Gene Expression And Human Genetics

工作概述：为了剖析基因重组的生化步骤，我们选择关注一个关键的早期步骤：同源亲本dna之间的同源配对和链交换。同源重组的一个基本问题是如何在两个dna之间寻找同源性。在目前所有的模型中，同源重组蛋白，如典型的大肠杆菌RecA蛋白，装载到由一个双工DNA产生的单链DNA上，并扫描另一个双工形成突触（配对）复合体。最终，DNA链被交换，形成一个新的异双工。同源配对和链交换是由RecA及其真核同源物Rad51和Dmc1介导的两个亲本DNA之间最早的接触，同源重组是在DNA双链断裂（DSBs）时启动的。在出芽酵母（Saccharomyces cerevisae）减数分裂中催化DSB形成的蛋白质是SPO11基因的产物。令人惊讶的是，Spo11同源物在秀丽隐杆线虫和黑腹线虫的突触中是不需要的，但在减数分裂重组中却是必需的。我们已经产生了SPO11小鼠敲除，以研究该基因在哺乳动物中的生物学功能。破坏小鼠SPO11导致不孕。精母细胞由于同源突触很少或没有同源突触而在粗成期发生阻滞并发生凋亡。最近，我们一直在进行DNA微阵列实验，以确定那些表达被DSB修饰的减数表达基因。在年轻的小鼠中，由于基因敲除导致细胞凋亡，在退行性变化开始之前，只有几十个基因在Spo11 -/-中与野生型相比有差异表达。受影响最大的基因是Hop2和Mnd1基因。这些是影响酵母中同源配对的基因的同源物。我们已经在老鼠体内产生了Hop2基因的敲除。它的减数分裂表型显示一个深刻的减数分裂阻滞，这是不同于任何以前看到的。与大多数具有减数分裂表型的基因敲除不同，这些小鼠没有任何类型的突触。也就是说，尽管大多数基因敲除都显示出一些非同源突触，但来自这些小鼠的精细胞几乎没有任何突触。染色体在一定程度上是紧致的，看起来Rad51和Dmc1都正常地装饰着，就好像它们处于突触的尖端，但不能向前推进。这一发现表明，Hop2蛋白可能在使减数分裂染色体聚集在一起的过程中发挥了迄今未被认识到的核心作用。在这一生物学假说的启发下，我们纯化并研究了25kda Hop2蛋白的生化特性。最令人欣慰的是，我们已经能够证明Hop2蛋白能够促进真正同源重组蛋白特征的链入侵（d环形成）和链交换反应。值得注意的是，就非常重要的起始链入侵活性而言，它比任何一种RecA同源物都更活跃；Dmc1介导链入侵较差，而Rad51仅在Rad54蛋白存在时介导链入侵。最后，我们已经能够证明Hop2可以作为Rad51进行链交换的辅助蛋白。因此，Hop2不仅是一种真正的新型重组酶，而且可能是减数分裂HR中启动同源间接触的关键和中心蛋白。在所有生物体中，同源重组与DNA的修复和复制密不可分，因此与细胞增殖及其控制密切相关。最后，我们利用全基因组cDNA阵列分析了丝裂霉素C （MMC）治疗后所有大肠杆菌orf基因表达水平的变化。在不同的MMC处理模式下进行了几个实验，分析了添加DNA损伤剂后大肠杆菌细胞在不同时间点的表达谱。总的来说，这些实验包括16种不同的杂交，对应大约70,000个单独的数据点。大约5-10%的基因在表达水平上表现出显著的变化。如前所述，DNA损伤后，几个lexa调控基因的表达水平升高。另一方面，大多数在表达水平上表现出显著变化的基因在DNA修复过程中没有被证明是可诱导的或被抑制的。人们试图根据对DNA损伤的反应对所有基因进行分类。利用基因表达数据的聚类分析，可以将所有基因划分为至少12个不同的聚类。在大约400个上调的基因中，大约100个基因的注释很差，或者根本没有注释。在这100个中，我们选择了大约50个编码中等大小的蛋白质，不是明显的膜蛋白，并显示出一些进化守恒。我们已经为大多数这些基因制作了基因缺失菌株，现在正在研究它们的表型，包括DNA代谢（与布兰代斯大学的苏·洛维特合作）和使用生物学表型阵列的一般中间代谢。此外，我们还启动了一个结构基因组学项目（与CARB的Galya Obmolova， Alex Teplyakov和Gary Gilliland合作），以确定这50个基因中尽可能多的蛋白质产物的结构。到目前为止，我们已经获得了其中6种蛋白质的分子结构。

Summary of work: In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step(s): homologous pairing and strand exchange between homologous parental DNAs. A fundamental problem in homologous recombination is how the search for homology between the two DNAs is carried out. In all current models a homologous recombination protein, such as the prototypical E. coli RecA protein, loads onto a single-strand DNA generated from one duplex DNA and scans another duplex to form a synaptic (pairing) complex. Eventually, DNA strands are exchanged and a new heteroduplex is formed. While homologous pairing and strand exchange are the earliest contacts between two parental DNAs mediated by RecA and its eukaryotic homologues, Rad51 and Dmc1, homologous recombination is initiated at DNA double-strand breaks (DSBs). The protein that catalyzes DSB formation in meiosis in the budding yeast, Saccharomyces cerevisae, is the product of the SPO11 gene. Surprisingly, Spo11 homologues are dispensable for synapsis in C. elegans and D. melanogaster yet required for meiotic recombination. We have generated a SPO11 mouse knock-out to investigate the biological function of this gene in mammals. Disruption of mouse SPO11 results in infertility. Spermatocytes arrest prior to pachytene with little or no homologous synapsis and undergo apoptosis. Recently, we have been conducting DNA microarray experiments to determine those meiotically expressed genes whose expression is modified by a DSB. In young mice, before degenerative changes have set in as a result of the apoptosis seen in the knockouts, there are only a few dozen genes that are differentially expressed in Spo11 -/- compared to wild type. Among the genes most affected are the Hop2 and Mnd1 genes. These are homologues of genes that affect homologous pairing in yeast. We have generated a knockout of the Hop2 gene in the mouse. Its meiotic phenotype shows a profound meiotic arrest that is unlike any seen previously. Unlike most knockouts with a meiotic phenotype these mice show no synapsis of any kind. That is, whereas most knockouts, show some willy-nilly non-homologous synapsis, spermatocytes from these mice are arrested without almost any synapsis. The chromosomes are somewhat compacted and appear normally decorated with both Rad51 and Dmc1, as if they are on the cusp of synapsis but fail to proceed forward. This finding suggests that the Hop2 protein might play a heretofore-unrecognized central role in bringing meiotic chromosomes together. Prompted by this biologically inspired hypothesis we have purified and studied the biochemical properties of the 25 Kda Hop2 protein. Most gratifyingly, we have been able to show that Hop2 protein can promote the strand invasion (D-loop formation) and strand exchange reactions characteristic of bona fide homologous recombination proteins. Remarkably, with respect to the very important initiating strand invasion activity it is more active than either RecA homologue; Dmc1, mediates strand invasion poorly and Rad51, mediates strand invasion only in the presence of the Rad54 protein. Finally, we have been able to show that Hop2 can act as an accessory protein for the strand exchange carried out by Rad51. Thus, Hop2 is not only a true novel recombinase but also may be the crucial and central protein in initiating interhomolog contacts in meiotic HR. In all organisms, homologous recombination is inextricably related to DNA repair and replication, hence cell proliferation and its control. Finally, we have used whole-genome cDNA arrays were used to analyze changes in the levels of gene expression of all E. coli ORFs after treatment with mitomycin C (MMC). Several experiments, which differ in the mode of MMC treatment, were performed, and expression profiles of E. coli cells at different time points after the addition of the DNA damaging agent were analyzed. As a whole, these experiments consist of 16 different hybridizations corresponding to about 70,000 individual data points. Around 5-10% of all genes show significant changes in their level of expression. As shown before, the expression level of several LexA-regulated genes was increased after DNA damage. On the other hand, most of those genes that show significant changes in their level of expression have not been shown previously to be inducible or repressed in the process of DNA repair. An attempt was made to classify all genes based on their responses to DNA damage. Using cluster analysis of the gene expression data it is possible to divide all the genes into at least 12 different clusters. Of the 400 or so upregulated genes about 100 were poorly annotated or not annotated at all. Of these 100 we have selected about 50 that encode for proteins of modest size, are not clearly membrane proteins and show some evolutionary conservation. We have made gene deletion strains for most of these genes and are now studying their phenotypes, both with regard to DNA metabolism (in collaboration with Sue Lovett at Brandeis) and general intermediate metabolism using the Biolog phenotypic arrays. In addition, we have initiated a structural genomics project (in collaboration with Galya Obmolova, Alex Teplyakov and Gary Gilliland at CARB) to determine the structure of as many as possible of the protein products of these 50 genes. So far we have obtained the molecular structure of six of these proteins. Understanding the evolutionary selective forces that fashioned the sex chromosomes from a putative ancestral autosome pair is a major problem in biology. In the last couple of years it has been reported that in C. elegans and D. melanogaster (Parisi et al. (2003) Science 299, 697) male-specific genes are underrepresented on the X-chromosome whereas the opposite is true in the mouse. Is the mouse really different? Our analysis of both EST and Affymetrix expression databases for different tissues indicates that testis-enriched and male-biased genes are significantly underrepresented on the mouse X-chromosome. Why the discrepancy between these results and the previous mouse study? Our analysis of the Spo11 adult mice microarray data provides a time line for the expression of testis genes. In effect, the Spo11 -/- adult testes represent a partial castration, that is, they are depleted for all cells after the arrest in meiosis I and enriched for earlier cells. We find that those genes expressed in late cells, including the majority of testis-enriched genes, are underrepresented on the X. However, early genes are overrepresented. These results reconcile all the data since the previously published mouse data pertained to early spermatogonial genes only.