Cloning and sequencing of an intronless mouse S-adenosylmethionine decarboxylase gene coding for a functional enzyme strongly expressed in the liver.
A genomic clone for a mouse S-adenosylmethionine decarboxylase (AdoMetDC) gene was isolated from a cosmid library. Surprisingly, the gene proved to be intronless. With the exception of three base substitutions (changing 2 amino acids in the deduced protein), the 1002-nucleotide sequence of the open reading frame was identical to that of mouse AdoMetDC cDNA. Moreover, the gene contained a poly(dA) tract at the 3' end and was flanked by 13-base pair direct repeats. Our findings suggest that this gene has arisen by retroposition, in which a fully processed AdoMetDC mRNA has been reverse transcribed into a DNA copy and inserted into the genome. By polymerase chain reaction, we positively identified the intronless gene in the mouse genome, and, by primer extension analysis, we proved the gene to be functional. Thus, its transcripts were found in many cell lines and tissues of the mouse and were particularly abundant in the liver. When the open reading frame of the intronless gene was expressed in Escherichia coli HT551, a strain with no AdoMetDC activity, it was found to encode a 38-kDa protein, corresponding to AdoMetDC proenzyme. Although the change of methionine 70 to isoleucine was close to the cleavage site at serine 68, this protein underwent proenzyme processing, generating a 31-kDa alpha subunit and an 8-kDa beta subunit. Importantly, the protein encoded by the intronless gene was functional, i.e. it catalyzed the decarboxylation of S-adenosylmethionine, and its specific activity was comparable with that of recombinant human AdoMetDC purified according to the same procedure.
Persson
K
K
Department of Cellular and Developmental Biology, University of Umeå, Sweden.
Holm
I
I
Heby
O
O
D012437:Adenosylmethionine Decarboxylase / Q000096:biosynthesis* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D015870:Gene Expression; D016678:Genome; D006801:Humans; D007438:Introns; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D013154:Spleen / Q000201:enzymology; D014158:Transcription, Genetic
D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012437:Adenosylmethionine Decarboxylase