عنوان

Transcriptional regulation of the prolactin gene in turkeys

TLpq304319845

انگلیسی

Transcriptional regulation of the prolactin gene in turkeys

[Thesis]

K. Kurima

E. A. Wong

Virginia Polytechnic Institute and State University

1996

118-118 p.

Ph.D.

Virginia Polytechnic Institute and State University

1996

Poor reproductive performance by turkey hens compared with chickens is partially due to the early cessation of egg production associated with the onset of incubation behavior. Prolactin (Prl) is involved in the induction and maintenance of incubation behavior in birds, and understanding the regulatory mechanism(s) of Prl gene expression will provide fundamental information to manipulate Prl production for better reproductive performance in turkey hens. To better understand the regulatory mechanism of Prl gene expression, the turkey Prl gene was isolated from a usd\lambdausd phage genomic library using a turkey Prl cDNA probe. The turkey Prl gene consists of five exons and spans approximately 6.7 kilobases (kb). The arrangement of the exons was found to be nearly identical to the rat and human Prl genes, showing the conserved feature of the gene among these species. Two regions similar to the binding sites for the transcription factor Pit-1/GHF-1 were found within 2 kb of the 5-flanking region of the Prl gene, while no estrogen response element (ERE) was found. This suggests that transcription of the turkey Prl gene may be directly regulated by Pit-1/GHF-1, and not by the estrogen receptor. In order to identify regulatory elements for turkey Prl gene expression, a rat pituitary-derived tumor cell line, GH3 and primary turkey pituitary cells were transiently transfected with luciferase reporter gene constructs containing the 5-flanking region of the turkey Prl gene. In the GH, cell system, the results indicated that negative-acting elements may be present between the 2.0 and 1.3 kb region and a positive-acting element between the 1.3 and 1.0 kb region of the turkey Prl gene. The decreased promoter activity resulting from the elimination of one or two Pit-1/GHF-1 binding sites strongly suggests that the Pit-1/GHF-1 transcription factor plays an essential role for turkey Prl gene expression. To test the regulatory elements in a native system, a primary turkey pituitary cell culture was prepared for transient transfection. Despite repeated trials, no consistent reporter gene expression was obtained from cells transfected with the reporter gene constructs containing the turkey Prl promoter. The results of the GH, cell transfection experiment indicated that the Pit-1/GHF-1 transcription factor plays an essential role for turkey Prl gene expression. Three cDNAs encoding turkey Pit-1/GHF-1 isoforms, referred to as tPit-1*, tPit-1usd\beta\sp*usd and tPit-1W*, were isolated from anterior pituitaries of turkey hens, and the ability of each tPit-1 isoform to activate the turkey Prl promoter was examined in mouse Ltk-cells. Both tPit-1* and tPit-1usd\beta\sp*usd isoforms use the transcription start site (tss) of exon 1, while tPit-1W* uses the tss of exon 2, thus, lacking exon 1. Relative to tPit-1*, tPit-1usd\beta\sp*usd has an additional 28 amino acids at the N-terminal domain due to the usage of an alternative splicing site 84 nt upstream from the splicing site of exon 2 used for tPit-1*. All three isoforms appeared to promote transcription of the turkey Prl gene, but tPit-1usd\beta\sp*usd and tPit-1W* activated the Prl promoter to a lesser extent than tPit-1*. Clearly defined differential trans-activation effects of these tPit-1 isoforms were not obtained in the mouse Ltk-cell system.

Biological sciences

Genetics

Molecular biology

E. A. Wong

K. Kurima

p

[Thesis]

276903

a

Y