We have constructed and cloned in bacteria
recombinant DNA molecules containing DNA sequences coding for the precursor of the alpha subunit of
thyrotropin (pre-
TSH-alpha).
Double-stranded DNA complementary to total
poly(A)+RNA derived from a mouse pituitary thyrotropic
tumor was prepared enzymatically, inserted into the Pst I site of the plasmid pBR322 by using
poly(dC).poly(dG) homopolymeric extensions, and cloned in Escherichia coli chi 1776. Cloned cDNAs encoding pre-
TSH-alpha were identified by their hybridization to pre-
TSH-alpha mRNA as determined by cell-free translations of hybrid-selected and hybrid-arrested
RNA. The nucleotide sequences of two cDNAs (510 and 480 base pairs) were determined with chemical methods and corresponded to much of the region coding for the alpha subunit and the
3' untranslated region of pre-
TSH-alpha mRNA. The sequence of the 5' end of the
mRNA was determined from
cDNA synthesized by using total
mRNA as template and a
restriction enzyme DNA fragment as primer. Together these sequences represented greater than 90% of the coding and noncoding regions of full-length pre-
TSH-alpha mRNA, which was determined to be 800 bases long. The amino acid sequence of the pre-
TSH-alpha deduced from the nucleotide sequence showed a NH2-terminal leader sequence of 24
amino acids followed by the 96-amino-acid sequence of the
apoprotein of
TSH-alpha. There is greater than 90% homology in the amino acid sequences among the murine, ruminant, and porcine alpha subunits and 75-80% homology among the murine, equine, and human alpha subunits. Several regions of the sequence remain absolutely conserved among all species, suggesting that these particular regions are essential for the
biological function of the subunit. The successful cloning of the alpha subunit of TSH will permit further studies of the organization of the genes coding for the
glycoprotein hormone subunits and the regulation of their expression.