Profiling of the BRCA1 transcriptome through microarray and ChIP-chip analysis

A role for BRCA1 in the direct and indirect regulation of transcription is well established. However, a comprehensive view of the degree to which BRCA1 impacts transcriptional regulation on a genome-wide level has not been defined. We performed genome-wide expression profiling and ChIP-chip analysis, comparison of which revealed that although BRCA1 depletion results in transcriptional changes in 1294 genes, only 44 of these are promoter bound by BRCA1. However, 27 of these transcripts were linked to transcriptional regulation possibly explaining the large number of indirect transcriptional changes observed by microarray analysis. We show that no specific consensus sequence exists for BRCA1 DNA binding but rather demonstrate the presence of a number of known and novel transcription factor (TF)- binding sites commonly found on BRCA1 bound promoters. Co-immunoprecipitations confirmed that BRCA1 interacts with a number of these TFs including AP2-a, PAX2 and ZF5. Finally, we show that BRCA1 is bound to a subset of promoters of genes that are not altered by BRCA1 loss, but are transcriptionally regulated in a BRCA1-dependent manner upon DNA damage. These data suggest a model, whereby BRCA1 is present on defined promoters as part of an inactive complex poised to respond to various genotoxic stimuli. © The Author(s) 2011. Published by Oxford University Press.

IMMUNOREACTIVITY TO 2 SPECIFIC REGIONS OF CHROMOGRANIN-A IN THE NERVOUS-SYSTEM OF ASCARIS-SUUM - AN IMMUNOCYTOCHEMICAL STUDY

Antisera to a highly conserved region of chromogranin A (sequence KELTAE) and to a hexapeptide (sequence KGQELE) adjacent to the putative C-terminus of pancreastatin, a peptide whose sequence is found within the chromogranin A molecule, have been used to examine the localisation of immunoreactivity (IR) to these peptides in Ascaris suum. IR to both peptides was found in the nerve rings and nerve cords. In addition, KGQELE-IR was also observed in the pharyngeal neurones and in a network of fibres on the surface of the female gonoduct. The staining was specific in that it could be abolished by preincubation of the antisera with the appropriate antigen. The two antisera appeared to be staining different subsets of neurones, suggesting that (at least) two peptides were being recognised. The widespread distribution of IR to both peptides throughout the nervous system of the parasite suggests that the peptides carrying the epitopes recognised by the antisera are of fundamental importance to the functioning of the parasite's nervous system.

Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family

A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116, Two copies of IS2112 were found in R, rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the l-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp, HA1 or in several Rhodococcus strains which do not utilize haloalkanes, IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064 which harbours genes encoding utilization of l-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA), Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

The Supremacy of the Sequence: Key Elements and Dimensions in the Process of Change

How single organizations manage the process of change and why only some of them are able to actually reach radical change are central questions in today’s theoretical debate. The role played by the process of change and its dimensions (namely, pace, sequence and linearity), however, has been poorly investigated. Drawing on archetype theory, this paper explores: (i) whether a specific pace of radical change exists; (ii) whether different outcomes of change are characterized by different sequences of change in key-structures and systems (iii) how the three dimensions of the process of change possibly interact. As an example of organizational change the study takes into consideration processes of accounting change in three departments of two Canadian and two Italian municipalities. The results suggest the supremacy of the sequence of implemented changes over the other two dimensions of the process in order to achieve a radical outcome of change.

A nine-base nucleotide sequence in the porcine circovirus type 2 (PCV2) nucleocapsid gene determines viral replication and virulence

Porcine circovirus type 2 (PCV2) was retrospectively identified by serology in swine populations as an asymptomatic infection at least 25 years prior to the first reported case of PCV2-associated postweaning multisystemit wasting syndrome (PMWS). To investigate the sudden emergence of PMWS, viral sequences were amplified from frozen archived (1970-1971) porcine tissues and the complete genome of archival PCV2 was determined. The ORF1 gene product (viral DNA replicase) was homologous to contemporary PCV2 ORF1. In ORF2 (viral nucleocapsid gene) archival PCV2, a consistent linear nine-base sequence difference at base positions 1331 through 1339 was observed.The deduced amino acid sequence from these base changes alters the nucleocapsid conformation within the second immunogenic epitope from a hydrophobic (contemporary PCV2) to a hydrophilic (archival PCV2) configuration.

Phylogenetic analysis of porcine circovirus type 2 (PCV2) pre- and post-epizootic postweaning multisystemic wasting syndrome (PMWS)

The porcine circovirus type 2 (PCV2) genome encodes three major open reading frames (ORFs) encoding the replicase proteins (ORF1), the viral capsid protein (ORF2), and a protein with suggested apoptotic activity (ORF3). Previous phylogenetic analyses of complete genome sequences of PCV2 from GenBank have demonstrated 95-100% intra-group nucleotide sequence identity. However, although these isolates were readily grouped into clusters and clades, there was no correlation between the occurrence of specific PCV2 genotypes and the geographic origin or health status of the pig. In the present study, a unique dataset from a field study spanning the years pre and post the recognition of postweaning multisystemic wasting syndrome (PMWS) in Sweden was utilized. Using this dataset it was possible to discriminate three Swedish genogroups (SG1-3) of PCV2, of which SG1 was recovered from a pig on a healthy farm ten years before the first diagnosis of PMWS in Sweden. The SG1 PCV2/ORF2 gene sequence has been demonstrated to exhibit a high genetic stability over time and has subsequently only been demonstrated in samples from pigs on nondiseased farms. In contrast, SG2 was almost exclusively found on farms that had only recently broken down with PMWS whereas the SG3 genogroup predominated in pigs from PMWS-affected farms. These results further support the results obtained from earlier in vitro and in vivo experimental models and suggest the association of specific PCV2 genogroups with diseased and nondiseased pigs in the field.

Reconstitution of O-specific lipopolysaccharide expression in Burkholderia cenocepacia strain J2315, which is associated with transmissible infections in patients with cystic fibrosis

Burkholderia cenocepacia is an opportunistic bacterium that infects patients with cystic fibrosis. B. cenocepacia strains J2315, K56-2, C5424, and BC7 belong to the ET12 epidemic clone, which is transmissible among patients. We have previously shown that transposon mutants with insertions within the O antigen cluster of strain K56-2 are attenuated for survival in a rat model of lung infection. From the genomic DNA sequence of the O antigen-deficient strain J2315, we have identified an O antigen lipopolysaccharide (LPS) biosynthesis gene cluster that has an IS402 interrupting a predicted glycosyltransferase gene. A comparison with the other clonal isolates revealed that only strain K56-2, which produced O antigen and displayed serum resistance, lacked the insertion element inserted within the putative glycosyltransferase gene. We cloned the uninterrupted gene and additional flanking sequences from K56-2 and conjugated this plasmid into strains J2315, C5424, and BC7. All the exconjugants recovered the ability to form LPS O antigen. We also determined that the structure of the strain K56-2 O antigen repeat, which was absent from the LPS of strain J2315, consisted of a trisaccharide unit made of rhamnose and two N-acetylgalactosamine residues. The complexity of the gene organization of the K56-2 O antigen cluster was also investigated by reverse transcription-PCR, revealing several transcriptional units, one of which also contains genes involved in lipid A-core oligosaccharide biosynthesis.

Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit

One of the most common pathways for the export of O-specific lipopolysaccharide (LPS) across the plasma membrane requires the participation of the Wzx protein. Wzx belongs to a family of integral membrane proteins that share little conservation in their primary amino acid sequence, making it difficult to delineate functional domains. This paper reports the cloning and expression in Escherichia coli K-12 of various Wzx homologues from different bacteria as FLAG epitope-tagged protein fusions. A reconstitution system for O16 LPS synthesis was used to assess the ability of each Wzx protein to complement an E. coli K-12 Deltawzx mutant. The results demonstrate that Wzx proteins from O-antigen systems that use N-acetylglucosamine or N-acetylgalactosamine for the initiation of the biosynthesis of the O repeat can fully complement the formation of O16 LPS. Partial complementation was seen with Wzx from Pseudomonas aeruginosa, a system that uses N-acetylfucosamine in the initiation reaction. In contrast, there was negligible complementation with the Wzx protein from Salmonella enterica, a system in which galactose is the initiating sugar. These results support a model whereby the first sugar of the O repeat can be recognized by the O-antigen translocation machinery.

Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1)

In previous studies the authors cloned and characterized the DNA sequence of the regions at both ends of the O7-specific lipopolysaccharide (LPS) biosynthesis cluster of Escherichia coli VW187 (O7:K1), and identified the biosynthetic genes for dTDP-rhamnose and GDP-mannose, as well as one of the candidate glycosyltransferases. In this work the complete DNA sequence of a 6.9 kb intervening region is presented. Seven new ORFs were identified. All the functions required for the synthesis and transfer of the O7 LPS were assigned on the basis of complementation experiments of transposon insertion mutants, and amino acid sequence homology to proteins involved in LPS synthesis of other bacteria. Of the seven ORFs, two encoded membrane proteins that were homologous to the O-antigen translocase (Wzx) and polymerase (Wxy), two were involved in the biosynthesis of dTDP-N-acetylviosamine, and the remaining three showed homologies to sugar transferases. The O antigen chain length regulator gene wzz was also identified in the vicinity of the O7 polysaccharide cluster. O7-specific DNA primers were designed and tested for serotyping of O7 E. coli strains.

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110:identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.

Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1)

The O7-specific lipopolysaccharide (LPS) in strains of Escherichia coli consists of a repeating unit made of galactose, mannose, rhamnose, 4-acetamido-2,6-dideoxyglucose, and N-acetylglucosamine. We have recently cloned and characterized genetically the O7-specific LPS biosynthesis region (rfbEcO7) of the E. coli O7:K1 strain VW187 (C. L. Marolda, J. Welsh, L. Dafoe, and M. A. Valvano, J. Bacteriol. 172:3590-3599, 1990). In this study, we localized the gnd gene encoding gluconate-6-phosphate dehydrogenase at one end of the rfbEcO7 gene cluster and sequenced that end of the cluster. Three open reading frames (ORF) encoding polypeptides of 275, 464, and 453 amino acids were identified upstream of gndEcO7, all transcribed toward the gnd gene. ORF275 had 45% similarity at the protein level with ORF16.5, which occupies a similar position in the Salmonella enterica LT2 rfb region, and presumably encodes a nucleotide sugar transferase. The polypeptides encoded by ORFs 464 and 453 were expressed under the control of the ptac promoter and visualized in Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels and by maxicell analysis. ORF464 expressed GDP-mannose pyrophosphorylase and ORF453 encoded a phosphomannomutase, the enzymes for the biosynthesis pathway of GDP-mannose, one of the nucleotide sugar precursors for the formation of the O7 repeating unit. They were designated rfbMEcO7 and rfbKEcO7, respectively. The RfbMEcO7 polypeptide was homologous to the corresponding protein in S. enterica LT2, XanB of Xanthomonas campestris, and AlgA of Pseudomonas aeruginosa, all GDP-mannose pyrophosphorylases. RfbKEcO7 was very similar to CpsG of S. enterica LT2, an enzyme presumably involved in the biosynthesis of the capsular polysaccharide colanic acid, but quite different from the corresponding RfbK protein of S. enterica LT2.

Genome-Wide Association Study of Multiplex Schizophrenia Pedigrees

OBJECTIVE The authors used a genome-wide association study (GWAS) of multiply affected families to investigate the association of schizophrenia to common single-nucleotide polymorphisms (SNPs) and rare copy number variants (CNVs). METHOD The family sample included 2,461 individuals from 631 pedigrees (581 in the primary European-ancestry analyses). Association was tested for single SNPs and genetic pathways. Polygenic scores based on family study results were used to predict case-control status in the Schizophrenia Psychiatric GWAS Consortium (PGC) data set, and consistency of direction of effect with the family study was determined for top SNPs in the PGC GWAS analysis. Within-family segregation was examined for schizophrenia-associated rare CNVs. RESULTS No genome-wide significant associations were observed for single SNPs or for pathways. PGC case and control subjects had significantly different genome-wide polygenic scores (computed by weighting their genotypes by log-odds ratios from the family study) (best p=10-17, explaining 0.4% of the variance). Family study and PGC analyses had consistent directions for 37 of the 58 independent best PGC SNPs (p=0.024). The overall frequency of CNVs in regions with reported associations with schizophrenia (chromosomes 1q21.1, 15q13.3, 16p11.2, and 22q11.2 and the neurexin-1 gene [NRXN1]) was similar to previous case-control studies. NRXN1 deletions and 16p11.2 duplications (both of which were transmitted from parents) and 22q11.2 deletions (de novo in four cases) did not segregate with schizophrenia in families. CONCLUSIONS Many common SNPs are likely to contribute to schizophrenia risk, with substantial overlap in genetic risk factors between multiply affected families and cases in large case-control studies. Our findings are consistent with a role for specific CNVs in disease pathogenesis, but the partial segregation of some CNVs with schizophrenia suggests that researchers should exercise caution in using them for predictive genetic testing until their effects in diverse populations have been fully studied.

Detecting sequence dependent transcriptional pauses from RNA and protein number time series

Background: Evidence suggests that in prokaryotes sequence-dependent transcriptional pauses a?ect the dynamics of transcription and translation, as well as of small genetic circuits. So far, a few pause-prone sequences have been identi?ed from in vitro measurements of transcription elongation kinetics.

Results: Using a stochastic model of gene expression at the nucleotide and codon levels with realistic parameter values, we investigate three di?erent but related questions and present statistical methods for their analysis. First, we show that information from in vivo RNA and protein temporal numbers is su?cient to discriminate between models with and without a pause site in their coding sequence. Second, we demonstrate that it is possible to separate a large variety of models from each other with pauses of various durations and locations in the template by means of a hierarchical clustering and a random forest classi?er. Third, we introduce an approximate likelihood function that allows to estimate the location of a pause site.

Conclusions: This method can aid in detecting unknown pause-prone sequences from temporal measurements of RNA and protein numbers at a genome-wide scale and thus elucidate possible roles that these sequences play in the dynamics of genetic networks and phenotype.

An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of ‘Pathogenic’, ‘Commensal’ and Antibiotic Resistant Strains

The Gram-positive bacterium Propionibacterium acnes is a member of the normal human skin microbiota and is associated with various infections and clinical conditions. There is tentative evidence to suggest that certain lineages may be associated with disease and others with health. We recently described a multilocus sequence typing scheme (MLST) for P. acnes based on seven housekeeping genes (http://pubmlst.org/pacnes). We now describe an expanded eight gene version based on six housekeeping genes and two ‘putative virulence’ genes (eMLST) that provides improved high resolution
typing (91eSTs from 285 isolates), and generates phylogenies congruent with those based on whole genome analysis. When compared with the nine gene MLST scheme developed at the University of Bath, UK, and utilised by researchers at Aarhus University, Denmark, the eMLST method offers greater resolution. Using the scheme, we examined 208 isolates from disparate clinical sources, and 77 isolates from healthy skin. Acne was predominately associated with type IA₁ clonal complexes CC1, CC3 and CC4; with eST1 and eST3 lineages being highly represented. In contrast, type IA₂ strains were recovered at a rate similar to type IB and II organisms. Ophthalmic infections were predominately associated with type IA₁ and IA₂ strains, while type IB and II were more frequently recovered from soft tissue and retrieved medical devices. Strains with rRNA mutations conferring resistance to antibiotics used in acne treatment were dominated by eST3, with some evidence for intercontinental spread. In contrast, despite its high association with acne, only a small number of resistant CC1 eSTs were identified. A number of eSTs were only recovered from healthy skin, particularly eSTs representing CC72 (type II) and CC77 (type III). Collectively our data lends support to the view that pathogenic versus truly commensal lineages of P. acnes may exist. This is likely to have important therapeutic and diagnostic implications.

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.