Molecular cloning, characterization, and expression in Escherichia coli of a cDNA encoding mammalian choline kinase.
A rat liver lambda gt11 cDNA library was screened with antibody against rat liver choline kinase. One of the putative phage clones obtained was found to express choline kinase activity in infected Escherichia coli cells and used for the isolation of fully extended cDNA clones by hybridization. The obtained clones had identical overlapping nucleotide sequences and gave a composite cDNA sequence of 2540 bases. Poly(A)+ tails were found at three different sites of the cDNA sequence. Within the sequence there was a large open reading frame encoding 435 amino acids with a molecular mass of 49,743 Da. When this open reading frame was placed after the trc promoter and introduced into E. coli cells, fully active choline kinase was produced. This enzyme was partially purified using a published choline kinase purification method and was found to mediate the phosphorylation of choline, N,N-dimethylethanolamine,N-monomethylethanolamine, and ethanolamine. Similar to the case with choline kinases from various sources, Km decreased as the number of N-methyl groups increased. The deduced amino acid sequence significantly resembled the yeast choline kinase sequence. It also showed local sequence similarity to protein kinases and some bacterial phosphotransferases. Northern blot analysis revealed that the cDNA probe hybridized to 2.8- and 1.8-kilobase RNA in various rat tissues. The sizes of the transcripts were fairly consistent with that of cDNA. The order of mRNA abundance was testis, brain, lung, kidney, and liver, but did not coincide with the order of the activity levels in these tissues. These results show that the cloned cDNA encodes one of the choline kinase isoforms present in mammalian tissues.
Uchida
T
T
Department of Biochemistry, Gunma University School of Medicine, Maebashi, Japan.
Yamashita
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002797:Choline Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D011994:Recombinant Proteins; D004247:DNA; D002797:Choline Kinase