Molecular Evolutional Study on Gene-expression of Enzymes Responsible for Uric Acid-degradation

为了开展研究项目，我们关注了不同活细胞中尿酸酶的差异表达。人们一直认为尿酸酶在鸡胚早期表达，而在后期和成年鸡中不表达。为了证明鸡胚中存在尿酸酶，我们试图在鸡胚早期检测到抗大鼠肝脏尿酸酶的交叉反应产物和与大鼠肝脏尿酸酶cDNA同源的cDNA。观察产物和cDNA。但在鸡胚中只表达了少量的尿酸酶，且难以确定其氨基酸序列以证实产物和cDNA。然后，我们将注意力转向了在成年期表达的大鼠肝脏尿酸酶，并打算研究尿酸酶基因的分子进化。以大鼠肝脏尿酸酶cDNA为探针，分离大鼠尿酸酶基因。从大鼠基因组文库中分离到5个含尿酸酶基因的克隆。该基因全长40kb，由8个外显子组成。所有外显子-内含子连接序列均符合GT/AG规则。结合限制性内切图谱和Southern blot分析，发现该酶是由一个拷贝基因编码的。通过引物延伸分析和Sl保护分析确定了大鼠尿酸酶基因的转录起始位点。分析表明对连续核苷酸的不同使用。主密码子位于第一个蛋氨酸密码子上游55个核苷酸处。5'侧区核苷酸序列分析显示存在TATA序列、CAAT序列和被直接重复包围的回文序列。典型的启动子序列可能参与了尿酸酶的肝脏特异性表达。

We paid attention to differential expressions of uricases in various living cells in order to carry out the research project. It has been supposed that uricase is expressed at the early stage of chicken embryo but that it is not at the later stage and in the adult chicken. In order to show the presence of uricase at the chicken embryo, we tried to detect a cross-reactive product against anti-rat liver uricase and a cDNA homologous to rat liver uricase cDNA in the early stage of chicken embryo. We observed the product and the cDNA. However, a little amount of uricase was expressed in chicken embryo and it was difficult to determine the amino acid sequence for proving the product and the cDNA. Then, we turned the attention to rat liver uricase which is expressed in the adult stage and intended to study the molecular evolution of uricase genes. We used rat liver uricase cDNA as a probe to isolate rat uricase gene. Five clones containing the uricase gene were isolated from rat genomic libraries. The gene spans 40 kb and consists of 8 exons. All the exon-intron junctional sequences conform to GT/AG rule. It was found that the enzyme is encoded by a single copy-gene from the coincidence between the restriction map and Southern blot analysis. The transcriptional initiation site in rat uricase gene was determined by primer-extension analyses and Sl protection analysis. The analyses indicated the differential use to consecutive nucleotides. The principal one is located 55 nucleotides upstream from the first methionine codon. Nucleotide sequence analysis of the 5'-flanking region showed the presences of a TATA sequence, a CAAT sequence and a palindromic sequence surrounded by a direct repeat. The typical promoter sequence could be involved in the liver-specific expression of uricase.