Zieler H A, Walberg M, Berg P
Department of Biochemistry, Stanford University School of Medicine, California, USA.
Mol Cell Biol. 1995 Jun;15(6):3227-37. doi: 10.1128/MCB.15.6.3227.
The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.
腺病毒E1A基因的蛋白质产物参与多种转录和细胞周期事件，涉及与人类细胞中存在的几种蛋白质相互作用，包括转录机制的部分成分以及细胞分裂的负调控因子，如Rb基因产物和p107。为了确定酿酒酵母中是否存在E1A的功能同源物，我们开发了一种针对依赖E1A生长的突变体的遗传筛选方法。该筛选基于菌落颜色扇形分析，可用于鉴定依赖含E1A质粒的维持和表达的突变体。利用这种筛选方法，我们分离出了五个依赖E1A的12S或13S cDNA表达才能生长的突变体。质粒洗牌分析证实，质粒依赖性表型是由于存在12S或13S E1A cDNA，并且两种形式的E1A对所有突变体生长的挽救效果同样良好。这五个突变体分为两类，分别命名为web1和web2（意为“非常需要E1A”）。用E1A突变形式进行的质粒洗牌分析表明，保守区域1（CR1）是挽救web1和web2 E1A依赖性酵母突变体生长所必需的，而N端的22个氨基酸只是部分必需；保守区域2（CR2）和C端则是可有可无的。web1和web2组突变体的表型均由单个基因缺陷导致，并且克隆了能完全互补两组突变体表型的酵母基因。WEB1基因序列编码一种1273个氨基酸的蛋白质，与SEC31相同，SEC31是一种参与内质网运输小泡出芽的蛋白质。WEB2基因编码一种与核酸依赖性ATP酶具有同源性的1522个氨基酸的蛋白质。缺失WEB1或WEB2均是致死的。E1A的表达无法挽救web1或web2无效等位基因的致死性，这意味着导致E1A依赖性的等位基因特异性突变。
