Steyn A J, Marmur J, Pretorius I S
Department of Microbiology, University of Stellenbosch, South Africa.
Yeast. 1996 Aug;12(10):925-37. doi: 10.1002/(SICI)1097-0061(199608)12:10%3C925::AID-YEA987%3E3.0.CO;2-0.
The expression in Saccharomyces cerevisiae and Schizosaccharomyces pombe of a cDNA copy of the Lipomyces kononenkoae IGC4052B alpha-amylase gene (LKA1), linked to the phosphoglycerate kinase gene (PGK1) promoter, resulted in the extracellular production of biologically active alpha-amylase (LKA1). However, transformation of S. cerevisiae and Schiz. pombe with a cosmid clone containing the complete genomic copy of LKA1, expressed from its native promoter, did not result in secretion of active alpha-amylase by any of the transformants. When the cDNA copy of LKA1 was expressed in S. cerevisiae under control of the wild-type L, kononenkoae promoter, biologically active alpha-amylase was secreted into the culture medium, indicating the recognition of the LKA1 promoter in S. cerevisiae. Sequence analysis of the GC-rich LKA1 promoter revealed canonical sequences that are homologous to the TATAAA, CAAT and CCAAT boxes and GCN4-binding sites that are present in several promoter sequences of S. cerevisiae. Primer extension analysis of LKA1 transcripts in L. kononenkoae indicated major initiation sites at nucleotides -64 and -65. S. cerevisiae and Schiz. pombe cells transformed with a plasmid containing the open reading frame of the genomic copy of LKA1, linked to the PGK1 promoter, did not produce alpha-amylase. Polymerase chain reaction mapping and sequence analysis revealed the presence of a 61-bp intron in the genomic copy of LKA1 that impaired synthesis of biologically active alpha-amylase in S. cerevisiae and Schiz. pombe. This intron contains donor, acceptor and branch sequences that correlate with the consensus sequences identified in the introns of split genes from Schiz. pombe and mammals. Pulsed-field gradient gel electrophoresis resolved at least eight chromosomal DNAs for L. kononenkoae IGC4052B and chromoblot analysis indicated that LKA1 is located on the second smallest chromosome, designated chromosome II.
将与磷酸甘油酸激酶基因（PGK1）启动子相连的科诺宁科脂酵母IGC4052Bα-淀粉酶基因（LKA1）的cDNA拷贝在酿酒酵母和粟酒裂殖酵母中表达，导致生物活性α-淀粉酶（LKA1）在细胞外产生。然而，用含有LKA1完整基因组拷贝且由其天然启动子表达的黏粒克隆转化酿酒酵母和粟酒裂殖酵母，任何转化子均未分泌出活性α-淀粉酶。当LKA1的cDNA拷贝在野生型科诺宁科脂酵母启动子的控制下在酿酒酵母中表达时，生物活性α-淀粉酶被分泌到培养基中，这表明酿酒酵母能够识别LKA1启动子。对富含GC的LKA1启动子的序列分析揭示了与酿酒酵母多个启动子序列中存在的TATAAA、CAAT和CCAAT框以及GCN4结合位点同源的规范序列。对科诺宁科脂酵母中LKA1转录本的引物延伸分析表明，主要起始位点位于核苷酸-64和-65处。用含有与PGK1启动子相连的LKA1基因组拷贝开放阅读框的质粒转化的酿酒酵母和粟酒裂殖酵母细胞未产生α-淀粉酶。聚合酶链反应图谱分析和序列分析表明，LKA1的基因组拷贝中存在一个61bp的内含子，该内含子损害了酿酒酵母和粟酒裂殖酵母中生物活性α-淀粉酶的合成。该内含子包含供体、受体和分支序列，这些序列与粟酒裂殖酵母和哺乳动物的断裂基因内含子中鉴定出的共有序列相关。脉冲场梯度凝胶电泳解析了科诺宁科脂酵母IGC4052B的至少八条染色体DNA，染色体印迹分析表明LKA1位于第二小的染色体上，命名为染色体II。
