Kuprina N Iu, Krol' E S, Koriabin M Iu, Bannikov V M, Kirillov A V, Zakhar'ev V M, Larionov V L
Mol Biol (Mosk). 1993 May-Jun;27(3):589-607.
Earlier we have identified the chl4-1 mutation in a screen for yeast mutants with increased loss of chromosome III and circular artificial minichromosome in mitosis. Mutation in the CHL4 gene leads to a 50-100-fold promotion in the rate of chromosome loss per cell division compared to the isogenic wild type strain. Detailed analysis of behaviour of the circular minichromosome marked by the CUP1 gene has shown that minichromosome nondisjunction (2:0 segregation) leading to an increase in the copy number of minichromosome in part of a cell population is the main reason of minichromosome instability in the mutant. The unique peculiarity of chl4-1 mutation is the ability of the strains carrying this mutation to stably maintain circular dicentric minichromosomes without any rearrangement during many generations. (In the wild type strains dicentric minichromosomes are extremely unstable. As a consequence of that there is a strong selection for cells harboring monocentric derivatives in a population of cells derived from a cell containing a dicentric plasmid). Introduction of the second centromere into one of the natural chromosomes (chromosomes II or III) in the chl4-1 mutant leads to the same dramatic consequences as that in the wild type strain (mitotic lag of cells harboring dicentric chromosomes and, as a result of that, selective pressure for cells harboring monocentric derivatives of dicentric chromosome). A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation. Nucleotide sequence analysis of CHL4 revealed a 1.4-kb open reading frame with a predicted 53-kDa protein sequence. Analyzing the sequence of the CHL4 protein we have found a region meeting the necessary requirements for the helix-turn-helix (HTH) structure. This region of the CHL4 protein has about 40% homology with the repressor of tryptophane operon (TrpR) of E. coli. A strain containing a null allele of CHL4 was viable under standard growth conditions, but had temperature-sensitive phenotype (conditional lethality at 34 degrees C). We suggest that the CHL4 gene product is one of the components of the segregation cell machinery.
此前，我们在一项针对酵母突变体的筛选中鉴定出chl4-1突变，这些突变体在有丝分裂过程中染色体III和环状人工微型染色体的丢失增加。与同基因野生型菌株相比，CHL4基因突变导致每个细胞分裂中染色体丢失率提高50至100倍。对由CUP1基因标记的环状微型染色体行为的详细分析表明，微型染色体不分离（2:0分离）导致部分细胞群体中微型染色体拷贝数增加，这是突变体中微型染色体不稳定的主要原因。chl4-1突变的独特之处在于，携带这种突变的菌株能够在许多代中稳定维持环状双着丝粒微型染色体而不发生任何重排。（在野生型菌株中，双着丝粒微型染色体极其不稳定。因此，在源自含有双着丝粒质粒的细胞群体中，对携带单着丝粒衍生物的细胞有强烈的选择作用。）在chl4-1突变体的一条天然染色体（染色体II或III）中引入第二个着丝粒会导致与野生型菌株相同的显著后果（携带双着丝粒染色体的细胞有丝分裂滞后，结果是对携带双着丝粒染色体单着丝粒衍生物的细胞有选择压力）。通过对chl4-1突变的互补作用分离出了CHL4的基因组克隆。CHL4的核苷酸序列分析揭示了一个1.4 kb的开放阅读框，其预测的蛋白质序列为53 kDa。分析CHL4蛋白的序列时，我们发现了一个符合螺旋-转角-螺旋（HTH）结构必要要求的区域。CHL4蛋白的这个区域与大肠杆菌色氨酸操纵子的阻遏物（TrpR）有大约40%的同源性。含有CHL4无效等位基因的菌株在标准生长条件下是有活力的，但具有温度敏感表型（在34℃时条件致死）。我们认为CHL4基因产物是分离细胞机制的组成部分之一。
