Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction

Genomics. 1989 Feb;4(2):198-205. doi: 10.1016/0888-7543(89)90300-5.

Abstract

The polymerase chain reaction was used to clone a full-length human methylmalonyl-CoA mutase cDNA from a human liver library by priming with sequences from the 5' end of a partial cDNA and sequences in the phage vector. The amino acid sequence predicted from the cDNA corresponds to the authentic amino acid sequences of peptide fragment from purified methylmalonyl-CoA mutase. The open reading frame of the cDNA encodes 742 amino acids (82,283 Da) comprising a 32 amino acid mitochondrial leader sequence and a mature protein of 710 amino acids (78,489 Da). The use of the polymerase chain reaction to "screen" the cDNA library represents a novel application of this technique. The full length will enable analysis of mutations underlying inherited methylmalonic acidemias caused by deficiency of the methylmalonyl-CoA mutase apoenzyme.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Gene Amplification
  • Humans
  • Isomerases / genetics*
  • Liver / enzymology
  • Methylmalonyl-CoA Mutase / genetics*
  • Molecular Sequence Data
  • Protein Conformation
  • RNA, Messenger / metabolism

Substances

  • RNA, Messenger
  • DNA
  • DNA-Directed DNA Polymerase
  • Isomerases
  • Methylmalonyl-CoA Mutase

Associated data

  • GENBANK/M22990