Comparative whole-genome hybridization reveals genomic islands in Brucella species

Brucella species are responsible for brucellosis, a worldwide zoonotic disease causing abortion in domestic animals and Malta fever in humans. Based on host preference, the genus is divided into six species. Brucella abortus, B. melitensis, and B. suis are pathogenic to humans, whereas B. ovis and B. neotomae are nonpathogenic to humans and B. canis human infections are rare. Limited genome diversity exists among Brucella species. Comparison of Brucella species whole genomes is, therefore, likely to identify factors responsible for differences in host preference and virulence restriction. To facilitate such studies, we used the complete genome sequence of B. melitensis 16M, the species highly pathogenic to humans, to construct a genomic microarray. Hybridization of labeled genomic DNA from Brucella species to this microarray revealed a total of 217 open reading frames (ORFs) altered in five Brucella species analyzed. These ORFs are often found in clusters (islands) in the 16M genome. Examination of the genomic context of these islands suggests that many are horizontally acquired. Deletions of genetic content identified in Brucella species are conserved in multiple strains of the same species, and genomic islands missing in a given species are often restricted to that particular species. These findings suggest that, whereas the loss or gain of genetic material may be related to the host range and virulence restriction of certain Brucella species for humans, independent mechanisms involving gene inactivation or altered expression of virulence determinants may also contribute to these differences.     

Recognition of 5'-YpG-3' sequences by coupled stacking/hydrogen bonding interactions with amino acid residues

The combined biochemical and structural study of hundreds of protein-DNA complexes has indicated that sequence-specific interactions are mediated by two mechanisms termed direct and indirect readout. Direct readout involves direct interactions between the protein and base-specific atoms exposed in the major and minor grooves of DNA. For indirect readout, the protein recognizes DNA by sensing conformational variations in the structure dependent on nucleotide sequence, typically through interactions with the phosphodiester backbone. Based on our recent structure of Ndt80 bound to DNA in conjunction with a search of the existing PDB database, we propose a new method of sequence-specific recognition that utilizes both direct and indirect readout. In this mode, a single amino acid side-chain recognizes two consecutive base-pairs. The 3'-base is recognized by canonical direct readout, while the 5'-base is recognized through a variation of indirect readout, whereby the conformational flexibility of the particular dinucleotide step, namely a 5'-pyrimidine-purine-3' step, facilitates its recognition by the amino acid via cation-pi interactions. In most cases, this mode of DNA recognition helps explain the sequence specificity of the protein for its target DNA.     

Three options for citation tracking: Google Scholar,Scopus and Web of Science

Background

Researchers turn to citation tracking to find the most influential articles for a particular topic and to see how often their own published papers are cited. For years researchers looking for this type of information had only one resource to consult: the Web of Science from Thomson Scientific. In 2004 two competitors emerged – Scopus from Elsevier and Google Scholar from Google. The research reported here uses citation analysis in an observational study examining these three databases; comparing citation counts for articles from two disciplines (oncology and condensed matter physics) and two years (1993 and 2003) to test the hypothesis that the different scholarly publication coverage provided by the three search tools will lead to different citation counts from each.

Methods

Eleven journal titles with varying impact factors were selected from each discipline (oncology and condensed matter physics) using the Journal Citation Reports (JCR). All articles published in the selected titles were retrieved for the years 1993 and 2003, and a stratified random sample of articles was chosen, resulting in four sets of articles. During the week of November 7–12, 2005, the citation counts for each research article were extracted from the three sources. The actual citing references for a subset of the articles published in 2003 were also gathered from each of the three sources.

Results

For oncology 1993 Web of Science returned the highest average number of citations, 45.3. Scopus returned the highest average number of citations (8.9) for oncology 2003. Web of Science returned the highest number of citations for condensed matter physics 1993 and 2003 (22.5 and 3.9 respectively). The data showed a significant difference in the mean citation rates between all pairs of resources except between Google Scholar and Scopus for condensed matter physics 2003. For articles published in 2003 Google Scholar returned the largest amount of unique citing material for oncology and Web of Science returned the most for condensed matter physics.

Conclusion

This study did not identify any one of these three resources as the answer to all citation tracking needs. Scopus showed strength in providing citing literature for current (2003) oncology articles, while Web of Science produced more citing material for 2003 and 1993 condensed matter physics, and 1993 oncology articles. All three tools returned some unique material. Our data indicate that the question of which tool provides the most complete set of citing literature may depend on the subject and publication year of a given article.

     

Regional differences in awareness and attitudes regarding genetic testing for disease risk and ancestry

Little is known about the lay public’s awareness and attitudes concerning genetic testing and what factors influence their perspectives. The existing literature focuses mainly on ethnic and socioeconomic differences; however, here we focus on how awareness and attitudes regarding genetic testing differ by geographical regions in the US. We compared awareness and attitudes concerning genetic testing for disease risk and ancestry among 452 adults (41% Black and 67% female) in four major US cities, Norman, OK; Cincinnati, OH; Harlem, NY; and Washington, DC; prior to their participation in genetic ancestry testing. The OK participants reported more detail about their personal ancestries (p = 0.02) and valued ancestry testing over disease testing more than all other sites (p < 0.01). The NY participants were more likely than other sites to seek genetic testing for disease (p = 0.01) and to see benefit in finding out more about one’s ancestry (p = 0.02), while the DC participants reported reading and hearing more about genetic testing for African ancestry than all other sites (p < 0.01). These site differences were not better accounted for by sex, age, education, self-reported ethnicity, religion, or previous experience with genetic testing/counseling. Regional differences in awareness and attitudes transcend traditional demographic predictors, such as ethnicity, age and education. Local sociocultural factors, more than ethnicity and socioeconomic status, may influence the public’s awareness and belief systems, particularly with respect to genetics.     

Concordant phylogeography and cryptic speciation in two Western Palaearctic oak gall parasitoid species complexes

Little is known about the evolutionary history of most complex multi‐trophic insect communities. Widespread species from different trophic levels might evolve in parallel, showing similar spatial patterns and either congruent temporal patterns (Contemporary Host‐tracking) or later divergence in higher trophic levels (Delayed Host‐tracking). Alternatively, host shifts by natural enemies among communities centred on different host resources could disrupt any common community phylogeographic pattern. We examined these alternative models using two Megastigmus parasitoid morphospecies associated with oak cynipid galls sampled throughout their Western Palaearctic distributions. Based on existing host cynipid data, a parallel evolution model predicts that eastern regions of the Western Palaearctic should contain ancestral populations with range expansions across Europe about 1.6 million years ago and deeper species‐level divergence at both 8–9 and 4–5 million years ago. Sequence data from mitochondrial cytochrome b and multiple nuclear genes showed similar phylogenetic patterns and revealed cryptic genetic species within both morphospecies, indicating greater diversity in these communities than previously thought. Phylogeographic divergence was apparent in most cryptic species between relatively stable, diverse, putatively ancestral populations in Asia Minor and the Middle East, and genetically depauperate, rapidly expanding populations in Europe, paralleling patterns in host gallwasp species. Mitochondrial and nuclear data also suggested that Europe may have been colonized multiple times from eastern source populations since the late Miocene. Temporal patterns of lineage divergence were congruent within and across trophic levels, supporting the Contemporary Host‐tracking Hypothesis for community evolution.     

The activity of the thiazide-sensitive Na(+)-Cl(-) cotransporter is regulated by protein phosphatase PP4

Cation transport in the distal mammalian nephron relies on the SLC12 family of membrane cotransporters that include the thiazide-sensitive Na(+)-Cl? cotransporter (NCC). NCC is regulated through a scaffold of interacting proteins, including the WNK kinases, WNK 1 and WNK 4, which are mutated in the hypertensive Gordon's syndrome. Dynamic regulation of NCC function by kinases must involve dephosphorylation by phosphatases, as illustrated by the role of PP1 and PP2B in the regulation of KCC members of the SLC12 family. There are 2 phosphorylation-controlled regulatory pathways for NCC: type 1, mediated by WNK4 and affecting trafficking to the surface membrane, and type 2, affecting intrinsic transporter kinetics by phosphorylation of conserved N-terminal S/T amino acids. Using the Xenopus oocyte expression system, we show that PP4 inhibits NCC activity - but not trafficking to the surface membrane - by a mechanism that requires phosphatase activity and a conserved N-terminal amino acid of NCC, threonine 58. This action is distinct from WNK4 regulation of membrane trafficking. In the mouse kidney, PP4 is selectively expressed in the distal nephron, including cells of the distal convoluted tubule cells, suggesting that PP4 may have a physiological role in regulating NCC and hence NaCl reabsorption in vivo.     

A targeted proteomic approach for the identification of tumor-associated membrane antigens using the ProteomeLab PF-2D in tandem with mass spectrometry

Mapping differential expression of soluble proteins has become fairly routine using chromatofocusing in combination with the reversed-phase HPLC (ProteomeLab PF-2D by Beckman Coulter Inc.); however, identification of membrane antigens has not been reported thus far. In this report, we demonstrate a targeted proteomic approach employing immunoprecipitation, prior to 2D-LC separation, in tandem with MS/MS that can be used to identify tumor-associated membrane antigens. This system is very sensitive and reproducible in that only 1/4th the amount of starting material is required for analysis as compared to gel-based analysis, and permits a focused environment for eliminating non-specific interactions leading to an accurate resolution of the cognate antigen. This system also circumvents the well-known limitations associated with gel-based approaches. This approach has been validated in the identification of ErB2/HER-2 and was subsequently used to identify CD44E as the cognate antigen for VB1-008, one of our fully human, tumor-specific, monoclonal antibodies.     

Repression of polymerase I-mediated gene expression at Trypanosoma brucei telomeres

The African trypanosome, Trypanosoma brucei, is a flagellated pathogenic protozoan that branched early from the eukaryotic lineage. Unusually, it uses RNA polymerase I (Pol I) for mono-telomeric expression of variant surface glycoprotein (VSG) genes in bloodstream-form cells. Many other subtelomeric VSG genes are reversibly repressed, but no repressive DNA sequence has been identified in any trypanosomatid. Here, we show that artificially seeded de novo telomeres repress Pol I-dependent gene expression in mammalian bloodstream and insect life-cycle stages of T. brucei. In a telomeric VSG expression site, repression spreads further along the chromosome and this effect is specific to the bloodstream stage. We also show that de novo telomere extension is telomerase dependent and that the rate of extension correlates with the expression level of the adjacent gene. Our results show constitutive telomeric repression in T. brucei and indicate that an enhanced, developmental stage-specific repression mechanism controls antigenic variation.     

REV1 and polymerase ζ facilitate homologous recombination repair

REV1 and DNA Polymerase ζ (REV3 and REV7) play important roles in translesion DNA synthesis (TLS) in which DNA replication bypasses blocking lesions. REV1 and Polζ have also been implicated in promoting repair of DNA double-stranded breaks (DSBs). However, the mechanism by which these two TLS polymerases increase tolerance to DSBs is poorly understood. Here we demonstrate that full-length human REV1, REV3 and REV7 interact in vivo (as determined by co-immunoprecipitation studies) and together, promote homologous recombination repair. Cells lacking REV3 were hypersensitive to agents that cause DSBs including the PARP inhibitor, olaparib. REV1, REV3 or REV7-depleted cells displayed increased chromosomal aberrations, residual DSBs and sites of HR repair following exposure to ionizing radiation. Notably, cells depleted of DNA polymerase η (Polη) or the E3 ubiquitin ligase RAD18 were proficient in DSB repair following exposure to IR indicating that Polη-dependent lesion bypass or RAD18-dependent monoubiquitination of PCNA are not necessary to promote REV1 and Polζ-dependent DNA repair. Thus, the REV1/Polζ complex maintains genomic stability by directly participating in DSB repair in addition to the canonical TLS pathway.     

Insight into molecular basis of curing of [PSI+] prion by overexpression of 104-kDa heat shock protein (Hsp104)

Yeast prions are a powerful model for understanding the dynamics of protein aggregation associated with a number of human neurodegenerative disorders. The AAA+ protein disaggregase Hsp104 can sever the amyloid fibrils produced by yeast prions. This action results in the propagation of "seeds" that are transmitted to daughter cells during budding. Overexpression of Hsp104 eliminates the [PSI+] prion but not other prions. Using biochemical methods we identified Hsp104 binding sites in the highly charged middle domain of Sup35, the protein determinant of [PSI+]. Deletion of a short segment of the middle domain (amino acids 129-148) diminishes Hsp104 binding and strongly affects the ability of the middle domain to stimulate the ATPase activity of Hsp104. In yeast, [PSI+] maintained by Sup35 lacking this segment, like other prions, is propagated by Hsp104 but cannot be cured by Hsp104 overexpression. These results provide new insight into the enigmatic specificity of Hsp104-mediated curing of yeast prions and sheds light on the limitations of the ability of Hsp104 to eliminate aggregates produced by other aggregation-prone proteins.