MATING TYPE SILENCING AND SWITCHING IN YEAST

酿酒酵母中的交配型基因座为位置效应调控提供了一个显著的例子。位于酵母染色体III末端的交配型调控基因HML和HMR被包装在异染色质中，这排除了它们的表达。我们计划探索异染色质沉积和维持在沉默位置的机制，并检查异染色质的结构，以确定它是如何阻止进入转录装置的。我们的实验计划包括：(1)DNA复制和异染色质形成之间的关系的确定；(2)染色质组装因子的体内分析；(3)提高异染色质持久性的组蛋白突变体的分离和分析；(4)沉默微环的分离和鉴定。这些结果应该有助于阐明影响X失活和染色体印记等不同事件的调控过程，这些事件是各种疾病的基础，包括一些散发性癌症。我们还计划研究酵母交配型相互转化的方向性机制。两个沉默的交配型盒内信息的激活是通过定向转座到一个表达位点发生的。供体盒的选择不是随机的，而是由细胞的交配类型决定的，通过激活或抑制染色体上的一个位置来激活手臂的整个远端部分进行重组。我们计划通过(1)确定三号染色体在体内的大规模组织；(2)确定激活染色体进行重组所需的蛋白质，来探索基因组远端区域选择性相互作用的机制基础。这些实验的结果应该有助于我们确定在执行发育计划时，基因组的远距离区域可以在功能上和选择性地相互作用。这些观察结果应该会在类似的过程中产生洞察力，例如在更大的真核生物中进行免疫球蛋白类别转换。

The mating type loci in Saccharomyces cerevisiae provide a striking example of position effect regulation. Mating type regulatory genes located at the ends of chromosome III in yeast in loci designated HML and HMR are packaged in heterochromatin, which precludes their expression. We plan to explore the mechanism by which heterochromatin is deposited and maintained at silent loci and to examine the structure of the heterochromatin to determine how it precludes access to the transcriptional apparatus. Our experimental plan includes (1) determination of the relationship between DNA replication and heterochromatin formation; (2) an in vivo analysis of chromatin assembly factors; (3) isolation and analysis of histone mutants that enhance heterochromatin persistence; and (4) isolation and characterization of silenced minicircles. These results should shed light on regulatory processes affecting such diverse events as X-inactivation and chromosome imprinting, which underlies various diseases including a number of sporadic cancers. We also plan to examine the mechanism underlying directionality of mating type interconversion in yeast. Activation of information within two silent mating type cassettes occurs by directed transposition to an expression locus. Choice of donor cassettes is not random but is dictated by the mating type of the cell through activation or inhibition of a site on the chromosome that activates the entire distal portion of the arm for recombination. We plan to explore the mechanistic basis of this selective interaction of distant regions of the genome by (1) determining the large scale organization of chromosome III in vivo; and (2) determining the proteins required for activation of the chromosome for recombination. The results of these experiments should help us define how distant regions of a genome can functionally and selectively interact in execution of a developmental program. These observations should yield insights in similar processes, such as immunoglobulin class switching, in larger eucaryote.