift mutations and all types of base substitutions. The analysis of mutational specificity indicates that more than a single mechanism is involved in the induction of mutation by thymine depletion. We suggest that deoxyribonucleoside triphosphate pool imbalances, the removal of uracil incorporated into DNA during thymine starvation, and the induction of recA-dependent DNA repair functions all may play a role in thymineless mutagenesis.     

rorA mutation of Escherichia coli K-12 affects the recB subunit of exonuclease V.

The ATP-dependent nuclease, exonuclease V, of Escherichia coli plays an important role in repair and recombination. The enzyme is composed of two subunits, one of which is the product of the recB and recC genes. In this communication it is shown by mapping and complementation experiments that the rorA mutation, which results in radiation sensitivity but not the loss of recombination ability, is an allele of the recB gene.     

Pretreatment tumor prostaglandin E2 concentration and cyclooxygenase-2 expression are not associated with the response of canine naturally occurring invasive urinary bladder cancer to cyclooxygenase inhibitor therapy

The purpose of this study was to determine the extent to which pretreatment prostaglandin E2 (PGE2) concentration and cyclooxygenase-2 (cox-2) expression could be used to predict the antitumor activity of cox inhibitor treatment in naturally occurring canine transitional cell carcinoma of the urinary bladder (TCC). Snap frozen tissues (to measure PGE2) and formalin-fixed TCC samples (for cox-2 immunohistochemistry) were obtained by cystoscopy or surgery. Complete tumor staging was performed before and after one month of treatment with the cox inhibitor, piroxicam (0.3 mg/kg q24 h po). The pretreatment PGE2 concentration ranged from 57 to 1624 ng/g of TCC tissue; n=18 dogs). Cox-2 immunoreactivity was observed in all TCC samples. There was no association between PGE2 concentration, cox-2 expression, and change in tumor volume with piroxicam treatment. In conclusion, cox-2 expression or PGE2 concentration alone, or the combination of the two was not useful in predicting response to piroxicam treatment in canine TCC.     

Hydraulic properties of layered soils influence survival of Rhodes grass (<Emphasis Type="Italic">Chloris gayana</Emphasis> Kunth.) during water stress

Survival of vegetation on soil-capped mining wastes is often impaired during dry seasons due to the limited amount of water stored in the shallow soil capping. Growth and survival of Rhodes grass (Chloris gayana) during soil drying on various layered capping sequences constructed of combinations of topsoil, subsoil, seawater-neutralised residue sand and low grade bauxite was determined in a glasshouse. The aim was to describe the survival of Rhodes grass in terms of plant and soil water relationships. The soil water characteristic curve and soil texture analysis was a good predictor of plant survival. The combination of soil with a high water holding capacity and low soil water diffusivity (e.g. subsoil with high clay contents) with soil having a high water holding capacity and high diffusivity (e.g. residue sand) gave best survival during drying down (up to 88 days without water), whereas topsoil and low grade bauxite were unsuitable (plants died within 18–39 days). Clayey soil improved plant survival by triggering a water stress response during peak evaporative water demand once residue sand dried down and its diffusivity fell below a critical range. Thus, for revegetation in seasonally dry climates, soil capping should combine one soil with low diffusivity and one or more soils with high total water holding capacity and high diffusivity.     

A window of opportunity: timing protein degradation by trimming of sugars and ubiquitins

Of the many post-translational modifications of proteins, ubiquitination and N-glycosylation stand out because they are polymeric additions. In contrast to single-unit modifications, the fate of the modified protein is determined by the dynamic equilibrium of polymerization versus depolymerization, rather than by the initial addition itself. Notably, it is the trimming of sugar chains and elongation of polyubiquitin that target the protein to degradation. Recent research suggests that, for each process, special receptors recognize chains that reach an appropriate length and commit the conjugated substrate for proteasomal disposal. We propose that the 'magic numbers' are loss of at least three mannose residues from the initial chain, or extension to at least four ubiquitins. Although these processes are compartmentalized to either side of the endoplasmic reticulum (ER) membrane, some proteins are sequentially subjected to both because they transverse this membrane for ER-associated degradation.     

Siblicide in the spotted hyena: analysis with ultrasonic examination of wild and captive individuals

Integrated field and laboratory studies of long-lived, large-bodiedmammals are rare but offer unique opportunities to elucidatethe behavioral ecology of these animals. Here, we used thisapproach to examine whether siblicide in spotted hyenas (Crocutacrocuta) is obligate or facultative. First, we tested predictionsof obligate and facultative hypotheses by using ultrasonographyto compare litter size before and after parturition and identifypotential causes of litter reduction. Second, we compared littersize and composition between wild and captive hyenas to assessvariation in offspring sex ratios. Third, we used demographicdata to compare survivorship among litters of various sizesand compositions. Fourth, we compared sex ratios within twinlitters born in the wild under conditions of high populationdensity and intense feeding competition with those born whenpopulation density and intensity of feeding competition werereduced. Our data were inconsistent with the obligate siblicidehypothesis. Litter reduction occurred during roughly one-thirdof pregnancies in both wild and captive hyenas, and all suchreductions among captives were due to fetal resorptions or stillbirths.Litter sizes and compositions differed little between wild andcaptive hyenas. However, sex ratios in twin litters varied inthe wild with intensity of feeding competition. In conjunctionwith captive data, long-term study of a wild hyena populationunder varying environmental conditions suggests facultativesiblicide is most likely to occur when feeding competition ismost intense, thus offering an ecological explanation for earlierconflicting reports on siblicide in this species.     

Apolipoprotein B synthesis in rat small intestine: regulation by dietary triglyceride and biliary lipid

Apolipoprotein B (apoB) synthesis rates have been determined, in vivo, in rat enterocytes. Following intralumenal administration of a pulse of [3H]leucine, newly synthesized apoB was quantitated by specific immunoprecipitation and compared to [3H]leucine incorporation into total, trichloroacetic acid-insoluble protein. ApoB synthesis rates were determined after acute administration of either 0.1 or 1 g of triglyceride to fasting animals. No differences were found at any time from 90 min to 6 hr after challenge and values were not different from the basal values established in fasted controls. Animals rechallenged with triglyceride after 8 days' intake of fat-free chow also failed to demonstrate a change in intestinal apoB synthesis rate. By contrast, enterocyte content of apoB appeared to fall, temporarily, with the onset of active triglyceride flux. Groups of animals were then subjected to external bile diversion for 48 hr, a maneuver designed to remove all lumenal sources of lipid. Jejunal apoB synthesis rates fell by 43% (from 0.76% +/- 0.14 to 0.43% +/- 0.12, P less than 0.001), a change that was completely prevented by continuous replacement with 10 mM Na taurocholate. The suppression of jejunal apoB synthesis, induced by prolonged bile diversion, was reversed after 14 hr, but not 8 hr, of intralumenal perfusion with 10 mM Na taurocholate. The addition of micellar fatty acid-monoolein to the perfusate for 4 hr produced no further change in apoB synthesis. Ileal apoB synthesis rates fell by 70% (from 0.61% +/- 0.15 to 0.18% +/- 0.10, P less than 0.001) following 48 hr external bile diversion, a change that was only partially prevented by continuous bile salt replacement. These results suggest that jejunal apoB synthesis demonstrates bile salt dependence but not regulation by acute triglyceride flux. The data further suggest that key aspects of the regulation of apoB synthesis by cellular lipid flux may be mediated independently in jejunal and ileal enterocytes.