Brian P. Dalrymple

Genome-Wide Analysis of the World's Sheep Breeds Reveals High Levels of Historic Mixture and Strong Recent Selection

Genomic structure in a global collection of domesticated sheep reveals a history of artificial selection for horn loss and traits relating to pigmentation, reproduction, and body size.

A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small regulatory RNAs characterized in chickens we used a deep sequencing approach developed by Solexa (now Illumina Inc.). We sequenced three small RNA libraries prepared from different developmental stages of the chicken embryo (days five, seven, and nine) to produce over 9.5 million short sequence reads. We developed a bioinformatics pipeline to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected almost all of the previously known chicken miRNAs and their respective miRNA* sequences. In addition we discovered 449 new chicken miRNAs including 88 miRNA candidates. Of these, 430 miRNAs appear to be specific to the avian lineage. Another six new miRNAs had evidence of evolutionary conservation in at least one vertebrate species outside of the bird lineage. The remaining 13 putative miRNAs appear to represent chicken orthologs of known vertebrate miRNAs. We discovered 39 additional putative miRNA candidates originating from miRNA generating intronic sequences known as mirtrons.

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 β 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 α (DnaE) subunit of E. coli DNA polymerase III to β at μM 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.

A Genome Wide Survey of SNP Variation Reveals the Genetic Structure of Sheep Breeds

The genetic structure of sheep reflects their domestication and subsequent formation into discrete breeds. Understanding genetic structure is essential for achieving genetic improvement through genome-wide association studies, genomic selection and the dissection of quantitative traits. After identifying the first genome-wide set of SNP for sheep, we report on levels of genetic variability both within and between a diverse sample of ovine populations. Then, using cluster analysis and the partitioning of genetic variation, we demonstrate sheep are characterised by weak phylogeographic structure, overlapping genetic similarity and generally low differentiation which is consistent with their short evolutionary history. The degree of population substructure was, however, sufficient to cluster individuals based on geographic origin and known breed history. Specifically, African and Asian populations clustered separately from breeds of European origin sampled from Australia, New Zealand, Europe and North America. Furthermore, we demonstrate the presence of stratification within some, but not all, ovine breeds. The results emphasize that careful documentation of genetic structure will be an essential prerequisite when mapping the genetic basis of complex traits. Furthermore, the identification of a subset of SNP able to assign individuals into broad groupings demonstrates even a small panel of markers may be suitable for applications such as traceability.

The sheep genome illuminates biology of the rumen and lipid metabolism

A genome for ewe and ewe Sheep-specific genetic changes underlie differences in lipid metabolism between sheep and other mammals, and may have contributed to the production of wool. Jiang et al. sequenced the genome of two Texel sheep, a breed that produces high-value meat, milk, and wool. The genome information will provide an important resource for livestock production and aid in the understanding of mammalian evolution. Science, this issue p. 1168 A genomic analysis of sheep explains specializations in digestive system physiology and wool production. Sheep (Ovis aries) are a major source of meat, milk, and fiber in the form of wool and represent a distinct class of animals that have a specialized digestive organ, the rumen, that carries out the initial digestion of plant material. We have developed and analyzed a high-quality reference sheep genome and transcriptomes from 40 different tissues. We identified highly expressed genes encoding keratin cross-linking proteins associated with rumen evolution. We also identified genes involved in lipid metabolism that had been amplified and/or had altered tissue expression patterns. This may be in response to changes in the barrier lipids of the skin, an interaction between lipid metabolism and wool synthesis, and an increased role of volatile fatty acids in ruminants compared with nonruminant animals.

High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis

A high osmolarity electroporation method has been developed for the efficient transformation of Bacillus subtilis and B. licheniformis. The presence of high concentrations of the osmoticums, sorbitol and mannitol, in the electroporation, growth and recovery media resulted in an approximately 5000-fold increase in the transformation efficiency of B. subtilis, with a maximum value of 1.4×106 transformants per μg DNA. The transformation efficiency increased with electric field strength in the range of 12–23 kV/cm using the high osmolarity method. This enhanced transformation efficiency correlated with improved cell survival rate, indicating that the high osmolarity media have some protective action on the electrically injured cells. The application of the optimised high osmolarity method to transformation of B. licheniformis resulted in an increase in transformation efficiency by approximately 400-folds, with a maximum value of 1.8×104 transformants per μg DNA. As with B. subtilis, the highest transformation efficiency of B. licheniformis was achieved at a very high field strength (21 kV/cm). These results indicate that a combination of high osmolarity and high field strength may be a useful approach to improving the transformation efficiency of some gram-positive bacterial species which are able to grow in a high osmolarity medium.

A differential wiring analysis of expression data correctly identifies the gene containing the causal mutation.

Transcription factor (TF) regulation is often post-translational. TF modifications such as reversible phosphorylation and missense mutations, which can act independent of TF expression level, are overlooked by differential expression analysis. Using bovine Piedmontese myostatin mutants as proof-of-concept, we propose a new algorithm that correctly identifies the gene containing the causal mutation from microarray data alone. The myostatin mutation releases the brakes on Piedmontese muscle growth by translating a dysfunctional protein. Compared to a less muscular non-mutant breed we find that myostatin is not differentially expressed at any of ten developmental time points. Despite this challenge, the algorithm identifies the myostatin ‘smoking gun’ through a coordinated, simultaneous, weighted integration of three sources of microarray information: transcript abundance, differential expression, and differential wiring. By asking the novel question “which regulator is cumulatively most differentially wired to the abundant most differentially expressed genes?” it yields the correct answer, “myostatin”. Our new approach identifies causal regulatory changes by globally contrasting co-expression network dynamics. The entirely data-driven ‘weighting’ procedure emphasises regulatory movement relative to the phenotypically relevant part of the network. In contrast to other published methods that compare co-expression networks, significance testing is not used to eliminate connections.

The development of a recombinant Babesia vaccine.

Crude extracts of Babesia bovis parasites were shown to induce levels of protection in susceptible cattle equivalent to that resulting from natural infection. The crude material was systematically fractionated and tested in numerous sequential vaccination/challenge experiments in adult cattle. Antigens in protective fractions were then purified by affinity chromatography with monoclonal antibodies. Three highly protective (more than 95% reduction in parasitaemias) antigens were thus identified. None of these antigens was immunodominant; a number of immunodominant antigens were identified and all were immunosuppressive and/or non-protective. The three protective antigens were cloned and expressed as either beta-galactosidase or glutathione-S-transferase (GST) fusion proteins. Two of these, GST-12D3 and GST-11C5, when used in combination were almost as protective as has been previously shown for the commercially available live attenuated vaccine. A short fragment of a third antigen (21B4) has also been shown to be protective. In two of the antigens, repetitive segments have been shown to be non-protective while the third antigen (12D3) does not contain repetitive domains. Homologues of these antigens exist in other Babesia species and it is anticipated that these may be candidate antigens for protective vaccines against those species.

Origin, Evolution, and Biological Role of miRNA Cluster in DLK-DIO3 Genomic Region in Placental Mammals

MicroRNAs (miRNAs) are a rapidly growing family of small regulatory RNAs modulating gene expression in plants and animals. In animals, most of the miRNAs discovered in early studies were found to be evolutionarily conserved across the whole kingdom. More recent studies, however, have identified many miRNAs that are specific to a particular group of organisms or even a single species. These present a question about evolution of the individual miRNAs and their role in establishing and maintaining lineage-specific functions and characteristics. In this study, we describe a detailed analysis of the miRNA cluster (hereafter mir-379/mir-656 cluster) located within the imprinted DLK-DIO3 region on human chromosome 14. We show that orthologous miRNA clusters are present in all sequenced genomes of the placental (eutherian) mammals but not in the marsupial (metatherian), monotreme (prototherian), or any other vertebrate genomes. We provide evidence that the locus encompassing this cluster emerged in an early eutherian ancestor prior to the radiation of modern placental mammals by tandem duplication of the ancient precursor sequence. The original amplified cluster may have contained in excess of 250 miRNA precursor sequences, most of which now appear to be inactive. Examination of the eutherian genomes showed that the cluster has been maintained in evolution for approximately 100 Myr. Analysis of genes that contain predicted evolutionarily conserved targets for miRNAs from this cluster revealed significant overrepresentation of the Gene Ontology terms associated with biological processes such as neurogenesis, embryonic development, transcriptional regulation, and RNA metabolism. Consistent with these findings, a survey of the miRNA expression data within the cluster demonstrates a strong bias toward brain and placenta samples from adult organisms and some embryonic tissues. Our results suggest that emergence of the mir-379/mir-656 miRNA cluster was one of the factors that facilitated evolution of the placental mammals. Overrepresentation of genes involved in regulation of neurogenesis among predicted miRNAs targets indicates an important role of the mir-379/mir-656 cluster in this biological process in the placental mammals.

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.