Power of a genome scan to detect and locate quantitative trait loci in cattle using dense single nucleotide polymorphisms.

There is increasing use of dense single nucleotide polymorphisms (SNPs) for whole-genome association studies (WGAS) in livestock to map and identify quantitative trait loci (QTL). These studies rely on linkage disequilibrium (LD) to detect an association between SNP genotypes and phenotypes. The power and precision of these WGAS are unknown, and will depend on the extent of LD in the experimental population. One complication for WGAS in livestock populations is that they typically consist of many paternal half-sib families, and in some cases full-sib families; unless this subtle population stratification is accounted for, many spurious associations may be reported. Our aim was to investigate the power, precision and false discovery rates of WGAS for QTL discovery, with a commercial SNP array, given existing patterns of LD in cattle. We also tested the efficiency of selective genotyping animals. A total of 365 cattle were genotyped for 9232 SNPs. We simulated a QTL effect as well as polygenic and environmental effects for all animals. One QTL was simulated on a randomly chosen SNP and accounted for 5%, 10% or 18% of the total variance. The power to detect a moderate-sized additive QTL (5% of the phenotypic variance) with 365 animals genotyped was 37% (p < 0.001). Most importantly, if pedigree structure was not accounted for, the number of false positives significantly increased above those expected by chance alone. Selective genotyping also resulted in a significant increase in false positives, even when pedigree structure was accounted for.

Analysis of developmentally regulated genes of the parasite Haemonchus contortus.

Differential regulation of gene expression in the development of Haemonchus contortus was analysed using RNA arbitrarily-primed PCR. A study of third-stage larval and adult H. contortus revealed large differences between the two stages; 32 and 30% unique third-stage larval and adult RNA arbitrarily-primed PCR products, respectively. This finding is consistent with a high degree of differential gene expression between these developmental stages. A number of adult products were sequenced, revealing 11 molecules to be similar to deposits within sequence databases. Four other molecules that did not have significant similarity to sequences in the databases may represent developmentally regulated genes specific to H. contortus. Northern analysis of the putative adult-expressed molecules with homologues in the databases confirmed that four were expressed only in adults, while four were expressed in both stages, but had different sized transcripts. This may reflect differential splicing, or expression of closely related but different molecules at different life cycle stages. Two molecules were present in mRNA populations from both stages, suggesting these were false stage-associated molecules. No transcript was detected for one molecule by Northern analysis, probably due to low level of expression. In situ hybridisation analysis was used to localise expression of transcripts in the adult parasite, in particular, to gain some insight into the nature of those molecules with no known predicted function.

Characterization of the mRNA encoding a proline-rich 37-kilodalton glycoprotein from the excretory-secretory products of Trichostrongylus colubriformis.

A glycoprotein, with apparent molecular weight in SDS-polyacrylamide gels of 37 kDa, has been isolated from the excretory-secretory (ES) products of the adult stage of Trichostrongylus colubriformis, a parasitic nematode. This protein is the major ES product recognized in immunoblots by lymph from a naturally infected sheep. A synthetic oligonucleotide, based on peptide sequence data from a digest of the purified protein was used to successfully screen a cDNA library. A cDNA clone was isolated which encoded a presumptive protein precursor of 220 amino acids that contained a 63 amino acid region of which more than 35% of the residues were proline, three peptide sequences determined from the natural component, and three potential N-glycosylation sites, consistent with the protein being isolated from the lectin-bound fraction of the adult ES products. The presumptive, processed, amino terminus encoded by the cDNA clone was preceded by a signal-like, hydrophobic-rich region of 16 amino acids.

Improved shuttle vectors for cloning and high-level Cu(2+)-mediated expression of foreign genes in yeast.

New yeast episomal vectors having a high degree of utility for cloning and expression in Saccharomyces cerevisiae are described. One vector, pYEULlacZ, is based on pUC19 and employs the pUC19 multiple cloning site for the selection of recombinants in Escherichia coli by lacZ inactivation. In addition, the vector contains two genes, URA3 and leu2-d, for selection of the plasmid in ura3 or leu2 yeast strains. The presence of the leu2-d gene appears to promote replication at high copy numbers. The introduction of CUP1 cassettes allows these plasmids to direct Cu(2+)-regulated production of foreign proteins in yeast. We show the production of a helminth antigen as an example of the vector application.

Characterization, cloning and host-protective activity of a 30-kilodalton glycoprotein secreted by the parasitic stages of Trichostrongylus colubriformis.

The helminth Trichostrongylus colubriformis is a parasitic nematode infecting the small intestine of sheep. We report the isolation and characterization of a 30-kDa glycoprotein capable of partially protecting guinea-pigs against the parasite. This glycoprotein is secreted by the L4 and adult parasitic stages of the worm. The sequence of three separate cDNA clones predicts the polypeptide to be about 15 kDa, with four N-linked carbohydrate chains and an internal disulphide bond. The clones also indicate the existence of sequence variability in this antigen. Limited sequence homology to a porcine intestinal peptide suggests an influence on host gut physiology.

Characterization of cDNA clones coding for muscle tropomyosin of the nematode Trichostrongylus colubriformis.

The host-protective antigen from detergent-solubilised extracts of the sheep intestinal helminth Trichostrongylus colubriformis has been identified as tropomyosin. Complementary DNA clones coding for T. colubriformis muscle tropomyosin have been isolated and characterised as the first step in obtaining recombinant protein to carry out more extensive vaccination trials. The clones represent an mRNA of 1544 bases, including a relatively long 5' untranslated sequence of 307 bases and a 3' non-coding region of 344 bases. The mRNA codes for a highly alpha-helical protein of 284 residues with a molecular weight of 33,000; characteristics typically observed for the muscle tropomyosins of higher organisms. The T. colubriformis protein has 58% sequence identity with rabbit and Drosophila melanogaster muscle tropomyosins, and the differences in the protein sequence are randomly distributed throughout the molecule. There is complete identity between the three sequences for the N-terminal 9 residues, the region believed to be essential for the polymerisation of tropomyosin molecules and for binding to actin and troponin.

Two families of abl-related transcripts in human haematopoietic cells differing in their homology to v-abl.

The transforming gene of the Abelson murine leukaemia virus, v-abl, contains two open reading frames (orf). The 5' orf encodes a tyrosine-specific protein kinase while the 3' orf has the capacity to code for an 18,000 Mr protein. However, no 3' orf product has yet been identified. Using probes capable of distinguishing between the 5' and 3' orfs of v-abl, we have examined the abl-related transcripts present in human haematopoietic cells and leukaemia-derived cell lines, including the chronic myeloid leukaemia-derived cell line K562. Our results indicate that transcripts of 6 kb, 7 kb and 8 kb (kilobase, 10(3) base-pairs) show strong homology to v-abl 5' protein kinase-encoding orf sequences, but are devoid of any sequences from the v-abl 3' orf. In addition, transcripts of 5 kb, 3 kb, 1.6 kb and 1.4 kb, reacting with both 5' orf and 3' orf probes, were observed. The latter species, with coding sequences from both the tyrosine kinase and the putative 18,000 Mr protein, must be transcribed from the human c-abl gene as this is apparently the only human gene containing sequences homologous to the v-abl 3' orf. The 6 kb, 7 kb and 8 kb transcripts may arise either from the c-abl gene through differential splicing, or from one of the three other regions of the human genome with sequences homologous to the 5' orf of v-abl. Examination of genomic DNA from the K562 cell line revealed that the amplification of abl-related sequences, which is presumed to result in the elevated levels of the 8 kb transcript found in this cell line, does not involve sequences homologous to the v-abl 3' orf. This lends credence to the idea that the 8 kb transcript may derive from an abl-related gene other than c-abl. While the significance of the 3' orf of v-abl remains unknown, the data presented strongly suggest the existence of at least two distinct abl-related proteins in human haematopoietic cells.

Changes in the expression of membrane antigens during the differentiation of chicken erythroblasts.

Chicken erythroblasts can be transformed by the avian retrovirus, avian erythroblastosis virus (AEV). Earlier studies have shown that the mechanism of transformation appears to involve a "block" in differentiation, in that when erythroblasts are transformed by a temperature-sensitive mutant of ts34 AEV and incubated at the nonpermissive temperature, the cells start to differentiate and produce hemoglobin. We have decided to use this system to isolate pure populations of chicken erythroblasts and raise monoclonal antibodies against their cell surface proteins. Three monoclonal antibodies were isolated and tested for their ability to bind to various hematopoietic cell types; two were shown to be erythroid-specific, whereas the other antibody bound to proliferating cells but not to erythrocytes or granulocytes. Of the erythroid-specific antibodies, one precipitated a 94,000 molecular weight protein, whereas the other precipitated a 11,000 molecular weight protein that was tentatively identified as hemoglobin. The use of this system and approach to identify and evaluate changes that occur during the differentiation is discussed.

Cell-surface glycoprotein synthesis during differentiation of chicken erythroblasts transformed by temperature-sensitive avian erythroblastosis virus.

Chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (ts34 AEV) have a greatly increased haemoglobin content (Graf, T., N. Ade and H. Beug: Nature 275, 496-501 (1978)) if allowed to grow for 3-5 days at the non-permissive temperature (41 degrees C), instead of the permissive temperature (35 degrees C) of the virus. Cell-surface molecular changes during this differentiation were investigated by examining the glycoproteins synthesized by a ts34-transformed erythroblast cell line. These cells synthesized a greatly increased amount of a 94,000 molecular weight erythrocyte cell-surface glycoprotein beginning 2-6 h after a shift in growth temperature from 35 degrees to 41 degrees C, consistent with the proposal that such a shift releases these transformed cells from a differentiation block.