Characterisation of the BRCT domains of the breast cancer susceptibility gene product BRCA1

The breast cancer susceptibility gene product BRCA1 is a tumour suppressor but the biochemical and biological functions that underlie its role in carcinogenesis remain to be determined. Here, we characterise the solution properties of the highly conserved C terminus of BRCA1, consisting of a tandem repeat of the BRCT domain (BRCT-tan), that plays a critical role in BRCA1-mediated tumour suppression. The overall free energy of unfolding of BRCT-tan is high (14.2 kcal mol(-1) at 20 degrees C in water) but unfolding occurs via an aggregation-prone, partly folded intermediate. A representative set of cancer-associated sequence variants was constructed and the effects on protein stability were measured. All of the mutations were highly destabilising and they would be expected to cause loss of function for this reason. Over half could not be purified in a soluble form, indicating that these residues are critical for maintaining structural integrity. The remaining mutants exhibited much greater aggregation propensities than the wild-type, which is most likely a consequence of their reduced thermodynamic stability relative to the partly folded intermediate. The mutations characterised here are located at different sites in the BRCT-tan structure that do not explain fully their effects on the protein's stability. Thus, the results indicate an important role for biophysical studies in assessing the significance of sequence variants and in determining how they cause disease.     

N-acyl-homoserine lactone inhibition of rhizobial growth is mediated by two quorum-sensing genes that regulate plasmid transfer

The growth of some strains of Rhizobium leguminosarum bv. viciae is inhibited by N-(3-hydroxy-7-cis tetradecenoyl)-L-homoserine lactone (3OH-C(14:1)-HSL), which was previously known as the small bacteriocin before its characterization as an N-acyl homoserine lactone (AHL). Tn5-induced mutants of R. leguminosarum bv. viciae resistant to 3OH-C(14:1)-HSL were isolated, and mutations in two genes were identified. These genes, bisR and triR, which both encode LuxR-type regulators required for plasmid transfer, were found downstream of an operon containing trb genes involved in the transfer of the symbiotic plasmid pRL1JI. The first gene in this operon is traI, which encodes an AHL synthase, and the trbBCDEJKLFGHI genes were found between traI and bisR. Mutations in bisR, triR, traI, or trbL blocked plasmid transfer. Using gene fusions, it was demonstrated that bisR regulates triR in response to the presence of 3OH-C(14:1)-HSL. In turn, triR is then required for the induction of the traI-trb operon required for plasmid transfer. bisR also represses expression of cinI, which is chromosomally located and determines the level of production of 3OH-C(14:1)-HSL. The cloned bisR and triR genes conferred 3OH-C(14:1)-HSL sensitivity to strains of R. leguminosarum bv. viciae normally resistant to this AHL. Furthermore, bisR and triR made Agrobacterium tumefaciens sensitive to R. leguminosarum bv. viciae strains producing 3OH-C(14:1)-HSL. Analysis of patterns of growth inhibition using mutant strains and synthetic AHLs revealed that maximal growth inhibition required, in addition to 3OH-C(14:1)-HSL, the presence of other AHLs such as N-octanoyl-L-homoserine lactone and/or N-(3-oxo-octanoyl)-L-homoserine lactone. In an attempt to identify the causes of growth inhibition, a strain of R. leguminosarum bv. viciae carrying cloned bisR and triR was treated with an AHL extract containing 3OH-C(14:1)-HSL. N-terminal sequencing of induced proteins revealed one with significant similarity to the protein translation factor Ef-Ts.     

Life history,ecology and longevity in bats

The evolutionary theory of aging predicts that life span should decrease in response to the amount of mortality caused by extrinsic sources. Using this prediction, we selected six life history and ecological factors to use in a comparative analysis of longevity among 64 bat species. On average, the maximum recorded life span of a bat is 3.5 times greater than a non-flying placental mammal of similar size. Records of individuals surviving more than 30 years in the wild now exist for five species. Univariate and multivariate analyses of species data, as well as of phylogenetically independent contrasts obtained using a supertree of Chiroptera, reveal that bat life span significantly increases with hibernation, body mass and occasional cave use, but decreases with reproductive rate and is not influenced by diet, colony size or the source of the record. These results are largely consistent with extrinsic mortality risk acting as a determinant of bat longevity. Nevertheless, the strong association between life span and both reproductive rate and hibernation also suggests that bat longevity is strongly influenced by seasonal allocation of non-renewable resources to reproduction. We speculate that hibernation may provide a natural example of caloric restriction, which is known to increase longevity in other mammals.     

Nonperturbing fluorescent labeling of polysaccharides

A fluorescent labeling procedure, which does not perturb macromolecular conformations, was employed to bind a rhodamine derivative to the reducing end of several water-soluble polysaccharides by reductive amination in the presence of sodium cyanoborohydride. Fluorescence correlation spectroscopy, atomic force microscopy, and size exclusion chromatography were used to demonstrate that the conformations of the polysaccharides schizophyllan, polygalacturonic acid (PGUA), succinoglycan, and several dextrans were maintained following the labeling procedure.     

Exogenous Slit2 does not affect ureteric branching or nephron formation during kidney development

In an attempt to elucidate the role of Slit2 in vertebrate kidney development, the effect of adding exogenous human Slit2 protein (hSlit2) to developing murine metanephric kidney explants was examined. To confirm the activity of the recombinant Slit2 protein, neurons from 8 day old chick sympathetic nerve chain dorsal root ganglia were cultured with hSlit2 protein, which induced significant neurite branching and outgrowth. Using kidney explants as a model system, metanephric development in the presence of hSlit2 protein was examined. Addition of hSlit2 up to a final concentration of 1 microg/ml had no detectable effect on the formation of nephrons or on branching morphogenesis of the ureteric tree after 2 or 4 days in culture, as assessed via immunofluorescence for the markers WT1 and calbindin 28K respectively. Similarly, maturation of the nephrogenic mesenchyme occurred in a phenotypically normal fashion. In situ analysis of the Slit receptors, Robo1 and Robo2, the vasculogenic markers VEGFA and Flk-1, and the stromal cell marker BF2 displayed no difference in comparison to controls.     

Risk assessment of GM plants: avoiding gridlock?

Cultivation of genetically modified crops is presently based largely on four crops containing few transgenes and grown in four countries. This will soon change and pose new challenges for risk assessment. A more structured approach that is as generic as possible is advocated to study consequences of gene flow. Hazards should be precisely defined and prioritized, with emphasis on quantifying elements of exposure. This requires coordinated effort between large, multidisciplinary research teams.     

NAD+-dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor

Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD(+)-dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD(+) as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperature-sensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD(+)-dependent DNA ligases.     

Genome-wide detection of LOH in prostate cancer using human SNP microarray technology

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.