Colcemid-resistant mutants of fission yeast have an altered cell cycle

Cell-cycle progression is altered in some colcemid-resistant mutants of fission yeast. The duration of particular stages of the cell cycle is different but total doubling time is unchanged from that of wild type. Cell-plate formation is prolonged relative to wild type but concomitant DNA synthesis is not affected. Separation of daughter cells is inhibited in some strains, giving rise to unusual cell morphologies. Control of cell division is altered in two ways: Critical size for division is increased. The probability of division a function of size is decreased.     

Citrate synthase mutants of Sinorhizobium meliloti are ineffective and have altered cell surface polysaccharides

The gltA gene, encoding Sinorhizobium meliloti 104A14 citrate synthase, was isolated by complementing an Escherichia coli gltA mutant. The S. meliloti gltA gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-type gltA with the gltA::kan allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different from the green fluorescence found with 104A14. High concentrations of arabinose (0.4%) or succinate (0. 2%) restored the green fluorescence to CSDX1. High-performance liquid chromatography analyses showed that CSDX1 produced partially succinylated succinoglycan. CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen. The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle.     

Phosphatase activity and phosphorus partitioning in nodules of developing mungbean

The acid and alkaline phosphatase activities were determined in bacteroid free fraction of nodules during development, using different phosphorylated substrates. Both enzymes change their substrate specificities with nodule development. Alkaline phosphatase, 20 days after sowing (DAS), showed negligible activity with ATP while at later stages maximum activity with ATP was observed. Invariably fructose 1,6 bisphosphate was a better substrate compared to fructose-6-phosphate and glucose-6-phosphate. Using Sephadex G-150 column chromatography, only one peak of acid phosphatase around Ve/Vo of 2.2 to 2.3 was observed at 20 and 30 DAS stages while at 40 DAS stage an additional ATP specific peak at around Ve/Vo of 2.9 was also observed. There was only one alkaline phosphatase peak at 20 and 30 DAS. However, at 40 DAS additional ATP specific peaks of phosphatases were observed at Ve/Vo of 1.4 and 2.6. Alkaline phosphatase could not be detected in the bacteroids whereas activity of acid phosphatase was about 5–7 % of that observed in the bacteroid free preparation. A low activity of both acid and alkaline phytases was observed at all stages of nodule development. However, phytic acid could not be detected. Increase in phosphorus content of water soluble organic phosphate at late stage of nodule development appears to be related with low level of phosphatase activity.     

Western sandpipers have altered migration tactics as peregrine falcon populations have recovered

The presence of top predators can affect prey behaviour, morphology and life history, and thereby can produce indirect population consequences greater and further reaching than direct depredation would have alone. Raptor species in the Americas are recovering since restrictions on the use of dichlorodiphenyltrichloroethane (DDT) and the implementation of conservation measures, in effect constituting a hemisphere-wide predator-reintroduction experiment, and profound effects on populations of their prey are to be expected. Here, we document changes in the behaviour of western sandpipers (Calidris mauri) at migratory stopover sites over two decades. Since 1985, migratory body mass and stopover durations of western sandpipers have fallen steadily at some stopovers in the Strait of Georgia, British Columbia. Comparisons between years, sites and seasons strongly implicate increasing danger from the recovery of peregrine falcons (Falco peregrinus) as a causal factor. A decade-long ongoing steep decline in sandpiper numbers censused on our study site is explained entirely by the shortening stopover duration, rather than fewer individuals using the site. Such behavioural changes are probably general among migratory shorebird species, and may be contributing to the widespread census declines reported in North America.     

Phenotypes of aquaporin mutants in genetically altered mice

The ability to move water across lipid membranes is crucial for nutrient intake, energy generation, waste excretion, and a myriad of other functions associated with life. Aquaporins, a family of integral membrane proteins, are now recognized as the channels responsible for transporting hydrophilic molecules, including water, across relatively impervious, hydrophobic cell membranes. A tremendous amount of work has been published on characterizing these proteins, which have been found in all bacteria, yeast, plants, and animals examined to date. In addition, an increasing number of mouse models with genetically altered aquaporin expression are being reported. This article will briefly review the basic biochemistry of aquaporins and then evaluate the use (and misuse) of mice in the quest for understanding the comparative pathophysiology of aquaporins in humans.     

Rhizobium meliloti mutants altered in ammonium utilization.

Derivatives of Rhizobium meliloti 2011 required trace amounts of glutamate to use ammonium as the nitrogen source for growth, although they could use serine as the sole nitrogen source. Specific activities of ammonium assimilatory enzymes were similar to those in strain Rm2011. The mutants were deficient in nitrogen fixation.     

Yeast killer mutants with altered double-stranded ribonucleic acid

Killer strains of Saccharomyces cerevisiae contain two species of double-stranded ribonucleic acid (dsRNA) with molecular weights estimated at 2.5 x 10(6) (L) and 1.4 x 10(6) (M). The M component appears to have a high adenine content. All mutants of killer which are defective for both the toxin and immunity functions lack the M dsRNA. One of these mutants has a novel dsRNA with a molecular weight of 5 x 10(5). Another class of killer mutants contains strains which are defective for either the toxin or the immunity function. They include temperature-sensitive killers, superkillers, and immunity-minus strains. The dsRNA profile of temperature-sensitive killers resembles that of the standard killer. The superkiller has 2.5 times more of the M dsRNA (1.4 x 10(6) daltons) than does the standard killer. Immunity-minus killers have, in addition to the two dsRNAs species of standard killer, a novel dsRNA with a molecular weight of 2.5 x 10(5). The data are consistent with the hypothesis that the M RNA controls toxin production. In addition, the two RNAs, L and M, seem to be regulated together. When the M RNA is missing, the amount of L is either greatly elevated or greatly reduced.     

Phe393 mutants of cytochrome P450 BM3 with modified heme redox potentials have altered heme vinyl and propionate conformations

It has been well established that the heme redox potential is affected by many different factors. Among others, it is sensitive to the proximal heme ligand and the conformation of the propionate and vinyl groups. In the cytochrome P450 BM3 heme domain, substitution of the highly conserved phenylalanine 393 results in a dramatic change in the heme redox potential [Ost, T. W. B., Miles, C. S., Munro, A. W., Murdoch, J., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 13421-13429]. We have used resonance Raman spectroscopy to characterize heme structural changes and modification of heme interactions with the protein matrix that are induced by the F393 substitutions and to determine their correlation with the heme redox potential. Our results show that the Fe-S stretching frequency of the 5-coordinated, high-spin ferric heme is not affected by the mutations, suggesting that the electron density in the Fe-S bond in this state is not affected by the F393 mutation and is not a good indicator of the heme redox potential. Substrate binding perturbs the hydrogen bonding between one propionate group and the protein matrix and correlates to both the size of residue 393 and the heme redox potential. However, heme reduction does not affect the conformation of the propionate groups. Although the conformation of the vinyl groups is not affected much by substrate binding, their conformation changes from mainly out-of-plane to predominantly in-plane upon heme reduction. The extent of these conformational changes correlates strongly with the size of the 393 residue and the heme redox potential, suggesting that steric interaction between this residue and the vinyl groups may be of importance in regulating the heme redox potential in the P450 BM3 heme domain. Further implications of our findings for the change in redox potential upon mutation of F393 will be discussed.     

Source-sink relations in maize mutants with starch-deficient endosperms

Partitioning and translocation of photosynthates were compared between a nonmutant genotype (Oh 43) of corn (Zea mays L.) and two starch-deficient endosperm mutants, shruken-2 (sh2) and brittle-1 (bt1), with similar genetic backgrounds. Steady-state levels of ¹⁴CO₂ were supplied to source leaf blades for 2-hour periods, followed by separation and identification of ¹⁴C-assimilates in the leaf, kernel, and along the translocation path. An average of 14.1% of the total ¹⁴C assimilated was translocated to normal kernels, versus 0.9% in sh2 kernels and 2.6% in btl kernels. Over 98% of the kernel ¹⁴C was in free sugars, and further analysis of nonmutant kernels showed 46% of this label in glucose and fructose. Source leaves of mutant plants exported significantly less total photosynthate (24.0% and 36.3% in sh2 and bt1 compared to 48.0% in the normal plants) and accumulated greater portions of label in the insoluble (starch) fraction. Mutant plants also showed lower percentages of photosynthate in the leaf blade and sheath below the exposed blade area. The starch-deficient endosperm mutants influence the partitioning and translocation of photosynthates and provide a valuable tool for the study of source-sink relations.     

Self-compatible B mutants in coprinus with altered pheromone-receptor specificities

A successful mating in the mushroom Coprinus cinereus brings together a compatible complement of pheromones and G-protein-coupled receptors encoded by multiallelic genes at the B mating-type locus. Rare B gene mutations lead to constitutive activation of B-regulated development without the need for mating. Here we characterize a mutation that arose in the B6 locus and show that it generates a mutant receptor with a single amino acid substitution (R96H) at the intracellular end of transmembrane domain III. Using a heterologous yeast assay and synthetic pheromones we show that the mutation does not make the receptor constitutively active but permits it to respond inappropriately to a normally incompatible pheromone encoded within the same B6 locus. Parallel experiments carried out in Coprinus showed that a F67W substitution in this same pheromone enabled it to activate the normally incompatible wild-type receptor. Together, our experiments show that a single amino acid replacement in either pheromone or receptor can deregulate the specificity of ligand-receptor recognition and confer a self-compatible B phenotype. In addition, we use the yeast assay to demonstrate that different receptors and pheromones found at a single B locus belong to discrete subfamilies within which receptor activation cannot normally occur.     

Escherichia coli formate dehydrogenase mutants with altered selenopolymer profiles

Four classes of Escherichia coli mutants deficient in either or both of their anaerobic selenium-containing formate dehydrogenases (FDH) were isolated. A class I mutant devoid of FDH_H activity specifically linked to benzyl viologen (BV) produced a small amount of the FDH_H 80,000 dalton selenopeptide. Three class II mutants were deficient in FDH_N activity specifically linked to phenazine methosulfate (PMS) and exhibited a selenopeptide doublet rather than the FDH_N 110,000 dalton selenosubunit. Three class III mutants were selenium incorporation deficient and did not exhibit either FDH activity or ⁷⁵Selabeled selenopolymers. A class IV mutant was devoid of PMS-linked FDH_N activity; neither its FDH_N 110,000 dalton selenosubunit nor its BV-linked FDH_H activity was fully regulated by nitrate.Abbreviations FDH formate dehydrogenase - BV benzyl viologen - MV methyl viologen - PMS phenazine methosulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Levallorphan-tolerant mutants of Excherichia coli with altered morphologies.

The penetration of levallorphan, a synthetic morphinan known to interact with the cytoplasmic membrane of E. coli, is seriously limited by the outer membrane. To select target-resistant mutants rather than outer membrane mutants, a two-step procedure was developed, which involved the selection by penicillin of an "intermediate" parental strain with a decreased penetration barrier and a subsequent positive selection of levallorphan-tolerant pseudo-revertant clones. Unlike the direct selection, this technique yielded various types of mutants in which the morphology, the septation ability, and the growth rate were greatly affected.     

New Salmonella typhimurium mutants with altered outer membrane permeability

We describe three new classes of Salmonella typhimurium mutants with increased sensitivity to hydrophobic agents. In contrast to many previously described mutants, the phage sensitivity pattern of these mutants did not give any indication of defective lipopolysaccharide. Furthermore, they had no detectable changes in their phospholipid or outer membrane protein composition, and their growth rate and cell morphology were normal. Class B mutants were nearly as sensitive to novobiocin, fusidic acid, erythromycin, rifampin, and clindamycin as are deep rough (heptoseless) mutants; in addition they were sensitive to methicillin, penicillin (to which heptoseless mutants are resistant), gentian violet, and anionic and cationic detergents. Class A and C mutants had less sensitive, but characteristic phenotypes. None of the three classes were sensitive to serum bactericidal action. The class B mutation mapped between map positions 7 and 11 on the S. typhimurium chromosome, and the class C mutation mapped between positions 5 and 7. The map position for the class A mutation remained undefined, but it was separate from the class B and C mutations and, like those, did not correspond to any gene loci known to participate in the synthesis of major outer membrane constituents.     

Isolation of Vibrio cholerae mutants with altered toxin production

V. cholerae multiple-labeled mutants 569B with altered toxin production have been obtained by the method of induced mutagenesis with the use of nitrosoguonidine. These mutants can be used for the genetic mapping of tox genes on the chromosome of V. cholerae.