A novel method for the cloning of chromosomal mutations in a single step: Isolation of two mutant alleles of envZ, an osmoregulatory gene from Escherichia coli

Summary We have developed a simple, rapid and powerful method for the cloning of chromosomal mutations from total cellular DNA in a single step using a plasmid carrying the clined wild-type locus of interest and a convenient selectable marker such as antibiotic resistance. This method relies upon the ability of the cloned wild-type gene to form a heteroduplex with the mutant chromosomal locus. The plasmid from primary transformants can be screened rapidly by size; more than 50% of plasmids of the correct size contained the mutant locus. When this method was used to clone two chromosomal mutations in the envZ gene of Escherichia coli, a locus which encodes a membrane-bound sensory protein involved in the osmoregulation of outer membrane porin biosynthesis, more than 50% of the retransformants from the plasmids selected by size were found to exhibit the mutant phenotype. Preliminary characterization of these mutant alleles is discussed. This novel and powerful method should be generally applicable in any system where the cloned locus is available.This work was presented at the 86th Annual Meeting of the American Society for Microbiology, March 1986, Washingnton, D.C.     

A dominant mutation in Escherichia coli OmpR lies within a domain which is highly conserved in a large family of bacterial regulatory proteins

Summary We have fortuitously created an in-frame insertion mutation in the cloned ompR gene of Escherichia coli in the course of an experiment involving linker insertion mutagenesis. According to the DNA sequence, the mutant protein has an insertion at the 53rd amino acid residue, which replaced the original valine, with the sequence Ala-Leu-Glu. The expression level of the mutant protein, OmpRX6, in a minicell system, is similar to that of the wild-type protein and the size of the mutant is slightly larger than the wild type by approxiately 300 daltons. This mutant was completely unable to activate porin expression as the wildtype does, and in addition, this phenotype was shown to be dominant over the wild type. Comparison of the amino acid sequence of OmpRX6 with those of a family of homologous bacterial regulatory proteins revealed that the mutation lies in a domain which is highly conserved among these proteins.     

Contributions of insects and droughts to growth decline of trembling aspen mixed boreal forest of western Canada

Insects, diseases, fire and drought and other disturbances associated with global climate change contribute to forest decline and mortality in many parts of the world. Forest decline and mortality related to drought or insect outbreaks have been observed in North American aspen forests. However, little research has been done to partition and estimate their relative contributions to growth declines. In this study, we combined tree‐ring width and basal area increment series from 40 trembling aspen (Populus tremuloides Michx.) sites along a latitudinal gradient (from 52° to 58°N) in western Canada and attempted to investigate the effect of drought and insect outbreaks on growth decline, and simultaneously partition and quantify their relative contributions. Results indicated that the influence of drought on forest decline was stronger than insect outbreaks, although both had significant effects. Furthermore, the influence of drought and insect outbreaks showed spatiotemporal variability. In addition, our data suggest that insect outbreaks could be triggered by warmer early spring temperature instead of drought, implicating that potentially increased insect outbreaks are expected with continued warming springs, which may further exacerbate growth decline and death in North America aspen mixed forests.     

Bivalve aquaculture‐environment interactions in the context of climate change

Coastal embayments are at risk of impacts by climate change drivers such as ocean warming, sea level rise and alteration in precipitation regimes. The response of the ecosystem to these drivers is highly dependent on their magnitude of change, but also on physical characteristics such as bay morphology and river discharge, which play key roles in water residence time and hence estuarine functioning. These considerations are especially relevant for bivalve aquaculture sites, where the cultured biomass can alter ecosystem dynamics. The combination of climate change, physical and aquaculture drivers can result in synergistic/antagonistic and nonlinear processes. A spatially explicit model was constructed to explore effects of the physical environment (bay geomorphic type, freshwater inputs), climate change drivers (sea level, temperature, precipitation) and aquaculture (bivalve species, stock) on ecosystem functioning. A factorial design led to 336 scenarios (48 hydrodynamic × 7 management). Model outcomes suggest that the physical environment controls estuarine functioning given its influence on primary productivity (bottom‐up control dominated by riverine nutrients) and horizontal advection with the open ocean (dominated by bay geomorphic type). The intensity of bivalve aquaculture ultimately determines the bivalve–phytoplankton trophic interaction, which can range from a bottom‐up control triggered by ammonia excretion to a top‐down control via feeding. Results also suggest that temperature is the strongest climate change driver due to its influence on the metabolism of poikilothermic organisms (e.g. zooplankton and bivalves), which ultimately causes a concomitant increase of top‐down pressure on phytoplankton. Given the different thermal tolerance of cultured species, temperature is also critical to sort winners from losers, benefiting Crassostrea virginica over Mytilus edulis under the specific conditions tested in this numerical exercise. In general, it is predicted that bays with large rivers and high exchange with the open ocean will be more resilient under climate change when bivalve aquaculture is present.     

Population density of the Réunion Grey White-eye Zosterops borbonicus within the summit ecosystems of Réunion,Mascarene Islands

Assessing population density is crucial for studying the ecology and evolutionary biology of species as well as for conservation purposes. Here we used point count methods to infer population density in a single-island endemic passerine bird, the Réunion Grey White-eye Zosterops borbonicus, that displays striking evidence of differentiation at a small spatial scale. Population density was estimated at 5.17 birds ha^?1 (CL 4.85–5.50), a value somewhat higher than previously believed. This estimation provides the first detailed estimation of bird population density in the vulnerable summit ecosystems of Réunion and will possibly allow a better understanding of the evolutionary causes of this plumage colour variation.     

Comparative effects of commercial Aroclors on rat liver enzyme activities

Hepatic DNA, RNA, protein and p-nitroanisole O-demethylase, aniline hydroxylase, nonspecific carboxylesterase, bromosulphophthalein-glutathione (BSP-GSH) conjugating enzyme and p-nitrophenol UDPglucuronyl transferase activities were measured in young Wistar male rats which had received intraperitoneal injections (50 mg/kg) of biphenyl and Aroclors 1016, 1221, 1232, 1242, 1248, 1254 and 1260, dissolved in peanut oil, for 3 consecutive days and assayed 96 h after the last injection. Biphenyl and all the Aroclors caused the same degree of enhancement of BSP-GSH conjugating enzyme. Decreased DNA content, increased RNA and protein content and the other enzymatic activities were related to the percent weight of chlorine and the chlorobiphenyl composition of the Aroclors. More marked effects were observed with the highly chlorinated Aroclor 1248, 1254, and 1260 mixtures which contained predominantly tetra-, penta-, hexa-, and higher-chlorinated biphenyls.     

Plasma neurotensin release and gastric emptying in the dumping syndrome

Seventy-three patients were studied after ingesting a liquid glucose meal, tagged with ¹¹³Indium. Nineteen of these patients were awaiting surgery for their duodenal ulcer, while 54 were studied postoperatively, 25 of whom experienced troublesome postprandial (dumping) symptoms in their daily lives. The radioactive marker emptied significantly faster in the symptomatic patients than in the symptomfree, pre and post-operative groups (initial emptying rate 3.45 ± 0.23, compared with 1.16 ± 0.19 and 1.27 ± 0.15% fall in counts/min respectively; p < 0.01). Initial (20 min) rises in the plasma concentrations of neurotensin-like immunoreactivity measured during the test correlated significantly with the rate of gastric emptying in all patients, being greatest in patients with dumping symptoms. Physiological concentrations of neurotensin have been shown to delay gastric emptying. The excessive rise in plasma neurotensin-like immunoreactivity in patients with dumping symptoms, presumably occuring as a result of the rapid passage of nutrients to the neurotensin-rich ileum, may possibly have a compensatory role in slowing further emptying from the stomach.