Molecular and genetic analysis of sex determination in Drosophila

9305074萨尔茨黑腹果蝇的性别是如何决定的，就像任何生物体的性别决定一样，人们也很清楚。然而，即使在这个系统中，这种决心是如何受到监管的仍然是一个谜。我们正在集中精力鉴定和鉴定维持二元开关基因SXL(SXL)开/关活动状态所需的基因。SXL的维持受“外显子跳跃”的调节，这是一种替代的RNA剪接。只有在雌性中，加工过的转录产物才能产生一种功能蛋白质。我们重点分析了性别决定基因SnF，它被用来建立SLX的女性特异性剪接模式。我们最近发现SNF与哺乳动物的SnRNP蛋白U1A和U2B具有同源性，这表明SNF基因产物是剪接位点选择所需机制的组成部分。我们将1)继续我们对SNF基因座的分子表征2)确定SNF蛋白是核的和/或细胞质的，以及它是否与多线染色体上的新转录有关3)通过定位与现有的SNF突变相关的分子损伤来确定现有的突变是简单的功能丧失突变还是具有异常功能的突变，并检查突变基因的多个副本的表型后果4)确定SNF可能是基本的一般剪接因子、基本的性别特异性剪接因子还是非必要的剪接“促进器”，通过产生和鉴定该位点的一个小缺陷5)通过在果蝇中表达人类基因来确定人类U1A蛋白和/或人U2B“蛋白在功能上是否与SNF相同，并确定两者是否可以补充SNF突变6)确定U1A的RNA识别基序(RRM)之间的差异。U2B“和SNF在功能上具有重要意义。鉴于已对人类U1A和U2B”蛋白进行了广泛的体外结构/功能研究，这些结构/功能研究应该特别有意义。通过鉴定和鉴定维持SXL活性所需的一般基因和性别特异性基因，我们将深入了解性别特异性剪接的调控机制。此外，我们的分析将对理解调控RNA剪接的一般机制有更广泛的影响，因为人们对剪接位点的选择知之甚少。***

9305074 Salz How the sex of Drosophila melanogaster is determined is as well understood as is sex determination in any organism. However, even in this system how such determination is regulated remains a mystery. We are concentrating on the identification and characterization of genes required for the maintenance of the ON/OFF activity state of the binary switch gene Sex-lethal (Sxl). Maintenance of Sxl is regulated by "exon-skipping", a type of alternative RNA splicing. Only in females does the processed transcription product yield a functional protein. We are focusing on the analysis of snf, a sex determination gene which is utilized to establish the female-specific splicing pattern of Slx. We have recently found that snf has homology to the mammalian snRNP proteins U1A and U2B", suggesting that the snf gene product is an integral component of the machinery required for splice site choice. We will 1) continue our molecular characterization of the snf locus 2) determine if the SNF protein is nuclear and/or cytoplasmic as well as whether it is associated with nascent transcripts on polytene chromosomes 3) determine whether the extant mutations are simple loss of function mutations or have aberrant functions by localizing the molecular lesions associated with the extant snf mutations and examine the phenotypic consequences of multiple copies of the mutant gene 4) determine whether snf is likely to be an essential general-splicing factor, an essential sex-specific splicing factor or an non-essential "facilitator" of splicing, by generating and characterizing a small deficiency of the locus 5)determine whether the human U1A protein and/or the human U2B" protein is functionally equivalent to snf by expressing the human genes in Drosophila and determine whether either can complement snf mutations 6) determine whether the differences between the RNA Recognition motifs (RRM) of U1A. U2B" and SNF are functionally significant. These structure/function studies should b e particularly informative in light of the extensive in vitro structure/function studies that have been carried out on the human U1A and U2B" proteins. %%% By identifying and characterizing both general and sex-specific genes required for maintenance of Sxl activity, we will gain insights into the mechanism regulating sex-specific splicing. Moreover, our analysis will have broader implications for the understanding of the mechanisms regulating RNA splicing in general since little is known about how splice site choices are made. ***