Fine mapping the KLK3 locus on chromosome 19q13.33 associated with prostate cancer susceptibility and PSA levels

Measurements of serum prostate-specific antigen (PSA) protein levels form the basis for a widely used test to screen men for prostate cancer. Germline variants in the gene that encodes the PSA protein (KLK3) have been shown to be associated with both serum PSA levels and prostate cancer. Based on a resequencing analysis of a 56?kb region on chromosome 19q13.33, centered on the KLK3 gene, we fine mapped this locus by genotyping tag SNPs in 3,522 prostate cancer cases and 3,338 controls from five case?Ccontrol studies. We did not observe a strong association with the KLK3 variant, reported in previous studies to confer risk for prostate cancer (rs2735839; P?=?0.20) but did observe three highly correlated SNPs (rs17632542, rs62113212 and rs62113214) associated with prostate cancer [P?=?3.41?×?10^?4, per-allele trend odds ratio (OR)?=?0.77, 95% CI?=?0.67?C0.89]. The signal was apparent only for nonaggressive prostate cancer cases with Gleason score <7 and disease stage <III (P?=?4.72?×?10^?5, per-allele trend OR?=?0.68, 95% CI?=?0.57?C0.82) and not for advanced cases with Gleason score >8 or stage ??III (P?=?0.31, per-allele trend OR?=?1.12, 95% CI?=?0.90?C1.40). One of the three highly correlated SNPs, rs17632542, introduces a non-synonymous amino acid change in the KLK3 protein with a predicted benign or neutral functional impact. Baseline PSA levels were 43.7% higher in control subjects with no minor alleles (1.61?ng/ml, 95% CI?=?1.49?C1.72) than in those with one or more minor alleles at any one of the three SNPs (1.12?ng/ml, 95% CI?=?0.96?C1.28) (P?=?9.70?×?10^?5). Together our results suggest that germline KLK3 variants could influence the diagnosis of nonaggressive prostate cancer by influencing the likelihood of biopsy.     

Microbial ecology of autothermal thermophilic aerobic digester (ATAD) systems for treating waste activated sludge

Despite their widespread use, our understanding of the microbial ecology of the autothermal thermophilic aerobic digesters (ATAD) used to dispose of sludge from wastewater treatment plants is poor. Applying both culture-dependent and molecular methods to two ATAD systems in Victoria, Australia treating different wastewaters revealed that their communities were highly specialized. Denaturing gradient gel electrophoresis (DGGE) profiling suggested differences in their population compositions and both changed over time. However, both showed low level biodiversity, and contained several novel bacterial populations. 16S rRNA clone library data and FISH analyses showed that Thermus thermophilus dominated both communities and that of a third ATAD plant in NSW (more than 90% of the total bacterial biovolume in repeated samples taken from each of the three ATAD plants). Culture-dependent methods also showed Geobacillus spp. were present in both Victorian communities. Nevertheless, the ecophysiology of these populations and their putative roles in sludge digestion remain unclear. FISH/microautoradiographic studies did not provide conclusive data elucidating which substrate/s T. thermophilus might utilize in the ATAD reactors.     

tmRNA regulates synthesis of the ArfA ribosome rescue factor

As Giardia lamblia is unable to synthesize cholesterol de novo, this steroid might be obtained from the host's intestinal milieu by endocytosis of lipoproteins. In this work, we identified a putative Giardia lamblia low-density lipoprotein receptor-related proteins (GlLRP), a type I membrane protein, which shares the substrate N-terminal binding domain and a FXNPXY-type endocytic motif with human LRPs. Expression of tagged GlLRP showed that it was localized predominantly in the endoplasmic reticulum, lysosomal-like peripheral vacuoles and plasma membrane. However, the FXNPXY-deleted GlLRP was retained at the plasma membrane suggesting that it is abnormally transported and processed. The low-density lipoprotein and chylomicrons interacted with GlLRP, with this interaction being necessary for lipoprotein internalization and cell proliferation. Finally, we show that GlLRP binds directly to the medium subunit of Giardia adaptor protein 2, indicating that receptor-mediated internalization occurs through an adaptin mechanism.     

Deletion of the RluD pseudouridine synthase promotes SsrA peptide tagging of ribosomal protein S7

RluD catalyses formation of three pseudouridine residues within helix 69 of the 50S ribosome subunit. Helix 69 makes important contacts with the decoding centre on the 30S subunit and deletion of rluD was recently shown to interfere with translation termination in Escherichia coli. Here, we show that deletion of rluD increases tmRNA activity on ribosomes undergoing release factor 2 (RF2)-mediated termination at UGA stop codons. Strikingly, tmRNA-mediated SsrA peptide tagging of two proteins, ribosomal protein S7 and LacI, was dramatically increased in ΔrluD cells. S7 tagging was due to a unique C-terminal peptide extension found in E. coli K-12 strains. Introduction of the rpsG gene (encoding S7) from an E. coli B strain abrogated S7 tagging in the ΔrluD background, and partially complemented the mutant's slow-growth phenotype. Additionally, exchange of the K-12 prfB gene (encoding RF2) with the B strain allele greatly reduced tagging in ΔrluD cells. In contrast to E. coli K-12 cells, deletion of rluD in an E. coli B strain resulted in no growth phenotype. These findings indicate that the originally observed rluD phenotypes result from synthetic interactions with rpsG and prfB alleles found within E. coli K-12 strains.     

The response of foetal annulus fibrosus cells to growth factors: modulation of matrix synthesis by TGF-β1 and IGF-1

The annulus fibrosus of the intervertebral disc is a complex radial-ply tissue that derives initially from segmental condensations of axial mesenchyme surrounding the notochord. These mesenchymal condensations differentiate into the early annulus fibrosus during foetal development—their outer part becoming fibrous, containing collagen type I; and their inner part cartilaginous, containing type II collagen and aggrecan. With post-natal growth and ageing, there is a switch from type I to type II collagen and an increase in proteoglycan synthesis in the outer annulus. This fibrocartilaginous metaplasia appears to occur in response to compressive loading of the tissue as occurs in tendons that wrap around bony pulleys, and driven by growth factors, such as TGF-β. In this study, using high-density micromass cultures, we have assessed the response of foetal outer annulus cells to growth factor stimulation with TGF-β1 and IGF-1, growth factors known to occur within the developing disc. We qualitatively and quantitatively describe the stimulatory effects of these growth factors, both alone and in combination, on the synthesis of sulphated glycosaminoglycan, and collagen types I and II by annulus cells. We show a potential role for TGF-β1 in pushing cells towards a fibrocartilaginous phenotype, with possible complementary effects of IGF-1.     

Alternative fates of paused ribosomes during translation termination

The bacterial tmRNA·SmpB system facilitates recycling of stalled translational complexes in a process termed "ribosome rescue." During ribosome rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which targets the tagged polypeptide for degradation. Translational pausing also induces a variety of recoding events such as frameshifts, ribosome hops, and stop codon readthrough. To examine the interplay between recoding and ribosome rescue, we determined the various fates of ribosomes that pause during translation termination. We expressed a model protein containing the C-terminal Asp-Pro nascent peptide motif (which interferes with translation termination) and quantified the protein chains produced by recoding and ssrA-peptide tagging. The nature and extent of translational recoding depended upon the codon for the C-terminal Pro residue, with CCU and CCC promoting efficient +1 frameshifting. In contrast, ssrA-peptide tagging was unaffected by C-terminal Pro coding. Moreover, +1 frameshifting was not suppressed by tmRNA·SmpB activity, suggesting that recoding and ribosome rescue are not competing events. However, cells lacking ribosomal protein L9 (ΔL9) exhibited a significant increase in recoding and a concomitant decrease in ssrA-peptide tagging. Pulse-chase analysis revealed that pre-termination ribosomes turn over more rapidly in ΔL9 cells, suggesting that increased recoding alleviates the translational arrest. Together, these results indicate that tmRNA·SmpB does not suppress transient ribosome pauses, but responds to prolonged translational arrest.     

Imputation of missing genotypes from sparse to high density using long-range phasing

Related individuals share potentially long chromosome segments that trace to a common ancestor. We describe a phasing algorithm (ChromoPhase) that utilizes this characteristic of finite populations to phase large sections of a chromosome. In addition to phasing, our method imputes missing genotypes in individuals genotyped at lower marker density when more densely genotyped relatives are available. ChromoPhase uses a pedigree to collect an individual's (the proband) surrogate parents and offspring and uses genotypic similarity to identify its genomic surrogates. The algorithm then cycles through the relatives and genomic surrogates one at a time to find shared chromosome segments. Once a segment has been identified, any missing information in the proband is filled in with information from the relative. We tested ChromoPhase in a simulated population consisting of 400 individuals at a marker density of 1500/M, which is approximately equivalent to a 50K bovine single nucleotide polymorphism chip. In simulated data, 99.9% loci were correctly phased and, when imputing from 100 to 1500 markers, more than 87% of missing genotypes were correctly imputed. Performance increased when the number of generations available in the pedigree increased, but was reduced when the sparse genotype contained fewer loci. However, in simulated data, ChromoPhase correctly imputed at least 12% more genotypes than fastPHASE, depending on sparse marker density. We also tested the algorithm in a real Holstein cattle data set to impute 50K genotypes in animals with a sparse 3K genotype. In these data 92% of genotypes were correctly imputed in animals with a genotyped sire. We evaluated the accuracy of genomic predictions with the dense, sparse, and imputed simulated data sets and show that the reduction in genomic evaluation accuracy is modest even with imperfectly imputed genotype data. Our results demonstrate that imputation of missing genotypes, and potentially full genome sequence, using long-range phasing is feasible.     

Population structure of <Emphasis Type="Italic">Apis cerana</Emphasis> in Thailand reflects biogeography and current gene flow rather than <Emphasis Type="Italic">Varroa</Emphasis> mite association

Concordance between the mitochondrial haplotypes of the Eastern honey bee, Apis cerana, and its ectoparasitic Varroa mites across the Isthmus of Kra in Thailand has suggested that local host–pathogen co-evolution may be responsible for the geographic distribution of particular genotypes. To investigate nuclear microsatellites population structure in A. cerana, single workers of A. cerana colonies from Thailand were genotyped at 18 microsatellite loci. The loci showed intermediate to high levels of heterozygosity and a range of allele numbers. The analyses confirmed a fundamental subdivision of the Thai A. cerana population into the “Asia Mainland” and “Sundaland” regions at the Isthmus of Kra. However, the nuclear microsatellite differentiation was less distinct than mtDNA haplotype differences, suggesting male-biased dispersal and population admixture. Overall, samples showed a weak isolation-by-distance effect. The isolated population on Samui island was most differentiated from the other samples. The results do not support our initial hypothesis of local host–pathogen co-evolution, which predicts a strict correspondence between the nuclear genome and the lineage of parasitic Varroa mite of the A. cerana samples, because the gene flow indicated by our nuclear microsatellite markers should also mix potential Varroa resistance alleles among subpopulations. Instead, our study suggests that the coincidental distribution of Varroa lineages and A. cerana population structure in Thailand are the result of biogeographic history and current migration patterns.     

The inheritance of resistance to Verticillium wilt caused by race 1 isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the lettuce cultivar La Brillante

Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. Complete resistance to race 1 isolates is available in Lactuca sativa cultivar (cv.) La Brillante and understanding the genetic basis of this resistance will aid development of new resistant cultivars. F₁ and F₂ families from crosses between La Brillante and three iceberg cultivars as well as a recombinant inbred line population derived from L. sativa cv. Salinas 88 × La Brillante were evaluated for disease incidence and disease severity in replicated greenhouse and field experiments. One hundred and six molecular markers were used to generate a genetic map from Salinas 88 × La Brillante and for detection of quantitative trait loci. Segregation was consistent with a single dominant gene of major effect which we are naming Verticillium resistance 1 (Vr1). The gene described large portions of the phenotypic variance (R ² = 0.49–0.68) and was mapped to linkage group 9 coincident with an expressed sequence tag marker (QGD8I16.yg.ab1) that has sequence similarity with the Ve gene that confers resistance to V. dahliae race 1 in tomato. The simple inheritance of resistance indicates that breeding procedures designed for single genes will be applicable for developing resistant cultivars. QGD8I16.yg.ab1 is a good candidate for functional analysis and development of markers suitable for marker-assisted selection.