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

Abstract

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 ...

Kidney fatty acid‐binding protein: Identification as α2U‐globulin

“…Furthermore, it was found that the amino acid composition of kidney FABP is very similar to that of the region spanning residues 10-162 (carboxyl-terminus) of urine cezu-globulin (table 1). These results are in agreement with the [18,19] three hydrolysates report of Charles [20] that ce2u-globulin in rat kidney lacked the 9 amino-terminal residues. A double-immunodiffusion test using anti-rat kidney FABP antibodies showed further that this protein was present only in male, but not in female, rat kidney ( fig.4).…”

“…Amino-terminal amino acid sequence of kidney FABP. The sequence of urine ~2u-globulin is based on the data reported in [19].…”

“…A computer search for sequence similarity in the National Biochemical Research Foundation Protein Sequence Database showed that this amino-terminal sequence is identical with the sequence from residues l0 to 29 of urine ce2o-globulin [18,19], as shown in fig.3. Furthermore, it was found that the amino acid composition of kidney FABP is very similar to that of the region spanning residues 10-162 (carboxyl-terminus) of urine cezu-globulin (table 1).…”

Identification of two human beta-tubulin isotypes.

“…However, no functional requirement for alternative Cterminal domains is known to exist in the case of 1-tubulin. On the other hand, addition of poly(A) at more than one site has been shown to be a feature of a number of eucaryotic genes (14,30,35,36).…”

α2-μ-Globulin Fragment (A2-f) from Kidneys of Male Rats forms Multimers in Vitro

“…The precursor protein, A2, is also a member of this family. The amino acid sequence of A2 was earlier determined using a combination of nucleic acid and protein sequencing techniques [9]. The Lipocalin family of proteins is very diverse at the sequence level yet displays highly conserved structures.…”