Human cytidine deaminase: purification of enzyme, cloning, and expression of its complementary DNA.
Cytidine (CR) deaminase was purified 47,000-fold to homogeneity from human placenta. The molecular mass of CR deaminase was estimated to be 48.7 kDa by gel filtration and 16.1 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that it contains three or four identical subunits. We determined the amino acid sequence of several peptide fragments and designed 5'-primers to amplify, by the polymerase chain reaction, a specific 364-base pair DNA fragment using human liver complementary DNA (cDNA) as the template. This DNA fragment, which contains the codons of one peptide, was used as a probe to screen a cDNA library from human liver. We isolated and sequenced a cDNA clone of 910 base pairs that contained a 5' nontranslated region, a 438-base pair coding region, and a 3' nontranslated region with a polyadenylated tail. The translated region of the clone contained a deduced sequence of 146 amino acids, with a predicted molecular mass of 16.2 kDa and the sequences of our peptides. The cDNA was ligated in pGEX vector and expressed in Escherichia coli. The expressed protein had a high CR deaminase activity and molecular mass of 16.3 kDa. These data demonstrate clearly that the open reading frame of our cDNA clone codes for a functional human CR deaminase. Polymerase chain reaction amplifications of gene-specific DNA fragments from human/rodent hybrid cells indicate the localization of CR deaminase gene to human chromosome 1. The cDNA for CR deaminase will be a useful molecular probe to investigate the importance of this enzyme in chemotherapy.
Laliberté
J
J
Département de Pharmacologie, Université de Montréal, Québec, Canada.
Momparler
R L
RL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002878:Chromosomes, Human, Pair 1; D003564:Cytidine Deaminase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology
D018076:DNA, Complementary; D003564:Cytidine Deaminase