Gene Shut-Off Mechanisms in Bacteriophage Infection

程序化的基因表达序列在生物学中普遍存在。没有一个是完全了解的，尤其是对基因被顺序关闭的机制知之甚少。这个项目将分析枯草芽孢杆菌通过噬菌体SPO1感染过程中的序列基因关闭。在最初的实验中，将研究两类基因，宿主rRNA基因，它们在感染过程中不会关闭，以及SPO1早期基因，它们在感染后几分钟就会完全关闭。首先，将测试这两种类型的启动子，以确认基因关闭行为的差异存在于它们的启动子中。来自每个类别代表的启动子将被分离并整合到宿主染色体中，每个启动子位于相同的位置，因此每个启动子控制相同报告基因的转录。含有不同克隆启动子的细胞将被野生型SPO1感染，每个启动子的关闭时间和程度将通过脉冲标记报告基因转录本来测量。通过克隆SPO1基因组中的某些启动子，并以同样的方式对它们进行分析，将探索不寻常碱基hmUra的可能作用。然后，将识别出推广者中导致一些站点被关闭而另一些站点不被关闭的站点。具有不同监管特异性的启动子之间将形成嵌合体，其监管将以上述相同的方式进行分析。在适当的情况下，将使用缺失分析来确认从嵌合体分析得出的结论。一旦这些初步实验完成，将对其他类别的基因代表进行类似的实验，这些基因在感染期间在不同的时间被关闭，因此对不同的关闭机制做出反应。然而，预计这些额外的实验不会在这一项目期内完成。当病毒感染细菌细胞时，基因表达会发生一系列复杂的变化。宿主细胞的部分(但不是全部)基因被关闭。病毒自身的基因是以非常严格的控制顺序启动的；偏离这一顺序会导致感染过程的部分或完全失败。作为这一严格控制的一系列事件的一部分，特定的病毒基因也在严格指定的时间被关闭。斯图尔特博士之前的一些研究表明，密切控制某些病毒基因的关闭对感染的成功至关重要，就像控制其他病毒基因的开启时间一样。在过去的30年里，人们对特定基因启动的机制有了很大的了解，但对特定基因关闭的机制几乎一无所知。这是一项旨在阐明其中一些重要但尚不清楚的机制的提议。

Programmed sequences of gene expression are ubiquitous in biology. None is completely understood, and, in particular, very little is known about mechanisms by which genes are sequentially shut off. This project will analyze sequential gene shut-off during infection of Bacillus subtilis by bacteriophage SPO1. In the initial experiments, two categories of genes will be studied, the host rRNA genes, which are not shut off during infection, and the SPO1 early genes, which are completely shut off by a few minutes after infection. First the two types of promoters will be tested to confirm that the difference in shut-off behavior of the genes resides in their promoters. Promoters from representatives of each category will be isolated and integrated into the host chromosome, each at the same site so each controls transcription of the same reporter gene. Cells containing the different cloned promoters will be infected by wild-type SPO1, and the time and extent of shut-off of each promoter will be measured by pulse-labeling the reporter gene transcripts. A possible role of the unusual base, hmUra, will be explored by cloning certain promoters in the SPO1 genome, and assaying them in the same way. Then, the sites within the promoters that cause some to be shut off, while others are not, will be identified. Chimeras will be formed between promoters with different regulatory specificities, and their regulation will be assayed in the same way as above. Where appropriate, deletion analysis will be used to confirm the conclusions drawn from analysis of chimeras. Once these initial experiments are completed, similar experiments will be performed with representatives of other categories of genes, which are turned off at different times during infection, and which therefore respond to different shut-off mechanisms. However, it is not anticipated that these additional experiments can be completed within this project period. When a virus infects a bacterial cell, a complex series of changes in gene expression occurs. Some, but not all, of the host cell's genes are turned off. The virus'es own genes are turned on in a very strictly controlled order; deviation from this order results in partial or complete failure of the infection process. As part of this strictly controlled series of events, particular viral genes are also turned off at closely specified times. It is emerging from some of Dr. Stewart's prior studies that close control of turn-off of certain viral genes is just as critical for the success of the infection as is control of the time of turn-on of other viral genes. Over the last 30 years a great deal has come to be know about mechanisms of specific gene turn-on, but as yet virtually nothing is known about mechanisms of specific gene turn-off. This is a proposal to elucidate some of these important but as yet obscure mechanisms.