Studies on the Adenosine Deaminase Gene from Nocardioides sp. J-326TK

Nocardioides sp. J-326TK의 Adenosine Deaminase Gene에 관한 연구

Adenosine deaminase gene from Nocardioides sp. J-326TK was cloned by polymerase chain reaction using primers (PI, PII and PIII) constructed from the highly conserved amino acid sequences among Escherichia coli, mouse and human. A PCR product of about 800bp, as expected from the sequence of E. coli adenosine deaminase gene, was obtained from Nocardioides sp. J-326TK chromosomal DNA double-digested with EcoRI and Pst I. DNA sequencing of the PCR product after cloning into pT7Blue T-vector shows 99.5% and 98.9% homologies in nucleotide and amino acid sequences, respectively, with the E. coli adenosine deaminase whereas 59.5% and 46.8% homologies with the human adenosine deaminase, indicating the evolutionarily relationship of these organisms.

Nocardioides sp. J-326TK의 adenosine deaminase gene을 분리하기 위하여 genomic DNA를 제한효소로 무작위적으로 절단하여 pBluscript KS에 ligation시켰다. 또한 hu-man과 mouse, E. cali 등의 adenosine deaminase gene의 보존적인 부위를 primer로 합성을 하여 PCR reaction을 행하였다. Genomic DNA를 cloning시킨 pKSN60은 5kb정도의 DNA를 포함하고 있으며 sourthern hybridization 등의 여러 확인 실험을 통하여 adenosine deaminase gene을 포함하고 있다는 알았다. PCR product를 cloning시켜 형성된 recombinant plasmid를 PCR reaction의 primer로서 pTBN20를 sequencing을 행하였다. 그 결과를 다른 ade-nosine deaminase gene의 서열과 비교를 하였는데 미생물인 E. coli와는 nucleotide sequence는 99.5%, amino acid sequence는 98.9%의 homology를 나타내고 human과는 각각 59.5%, 46.8%의 homolosy를 나타내었다.