Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GAPDH mRNA which was at least 50% pure. Double-stranded cDNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select complementary RNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by (1) comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, (2) precipitation with specific anti-GAPDH antiserum, (3) cyanylation fingerprinting, and (4) AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert [Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560]. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5' end, the entire coding region, and 230 nucleotides of the 3'-noncoding region including a poly(A) addition signal (AATAAA) and the first five residues of the poly(A) tail.

The complete nucleotide sequence of human serum albumin mRNA has been determined from recombinant cDNA clones and from a primer-extended cDNA synthesis on the mRNA template. The sequence is composed of 2078 nucleotides, starting upstream from a potential ribosome binding site in the 5' untranslated region. It contains all the translated codons and extends into the poly(A) at the 3' terminus. Part of the translated sequence codes for a hydrophobic prepeptide, Met-Lys-Trp-ValThr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr-Ser, followed by a basic propeptide, Arg-Gly-Val-Phe-Arg-Arg. These signal peptides are absent from mature normal serum albumin and, so far, have not been identified in their nascent state in humans. A remaining 1755 nucleotides of the translated mRNA sequence code for 585 amino acids, which are in agreement, with few exceptions, with the published amino acid sequence for human serum albumin. The mRNA sequence verifies and refines the repeating homology in the triple-domain structure of the serum albumin molecule.

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