Gene Expression And Human Genetics

工作总结：为了剖析基因重组中涉及的生物化学步骤，我们选择关注一个关键的早期步骤：同源配对和同源亲本DNA之间的链交换。同源重组中的一个基本问题是如何进行两个DNA之间的同源性搜索。在目前的所有模型中，同源重组蛋白，如原型E。coli RecA蛋白，加载到由一个双链体DNA产生的单链DNA上，并扫描另一个双链体以形成突触（配对）复合物。最终，DNA链交换并形成新的异源双链体。虽然同源配对和链交换是RecA及其真核同源物Rad 51和Dmc 1介导的两个亲本DNA之间的最早接触，但同源重组始于DNA双链断裂（DSB）。在芽殖酵母（Saccharomyces cerevisae）减数分裂中催化DSB形成的蛋白质是SPO 11基因的产物。令人惊讶的是，Spo 11同源物在C. elegans和D.但仍是减数分裂重组所必需的。我们已经产生了一个SPO 11基因敲除小鼠，以研究该基因在哺乳动物中的生物学功能。小鼠SPO 11的破坏导致不育。精母细胞在粗线期之前停滞，很少或没有同源突触，并经历凋亡。最近，我们一直在进行DNA微阵列实验，以确定那些减数分裂表达的基因，其表达被DSB修饰。在年轻的小鼠中，在基因敲除中观察到的细胞凋亡导致退行性变化之前，与野生型相比，Spo 11-/-中只有几十个基因差异表达。受影响最大的基因是Hop 2和Mnd 1基因。这些是影响酵母中同源配对的基因的同源物。我们已经在小鼠中产生了Hop 2基因的敲除。它的减数分裂表型显示出深刻的减数分裂停滞，这是不同于以往任何看到的。与大多数具有减数分裂表型的基因敲除小鼠不同，这些小鼠没有显示任何类型的突触。也就是说，尽管大多数基因敲除小鼠显示出一些非同源突触，但这些小鼠的精母细胞几乎没有任何突触。染色体有些紧凑，看起来正常地被Rad 51和Dmc 1装饰，就好像它们在突触的尖端，但不能继续前进。这一发现表明Hop 2蛋白可能在将减数分裂染色体聚集在一起方面发挥了迄今未被认识的中心作用。基于这一生物学上的假设，我们纯化并研究了25 Kda Hop 2蛋白的生化特性。最令人满意的是，我们已经能够表明，Hop 2蛋白可以促进真正的同源重组蛋白的特征链侵入（D-环形成）和链交换反应。值得注意的是，对于非常重要的起始链侵入活性，它比RecA同源物更活跃; Dmc 1介导链侵入较差，Rad 51仅在Rad 54蛋白存在下介导链侵入。最后，我们已经能够表明，Hop 2可以作为辅助蛋白的链交换进行Rad 51。因此，Hop 2不仅是一个真正的新的重组酶，但也可能是在启动同源物间的接触在减数分裂HR. Deletion的MND 1基因在酵母中的结果非常相似的表型中观察到的一个hop 2突变体的关键和中心蛋白。此外，这两种蛋白质可以从酵母减数分裂提取物中免疫沉淀。我们已经能够证实，这两种蛋白质可以从小鼠睾丸提取物的免疫共沉淀，此外，纯化的小鼠Hop 2和Mnd 1蛋白在体外相互作用，形成一个稳定的异源二聚体。最近，我们已经表明，这种异源二聚体，而不是个别蛋白本身，大大刺激Rad 51和Dmc 1促进链侵入，在后一种情况下，35倍，使近70%的超螺旋底物转化为D-环。此外，我们可以用纯化的蛋白质证明Rad 51和Dmc 1都与Hop 2/Mnd 1异二聚体相互作用。因此，似乎Mnd 1可以作为伴侣，确保Hop 2配对活性仅在真核RecA同源重组酶Rad 51和Dmc 1存在下引发。 了解从一对假定的祖先常染色体中形成性染色体的进化选择力是生物学中的一个主要问题。在过去的几年里，据报道，在C。elegans和D.黑腹果蝇（Parisi等，（2003）Science 299，697）雄性特异性基因在X染色体上代表不足，而在小鼠中则相反。老鼠真的不同吗？我们对不同组织的EST和Affyestin表达数据库的分析表明，睾丸富集和雄性偏向基因在小鼠X染色体上的代表性显着不足。为什么这些结果与之前的小鼠研究结果存在差异？我们对Spo 11成年小鼠微阵列数据的分析提供了睾丸基因表达的时间线。实际上，Spo 11-/-成体睾丸代表部分去势，也就是说，它们在减数分裂I停滞后耗尽所有细胞，并富集早期细胞。我们发现那些在晚期细胞中表达的基因，包括大多数睾丸富集基因，在X染色体上的表达不足。然而，早期基因被过度代表。这些结果调和了所有的数据，因为以前发表的小鼠数据只涉及早期精原细胞基因。

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. Deletion of the MND1 gene in yeast results in a very similar phenotype to that observed in a hop2 mutant. Furthermore, the two proteins could be immunoprecipitated from yeast meiotic extracts. We have been able to confirm that the two proteins could be coimmunoprecipitated from mouse testes extracts and that, in addition, purified mouse Hop2 and Mnd1 proteins interact in vitro and form a stable heterodimer. Recently, we have shown that this heterodimer, but not the individual proteins by themselves, greatly sstimulates Rad51- and Dmc1-promoted strand invasion, in the latter case by 35-fold so that almost 70% of the supercoiled substrate is converted to D-loops. Furthermore, we can demonstrate with purified proteins that both Rad51 and Dmc1 interact with the Hop2/Mnd1 heterodimer. Thus, it appears that Mnd1 may act as a chaperone that ensures that the Hop2 pairing activity is only elicited in the presence of the eukaryotic RecA-homologous recombinases, Rad51 and Dmc1. 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.