Varma A, Kwon-Chung K J
Molecular Microbiology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Gene. 1999 May 31;232(2):155-63. doi: 10.1016/s0378-1119(99)00132-8.
The GPD gene encoding glyceraldehyde-3-phosphate dehydrogenase was isolated from Cryptococcus neoformans, a heterobasidiomycetous yeast that is pathogenic to humans. The gene contains 11 introns, differing from the conserved intron positions found in the GPD genes of Basidiomycetes. The predicted amino-acid sequence of this gene is extremely similar to that reported from GPD proteins of other basidiomycetes. The promoter region of the C. neoformans GPD gene was similar to those of other basidiomycetes. Plasmid constructs containing up to 1600 base pairs upstream of the native GPD open reading frame were used to express either the native URA5 gene in a ura5 mutant or the heterologous hphI gene (a bacterial gene that confers resistance to the aminoglycoside hygromycin) in a wild-type strain of C. neoformans. Transformation frequencies resulting from the plasmid-borne Gpdp::URA5 gene were at levels similar to those of the native URA5, which suggested that all the sequences necessary for proper expression were present. Transformation frequencies using the Gpdp::hphI gene constructs were poor. However, addition of DNA sequences flanking the 3'-end of an native C. neoformans gene significantly improved the transformation frequencies resulting from the expression of the heterologous hphI gene.
编码甘油醛-3-磷酸脱氢酶的GPD基因是从新型隐球菌中分离出来的，新型隐球菌是一种对人类致病的异担子菌酵母。该基因含有11个内含子，与担子菌纲GPD基因中保守的内含子位置不同。该基因预测的氨基酸序列与其他担子菌纲GPD蛋白报道的序列极为相似。新型隐球菌GPD基因的启动子区域与其他担子菌纲的相似。含有天然GPD开放阅读框上游多达1600个碱基对的质粒构建体用于在ura5突变体中表达天然URA5基因，或在新型隐球菌野生型菌株中表达异源hphI基因（一种赋予对氨基糖苷类潮霉素抗性的细菌基因）。由质粒携带的Gpdp::URA5基因产生的转化频率与天然URA5的水平相似，这表明存在正确表达所需的所有序列。使用Gpdp::hphI基因构建体的转化频率较低。然而，添加新型隐球菌天然基因3'端侧翼的DNA序列显著提高了异源hphI基因表达产生的转化频率。
