CONTROL OF GENOME SIZE

这个项目的目标是描述酿酒酵母倍性受到限制的机制。倍性高达6n的酵母很容易产生，然而，使用低分辨率技术来产生倍性高于6n(大于6n)的活性酵母是不可能的。在其他真核细胞中，多倍体的局限性也以一种细胞类型特有的方式出现。许多肝细胞为4N，但达到8N倍性的数量有限。此外，大鼠肝癌细胞的增殖活性与其倍性成反比。相比之下，巨核细胞可以高达64n。本研究建议的具体目标如下：(1)准确确定酵母多倍体增加的限度。互补标记将被用来从使用大量亲本细胞的杂交中选择可行的8n多倍体。将确定8n多倍体出现的频率(如果在这些菌株中8n多倍体是可能的)。将仔细检查分离的8n酵母的表型。(2)确定限制多倍体的基因。已知有17个基因具有倍性调节的表达。通过过度表达那些在多倍体中被抑制的基因和干扰那些被诱导的基因，将分析每个基因的表达变化在防止存活超过6n的菌株中可能发挥的作用。(3)在比较n×n和多倍体杂交时，确定在接合过程中基因表达模式的差异。全基因组mRNA表达微阵列将用于分析n×n、2n×2n和4n×4n杂交的酵母。这些杂交组合之间差异表达的基因将被识别和进一步检测。

The goals of this project are to characterize the mechanisms by which ploidy is limited in Saccharomyces cerevisiae. Yeast with ploidies as high as 6n have been easily generated, however, it has not been possible using low-resolution techniques to generate viable yeast with a ploidy above 6n (greater than 6n). Limitations in polyploidy are also seen in other eukarytoic cells in a cell-type specific manner. Many liver hepatocytes are 4n, but, the number that achieve a ploidy of 8n are limited. In addition, the proliferative activity of rat liver carcinoma cells decreases in an inverse proportion to their ploidy. In contrast, megakaryocytes can reach as high as 64n. The specific aims of this research proposal are the following: (1) Accurately identify the limits on increasing polyploidy in yeast. Complementary markers will be used to select for viable 8n polyploids from crosses using large numbers of parental cells. The frequency with which 8n polyploids arise (if 8n polyploidy is possible in these strains) will be determined. The phenotypes of isolated 8n yeast will be carefully examined. (2) Identify genes that are involved in limiting polyploidy. Seventeen genes are known to have ploidy-regulated expression. The role that the altered expression of each may play in the prevention of viable greater than 6n strains will be analyzed by overexpressing those that are repressed in polyploids and disrupting those that are induced. (3) Identify differences in the patterns of genes expressed during conjugation when comparing n x n and polyploid crosses. Whole genome mRNA expression microarrays will be used to analyze yeast in n x n, 2n x 2n, and 4n x 4n crosses. Genes that are differentially expressed between these crosses will be identified and examined further.