We describe a simple cloning procedure for a%.-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 cDNA probes prepared from hepatic male and female mRNA, respectively. Almost all of the selected "male-specific" clones were later shown to contain a2,-globulin sequences. This cloned au-globulin cDNA has been shown to specifically hybridize to male rat liver RNA, which, when isolated and translated in vitro, codes for a 21,000-dalton protein (pro-a2u-globulin) immunologically identical to auglobulin. When translation occurs in the presence of pancreatic microsomes this in vitro synthesized pro-a2u-globulin is processed to the 19,000-dalton mature form of a2u-globulin. The nucleotide sequence of the a2u-globulin cDNA has been determined, thus elucidating the complete amino acid sequence of a2.-globulin and most of the hydrophobic "leader" sequence of pro-a2u-globulin.The amino acid sequence deduced from the cDNA is in agreement with the partial sequence that we previously determined by sequential Edman degradation ofthe purified-protein. a2u-Globulin cDNA clones contain within the.3'-untranslated region one or both of the two putative polyadenylylation/transcription termination sites (A-A-T-A-A-A and A-A-T-T-A-A-A). Either of these can be used, generating a2u-globulin mRNA species of two lengths. A codon usage analysis of the cDNA showed that, although all six leucine codons are used for the 14 leucine residues in mature auglobulin, the seven 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 (-turn conformation, as the probable site of glycosylation for this glycoprotein.ac2u-Globulin is synthesized in the liver of normal adult male rats, secreted into the bloodstream, and excreted in the urine (1), and represents ':'1% of total hepatic protein synthesis (2). Female liver, male kidney, and several hepatomas do not synthesize a2u-globulin and have no detectable a2,,-globulin mRNA sequences (2-4). The biosynthesis of a2u-globulin is also under complex multihormonal control: androgens, glucocorticoids, thyroid hormone, insulin, and growth hormone stimulate and estrogens repress its synthesis (5-9). This regulation in vivo has been shown to occur via modulation of the rate of synthesis of hepatic a2u-globulin mRNA (5, 10). The existence of both glycosylated and nonglycosylated forms of a2,-globulin has been reported (11,12), as well as induction of the glycosylated form by glucocorticoids (13,14). a2u-Globulin is first synthesized as the 21,000-dalton precursor pro-a2u-globulin, which is then processed to the mature form of the protein (15).As part of our ongoing research into the regulation of a2u-globulin synthesis, we have cloned the cDNA ...

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When liver cells undergo malignant transformation, certain genes cease being expressed. We Preparation and Isolation of p41 Clone. The cDNA was prepared from male hepatic poly(A)-RNA and inserted into the Pst I site of pBR322 by the oligo(dA-dT) "tailing" method (7). After transformation of Escherichia coli X1776 and culture under P2-EKI containment conditions, clones that were both ampicillin sensitive and tetracycline resistant were screened with cDNA that was specific to livers of male rats (7-9). Of the clones that hybridized to this probe, the one containing the largest inserted sequence (p41) was selected for further study. * Hybridization and Translation of mRNA Complementary to p41. The procedure used for isolating liver mRNA from male rats complementary to the cDNA insert in clone p41 was from the work of Ricciardi et aL (10) and Woolford and Rosbash (11). Briefly, plasmid DNA from a 100-ml culture was prepared by the method of Katz et aL (12), denatured by successive heat and alkali treatments, bound to nitrocellulose discs, and hybridized at 50°C in a medium consisting of 0.4 M NaCl, 30 mM 1,4-piperazinediethanesulfonic acid (Pipes; pH 6.5), 0.2% NaDodSO4, 50 ,g of yeast tRNA, and 25 ,ug of liver poly(A)-RNA from male rats. After 16 hr of hybridization, filters were removed, washed to remove nonspecifically bound RNA, and boiled in water to elute the specific mRNA. The RNA solution was then treated with phenol, washed, lyophilized, and finally dissolved in water. Half of this RNA solution was used to program a wheat germ-based translation system (13,14). The L-[LS]methioninelabeled translation products were displayed on a 12.5% polyacrylamide/NaDodSO4 gel (15) supplemented with 6 M urea, after which the gel was fluorographed (16) and exposed to x-ray film at -70°C.Hybridization of p41 DNA with Excess RNA. The hybridization mixture consisted of 0.4 M NaCl, 65% (vol/vol) formamide, 30 mM Pipes (pH 6.5), 0.2% NaDodSO4, 10 ,ug of denatured E. coli DNA, -6000 cpm of 32P-labeled Ava II fragment of clone p41 (~" 160 pg), and various concentrations of total Abbreviations: kbp, kilobase pair; Pipes, 1,4-piperazinediethanesulfonic acid.

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