X-CHROMOSOME INACTIVATION IN OPOSSUM DEVELOPMENT

我们的长期目标是发展对细胞 以及与基因活性调节有关的亚细胞事件 哺乳动物的发育，并最终了解分子 控制正常和异常基因表达的过程 发育过程。我们的方法是利用 X染色体失活模式(随机与确定)之间 真兽类和变态类哺乳动物作为比较研究的模式 X连锁Virginiana(弗吉尼亚负鼠：唯一已知的变态动物 在一个X连锁的基因座上表现出更多的变异)和单胞胎 家鼠(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以及 父本和母本来源的X连锁基因在 胚胎发育和出生后早期发育的关键阶段。 具体地说，目标是(1)确定 GPD和PGK-A基因(编码葡萄糖-6-磷酸)的表达 脱氢酶和磷酸甘油酸激酶-A分别对母体的影响 和父系X染色体在幼年新生儿的组织和器官中， 以及中晚期的组织、器官和胚外膜 (2)测定维吉尼亚石斑鱼的妊娠胚胎 母本和父本GPD和PGK-a基因的差异表达 发生在维吉尼亚锥虫的早期胚胎阶段，如果是这样的话，什么时候可以 首先被检测到；以及(3)确定一个后期复制的X 染色体(表示不活跃状态)存在于胚胎和 妊娠早期和中期的胚外组织 Virginiana和M.home tica，如果是这样的话，将复制时间与 GPD和PGK-A的表达模式。已知的雌性胚胎 发育阶段，在金线藻的情况下，特异性GPD和 PGK-A基因型，将由实验室杂交产生。电泳法 方法将被用来推断在维吉尼亚圆盘藻中的相对水平 母本和父本GPD和PGK-A等位基因的整体表达 早期胚胎，2)胚外膜和老年胚胎本身 胚胎；3)新生囊幼体的各种组织器官。这个 5-BrdU标记技术将用于确定存在/不存在 在两个胚胎阶段的细胞中都有一个晚期复制的X染色体 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.