Molecular cloning of a cDNA encoding arylalkylamine N-acetyltransferase from the testicular system of Periplaneta americana: primary protein structure and expression analysis.
DNA encoding a fragment of putative arylalkylamine N-acetyltransferase (NAT) of the American cockroach, Periplaneta americana, was amplified by PCR with degenerate primers based on the two peptides previously purified from the testicular system of this species. A full clone was obtained by RACE-PCR. The clone consisted of 89 bp 5'-UTR, 753-bp open reading frame and 712-bp 3'-UTR. The amino acid sequence of 251 residues deduced from this ORF corresponded to the predicted molecular mass of 28.5 kDa. The predicted amino acid sequence had an overall identity of 35% with NAT1 and 27% with NAT2 of Drosophila melanogaster, respectively. Structural analysis revealed that NAT from P. americana contained two motifs characteristic of the NAT superfamily and three conserved regions (C/c-1, D/c-1, D/c-2) distinguishing aaNAT subfamily. Northern blot analysis showed that the mRNA of approximately 1.5 kb was transcribed at a high level in the testicular system, and corresponded to the length of the cDNA, i.e., 1,554 bp. Significant levels of NAT transcript were also detected in the midgut, ovary and the accessory glands and at much lower levels in the fat body and brain. Southern blot analysis suggested the presence of a single copy of the cloned gene in the genome.
Bembenek
Jadwiga
J
Division of Molecular Science, Graduate School of Science and Technology, Kobe University, Kobe, Japan. jadwigabembenek@yahoo.com
Sakamoto
Katsuhiko
K
Takeda
Makio
M
D000595:Amino Acid Sequence; D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010526:Periplaneta / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013737:Testis / Q000201:enzymology*
D018076:DNA, Complementary; D012333:RNA, Messenger; D047068:Arylalkylamine N-Acetyltransferase