Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction.
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.
Jansen
R
R
Howard Hughes Medical Institute, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Kalousek
F
F
Fenton
W A
WA
Rosenberg
L E
LE
Ledley
F D
FD
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005784:Gene Amplification; D006801:Humans; D007535:Isomerases / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008765:Methylmalonyl-CoA Mutase / Q000235:genetics*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism
D012333:RNA, Messenger; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D007535:Isomerases; D008765:Methylmalonyl-CoA Mutase