X-CHROMOSOME INACTIVATION IN OPOSSUM DEVELOPMENT

长期目标是了解细胞的 和亚细胞事件有关的基因活性的调节， 哺乳动物的发展，并最终了解分子 控制基因表达的过程，在正常和异常 发展过程。 方法是利用二分法， X染色体失活模式（随机与确定） 真兽目和后兽目哺乳动物作为比较研究的模式， X连锁弗吉尼亚负鼠（Virginia opossum）：已知的唯一一种 在一个以上的X连锁基因位点上表现出变异）和Monodelphis 南极负鼠（灰色，短尾负鼠），将作为模型系统， 建立X染色体复制的模式和时间， 父源性和母源性X连锁基因的表达 胚胎发生和出生后早期发育的关键阶段。 具体而言，目标是（1）确定 表达Gpd和Pgk-A基因（编码葡萄糖-6-磷酸 脱氢酶和磷酸甘油酸激酶-A，分别对母体 和父方X染色体在新生育儿袋幼仔的组织和器官中， 以及中晚期的组织、器官和胚外膜 D.妊娠胚胎（2）确定是否 母本和父本Gpd和Pgk-a基因的差异表达 发生在D.弗吉尼亚州，如果是这样，当它可以 首先被检测到;和（3）确定是否一个晚期复制的X 染色体（指示非活性状态）存在于胚胎中， 妊娠早、中期的胚外组织; virginiana和M.如果是，则将复制时间与 Gpd和Pgk-A表达模式。 已知的女性胚胎 发育阶段，D. virginiana、特异性Gpd和 Pgk-A基因型将通过实验室杂交产生。 电泳 方法将被用来推导，在D.弗吉尼亚州，相对水平 在1）整体中的母本和父本Gpd和Pgk-A等位基因的表达 早期胚胎，2）胚外膜和胚胎本身在老年人 胚胎; 3）初生囊幼的各种组织器官。 的 5-BrdU标记技术将用于确定是否存在 晚期复制的X染色体在胚胎阶段的细胞中， 物种

The long-term objectives are to develop an understanding of the cellular and subcellular events pertinent to the regulation of gene activity in mammalian development, and ultimately to understand the molecular processes that control gene expression in normal and abnormal developmental processes. The approach is to exploit the dichotomy in patterns of X-chromosome inactivation (random vs. determinate) between eutherian and metatherian mammals as comparative probes into patterns of X-linked virginiana (Virginia opossum: the only metatherian known to exhibit variation at more tan one X-linked gene locus) and Monodelphis domestica (gray, short-tailed opossum), will serve as model systems to establish the patterns and timing of X-chromosome replication and the expression of paternally and maternally derived X-linked genes during critical stages of embryogenesis and early postnatal development. Specifically, the aims are (1) to determine the relative levels of expression of the Gpd and Pgk-A genes (encode glucose-6-phosphate dehydrogenase and phosphoglycerate kinase-A, respectively on the maternal and paternal X chromosomes in tissues and organs of newborn pouch young, and in tissues, organs, and extraembryonic membranes of mid to late gestational embryos of D. virginiana; (2) to determine whether differential expression of the maternal and paternal Gpd and Pgk-a genes occurs in early embryonic stages of D. virginiana, and if so, when it can first be detected; and (3) to establish whether a late replicating X chromosome (indicative of the inactive state) is present in embryonic and extraembryonic tissues during early and mid gestational stages in D. virginiana and M. domestica, and if so, to relate replication timing to pattern of Gpd and Pgk-A expression. FEmale embryos of known developmental stage, and in the case of D. virginiana, specific Gpd and Pgk-A genotypes, will be produced by laboratory crosses. Electrophoretic methods will be used to deduce, in D. virginiana, the relative levels of expression of the maternal and paternal Gpd and Pgk-A alleles in 1) whole early embryos, 2) extraembryonic membranes and embryo proper in older embryos, and 3) various tissues and organs of newborn pouch young. the 5-BrdU-labeling technique will be used to establish the presence/absence of a late replicating X chromosome in cells of embryonic stages in both species.