Determining the start codons of aadA2 gene in class 1 integron / 中华微生物学和免疫学杂志

ABSTRACT

ObjectiveTo determine whether aadA2 gene can be translated from the ATG triplet,which there was no plausible ribosome binding site preceding it,and synthetized a functional protein in class 1 integron.MethodsSite-specific mutagenesis was used to construct aadA2 gene cassette with different start codons,together with their upstreamed promoters of variable regions were cloned into plasmid pACYC184 respective.The constructed plasmids were then transfored into Escherichia coli JM109,Western blot was used to detect the translation products of aadA2 gene with different start codons.Broth microdilution method was used to detect the minimal inhibitory concentrations to streptomycin in Escherichia coli JM109 containing aadA2 gene with different start codons.ResultsaadA2 gene can initiate translation from both ATG and GTG triplets in aminoacyl -3-adenylyltransferase protein synthesis,though there was no plausible ribosome binding site preceding the ATG triplet.Besides GTG and ATG triplets,there was other start codon downstream of the GTG triplet in aadA2 gene.The translated products that initiated from the start codons that described above were all functional AAD(3) proteins that can be detected by anti- aminoacyl -3-adenylyltransferase polyclonal antisera in Western blot and conferred different resistance levels to streptomycin in E.coli.ConclusionWhen inserted as the first gene cassette in class 1 integron,aadA2 gene can initiate translation from ATG triplet and synthetized a functional protein,though there was no plausible ribosome binding site preceding it.This structural characterization of class 1 integron can initiate translation of the open reading frame harbored in gene cassette that integrated into class 1 integron,though there was no plausible RBS preceding the start codon.This make class 1 integron be more convenience to express the genes that capture from environment.