Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA.

J. I. Gordon, A. T. Burns, J. L. Christmann, R. G. Deeley

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44 Scopus citations

Abstract

A scheme is presented for cloning a double-stranded cDNA molecule that codes for a portion of chicken preproalbumin. This method, which does not require pure mRNA or cDNA, has widespread applicability. Chicken preproalbumin was identified as a Mr = 72,000 polypeptide by immunoprecipitation of proteins synehesized in a wheat germ cell-free translation system from total, guanidine.HCl-extracted, rooster liver RNA. After removal of the bulk of the ribosomal RNA by poly(U)-Sephadex G-10 chromatography, albumin mRNA was enriched approximately 2-fold by centrifugation through low salt, isokinetic sucrose gradients, until it represented about 30% of the mRNA sequences present. Double-stranded cDNA prepared from this mRNA was then inserted into the Pst 1 site of the plasmid PBR322 by the "G-C tailing" technique and the recombinant DNA was used to transform Echerichia coli stran X1776. Transformants containing putative albumin DNA sequences were identified by colony hybridization with a cDNA probe that was highly enriched for albumin cDNA sequences. This probe was isolated by hybridizing the partially purified RNA preparation to its cDNA, under conditions of RNA excess, to a R0t value such that only the most abundant cDNA sequences had hybridized. Unhybridized, less abundant, sequences were destroyed by subsequent S1 nuclease digestion. The identity of clones that hybridized to this abundant class cDNA was established by DNA-mRNA hybrid-arrested cell-free translation. Hybridization of nick-translated, albumin-containing, plasmid DNA to total liver poly(A)+ RNA, that had been separated on methyl mercury agarose gels and transferred to diazobenzyloxymethyl paper, established that avian albumin mRNA has a molecular weight of 850,000. This molecular weight corresponds to approximately 2,600 nucleotides, or 600 nucleotides longer than the size required to code for the preproalbumin polypeptide.

Original languageEnglish
Pages (from-to)8629-8639
Number of pages11
JournalJournal of Biological Chemistry
Volume253
Issue number23
StatePublished - Dec 10 1978

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