GENETIC RECOMBINATION IN YEAST

这项工作的目标是识别和描述特殊的 刺激近乎相同的重复序列间重组的机制 某些多基因家族的单位。这样的机制可能对 维持核糖体重复单位之间的序列同源性 RNA基因和组蛋白基因。此外，他们还将发挥重要的作用 在这些多基因家族进化中的作用。 酵母核糖体DNA片段，刺激酿酒酵母 侧翼序列中的重组已被鉴定。精确的DNA 该片段中刺激重组所需的序列 将会被确定。此片段的子克隆最初将用于 对重要序列进行定位。该片段的体外诱变作用 将被用来准确地识别导致这种情况的序列 活动。来自亚克隆的初步数据表明，高效率 核糖体RNA前体的起始点转录为 刺激交流所需的。因此，我们将尝试关联 该序列对重组的刺激作用 在片段中启动。反式作用突变，特别影响 该片段的重组刺激活性将被分离出来。 这些突变的基因和生化特征将是 进一步研究重组的机制--刺激 活动。这一特征将包括分析这些因素的影响 核糖体DNA转录的突变。编码该基因的 反式作用功能将被克隆。这些基因和它们的产物将 然后用体内和体外相结合的方法来表征 确定他们的行动模式的技术。影响的参数 这一重组热点的活动也将严格 特色化的。要研究的参数包括DNA的距离 热点在其上刺激重组，并且无论是一个还是两个 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 在其他生物体的多基因家族中也将进行研究。 莱维氏并殖吸虫和黑腹吸虫的5S核糖体DNA片段 将研究组蛋白基因刺激重组的能力 放在酵母里。如果这些序列刺激重组，研究确定 它们发挥作用的机制将被启动。

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.