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

我们注意到尿酸酶在不同活细胞中的差异表达，以进行这一研究项目。已有的研究认为尿酸酶在鸡胚胎早期表达，但在后期和成年鸡中不表达。为了证明尿酸酶在鸡胚中的存在，我们试图在鸡胚胎早期检测到一个抗大鼠肝尿酸酶交叉反应产物和一个与大鼠肝尿酸酶基因同源的cDNA。我们观察了产物和基因的表达。然而，尿酸酶在鸡胚中有少量表达，很难确定其氨基酸序列以证明产物和基因的真实性。然后，我们将注意力转向在成体阶段表达的大鼠肝尿酸酶，旨在研究尿酸酶基因的分子进化。以大鼠肝尿酸酶基因为探针，分离大鼠尿酸酶基因。从大鼠基因组文库中分离到5个含有尿酸酶基因的克隆。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。限制性内切酶图谱与Southern杂交结果一致，发现该酶由单一拷贝基因编码。通过引物延伸分析和Sl保护分析确定大鼠尿酸酶基因的转录起始点。分析表明，连续核苷酸的不同用途。主密码子位于甲硫氨酸第一密码子上游55个核苷酸。5‘-侧翼区的核苷酸序列分析表明，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.