Holland M J, Holland J P, Thill G P, Jackson K A
J Biol Chem. 1981 Feb 10;256(3):1385-95.
Segments of yeast genomic DNA containing two enolase structural genes have been isolated by subculture cloning procedures using a cDNA hybridization probe synthesized from purified yeast enolase mRNA. Based on restriction endonuclease and transcriptional maps of these two segments of yeast DNA, each hybrid plasmid contains a region of extensive nucleotide sequence homology which forms hybrids with the cDNA probe. The DNA sequences which flank this homologous region in the two hybrid plasmids are nonhomologous indicating that these sequences are nontandemly repeated in the yeast genome. The complete nucleotide sequence of the coding as well as the flanking noncoding regions of these genes has been determined. The amino acid sequence predicted from one reading frame of both structural genes is extremely similar to that determined for yeast enolase (Chin, C. C. Q., Brewer, J. M., Eckard, E., and Wold, F. (1981) J. Biol. Chem. 256, 1370-1376), confirming that these isolated structural genes encode yeast enolase. The nucleotide sequences of the coding regions of the genes are approximately 95% homologous, and neither gene contains an intervening sequence. Codon utilization in the enolase genes follows the same biased pattern previously described for two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes (Holland, J. P., and Holland, M. J. (1980) J. Biol. Chem. 255, 2596-2605). DNA blotting analysis confirmed that the isolated segments of yeast DNA are colinear with yeast genomic DNA and that there are two nontandemly repeated enolase genes per haploid yeast genome. The noncoding portions of the two enolase genes adjacent to the initiation and termination codons are approximately 70% homologous and contain sequences thought to be involved in the synthesis and processing messenger RNA. Finally there are regions of extensive homology between the two enolase structural genes and two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes within the 5- noncoding portions of these glycolytic genes.
利用从纯化的酵母烯醇化酶mRNA合成的cDNA杂交探针，通过亚克隆程序分离出了包含两个烯醇化酶结构基因的酵母基因组DNA片段。基于这两个酵母DNA片段的限制性内切酶图谱和转录图谱，每个杂交质粒都包含一个与cDNA探针形成杂交体的广泛核苷酸序列同源区域。这两个杂交质粒中位于该同源区域两侧的DNA序列是非同源的，表明这些序列在酵母基因组中不是串联重复的。已经确定了这些基因的编码区以及侧翼非编码区的完整核苷酸序列。从两个结构基因的一个阅读框预测的氨基酸序列与酵母烯醇化酶的序列极其相似（Chin, C. C. Q., Brewer, J. M., Eckard, E., and Wold, F. (1981) J. Biol. Chem. 256, 1370 - 1376），证实这些分离的结构基因编码酵母烯醇化酶。这些基因编码区的核苷酸序列大约95%同源，且两个基因都不包含间隔序列。烯醇化酶基因中的密码子使用遵循与先前描述的两个酵母甘油醛 - 3 - 磷酸脱氢酶结构基因相同的偏向模式（Holland, J. P., and Holland, M. J. (1980) J. Biol. Chem. 255, 2596 - 2605）。DNA印迹分析证实，分离的酵母DNA片段与酵母基因组DNA共线性，并且每个单倍体酵母基因组中有两个非串联重复的烯醇化酶基因。与起始和终止密码子相邻的两个烯醇化酶基因的非编码部分大约70%同源，并且包含被认为参与信使RNA合成和加工的序列。最后，在这些糖酵解基因的5′非编码部分内，两个烯醇化酶结构基因与两个酵母甘油醛 - 3 - 磷酸脱氢酶结构基因之间存在广泛的同源区域。
