Sequence evaluation of four pooled-tissue normalized bovine cDNA libraries and construction of a gene index for cattle.

An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter- and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.

Interleukin-8 haplotype structure from nucleotide sequence variation in commercial populations of U.S. beef cattle.

The aim of the present study was twofold: first, to design a panel of 96 sires that reflects the breadth of genetic diversity in U.S. beef cattle, and second, to use this panel to discover nucleotide sequence diversity and haplotype structures of interleukin (IL)-8 in commercial populations. The latter is a requisite for epidemiological studies designed to test whether IL8 alleles are risk factors for acquiring or maintaining bacterial infections in production environments. IL-8 encodes a proinflammatory cytokine that plays a central role in cell-mediated immunity by attracting and activating neutrophils in the early stages of host defense against bacterial invasion. Seven single-nucleotide polymorphism (SNP) markers were identified by sequencing two IL8 DNA segments amplified from the panel of 17 popular cattle breeds (MARC beef cattle diversity panel, version 2.1). Assays for automated genotype scoring by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) were developed to independently verify the seven SNP alleles in the 96 bulls and 313 cattle from the MARC reference population. Five haplotype structures, spanning the two IL8 DNA segments, were unambiguously defined for the set of seven IL8 SNPs. Based on the breadth of germplasm in bovine diversity panel, the five haplotype structures for IL8 are estimated to represent >98% of those present in these DNA segments in commercial populations of U.S. beef cattle. The frequencies of the five respective haplotypes in the eight Angus sires of the diversity panel (0.75, 0.25, 0.00, 0.00. 0.00) were similar to those scored in 150 purebred Angus cattle from six herds in four Midwestern states (0.82, 0.18, 0.01, 0.00 0.00), suggesting that the diversity panel may also be useful for estimating allele frequencies in commercial populations.

Estimation of DNA sequence diversity in bovine cytokine genes.

DNA sequence variation provides the fundamental material for improving livestock through selection. In cattle, single nucleotide polymorphisms and small insertions/deletions (collectively referred to here as SNPs) have been identified in cytokine genes and scored in a reference population to determine linkage map positions. The aim of the present study was twofold: first, to estimate the SNP frequency in a reference population of beef cattle, and second, to determine cytokine haplotypes in a group of sires from commercial populations. Forty-five SNP markers in DNA segments from nine cytokine gene loci were analyzed in 26 reference parents. Comparison of all 52 haploid genomes at each PCR amplicon locus revealed an average of one SNP per 143 bp of sequence, whereas comparison of any two chromosomes identified heterozygous sites, on average, every 443 bp. The combination of these 45 SNP genotypes was sufficient to uniquely identify each of the 26 animals. The average number of haplotype alleles (4.4) per PCR amplicon (688 bp) and the percentage heterozygosity among founding parents (50%) were similar to those for microsatellite markers in the same population. For 49 sires from seven common breeds of beef cattle, SNP genotypes (1,225 total) were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) at three amplicon loci. All three of the amplicon haplotypes were correctly deduced for each sire without the use of parent or progeny genotypes. The latter allows a wide range of genetic studies in commercial populations of cattle where genotypic information from relatives may not be available.

Linkage mapping and comparative analysis of bovine expressed sequence tags (ESTs).

Bovine expressed sequence tags (ESTs) containing microsatellites are suitable markers for both linkage and comparative maps. We isolated clones from a bovine fetal thigh skeletal muscle cDNA library that were positive for a (CA)10 probe. Thirty individual clones were isolated and characterised by sequencing. Sequences from the 5' and 3' ends of a clone were considered as separate ESTs until a contiguous sequence was identified. A total of 47 ESTs were sequenced from the 5' and/or 3' ends and full sequence was obtained for the 30 clones. BLAST nucleotide analysis identified significant homology to known mammalian coding regions for 31 of the bovine ESTs, 30 of which also matched human ESTs or sequence-tagged sites (STS). The remaining 16 bovine ESTs represented novel transcripts. Microsatellites were isolated in 27 of the ESTs, 11 of which were developed into markers and placed on the MARC bovine linkage map. Human cytogenetic map positions were available for 20 of the 30 human EST orthologs, and a putative bovine map position for 17 of the sequences could be inferred using comparative mapping data. These results demonstrated that mapping bovine ESTs containing microsatellites is a plausible strategy to increase the density of gene markers on the bovine linkage and comparative maps.

Canine thyrotropin beta-subunit gene: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and transient expression in the Chinese hamster ovary cells.

The gene encoding the mature beta subunit of canine thyroid stimulating hormone (cTSH beta) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSH beta purified from E. coli were generated. The gene fragment that encodes mature TSH beta was cloned from the canine genomic DNA by direct polymerase chain reaction (PCR) using primers that were designed based on the consensus sequences from other species. The resulting 891 basepairs (bp) of genomic DNA consisted of two coding exons of the canine TSH beta gene and an intron of 450 bp. The two exons, which encode the mature cTSH beta subunit, was joined together by an overlap PCR and was expressed in E. coli as 6xHis-tagged protein. The purified recombinant cTSH beta with a molecular weight of about 15 kDa was recognized by the polyclonal antibodies prepared against the native canine TSH in Western blot. Monoclonal antibodies were raised against the purified cTSH beta and subsequently characterized. For transient expression in CHO cells that are permanently transfected with the bovine common alpha gene, a 60-oligonucleotide signal peptide coding sequence was added to the 5' end of the cTSH beta gene before it was cloned into the mammalian expression vector pRSV and used to transfect CHO cells. The medium from these transfected cells, presumably containing the bovine alpha and canine TSH beta in heterodimeric confirmation, exhibited TSH bioactivity as indicated by the stimulation of cAMP production in the cultured FRTL-5 thyrocytes.

Single nucleotide polymorphism (SNP) discovery and linkage mapping of bovine cytokine genes.

Polymorphic markers at bovine gene loci facilitate the integration of cattle genetic maps with those of humans and mice. To this end, 31 single nucleotide polymorphism (SNP) markers were developed for seven bovine chemokine genes. Loci were amplified from bovine genomic DNA by the polymerase chain reaction, and candidate amplicons were sequenced to determine their identity. Amplified loci from 24 founding parents and select progeny from a beef cattle reference population were sequenced and analyzed for SNPs. SNP haplotype alleles were determined by examining segregation patterns and used to establish the locus position on the bovine linkage map. Loci for growth-related proteins (GRO3, GRO1, and GROX) were clustered with the related CXC chemokine genes, interleukin (IL) 8, and epithelial cell inflammatory protein 1, at 84 cM from the centromeric end of the bovine chromosome (BTA) 6 linkage group. Bovine loci for a cluster of IL8 receptors, a stromal cell-derived factor 1, interferon-gamma, and tumor necrosis factor-alpha were mapped at 90, 55, 59, and 34 cM, respectively, from the centromeric ends of the BTA 2, 28, 5, and 23 linkage groups. The positions of these bovine loci were compared with those of orthologous loci on the human map to refine the boundaries of conserved synteny. These seven loci provide examples of SNP development in which the efficiency was largely dependent on the availability of bovine genomic or cDNA sequence. The polymorphic nature of these SNP haplotype markers suggests that they will be useful for mapping complex traits in cattle, such as resistance to infectious disease.

Construction and use of a multi-competitor gene for quantitative RT-PCR using existing primer sets.

We describe a simple and inexpensive method for the construction of multi-competitor molecules for use as internal standards in quantitative RT-PCR. The construction involves the linking and annealing of 20mer PCR primers with complementary 40mers using either a step-wise or bulk process. The entire construct is then ligated and amplified by PCR prior to cloning. Using this approach, we have constructed a gene containing priming sites for 18 different products of immunological interest, including murine cytokines and cell surface markers, as well as murine beta-actin and T. cruzi rRNA. The cost of production of the competitor is minimized by use of a high-throughput multi-oligonucleotide synthesizer for production of the individual components of the synthetic gene, and by use of the same oligonucleotides in gene construction and as primers for the RT-PCR reactions. This procedure can be applied to the production of other polycompetitor molecules as well as to the construction of other types of synthetic genes.