Cloning and Characterization of Porcine CArG Binding Factor A Expression in the Anterior Pituitary

Abstract

Abstract. CArG binding factor A (CBF-A) is a transcription factor first isolated from mouse C2 myogenic cells. Several lines of evidence indicate that CBF-A is also present in the anterior pituitary lobe and participates in the process of development and cell transformation. This study was performed to clone porcine CBF-A and to investigate its roles in the porcine anterior pituitary lobe. The predicted amino acid sequence of porcine CBF-A showed a unique insertion of five TG-repeats in the N-terminal region in comparison with those of other mammals, whereas the other regions appeared to be mostly conserved including two RNA recognition motifs in the middle region. Investigation of the expression of CBF-A gene during porcine pituitary development by RT-PCR showed an exclusive and temporary decrease in expression level shortly after birth in both sexes that was gradually but insufficiently restored. The expression of fluorescence protein-fused CBF-A in CHO cells demonstrated that CBF-A is located in the nuclei. We examined whether CBF-A regulates the expression of pituitary hormone genes in CHO cells and found that CBF-A significantly stimulated the promoter activity of growth hormone and prolactin by about 2-fold but did not stimulate the LHβ gene. The specific DNA binding ability of porcine CBF-A was examined using serial oligonucleotides, CArG box and CC(W)0-6GG (W=A or T). As a result, porcine CBF-A was shown to have a high binding affinity for double-and single-stranded CC(W)6GG but no affinity for the known sequences of the CBF-A-target genes. Accordingly, this study demonstrated that porcine CBF-A may play a role in regulating at least two pituitary hormone genes, GH and PRL.

Molecular Cloning of Paired Related Homeobox 2 (Prx2) as a Novel Pituitary Transcription Factor

“…The isolation of total RNAs from porcine embryos and postnatal pituitaries of both sexes and construction of cDNAs have been described previously [24,25]. The specific PCR primer sets used were: porcine Prx2 (5'-CAGGTCTGGTTTCAGAACCGC-3', forward, and 5'-TGTCAGTTCACTGTGGGCACC-3', reverse) and Cyclophilin A (5'-TGGTGACTTYACACGCCATAAT-3', forward and 5'-ATTCCTGGACCRAAACGCTCC-3', reverse).…”

Molecular Cloning and Characterization of Porcine Homeodomain Transcription Factor Msx1

“…Total RNAs were extracted from porcine anterior pituitary glands collected on fetal days 40, 50, 65, 82, 95 and 110 (f40, f50, f65, f82, f95 and f110) and postnatal days 8, 60, 160 and 230 (p8, p60, p160 and p230) from German Landrace male and female pigs [10] and from several pituitary tumor derived cell lines [11]. Specific PCR primer sets were synthesized as follows: Msx1, the forward primer was 5'-AAGTTCCGCCAGAAGCAGTAC-3' and the reverse primer was 5'-TCAGGTGGTACATGCTGTAGC-CTAC-3', and for cyclophilin A, he forward primer was 5'-TGGTGACTTCACACGCCATAATG-3'and the reverse primer was 5'-ATTCCTGGACCRAAAACGCTCC-3.…”