GENETIC RECOMBINATION IN YEAST

这项工作的目标是确定和描述特殊的 在几乎相同的重复序列之间刺激重组的机制 某些多基因家族的单位。 这些机制可能对以下方面很重要： 维持核糖体的重复单元之间的序列均一性 RNA基因和组蛋白基因。 此外，他们还将发挥重要作用， 在这些多基因家族的进化中起着重要作用。 酵母S.酿酒酵母， 已经鉴定了侧翼序列中的重组。 精确的DNA 该片段内刺激重组所需的序列 将被确定。 该片段的亚克隆最初将用于 定位重要序列。 片段的体外诱变 将被用来精确地识别导致这一现象的序列 活动 来自亚克隆的初步数据表明， 从核糖体RNA前体的起始位点开始的转录是 需要促进交流。 因此，我们将尝试关联 通过该序列刺激与转录的重组 在碎片中开始。 反式作用突变， 将分离该片段的重组刺激活性。 这些突变将被遗传和生物化学表征， 进一步研究重组刺激的机制， 活动 这种表征将包括测定这些 核糖体DNA转录的突变。 编码基因 将克隆反式作用功能。 这些基因及其产物将 然后使用体内和体外的组合来表征 确定其作用方式的技术。 影响 这种重组热点的活动也将受到严格的限制。 表征了 待研究的参数包括DNA的距离 热点刺激重组的区域，以及 重组基因必须受到热点的作用。 确定这种重组刺激序列是否发生的研究 在其他生物的多基因家族中也将进行。 5S核糖体DNA片段。laevis和D. melanogaster 将研究组蛋白基因刺激重组的能力 在酵母中。 如果这些序列刺激重组，研究确定 将启动它们运作的机制。

The objective of the work is to identify and characterize special mechanisms for stimulating recombination among the nearly identical repeat units of certain multigene families. Such mechanisms may be important for maintaining sequence homogeneity among the repeat units of the ribosomal RNA genes and the histone genes. In addition, they would play an important role in the evolution of these multigene families. A fragment of the ribosomal DNA of the yeast S. cerevisiae that stimulates recombination in flanking sequences has been identified. The precise DNA sequence within this fragment that is required to stimulate recombination will be determined. Subclones of this fragment will be used initially to localize the important sequences. In vitro mutagenesis of the fragment will be used to precisely identify the sequences responsible for this activity. Preliminary data from subclones suggests that highly efficient transcription from the initiation site for the ribosomal RNA precursor is required to stimulate exchange. Therefore, we will attempt to correlate the stimulation of recombination by this sequence with transcription initiated in the fragment. Trans-acting mutations that specifically affect the recombination-stimulatory activity of this fragment will be isolated. These mutations will be genetically and biochemically characterized to further study the mechanism responsible for the recombination-stimulatory activity. This characterization will include assaying the effect of these mutation on ribosomal DNA transcription. The genes encoding the trans-acting functions will be cloned. These genes and their products will then be characterized using a combination of in vivo and in vitro techniques to determine their mode of action. Parameters that affect the activity of this recombination hotspot will also be rigorously characterized. The parameters to be studied include the distance of DNA over which the hotspot stimulates recombination and whether one or both of the recombining genes must be acted on by the hotspot. Studies to determine whether such recombination-stimulatory sequences occur in the multigene families of other organisms will also be performed. Fragments of the 5S ribosomal DNA from X. laevis and the D. melanogaster histone genes will be studied for their ability to stimulate recombination in yeast. If these sequences stimulate recombination, studies to determine the mechanism by which they function will be initiated.