Zou J, Scocca J R, Krag S S
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205.
Arch Biochem Biophys. 1995 Mar 10;317(2):487-96. doi: 10.1006/abbi.1995.1192.
The gene gpt encoding uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosaminylphosphoryltransferase (L-G1PT) was isolated by screening a Schizosaccharomyces pombe genomic DNA library in lambda phage under low-stringency hybridization using the Saccharomyces cerevisiae gene ALG7 as probe. Sequencing 2.4 kb of S. pombe DNA revealed a 1338-bp open reading frame (ORF) encoding a hydrophobic protein of 446 amino acids with a predicted molecular weight of 49,852. The S. pombe protein was 50% identical to the S. cerevisiae protein and 43% identical to the protein from Chinese hamster ovary (CHO) cells. Overexpression of the gpt gene in S. pombe cells increased resistance to tunicamycin 25-fold and increased the specific activity of the enzyme in isolated cell membranes 13-fold. This was accompanied by a 50-fold increase in poly(A)+ RNA hybridizing to the gpt probe. Northern analysis indicated a single 1.8-kb message is transcribed from the gpt gene. The gpt gene is essential for viability of S. pombe. Cells containing a disrupted ORF could be rescued by an expression plasmid containing either the intact S. pombe gpt ORF or the CHO L-G1PT cDNA. The S. pombe gpt gene was mapped to chromosome 2 near top1 and ade1.
通过使用酿酒酵母基因ALG7作为探针，在低严谨度杂交条件下筛选λ噬菌体中的粟酒裂殖酵母基因组DNA文库，分离出了编码尿苷二磷酸N-乙酰-D-葡糖胺：多萜醇磷酸N-乙酰葡糖胺磷酸转移酶（L-G1PT）的gpt基因。对2.4 kb的粟酒裂殖酵母DNA进行测序，发现一个1338 bp的开放阅读框（ORF），编码一个由446个氨基酸组成的疏水蛋白，预测分子量为49,852。粟酒裂殖酵母蛋白与酿酒酵母蛋白的同一性为50%，与中国仓鼠卵巢（CHO）细胞的蛋白同一性为43%。gpt基因在粟酒裂殖酵母细胞中的过表达使对衣霉素的抗性提高了25倍，并使分离细胞膜中该酶的比活性提高了13倍。这伴随着与gpt探针杂交的聚腺苷酸加尾（poly(A)+）RNA增加了50倍。Northern分析表明，gpt基因转录出一条单一的1.8 kb信使RNA。gpt基因对粟酒裂殖酵母的生存能力至关重要。含有破坏的ORF的细胞可以被含有完整的粟酒裂殖酵母gpt ORF或CHO L-G1PT cDNA的表达质粒拯救。粟酒裂殖酵母gpt基因被定位到2号染色体上靠近top1和ade1的位置。
