MATING TYPE SILENCING AND SWITCHING IN YEAST

酿酒酵母的交配型基因座是位置效应调控的一个显著例子。交配型调节基因位于酵母III染色体末端的HML和HMR位点，它们被包装在异染色质中，因此无法表达。我们计划探索异染色质沉积和维持在沉默位点的机制，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)确定DNA复制与异染色质形成之间的关系；(2)染色质组装因子的体内分析；(3)增强异染色质持久性的组蛋白突变体的分离和分析；(4)沉默微圆的分离与表征。这些结果应该阐明影响诸如x失活和染色体印记等不同事件的调控过程，这些事件是包括一些散发性癌症在内的各种疾病的基础。我们还计划研究酵母中交配型相互转换的方向性机制。两个沉默交配型磁带中的信息激活是通过直接转置到一个表达位点发生的。供体磁带的选择不是随机的，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点，激活手臂的整个远端部分进行重组。我们计划通过(1)确定III号染色体在体内的大规模组织来探索基因组远端区域这种选择性相互作用的机制基础；(2)确定激活染色体进行重组所需的蛋白质。这些实验的结果应该有助于我们确定基因组的遥远区域在执行发育程序时能够在功能上和选择性地相互作用。这些观察结果应该对类似的过程，如免疫球蛋白类转换，在更大的真核生物中产生见解。

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.