Cloning of the full length pig PIT1 (POU1F1) CDNA and a novel alternative PIT1 transcript, and functional studies of their encoded proteins.
PIT1 is an essential regulatory gene of growth hormone (GH), prolactin (PRL) and thyrotropin beta subunit (TSHbeta). Previously, a partial pig PIT1 cDNA and a genomic clone of the entire 3' end of the PIT1 gene was isolated, and polymorphisms at PIT1 were associated with several performance traits in the pig. In order to understand the biological function of the pig PIT1 gene and its possible application in swine genetics, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to complete the cloning of the full length cDNA for pig PIT1. The pig PIT1 cDNA and its deduced protein sequence have approximately 90% and 95% identity, respectively, with the PIT1 cDNA and protein of other mammals (human, bovine, sheep and rodents). Surprisingly, sequence comparison to other pig PIT1 sequences indicated only approximately 93% identity. Additional sequencing confirmed our sequence, and identified a new polymorphism in exon 4. Phylogenetic analysis of several mammalian PIT1 sequences indicates sequencing errors may account for the discrepancies observed in the other pig sequences reported. Several PIT1 alternative spliced forms were also identified by RT-PCR. They were the delta3PIT1 (missing entire exon 3), delta4PIT1 (missing entire exon 4) and PIT1beta (additional 26 amino acids inserted in front of exon 2) transcripts. The delta4PIT1 and PIT1beta transcripts have been found to encode functionally different proteins in rodents. The delta3PIT1 transcript is a novel isoform of PIT1. Potentially different functions between pig delta3PIT1 and PIT1 were analyzed by expressing these proteins in bacteria. The E. coli-expressed PIT1 and delta3PIT1 proteins were used with rat growth hormone (rGH) and rat prolactin (rPRL) promoter DNA in DNA mobility shift assays. The results showed that pig PIT1 can specifically bind rGH and rPRL promoter regions, but that the pig delta3PIT1 cannot, even at very high protein concentrations. Possible protein-protein interactions between delta3PIT1 and PIT1 were tested by mixing protein extracts before the gel shift assay, and the results showed that delta3PIT1 protein did not affect PIT1 binding to its target DNA. These data demonstrate the functionality of the PIT1 cDNA cloned in this study, and identify a novel delta3PIT1 transcript which encodes a protein that cannot bind rGH/rPRL target sequences.
Yu
T P
TP
Department of Animal Science, Iowa State University, Ames 50011, USA.
Sun
H S
HS
Wahls
S
S
Sanchez-Serrano
I
I
Rothschild
M F
MF
Tuggle
C K
CK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D011388:Prolactin / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012377:Rodentia; D017422:Sequence Analysis, DNA; D013552:Swine; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics*
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D011388:Prolactin