Cloning and Sequence Analysis of a cDNA for the β Subunit of Chicken Thyroid-Stimulating Hormone

Abstract

The β subunit of thyroid-stimulating hormone (TSHβ) has been isolated and sequenced in many species, including several mammals and the frog, but not in any avian species. Therefore, the objective of this study was to isolate and sequence a cDNA for chicken TSHβ. Degenerate oligonucleotide primers were designed, based on conserved regions of TSHβ from four other species, and used for reverse transcription and polymerase chain reaction amplification of a cDNA fragment from total cellular RNA of pituitary glands from 7-day-old chicks. The remaining sequence was completed by rapid amplification of cDNA ends. The predicted amino acid sequence was 70.4% identical between bovine and chicken, 69.6% identical between chicken and rat, and 57.4% identical between chicken and frog. To test for tissue specificity of the cDNA, total cellular RNA samples from testicle, liver, pituitary, lung, and heart were analyzed by Northern blot. The32P-labeled antisense riboprobe hybridized to an RNA species of approximately 600–700 bases in pituitary RNA alone, corresponding with the length of TSHβ mRNA in other species. Gene expression in Day 1 posthatch chickens was then analyzed by ribonuclease protection assay. Anterior pituitary cells of Day 1 chickens were treated for 20 to 24 hr in serum-free medium alone or with medium containing either thyrotropin-releasing hormone (TRH) (10−8M) or triiodothyronine (T3) (10−9M). The RNA was then harvested from these cells and hybridized with a32P-labeled antisense riboprobe. Treatment with TRH had no effect on TSHβ mRNA levels, while T3significantly decreased (P<0.05;n=6 trials) TSHβ mRNA levels by 45%. Taken together these results indicate that the cDNA sequence derived represents chicken TSHβ mRNA, and that TSHβ gene expression is downregulated by thyroid hormones as it is in mammals.