Cloning bovine interferon-tau genes and characterizing their transcriptional expression during early pregnancy

Abstract

The interferon-tau (IFN-τ) are major cytokines that play a pivotal role in maternal recognition of pregnancy in ruminants. They block the luteolytic secretion of prostaglandin F2α. thereby extending the functional life span of corpus luteum. allowing pregnancy to proceed. In spite of ample evidence indicating that these bovine IFNτ(bIFN-i) are encoded by multicopy genes, only a few promoters had been cloned and characterized to any extent. Therefore, several different bIFN-τ genes were cloned, and their expression patterns were analyzed during early pregnancy in cattle. Screening a bovine genomic library resulted in four putative clones: sequencing revealed that one of them was closely related to bIFN-ω and the rest were bIFN-τ genes. All of the clones lacked premature stop codons in coding regions. The newly cloned bIFN-τ contained approximately 2.3 kilobase (kb) of promoter region. 1.0 kb of coding region, and the first putative poly (A) signal, irrespective of subtype. Substantial sequence differences were found among the subtypes, confirming the multiplicity of bIFN-τ genes. Mismatches in the coding region which lead to amino acid differences were also notable indicating that the biological properties of different subtype gene products may be different. Most severe mismatches were found in 5' ends of promoters, which had not been cloned before; thus, these regions were subjected to further study. To study the expression pattern of each subtype gene, RNase protection assays (RPA) were applied to various developmental stages of bovine embryos. Overall transcriptional expression dropped markedly between day 16 and 25 regardless of subtype. However, one of the new clones. IFN-τb1 showed minimal expression compared to other clones. This particular clone showed extreme mismatching in first 500 base pairs of the 5' end. suggesting that this region may be an enhancer. In addition, a polymorphism was observed in one new clone, IFN-τb2 in an RPA using different embryos. These results indicated that bIFN-τ subtype genes are not expressed equally. The 500 bp gene fragment from a highly expressed subtype was further studied to test whether it functioned as a tissue specific enhancer. An in vitro transcription assay utilizing various cell nuclei and DNA templates revealed the 500 bp fragment increased transcription in vitro approximately 2-fold irrespective of DNA constructs and nuclei sources, suggesting that it works as a general, rather than a tissue specific enhancer. Together, these data support the hypotheses that the bIFN-τ are encoded by several subtype genes with substantial sequence differences in both promoter and coding regions, resulting in differential expression during early embryonic development, and different biological potencies of the gene products.