Cloning and screening with nanogram amounts of immunopurified mRNAs: cDNA cloning and chromosomal mapping of cystathionine beta-synthase and the beta subunit of propionyl-CoA carboxylase

Abstract

We have developed conditions for efficient cDNA cloning of nanogram amounts of purified mRNAs coding for cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and for the cytosolic precursors of mitochondrial ornithine transcarbamylase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) and the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.3]. The three mRNAs, prepared by sequential immunoselection from the same batch of rat liver polysomes, were pooled (20 ng each), and cDNA was synthesized by using avian reverse transcriptase. The second DNA strand was prepared by "nick-translation repair" of the cDNA . mRNA hybrid with RNase H, polymerase I, and DNA ligase from Escherichia coli. The double-stranded (ds) DNA was tailed with deoxycytidine residues, annealed with Pst I-cut/dG-tailed pBR322, and used to transform E. coli. The library generated by this three-step procedure contained 5000 independent colonies. A 550-base-pair (bp) cDNA clone of the beta subunit of propionyl-CoA carboxylase was detected by hybrid-selected translation; it was then used to screen the library for longer cDNAs. Two hybridizing cDNAs, 1200 and 1000 bp long with a 200-bp overlap, representing together a full-length copy of the coding region and 446 bp of 3' untranslated sequence, were recovered. Each plasmid mapped to the region q13.3----q22 of human chromosome 3. Cystathionine beta-synthase clones were obtained by screening the library with a single-stranded [32P]cDNA prepared directly from the highly purified synthase mRNA by reverse transcriptase. The longest hybridizing cDNA of 1700 bp was used in hybrid-selected translation and detected a polypeptide of 63 kDa, identical in size to rat liver synthase. In situ hybridization of this cDNA to q22 of human chromosome 21 confirmed two previous tentative assignments of the synthase locus to this chromosome.