Biomedical subjects

Brian P Dalrymple

Publications and source records attributed to Brian P Dalrymple.

11 recordsLinked to original sources

PubMed2006

A gene coexpression network for bovine skeletal muscle inferred from microarray data.

We present the application of large-scale multivariate mixed-model equations to the joint analysis of nine gene expression experiments in beef cattle muscle and fat tissues with a total of 147 hybridizations, and we explore 47 experimental conditions or treatments. Using a correlation-based method, we constructed a gene network for 822 genes. Modules of muscle structural proteins and enzymes, extracellular matrix, fat metabolism, and protein synthesis were clearly evident. Detailed analysis of the network identified groupings of proteins on the basis of physical association. For example, expression of three components of the z-disk, MYOZ1, TCAP, and PDLIM3, was significantly correlated. In contrast, expression of these z-disk proteins was not highly correlated with the expression of a cluster of thick (myosins) and thin (actin and tropomyosins) filament proteins or of titin, the third major filament system. However, expression of titin was itself not significantly correlated with the cluster of thick and thin filament proteins and enzymes. Correlation in expression of many fast-twitch muscle structural proteins and enzymes was observed, but slow-twitch-specific proteins were not correlated with the fast-twitch proteins or with each other. In addition, a number of significant associations between genes and transcription factors were also identified. Our results not only recapitulate the known biology of muscle but have also started to reveal some of the underlying associations between and within the structural components of skeletal muscle.

Adipose Tissue↗

PubMed2006

Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

Amino Acid Motifs↗

PubMed2006

Simultaneous identification of differential gene expression and connectivity in inflammation, adipogenesis and cancer.

MOTIVATION: Biological differences between classes are reflected in transcriptional changes which in turn affect the levels by which essential genes are individually expressed and collectively connected. The purpose of this communication is to introduce an analytical procedure to simultaneously identify genes that are differentially expressed (DE) as well as differentially connected (DC) in two or more classes of interest. RESULTS: Our procedure is based on a two-step approach: First, mixed-model equations are applied to obtain the normalized expression levels of each gene in each class treatment. These normalized expressions form the basis to compute a measure of (possible) DE as well as the correlation structure existing among genes. Second, a two-component mixture of bi-variate distributions is fitted to identify the component that encapsulates those genes that are DE and/or DC. We demonstrate our approach using three distinct datasets including a human systemic inflammation oligonucleotide data; a spotted cDNA data dealing with bovine in vitro adipogenesis and SAGE database on cancerous and normal tissue samples.

Adipogenesis↗

PubMed2005

Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs.

BACKGROUND: The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin. RESULTS: To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene. CONCLUSION: We have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.

Animals↗

PubMed2004

Validation of alternative methods of data normalization in gene co-expression studies.

MOTIVATION: Clusters of genes encoding proteins with related functions, or in the same regulatory network, often exhibit expression patterns that are correlated over a large number of conditions. Protein associations and gene regulatory networks can be modelled from expression data. We address the question of which of several normalization methods is optimal prior to computing the correlation of the expression profiles between every pair of genes. RESULTS: We use gene expression data from five experiments with a total of 78 hybridizations and 23 diverse conditions. Nine methods of data normalization are explored based on all possible combinations of normalization techniques according to between and within gene and experiment variation. We compare the resulting empirical distribution of gene x gene correlations with the expectations and apply cross-validation to test the performance of each method in predicting accurate functional annotation. We conclude that normalization methods based on mixed-model equations are optimal.

Algorithms↗

PubMed2004

Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins.

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

Amino Acid Motifs↗

PubMed2004

An interactive bovine in silico SNP database (IBISS).

An interactive bovine in silico SNP (IBISS) database has been created through the clustering and aligning of bovine EST and mRNA sequences. Approximately 324,000 EST and mRNA sequences were clustered to produce 29,965 clusters (producing 48,679 consensus sequences) and 48,565 singletons. A SNP screening regime was placed on variations detected in the multiple sequence alignment files to determine which SNPs are more likely to be real rather than sequencing errors. A small subset of predicted SNPs was validated on a diverse set of bovine DNA samples using PCR amplification and sequencing. Fifty percent of the predicted SNPs in the "putative >1" category were polymorphic in the population sampled. The IBISS database represents more than just a SNP database; it is also a genomic database containing uniformly annotated predicted gene mRNA and protein sequences, gene structure, and genomic organization information.

Animals↗

PubMed2002

Cellular localisation of the clamp protein during DNA replication.

The beta subunit of Escherichia coli DNA polymerase III holoenzyme was fused to the green fluorescent protein GFP. The gene fusion under the control of the heterologous lac promoter was used to replace the wild-type allele in the chromosome. The formation of GFP-beta fluorescent foci in GFP-beta expressing cells required DNA replication and their number per cell was dependent on cell growth. Examination of GFP-beta foci in a synchronous round of replication suggested that DNA replication was accompanied by the recruitment of GFP-beta foci near the midcell, followed by the rapid migration of the foci in opposite directions to the 1/4 and 3/4 positions during DNA replication.

Chromosomes, Bacterial↗

PubMed1999

16S rDNA sequencing of Ruminococcus albus and Ruminococcus flavefaciens: design of a signature probe and its application in adult sheep.

The ruminococci are an important group of fibrolytic bacteria inhabiting the rumen. Seventeen strains of presumptively identified Ruminococcus were evaluated by a combination of nearly complete and partial 16S rDNA sequence that identified all strains as either Ruminococcus albus or Ruminococcus flavefaciens. All sequences fell into cluster IV of the clostridia, while other species of ruminococci (e.g. Ruminococcus obeum, Ruminococcus gnavus, Ruminococcus lactaris) fall into cluster XIVa of the clostridia. Ruminococcus cluster IV sequences were used to design a 16S rRNA oligonucleotide probe to assess the relative abundance of target populations in a stable ruminal environment. A stable population (animals fed eight times per day) was established in sheep so that statistically robust comparisons could be made in the absence of variation due to diurnal rumen fluctuations. The steady state populations were sampled six times over a 24 d period and direct microscopic counts (DC), total culturable counts (TCC), and total cellulolytic counts (CEL) were determined. DC and culturable data (TCC and CEL) were compared with relative abundance estimates of Ruminococcus IV and Fibrobacter succinogenes. A combination of the Ruminococcus and F. succinogenes probes accounted for 4.0% of the bacterial population and cellulolytic bacteria (measured by most-probable numbers) were 5.2% of the total culturable count. These data suggest that a major portion of the Ruminococcus and Fibrobacter diversity has been cultured and is represented by available sequences. Steady state populations were measured over several days in three sheep and an estimate of variation in DC, TCC, CEL and 16S-based data were obtained. These variance estimates could be used to determine the theoretical sample sizes required to obtain statistically significant differences under different experimental conditions.

Animals↗

PubMed1997

Expression of a Butyrivibrio fibrisolvens E14 gene (cinB) encoding an enzyme with cinnamoyl ester hydrolase activity is negatively regulated by the product of an adjacent gene (cinR).

A second cinnamoyl ester hydrolase (CEH) encoding gene (cinB) has been characterized from the ruminal bacterium Butyrivibrio fibrisolvens E14. CinB is more similar to CinA (previously named CinI) (28% amino acid identify), the first CEH described from B. fibrisolvens E14, than either of the enzymes are to any other member of the family of hydrolases to which they belong. Upstream of cinB, and in the opposite orientation, is a gene (cinR) encoding a protein with substantial similarity to members of the MarR family of negative regulators of bacterial gene expression. By alignment of these sequences, a possible helix-turn-helix DNA-binding domain has been identified. CinR was expressed at a high level in Escherichia coli using the lac promoter. In E. coli CinR repressed the expression of CinB, but had no effect on the expression of CinA. In gel mobility-shift assays, CinR bound specifically to the cinR-cinB intergenic region. Two identical 16 nucleotide inverted repeats adjacent to the putative PcinR and PcinB promoters are likely binding sites for CinR. The addition of FAXX (O-[5-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1,3)- O-beta-D-xylopyranosyl-(1,4)-D-xylopyranose) and Fara [5-O-(trans-feruloyl)-arabinofuranose], but not xylobiose, ferulic acid and a number of other soluble components of hemicellulose, inhibited the binding of CinR to DNA.

Amino Acid Sequence↗

PubMed1997

Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases.

Acetylesterase and cinnamoyl ester hydrolase activities were demonstrated in culture supernatant of the anaerobic ruminal fungus Neocallimastix patriciarum. A cDNA expression library from N. patriciarum was screened for esterases using beta-naphthyl acetate and a model cinnamoyl ester compound. cDNA clones representing four different esterase genes (bnaA-D) were isolated. None of the enzymes had cinnamoyl ester hydrolase activity, but two of the enzymes (BnaA and BnaC) had acetylxylan esterase activity, bnaA, bnaB and bnaC encode proteins with several distinct domains. Carboxy-terminal repeats in BnaA and BnaC are homologous to protein-docking domains in other enzymes from Neocallimastix species and another anaerobic fungus, a Piromyces sp. The catalytic domains of BnaB and BnaC are members of a recently described family of Ser/His active site hydrolases [Upton, C. & Buckley, J.T. (1995). Trends Biochem Sci 20, 178-179]. BnaB exhibits 40% amino acid identity to a domain of unknown function in the CelE cellulase from Clostridium thermocellum and BnaC exhibits 52% amino acid identity to a domain of unknown function in the XynB xylanase from Ruminococcus flavefaciens. BnaA, whilst exhibiting less than 10% overall amino acid identity to BnaB or BnaC, or to any other known protein, appears to be a member of the same family of hydrolases, having the three universally conserved amino acid sequence motifs. Several other previously described esterases are also shown to be members of this family, including a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. However, none of the other previously described enzymes with acetylxylan esterase activity are members of this family of hydrolases.

Amino Acid Sequence↗