Global analysis of gene expression in the estrogen induced pituitary tumor of the F344 rat

The F344 rat rapidly forms large prolactinomas in response to chronic estrogen treatment. To identify genes expressed in the course of this estrogen induced pituitary tumor growth, we performed microarray analysis on the F344 rat pituitary after chronic estrogen treatment and on untreated controls. At a significance level set to minimize type I error, some 72 genes were found to be differentially expressed between estrogen treated and untreated. Of those genes, 70 have not been reported previously as being affected by estrogen in the F344 rat pituitary. Since many other investigators have studied the effect of estrogen on specific gene expression in rat pituitary, we also examined the mRNA expression of the 36 genes that have been previously reported as having their expression affected by estrogen in the rat pituitary. Of these, 13 were found to have their expression affected by estrogen treatment in the same direction as had been reported by others.     

The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation

BACKGROUND: Acetylation of histone H3 lysine 56 (K56Ac) occurs transiently in newly synthesized H3 during passage through S phase and is removed in G2. However, the physiologic roles and effectors of K56Ac turnover are unknown. RESULTS: The sirtuins Hst3p and, to a lesser extent, Hst4p maintain low levels of K56Ac outside of S phase. In hst3 hst4 mutants, K56 hyperacetylation nears 100%. Residues corresponding to the nicotinamide binding pocket of Sir2p are essential for Hst3p function, and H3 K56 deacetylation is inhibited by nicotinamide in vivo. Rapid inactivation of Hst3/Hst4p prior to S phase elevates K56Ac to 50% in G2, suggesting that K56-acetylated nucleosomes are assembled genome-wide during replication. Inducible expression of Hst3p in G1 or G2 triggers deacetylation of mature chromatin. Cells lacking Hst3/Hst4p exhibit many phenotypes: spontaneous DNA damage, chromosome loss, thermosensitivity, and acute sensitivity to genotoxic agents. These phenotypes are suppressed by mutation of histone H3 K56 into a nonacetylatable residue or by loss of K56Ac in cells lacking the histone chaperone Asf1. CONCLUSIONS: Our results underscore the critical importance of Hst3/Hst4p in controlling histone H3 K56Ac and thereby maintaining chromosome integrity.     

Evolution of an agriculture-associated disease causing Campylobacter coli clade: evidence from national surveillance data in Scotland

The common zoonotic pathogen Campylobacter coli is an important cause of bacterial gastroenteritis worldwide but its evolution is incompletely understood. Using multilocus sequence type (MLST) data of 7 housekeeping genes from a national survey of Campylobacter in Scotland (2005/6), and a combined population genetic-phylogenetics approach, we investigated the evolutionary history of C. coli. Genealogical reconstruction of isolates from clinical infection, farm animals and the environment, revealed a three-clade genetic structure. The majority of farm animal, and all disease causing genotypes belonged to a single clade (clade 1) which had comparatively low synonymous sequence diversity, little deep branching genetic structure, and a higher number of shared alleles providing evidence of recent clonal decent. Calibration of the rate of molecular evolution, based on within-species genetic variation, estimated a more rapid rate of evolution than in traditional estimates. This placed the divergence of the clades at less than 2500 years ago, consistent with the introduction of an agricultural niche having had an effect upon the evolution of the C. coli clades. Attribution of clinical isolate genotypes to source, using an asymmetric island model, confirmed that strains from chicken and ruminants, and not pigs or turkeys, are the principal source of human C. coli infection. Taken together these analyses are consistent with an evolutionary scenario describing the emergence of agriculture-associated C. coli lineage that is an important human pathogen.     

The Minimal Autoinhibited Unit of the Guanine Nucleotide Exchange Factor Intersectin

Intersectin-1L is a member of the Dbl homology (DH) domain guanine nucleotide exchange factors (GEF) which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E)) being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E)-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E)-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain.     

Morphological determinants of signal carrier frequency in katydids (Orthoptera): a comparative analysis using biophysical evidence of wing vibration

Male katydids produce mating calls by stridulation using specialized structures on the forewings. The right wing (RW) bears a scraper connected to a drum‐like cell known as the mirror and a left wing (LW) that overlaps the RW and bears a serrated vein on the ventral side, the stridulatory file. Sound is generated with the scraper sweeping across the file, producing vibrations that are amplified by the mirror. Using this sound generator, katydids exploit a range of song carrier frequencies (CF) unsurpassed by any other insect group, with species singing as low as 600 Hz and others as high as 150 kHz. Sound generator size has been shown to scale negatively with CF, but such observations derive from studies based on few species, without phylogenetic control, and/or using only the RW mirror length. We carried out a phylogenetic comparative analysis involving 94 species of katydids to study the relationship between LW and RW components of the sound generator and the CF of the male's mating call, while taking into account body size and phylogenetic relationships. The results showed that CF negatively scaled with all morphological measures, but was most strongly related to components of the sound generation system (file, LW and RW mirrors). Interestingly, the LW mirror (reduced and nonfunctional) predicted CF more accurately than the RW mirror, and body size is not a reliable CF predictor. Mathematical models were verified on known species for predicting CF in species for which sound is unknown (e.g. fossils or museum specimens).     

Allosteric interaction of nucleotides and tRNA(ala) with E. coli alanyl-tRNA synthetase

Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution. ATP binding to full length enzyme (ARS875) and to an N-terminal construct (ARS461) is endothermic (ΔH = 3-4 kcal mol(-1)) with stoichiometries of 1:1 for ARS461 and 2:1 for full-length dimer. Binding of aminoacyl-adenylate analogues, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (ASAd) and 5'-O-[N-(L-glycinyl)sulfamoyl]adenosine (GSAd), are exothermic; ASAd exhibits a large negative heat capacity change (ΔC(p) = 0.48 kcal mol(-1) K(-1)). Modification of alanyl-tRNA synthetase with periodate-oxidized tRNA(ala) (otRNA(ala)) generates multiple, covalent, enzyme-tRNA(ala) products. The distribution of these products is altered by ATP, ATP and alanine, and aminoacyl-adenylate analogues (ASAd and GSAd). Alanyl-tRNA synthetase was modified with otRNA(ala), and tRNA-peptides from tryptic digests were purified by ion exchange chromatography. Six peptides linked through a cyclic dehydromoropholino structure at the 3'-end of tRNA(ala) were sequenced by mass spectrometry. One site lies in the N-terminal adenylate synthesis domain (residue 74), two lie in the opening to the editing site (residues 526 and 585), and three (residues 637, 639, and 648) lie on the back side of the editing domain. At least one additional modification site was inferred from analysis of modification of ARS461. The location of the sites modified by otRNA(ala) suggests that there are multiple modes of interaction of tRNA(ala) with the enzyme, whose distribution is influenced by occupation of the ATP binding site.     

Temporal variation and host association in the Campylobacter population in a longitudinal ruminant farm study

Campylobacter jejuni and C. coli were quantified and typed, using multilocus sequence typing (MLST), from fecal samples collected from a mixed cattle and sheep farm during summer. Cattle had a significantly higher prevalence than sheep (21.9% [74/338] and 14.0% [30/214], respectively), but both decreased over time. There were no differences in the average Campylobacter concentrations shed by cattle (600 CFU g(-1)) and sheep (820 CFU g(-1)), although sheep did show a significant temporal reduction in the number of Campylobacter organisms shed in their feces. A total of 21 different sequence types (STs) (97.7% C. jejuni, 2.3% C. coli) were isolated from cattle, and 9 different STs were isolated from sheep (40.6% C. jejuni, 59.4% C. coli). The Campylobacter population in cattle was relatively stable, and the frequencies of genotypes isolated showed little temporal variation. However, the composition of subtypes isolated from sheep did show significant temporal differences. The cattle and sheep consistently showed significant differences in their carriage of Campylobacter species, STs, and CCs despite the fact that both were exposed to the same farming environment. This work has highlighted the patterns of a Campylobacter population on a ruminant farm by identifying the existence of both temporal and between-host variations.     

Is the prevalence and shedding concentrations of E. coli O157 in beef cattle in Scotland seasonal?

The prevalence of Escherichia coli O157 in Scottish beef cattle at abattoir was found to be greater during the cooler months [11.2% (95% CI, 8.4-13.9%)] compared to the warmer months [7.5% (95% CI, 5.4-9.6%)]; the reverse of seasonality of human infections. However, high shedding beef cattle (excreting 10(-4) g(-1)) appear to shed greater concentrations of E. coli O157 in the warmer months which may partly explain increased human infection seasonality at this time.     

Outlier SNP markers reveal fine‐scale genetic structuring across European hake populations (Merluccius merluccius)

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome‐wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large‐ and fine‐scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F_CT = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F_CT range 0.275–0.705) and fine‐scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.