UMP synthase activity expressed in deficient hamster cells by separate transferase and decarboxylase proteins or by linker-deleted bifunctional protein.
UMP synthase activity expressed in deficient hamster cells by separate transferase and decarboxylase proteins or by linker-deleted bifunctional protein.
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UMP 合酶活性在缺陷仓鼠细胞中通过单独的转移酶和脱羧酶蛋白或通过连接子缺失的双功能蛋白表达。
DOI：
10.1007/bf02254768
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Suttle,DP
中科院分区：
文献类型：
--
作者：
Lin,T;Suttle,DP
通过聚合酶链反应技术产生编码双功能蛋白 UMP 合酶的乳清酸磷酸核糖基转移酶 (OPRT) 和乳清酸脱羧酶 (ODC) 结构域的人 UMP 合酶 cDNA 片段，并将其克隆到真核表达载体中。将单独的 OPRT 和 ODC 载体与选择性标记一起共转染到需要外源尿苷生长的 UMP 合酶缺陷仓鼠细胞 (Urd−C) 中。分离在不添加尿苷的培养基中选择存活的转染Urd-C细胞，并将其命名为转移酶脱羧酶Urd-C (TDU)。所有选定的集落均含有与 OPRT 和 ODC 表达载体相对应的 DNA。与其他 TDU 系相比，两个细胞系（TDU3 和 TDU5）将更多的 OPRT 和 ODC 载体拷贝整合到其基因组中。使用 UMP 合酶特异性多克隆抗血清在蛋白质印迹上检测到 28.6-kDa ODC 蛋白条带和 24.4-kDa OPRT 条带。 TDU系的OPRT活性高达对照CHL细胞OPRT活性的8.7倍，ODC活性高达对照水平的12.5倍。单功能蛋白中的 OPRT 和 ODC 活性均比双功能 UMP 合酶蛋白中的热稳定性差。在 45°C 下，单功能 OPRT 蛋白的稳定性低于 ODC 蛋白。转染细胞在 6-氮杂苷中的生长导致单功能 ODC 蛋白的活性和温度稳定性显着增加。通过删除连接两个催化结构域的可疑接头区域，构建了 UMP 合酶双功能蛋白。删除连接子的 UMP 合酶在 OPRT 或 ODC 活性或温度稳定性方面没有表现出显着变化。双功能蛋白稳定性的增加可能是其在哺乳动物细胞中进化选择的一个因素。
Segments of the human UMP synthase cDNA coding for the orotate phosphoribosyl transferase (OPRT) and orotidylate decarboxylase (ODC) domains of the bifunctional protein UMP synthase were produced by polymerase chain reaction techniques and cloned into a eukaryotic expression vector. The separate OPRT and ODC vectors, along with a selectable marker, were cotransfected into UMP synthase-deficient hamster cells (Urd−C) that require exogeneous uridine for growth. Transfected Urd−C cells surviving selection in media without added uridine were isolated and designated transferase decarboxylase Urd−C (TDU). All of the selected colonies contained DNA corresponding to the OPRT and ODC expression vectors. Two cell lines (TDU3 and TDU5) integrated many more copies of the OPRT and ODC vectors into their genomes compared to the other TDU lines. A 28.6-kDa ODC protein band and a 24.4-kDa OPRT band were detected on western blots with UMP synthase-specific polyclonal antiserum. The OPRT activity of the TDU lines was up to 8.7 times the OPRT activity of control CHL cells, and the ODC activity was up to 12.5 times control levels. Both OPRT and ODC activities in the monofunctional proteins were less heat stable than in the bifunctional UMP synthase protein. The monofunctional OPRT protein was less stable than the ODC protein at 45°C. Growth of transfected cells in 6-azauridine resulted in striking increases in activity and temperature stability for the monofunctional ODC protein. A UMP synthase bifunctional protein was constructed with a deletion of the suspected linker region joining the two catalytic domains. The linker-deleted UMP synthase showed no significant change in either OPRT or ODC activity or temperature stability. The increased stability of the bifunctional protein may be a factor in its evolutionary selection in mammalian cells.