Molecular genetic study of hereditary orotic aciduria and UMP synthase

1.从日本癌症研究资源库获得了一个EMBL-3基因组DNA文库。用人UMP合酶cDNA筛选了大约6 × 10^6个噬菌体克隆，用EcoRI或hind Ⅲ消化6个阳性克隆并作图。三个克隆相互重叠，形成最长的连续序列，描述了一个单拷贝基因，跨度约15 kb。外显子/内含子边界的核苷酸序列数据显示，所有内含子开始于5 'GT，结束于3' AG末端。对人UMP合成酶的5'端侧翼序列进行了测定，该序列位于推测的转录起始位点上游约1.0kb处。没有一致的TATA或CAAT框。而持家基因启动子区5 '端附近区域则与此相反.将UMP合成酶cDNA克隆到pVL 1392杆状病毒表达载体中。在Sf 21昆虫细胞中表达的cDNA产生了约52 kD的蛋白质和UMP合酶活性的显着上升。重组蛋白经FPLC系统纯化至均一。通过超声破碎约4x10^8个细胞，将上清液上样至羟基磷灰石柱，然后在MonoQ柱上进行离子交换色谱。最终产物经SDS聚丙烯酰胺凝胶电泳显示为52 kD的单一条带。UMP合成酶基因的第109位密码子发生T → G颠换，导致缬氨酸→甘氨酸的突变，该突变已在我所诊断的遗传性乳清酸尿症患者中发现。通过杆状病毒系统表达的突变的UMP合酶表现出UMP合酶活性的显著降低。

1. A lambdaEMBL-3 genomic DNA library was obtained from the Japanese Cancer Research Resources Bank. Approximately 6x10^6 phage clones were screened with the human UMP synthase cDNA.Six positive clones were digested with EcoRI or hindIII and mapped. Three clones overlapping one another and forming the longest continuous sequence describe a single-copy gene which spans approximately 15 kb. Ncleotide sequence data of the exon/intron boundaries show that all introns begin with a 5'GT and conclude with a 3'AG terminus. The 5' flanking region of human UMP synthase was also sequenced about 1.0kb upstream of the putative transcription initiation site. There were no consensus TATA or CAAT boxes. However, the region around and immediately 5'of the promotor region of house keeping genes.2. The UMP synthase cDNA was cloned into pVL1392 baculovirus expression vector. Expression of the cDNA in Sf21 insect cells yielded a protein of approximately 52 kD and a significant rise in UMP synthase activity. The recombinant protein was purified to homogeneity using FPLC system. Approximately 4x10^8 cells were disrupted by sonication and the supernatant was applied to a hydroxylapatite column followed by an ion exchange chromatography on a MonoQ column. SDS polyacrylamide gel electrophoresis of the final product demonstrated a single band of 52 kD.3. The T to G transversion causing a valine to glycine change at codon 109 was introduced to the UMP synthase cDNA.This mutation had been described in a hereditary orotic aciduria patient diagnosed at our institute. The expressed mutated UMP synthase by the baculovirus system demonstrated a marked reduction in UMP synthase activity.