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B P Dalrymple

Publications and source records attributed to B P Dalrymple.

At least 19 recordsLinked to original sources
PubMed2005

The transcriptional landscape of the mammalian genome.

This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

3' Untranslated Regions↗
PubMed2004

A rapid method for computationally inferring transcriptome coverage and microarray sensitivity.

MOTIVATION: There are many different gene expression technologies, including cDNA and oligo-based microarrays, SAGE and MPSS. For each organism of interest, coverage of the transcriptome and the genome will be different. We address the question of what level of coverage is required to exploit the sensitivity of the different technologies, and what is the sensitivity of the different approaches in the experimental study. RESULTS: We estimate the transcriptome coverage by randomly sampling transcripts from a pre-defined tag-to-gene mapping function. For a given microarray experiment, we locate the thresholds in intensities that define the distribution of transcript abundance. These values are compared against the distribution obtained by applying the same thresholds to the intensities from differentially expressed genes. The ratio of these two distributions meets at the equilibrium defining sensitivity. We conclude that a collection of approximately 340,000 sequences is adequate for microarrays, but not large enough for maximum utilization of tag-based technologies. In the absence of large-scale sequencing, the majority of the tags detected by the latter approaches will remain unidentified until the genome sequence is available.

Algorithms↗
PubMed2003

A mixture model-based cluster analysis of DNA microarray gene expression data on Brahman and Brahman composite steers fed high-, medium-, and low-quality diets.

The objective of this study is to explore aspects of the statistical analysis of gene expression response at the muscle tissue level to varying levels of energy and protein in the diet. Eleven Brahman and Brahman composite steers (weighing 302 +/- 9.8 kg, on average) were allocated randomly into high- (HIGH), medium- (MED), and low- (LOW) quality forage diets for 27 d. After this period, a biopsy of the longissimus dorsi muscle was taken from each animal and total RNA was extracted to generate the labeled target for microarray experimentation. These targets were hybridized to a complementary DNA (cDNA) microarray of 9,274 probes from cattle muscle and subcutaneous fat cDNA libraries. After edits, 151,904 expression intensity levels of 4,747 genes were analyzed. Emphasis was given to the choice of power transformation of the intensity channel readings and to the consistency of readings within each diet quality group. The statistical approach to isolate differentially expressed genes was based on model-based clustering via a mixture of normal distributions estimated through maximal likelihood. The base-2 logarithm was found to be the optimal power transformation to normalize gene intensity levels. A two-sample t-statistic was defined as a measure of possible differential expression. For each of the three diet contrasts, HIGH vs. LOW, HIGH vs. MED, and MED vs. LOW, three clusters were found, two of which contained more than 94% genes with almost no altered gene expression levels, whereas the third cluster contained the remaining genes with a differential expression. Results from the HIGH vs. LOW contrast identified 27 genes with a greater than 95% posterior probability of belonging to the cluster of differentially expressed genes.

Animal Feed↗
PubMed2002

Transformation and expression of an anaerobic fungal xylanase in several strains of the rumen bacterium Butyrivibrio fibrisolvens.

AIMS: To obtain reliable transformation of a range of Butyrivibrio fibrisolvens strains and to express a Neocallimastix patriciarum xylanase gene in the recipients. METHODS AND RESULTS: Eight strains (H17c, E14, LP1309, LP1028, AR11a, OB156, LP210B and LP461A) of Bu. fibrisolvens were transformed by the Gram-positive vector pUB110. A xylanase expression/secretion cassette containing Bu. fibrisolvens promoter and signal peptide elements fused to catalytic domain II of the N. patriciarum xylanase A cDNA (xynANp) was inserted into pUB110 to create the plasmid pUBxynA. pUBxynA was used to transform seven of the Bu. fibrisolvens strains transformed by pUB110. In strain H17c pUBxynA, which produced native xylanase, 2.46 U mg-1 total xylanase activity was produced with 45% extracellular xylanase. In strain H17c pUMSX, 0.74 U mg-1 total xylanase activity was produced with 98% extracellular xylanase. H17c pUBxynA exhibited increased (28.7%) degradation of neutral detergent fibre compared with unmodified H17c; however, progressive loss of pUBxynA was observed in long-term cultivation. CONCLUSIONS: A stable transformation system was developed that was applicable for a range of Bu. fibrisolvens strains and high levels of expression of a recombinant xylanase were obtained in H17c which lead to increased fibre digestion. SIGNIFICANCE AND IMPACT OF THE STUDY: This stable transformation system with the accompanying recombinant plasmids will be a useful tool for further investigation aimed at improving ruminal fibre digestion.

Anaerobiosis↗
PubMed2001

A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems.

The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. The clamp proteins also participate in a variety of dynamic and competing protein-protein interactions. However, clamp-protein binding sequences have not so far been identified in the eubacteria. Here we show from three lines of evidence, bioinformatics, yeast two-hybrid analysis, and inhibition of protein-protein interaction by modified peptides, that variants of a pentapeptide motif (consensus QL[SD]LF) are sufficient to enable interaction of a number of proteins with an archetypal eubacterial sliding clamp (the beta subunit of Escherichia coli DNA polymerase III holoenzyme). Representatives of this motif are present in most sequenced members of the eubacterial DnaE, PolC, PolB, DinB, and UmuC families of DNA polymerases and the MutS1 mismatch repair protein family. The component tripeptide DLF inhibits the binding of the alpha (DnaE) subunit of E. coli DNA polymerase III to beta at microM concentration, identifying key residues. Comparison of the eubacterial, eukaryotic, and archaeal sliding clamp binding motifs suggests that the basic interactions have been conserved across the evolutionary landscape.

Amino Acid Sequence↗
PubMed2000

Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin.

The substrate specificity of porcine pepsin has been altered by site-directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys-Pro-Xaa-Yaa-Phe*Nph-Arg-Leu (where Xaa is Ile or Pro, Yaa is Glu. Leu, Gln or Lys, Nph is p-nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two- to fourfold higher kcat/Km, compared with wild-type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild-type pepsin cleaved a peptide corresponding to the carboxy-terminal telopeptide region of bovine type I collagen alpha1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp-Leu, Leu-Ser, and Phe-Leu) and at a significantly lower rate at Ser-Phe. However, Thr222Val/Glu287Met cleaved site Ser-Phe at a rate 20-fold higher than the wild-type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu-Ser (similar to cathepsin D) and at a rate 23-fold higher than the wild-type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage.

Amino Acid Sequence↗
PubMed1999

An acetylxylan esterase and a xylanase expressed from genes cloned from the ruminal fungus Neocallimastix patriciarum act synergistically to degrade acetylated xylans.

A Neocallimastit patriciarum acetylxylan esterase (BnaA) was expressed from the cloned gene in Escherichia coli. Purified recombinant BnaA efficiently released acetate from soluble acetylated birchwood xylan (ABX), with a specific activity of 76 U mg-1. In contrast, release of acetate was very inefficient from the insoluble substrates, spear grass and delignified spear grass. Addition of a recombinant xylanase, XynA, also expressed from a cloned N. patriciarum gene, had no effect on the release of acetate from ABX. However, the combination of recombinant BnaA and XynA released more acetate from spear grass and delignified spear grass than did BnaA alone. Significantly more reducing sugar was also released from all three substrates by the combination of recombinant XynA and BnaA than by XynA alone. Thus the extent of digestion of acetylated xylans by XynA appears to be limited by the acetylation. In this system BnaA does not appear to increase the rate of cleavage of insoluble substrates by XynA, but probably allows the release of shorter xylose oligomers from already solubilised acetylated xylan polymers.

Acetates↗
PubMed1999

Distribution and evolution of the xylanase genes xynA and xynB and their homologues in strains of Butyrivibrio fibrisolvens.

The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B. fibrisolvens.

Animals↗
PubMed1997

Improvement of expression and secretion of a fungal xylanase in the rumen bacterium Butyrivibrio fibrisolvens OB156 by manipulation of promoter and signal sequences.

Promoters and signal sequences for expression and secretion of a fungal xylanase encoded by a modified Neocallimastix patriciarum xynA cDNA in the rumen bacterium, Butyrivibrio fibrisolvens OB156, were investigated. Successful expression of the fungal xylanase in OB156 was obtained using the putative xylanase promoter from B. fibrisolvens strain 49. Replacing the putative -35 region sequence (TTGCAC) of the xylanase promoter with the sequence TTGACA by mutagenesis reduced the fungal xylanase expression level 4-fold in OB156, indicating that this B. fibrisolvens strain did not efficiently recognise the E. coli consensus -35 sequence. Reduction of the spacer length between the -35 and -10 regions of the xylanase promoter from 18 to 17 base-pairs (bp) considerably increased the expression levels of the fungal enzyme in both E. coli and OB156. Insertion of a pUB110 mob promoter upstream of the xylanase promoter also significantly improved the fungal xylanase expression. Secretion of the fungal xylanase mediated by the alpha-amylase signal peptide from B. fibrisolvens strain H17c was efficient in E. coli, but very poor in OB156. An increase in the hydrophobicity of the signal sequence resulted in a 4-fold increase in the extracellular portion of the fungal xylanase in OB156, indicating marked improvement in xylanase secretion efficiency. The recombinant plasmids and xylanase expression/secretion cassettes were found to be stable in OB156 after prolonged cultivation (100 generations) in the absence of antibiotic selection. These results suggest that the rumen bacterium B. fibrisolvens can be manipulated to produce and secrete a eukaryotic extracellular protein with stable maintenance of the expression cassette in plasmid form.

Amino Acid Sequence↗
PubMed1997

PCR methods for the discrimination of Babesia bovis isolates.

Three different polymerase chain reaction assays for the typing of isolates of Babesia bovis have been developed and compared with a hybridisation based method. Primers were designed within conserved regions flanking the variable length tandem repeats of the Bv80 and BvVA1 genes. For the long array of repeats in BvVA1, up to 7.5 kb, a modified long template PCR method was developed. The assays were compared using ten independent isolates of Babesia bovis. Using the BvVA1 and Bv80 PCR assays, 13 and 10 genotypes could be discriminated, respectively, with some isolates containing several genotypes. Combining the two PCR assays, 17 genotypes were identified within the ten Babesia bovis isolates. Whilst simpler and requiring less DNA, the BvVA1 PCR analysis exhibited significant bias towards some genotypes of the BvVA1 repeats. Further discrimination of BvVA1 PCR products was achieved using AccI digests producing population specific ladders. Genomic DNA fingerprints were also generated by PCR of DNA using an arbitrary primer (randomly amplified polymorphic DNA, RAPD) revealing polymorphic genotypes that were isolate specific. No amplification of host DNA resulted from any of the three PCR procedures. Babesia bigemina DNA was not amplified by the Bv80 or BvVA1 primers. Applications demonstrating changes in composition of populations of Babesia bovis parasites during attenuation and prolonged culture maintenance are described.

Animals↗
PubMed1996

Cloning of a gene encoding cinnamoyl ester hydrolase from the ruminal bacterium Butyrivibrio fibrisolvens E14 by a novel method.

A gene (cinI) encoding a cinnamoyl ester hydrolase (CEH) has been isolated from the ruminal bacterium, Butyrivibrio fibrisolvens E14, using a model substrate, MUTMAC [4-methylumbelliferoyl (p-trimethylammonium cinnamate chloride)]. CinI has significant amino-acid similarities with members of a large and diverse family of hydrolases with a serine/aspartic acid/histidine catalytic triad. Our analyses identified two previously unclassified amino acid sequences, the amino-terminal domain of unknown function in XynZ from Clostridium thermocellum and XynC, an acetylxylan esterase from Caldicellulosiruptor saccharolyticus, as members of the same family of hydrolases. A previously described esterase with CEH activity, XylD from Pseudomonas fluorescens ssp. cellulosa, is not similar to CinI. CinI was expressed at high levels in the periplasmic fraction of E. coli TOPP2 and released ferulic acid from Fara [5-O-(trans-feruloyl)-arabinofuranose] prepared from wheat bran.

Amino Acid Sequence↗
PubMed1994

Sequencing and expression of the aroA gene from Dichelobacter nodosus.

The aroA locus of the Gram- pathogen Dichelobacter nodosus, which encodes 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, has been sequenced and expressed in Escherichia coli. The gene is located on a 1.48-kb DraI-HindIII fragment located directly upstream and in opposite transcriptional orientation to the gene encoding the fimbrial structural subunit. The deduced open reading frame is 1329 nucleotides in length, which encodes a protein of 443 amino acids (aa) with a calculated M(r) of 47,413, which was visualized in E. coli minicells, under the control of its native promoter. This derived aa sequence displays significant similarities with the sequences of the aroA gene products from a variety of microorganisms.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1993

Molecular variation and diversity in candidate vaccine antigens from Babesia.

Recombinant vaccines are being developed against a number of species of protozoan parasites in the genus Babesia. Protozoan parasites are notorious for their diversity of strains and their ability to express families of equivalent, but antigenically distinct, surface proteins. In order to reduce the likelihood of evasion of the immune response induced by a recombinant vaccine, ideal components should be essential proteins encoded by single copy genes. The proteins should also have a limited ability to tolerate polymorphism in amino acid sequence, especially in critical epitopes. While little is known about the function of the candidate protective antigens, there is now considerable information concerning the variation of a number of candidate vaccine antigens from several species of Babesia. Four of the well studied antigens are all members of multi-gene families. The members of the VMSA gene family of Babesia bovis are also highly polymorphic in sequence. The members of the Bv60/p58 family of rhoptry protein homologues exhibit more limited polymorphism within a single species of Babesia. However, comparison of the sequences of the equivalent proteins and the organisation of the corresponding genes from B. bovis, Babesia bigemina, Babesia canis and Babesia ovis suggests that members of this family have the potential to acquire and to tolerate substantial polymorphism in amino acid sequence. The choice of protein, and particular region of the protein, suitable for incorporation in a recombinant vaccine may require extensive analysis of the genetic systems encoding the candidate antigens.

Amino Acid Sequence↗
PubMed1993

Characterisation of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, Babesia ovis and Babesia canis.

A monoclonal antibody that had been raised against a protease-containing fraction of Babesia bovis, and shown to bind to a protein located in the rhoptries, was used to screen a B. bovis cDNA expression library. The sequence of the protein encoded by a positive clone was almost identical to the equivalent region of a previously described B. bovis 60-kDa rhoptry protein (Bv60). A tandem repeat of the gene encoding Bv60 was identified in all Australian isolates of B. bovis examined. Genes encoding homologous of Bv60 were cloned from Babesia ovis and Babesia canis. In B. ovis, 5 closely linked genes were identified. Four of these genes appeared to encode very similar proteins (Bo60.1-4). The protein (Bo60.5) encoded by the fifth B. ovis gene had 72% amino acid identity to Bo60.1-4 in the amino-terminal 306 amino acids, but no significant similarities in the carboxy-terminal region. In B. canis one gene (Bc60.2) was sequenced and a second closely linked gene was identified. A further member of the family, p58, has also been described previously from Babesia bigemina. Tandemly repeated genes subject to extensive gene conversion appear to be a feature of this family of babesial rhoptry protein homologous. No proteins significantly related to any members of the gene family were identified in a search of translated DNA and protein sequence databases. Thus the function of this family of proteins remains a matter for speculation.

Amino Acid Sequence↗
PubMed1993

Cloning and characterisation of cDNA clones encoding two Babesia bovis proteins with homologous amino- and carboxy-terminal domains.

A dextran sulphate protein (DSP) fraction derived from Babesia bovis has previously been shown to induce a protective immune response in cattle. A B. bovis cDNA library was screened with both the complete anti-DSP serum and a subfraction of the anti-DSP serum affinity purified on a native B. bovis protein of approx. 80 kDa. cDNA clones encoding two different B. bovis proteins were identified. The product of one gene, Bv80, has a single divergent copy of a sequence of 149 amino acids (approx. 30% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are separated by an array of short variant repeat sequences rich in proline and glutamic acid. The product of the other gene, BvVAl (homologous to the previously described 225-kDa B. bovis protein)[19], is predicted to have a single divergent copy of a sequence of 170-171 amino acids (approx. 35% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are also separated by an array of repeats. The 73-amino acid repeat unit of this array is composed of a number of variant derivatives of shorter repeat units. Detailed analysis of genomic clones flanking two alleles of the gene encoding BvVAl/225 kDa identified further members of a multi-gene family. This region of the genome of B. bovis has been subject to a large number of amplification processes.

Amino Acid Sequence↗
PubMed1992

Characterization of a cDNA clone from the haemoparasite Babesia bovis encoding a protein containing an "HMG-Box".

The complete nucleotide sequence of a Babesia bovis cDNA clone encoding a protein containing an HMG-Box has been determined. The predicted protein of 97 amino acids has a molecular weight of 11,116. It exhibits approximately 45% overall amino acid identity with the Saccharomyces cerevisiae non-histone protein 6A (NHP6A) and approximately 57% identity in the HMG-Box. The B. bovis protein has been designated NHP1. Like HNP6A, and unlike most other HMG1 homologues, NHP1 does not have a basic or an acidic carboxy-terminal domain. The amino acid sequence of HNP1 is much less similar to HMG1 homologues of another protozoan, Tetrahymena thermophila, than to the HMG1 homologues identified in S. cerevisiae, plants and vertebrates. This suggests that the T. thermophila proteins may not be true HMG1 homologues, or that they may be evolving at a much faster rate.

Amino Acid Sequence↗