Sequence, regulation, and functions of fis in Salmonella typhimurium.

The fis operon from Salmonella typhimurium has been cloned and sequenced, and the properties of Fis-deficient and Fis-constitutive strains were examined. The overall fis operon organization in S. typhimurium is the same as that in Escherichia coli, with the deduced Fis amino acid sequences being identical between both species. While the open reading frames upstream of fis have diverged slightly, the promoter regions between the two species are also identical between -49 and +94. Fis protein and mRNA levels fluctuated dramatically during the course of growth in batch cultures, peaking at approximately 40,000 dimers per cell in early exponential phase, and were undetectable after growth in stationary phase. fis autoregulation was less effective in S. typhimurium than that in E. coli, which can be correlated with the absence or reduced affinity of several Fis-binding sites in the S. typhimurium fis promoter region. Phenotypes of fis mutants include loss of Hin-mediated DNA inversion, cell filamentation, reduced growth rates in rich medium, and increased lag times when the mutants are subcultured after prolonged growth in stationary phase. On the other hand, cells constitutively expressing Fis exhibited normal logarithmic growth but showed a sharp reduction in survival during stationary phase. During the course of these studies, the sigma 28-dependent promoter within the hin-invertible segment that is responsible for fljB (H2) flagellin synthesis was precisely located.     

Phylogenetic relationships among transposon-like elements in human and primate DNA

A THE-1 sequence in intron 7 of the human dystrophin gene has been found to represent a new subfamily of THE-1 elements. The sequence is closely related to the MstII family of repetitive sequences and is more like single-copy sequences found in the galago genome than any other THE-1 sequence previously reported. This new THE-1 sequence has been compared with two other complete THE-1 sequences and three related long-terminal repeat elements that we have previously found in intron 7 of the dystrophin gene, and with members of the same family from elsewhere in the primate genome. Parsimony and deletion analysis show that the cluster of THE-1 sequences in intron 7 of the dystrophin gene has arisen from at least three individual insertion events, rather than from the insertion and duplication of a single progenitor sequence. Correspondence to: G.B. Petersen     

Inositol lipid-mediated signalling in response to endothelin and ATP in the mammalian testis

Molecular and Cellular Biochemistry - The testis is a complex organ in which local control is achieved by signalling between its constituent cells. Herein we describe the responses of cultured rat...     

Role of arginine-43 and arginine-69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites

The Hin recombinase mediates the site-specific inversion of a segment of the Salmonella chromosome between two flanking 26 bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4 bp added to the centre of the site, unlike wild-type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA-binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.     

A motile but non-swarming mutant of Proteus mirabilis lacks FlgN, a facilitator of flagella filament assembly

A Tn phoA mutant of Proteus mirabilis was isolated, which had lost the ability to swarm, yet was still motile. The transposon had inserted into flgN , a flagella gene encoding a 147-amino-acid protein of undefined function. Proteus flgN is arranged in an operon with the class III anti-σ²⁸ gene, flgM , flanked by the class II genes, flgA , flgBCD and flhBA , and a novel putative virulence-related gene. The flgN mutation caused a substantial reduction in cell surface-associated flagellin, particularly during differentiation to the normally hyperflagellated swarm cell. This was not due to an effect on flagella gene expression or a typical defect in the flagella export apparatus as there was no class III gene downregulation by FlgM feedback, or intracellular flagellin accumulation. Loss of FlgN nevertheless caused a severe reduction in the incorporation of pulse-labelled flagellin into the membrane/flagellum fraction of differentiating cells. Substantial amounts of both non-oligomeric flagellin and flagellin degradation products appeared in the extracellular medium, although the few mature filaments made by the mutant were no more sensitive to proteolysis than those of the wild type. FlgN appeared soluble and active in the cytosol. The data suggest that the function of FlgN is to facilitate the initiation of flagella filament assembly, a role that may be especially critical in attaining the much higher concentration of surface flagellin required for swarming. Proteus FlgN has leucine zipper-like motifs arranged on potential amphipathic helices, a feature conserved in cytosolic chaperones for the exported substrates of flagella-related type III virulence systems. While gel filtration of FlgN from the soluble cell fraction did not establish an interaction with flagellin, it indicated that FlgN may associate with an unknown component and/or form an oligomer.     

Rapid separation of reduction products of 2,4,6-trinitrotoluene using TLC

Silica gel TLC methods were developed for the separation of 2,4,6-trinitrotoluene (TNT) in mixtures with possible reduction products. The methods employed repeated elutions with simple binary or ternary solvent systems in either one or two dimensional modes. The resolved analytes include TNT, selected amino derivatives (2-amino-4,6-di-nitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene) and known hydroxylamino derivatives (2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene and 2,4-dihydroxylamino-6-nitrotoluene).     

Carbon starvation of Salmonella typhimurium does not cause a general increase of mutation rates.

Mutation rates in bacteria can vary depending on the genetic target studied and the specific growth conditions of the cells. Here, two different methods were used to determine how rates of mutation to antibiotic resistance, auxotrophy, and prototrophy were influenced by carbon starvation on agar plates. The rate of mutation to rifampin resistance was increased by starvation as measured by fluctuation tests, similar to what has been reported previously for Escherichia coli. In contrast, the rates of mutation to various types of auxotrophy were unaffected or decreased as measured by both fluctuation tests and a repeated-streaking procedure. Similarly, the rates of reversion to prototrophy of his and lac nonsense and missense mutations were unaffected by starvation. Thus, mutation rates of different genetic targets can be affected differently by starvation and we conclude that carbon starvation is not generally mutagenic in Salmonella typhimurium.     

Comparative evolutionary rates of introns and exons in murine rodents

Analysis of DNA sequences of 132 introns and 140 exons from 42 pairs of orthologous genes of mouse and rat was used to compare patterns of evolutionary change between introns and exons. The mean of the absolute difference in length (measured in base pairs) between the two species was nearly five times as high in the case of introns as in the case of exons. The average rate of nucleotide substitution in introns was very similar to the rate of synonymous substitution in exons, and both were about three times the rate of substitution at nonsynonymous sites in exons. G+C content of introns and exons of the same gene were correlated; but mean G+C content at the third positions of exons was significantly higher than that of introns or positions 1–2 of exons from the same gene. G+C content was conserved over evolutionary time, as indicated by strong correlations between mouse and rat; but the change in G+C content was greatest at position 3 of exons, intermediate in introns, and lowest at positions 1–2 in introns. Received: 23 December 1996 / Accepted: 1 April 1997     

Investigation of the dynamics of the spread of human immunodeficiency virus to brain and other tissues by evolutionary analysis of sequences from the p17gag and env genes.

The time of spread of human immunodeficiency virus type 1 (HIV-1) from lymphoid to nonlymphoid tissues in the course of infection was investigated by sequence comparisons of variants infecting a range of lymphoid and nonlymphoid tissues from three individuals with AIDS in the pl7gag gene and regions flanking the V1/V2 hypervariable regions. Phylogenetic analysis in both regions revealed several lineages in each individual that contained sequences from both lymphoid and nonlymphoid tissues such as the brain. This observation contrasts strongly with the previously described organ-specific sequences in the V3 region in this study population and other investigations. Although individual pairwise comparisons of relatively short sequences such as p17gag are subject to considerable stochastic error, we found that the diversity of gag sequences in variants from lymphoid tissue was consistently lower than that found among variants amplified from the brain. By estimating mean synonymous pairwise distances in the p17gag region, we were able to make an approximate calculation of the ages of populations in different tissues. Those from lymphoid tissue ranged from 2.65 to 5.6 years in the three study subjects, compared with 4.1 to 6.2 years for variants in the brain. Indeed, variants infecting the brain were no more closely related to each other than they were to variants infecting other tissues in the body. In two of the three individuals, these times of divergence indicate that infection of the brain may have occurred as an early event in the progression to disease, preceding the onset of AIDS by several years. This study is the first in which it was possible to estimate times of diversification in different tissues in vivo and is of importance in understanding the dynamics of the spread of HIV-1 into nonlymphoid tissues and its possible adaptation for replication in different cell types.