ABSTRACT
Steroid receptors are key transcriptional regulators of mammary growth, development and lactation. Expression of estrogen receptors alpha (ERalpha) and beta (ERbeta), progesterone receptor (PR), and estrogen-related receptor alpha-1 (ERRbeta) have been evaluated in bovine mammary gland. The ERRalpha is an orphan receptor that, in other species and tissues, appears to function in the regulation of estrogen-response genes including lactoferrin and medium chain acyl-CoA dehydrogenase and in mitochondrial biogenesis. Expression of ERalpha, ERbeta, PR and ERRalpha was characterized in mammary tissue obtained from multiple stages of bovine mammary gland development using quantitative real-time RT-PCR. Expression was evaluated in prepubertal heifers, primigravid cows, lactating non-pregnant cows, lactating pregnant cows and non-lactating pregnant cows (n=4 to 9 animals/stage). In addition, ERalpha, ERbeta, PR and ERRalpha were mapped to chromosomes 9, 10, 15 and 29 respectively, by linkage and radiation hybrid mapping. Results indicated that expression of ERalpha, PR and ERRalpha was largely coordinately regulated and they were present in significant quantity during all physiological stages evaluated. In contrast, ERbeta transcripts were present at a very low concentration during all stages. Furthermore, no ERbeta protein could be detected in bovine mammary tissue by immunohistochemistry. The ERalpha and PR proteins were detected during all physiological states, including lactation. Our results demonstrate the presence of ERalpha, PR and ERRalpha during all physiological stages, and suggest a functional role for ERRalpha and a relative lack of a role for ERbeta in bovine mammary gland development and lactation.
Subject(s)
Estrogen Receptor alpha/genetics , Estrogen Receptor beta/genetics , Mammary Glands, Animal/growth & development , Mammary Glands, Animal/metabolism , Pregnancy, Animal/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Estrogen/genetics , Receptors, Progesterone/genetics , Animals , Cattle , Chromosome Mapping , Chromosomes, Mammalian , Female , Immunohistochemistry/methods , Lactation/metabolism , Pituitary Gland/metabolism , Pregnancy , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , ERRalpha Estrogen-Related ReceptorABSTRACT
Sequence-based gene expression data are used to interpret results from functional genomic and proteomics studies. Although more than 300 000 bovine-expressed sequence tags (ESTs) are available in public databases, a more thorough and directed sampling of the expressed genome is needed to identify new transcripts and improve assembly and annotation of existing transcript sequences. Accordingly, we examined the utility of constructing cDNA libraries synthesized by arbitrarily primed RT-PCR of mRNA from tissues not well represented in the publicly available bovine EST database. A total of 33 cDNA libraries were constructed from healthy and infected mammary gland tissues of Brazilian Gir and Holstein cattle. This series of libraries was used to generate 6481 open reading frame-expressed sequence tags (ORESTES) that assembled into 1798 unique sequence elements of which, 1157 did not significantly match sequence assemblies available in the Bos taurus gene index. However, a total of 264 of these 1157 sequence elements aligned with mouse and human expressed sequences demonstrating that ORESTES is an effective resource for discovery of novel expressed sequences in cattle. Furthermore, comparison of the alignment position of bovine ORESTES-derived sequence elements to human gene reference sequences suggested that the priming events for cDNA synthesis more often occurred at the central portion of a transcript, which may have contributed to the relatively high rate of novel sequence discovery.
Subject(s)
Cattle/genetics , Expressed Sequence Tags , Gene Library , Mammary Glands, Animal/chemistry , Open Reading Frames/genetics , RNA, Messenger/genetics , Animals , Base Sequence , DNA Primers , Gene Expression , Humans , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
BoLA-DRB3 is a gene of the major histocompatibility complex (MHC) in cattle. The product of the BoLA-DRB3 gene is a beta chain of an MHC class II molecule, a glycoprotein expressed on the surface of antigen-presenting cells (APCs). Responses of CD4+ T lymphocytes to peptides are dependent on the presentation of peptide ligands bound to class II molecules on APCs. Genotyping of the BoLA-DRB3 gene is relatively complex due to the extensive polymorphism of this locus. Current techniques for assignment of genotypes are polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), direct sequencing of PCR products, cloning-sequencing, polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP), and denaturant-gradient gel electrophoresis. These techniques are time-consuming, do not discriminate all possible alleles, or are not readily reproducible. The objective of this study was to genotype BoLA-DRB3 using temperature-gradient gel electrophoresis (TGGE) to separate alleles before sequencing. PCRs using 28 DNA samples from Gir Dairy cattle (a Brazilian breed of Bos indicus) were submitted to TGGE. New PCR products were generated from separated alleles, purified, and sequenced. Allele separation was possible in 21 out of 26 heterozygote samples (81%). Results indicate that two sequence reads (forward and reverse) were sufficient for accurate genotyping of BoLA-DRB3 alleles. Separation of alleles by TGGE provides high-throughput, reliable typing of BoLA-DRB3, which is critical in disease association studies in cattle.
Subject(s)
Alleles , Genotype , Histocompatibility Antigens Class II/genetics , Animals , Antigen-Presenting Cells/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cattle , Cloning, Molecular , DNA/chemistry , Electrophoresis , Exons , Glycoproteins/chemistry , Heterozygote , Humans , Peptides/chemistry , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNA , TemperatureABSTRACT
Brazilian dairy Gir (Bos indicus) cattle are a tropical, well-adapted breed, for which no information on the major histocompatibility complex (MHC) is presently available. The second exon of the bovine lymphocyte antigen (BoLA-DRB3) was amplified by polymerase chain reaction (PCR) of DNA samples from 28 Brazilian dairy Gir cows. Two experimental genotyping approaches were used: direct sequencing of PCR gene products (PCR-DS) and sequencing of cloned PCR fragments (S-CLO). Results demonstrate the viability of both typing approaches. PCR-DS allowed typing of 39% of the animals while the remainder were genotyped by S-CLO. Seventeen BoLA-DRB3 alleles were assigned, including some that were only recently described for zebu cattle. Allelic frequencies ranged from 0.02 to 0.18. The most frequent alleles were *3601 (frequency = 0.18), *2201 (0.14) and *2101 (0.11).
