Gene Shut-Off Mechanisms in Bacteriophage Infection

基因表达的程序化序列在生物学中无处不在。没有一个是完全被理解的，特别是，对于基因顺序关闭的机制知之甚少。本项目将分析枯草芽孢杆菌被噬菌体SPO1感染时的序列基因关闭。在最初的实验中，我们将研究两类基因：在感染过程中不被关闭的宿主rRNA基因和在感染后几分钟就完全关闭的SPO1早期基因。首先，将对两种启动子进行测试，以确认基因关闭行为的差异存在于它们的启动子中。每一类启动子的代表将被分离并整合到宿主染色体中，每一类启动子位于同一位点，因此每一类启动子控制同一报告基因的转录。含有不同克隆启动子的细胞将被野生型SPO1感染，并通过脉冲标记报告基因转录物来测量每个启动子关闭的时间和程度。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，将探索这种不寻常的碱基hmUra的可能作用。然后，将确定启动子内导致一些关闭而另一些没有关闭的位点。嵌合体将在具有不同调控特异性的启动子之间形成，它们的调控将按上述方法进行分析。在适当的情况下，将使用缺失分析来确认从嵌合体分析中得出的结论。一旦这些初步实验完成，将对其他类型基因的代表进行类似的实验，这些基因在感染期间的不同时间被关闭，因此对不同的关闭机制作出反应。但是，预计这些额外的实验不能在本项目期间完成。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。宿主细胞的一些（但不是全部）基因被关闭了。病毒自身的基因以一种非常严格控制的顺序被激活；偏离这个顺序会导致感染过程部分或完全失败。作为这一系列严格控制的事件的一部分，特定的病毒基因也会在严格规定的时间关闭。斯图尔特博士之前的一些研究表明，严格控制某些病毒基因的关闭，与控制其他病毒基因的开启时间一样，对感染的成功至关重要。在过去的30年里，人们对特定基因开启的机制已经了解了很多，但对特定基因关闭的机制几乎一无所知。本文旨在阐明其中一些重要但尚不清楚的机制。

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.