Cloning and sequence of several alpha 2u-globulin cDNAs.

Publisher Website
Google Scholar
Abstract
A simple cloning procedure is described for .alpha.2u-globulin that requires neither enrichment of mRNA for cloning nor purification of a specific probe for screening recombinant colonies. Total adult male liver poly(A)+RNA was used as template for cloning and the subsequent recombinant colonies were screened by comparing hybridization to radioactive c[complementary]DNA probes prepared from hepatic male and female mRNA, respectively. Almost all of the selected male-specific clones contained .alpha.2u-globulin sequences. This cloned .alpha.2u-globulin cDNA specifically hybridizes to male rat liver RNA, which, when isolated and translated in vitro, codes for a 21,000 dalton protein (pro-.alpha.2u-globulin) immunologically identical to .alpha.2u-globulin. When translation occurs in the presence of pancreatic microsomes this in vitro synthesized pro-.alpha.2u-globulin is processed to the 19,000 dalton mature form of .alpha.2u-globulin. The nucleotide sequence of the .alpha.2u-globulin cDNA was determined, elucidating the complete amino acid sequence of .alpha.2u-globulin and most of the hydrophobic leader sequence of pro-.alpha.2u-globulin. The amino acid sequence deduced from the cDNA agrees with the partial sequence previously determined by sequential Edman degradation of the purified protein. .alpha.2u-Globulin cDNA clones contain within the 3''-untranslated region 1 or both of the 2 putative polyadenylylation/transcription termination sites. Either can be used, regenerating .alpha.2u-globulin mRNA species of 2 lengths. A codon usage analysis of the cDNA showed that, although all 6 leucine codons are used for the 14 leucine residues in mature .alpha.2u-globulin, the 7 leucines in the partial leader sequence reported are all encoded by the same codon, CTG. The primary amino acid sequence contains a unique Asn-Gly-Ser sequence, likely to be in .beta.-turn conformation, as the probable site of glycosylation for this glycoprotein.