FUNCTIONAL ANALYSES OF DNA PKCS AND ITS SPLICE VARIANTS

在我们对SCID马驹DNA-PKcs缺陷进行鉴定的过程中， 我们观察到转录本区域内广泛的剪接变异 编码磷脂酰肌醇3激酶(PI3K)结构域。完全相似 在人类DNA-PKcs中也观察到剪接变异。变异的一个来源是 单个外显子的差异插入恰好位于该区域的近端 编码保守的蛋白激酶基序的转录本。附加变型 是通过内含子保留来实现的。相当大比例的DNA-PKcs 转录本在PI3K结构域中保留一个短内含子，导致 在激酶中所谓的催化部位之前插入终止密码子 域。保留内含子的转录本将编码截短形式的 预测质量为~441kD(而不是~469kD)的DNA-PKcs 理论上缺乏蛋白激酶活性。这份成绩单是用所有的 到目前为止被检查过的物种，并且是高度保守的。我们有初步的 这份文字记录被翻译的证据。此外，DNA-PK的分析 细胞中的活动，表达高水平的该转录本，编码 该激酶的调节形式。最后，这些DNA-PKcs剪接产物的表达 静息细胞中的变体明显高于周期细胞中的变体，这表明 DNA-PKcs与细胞周期进程之间的联系。 拟议研究的假设是，由于每个DNA-PKcs转录本 编码一种结构上不同的蛋白质，每种形式都有不同的 功能。拟议研究的总体目标是确定 利用生物化学和遗传学方法研究每个DNA-PKcs的功能。

During our characterization of the DNA-PKcs defect in SCID foals, we observed extensive splice variation within the region of the transcript encoding the phosphatidyl inositol 3 kinase (PI3K) domain. Completely analogous splice variation is also observed in human DNA-PKcs. One source of variation is the differential insertion of a single exon just proximal to the region of the transcript encoding the conserved protein kinase motifs. Additional variation is accomplished by intron retention. A significant percentage of DNA-PKcs transcripts retain a short intron within the PI3K domain resulting in the insertion of a stop codon prior to the purported catalytic site in the kinase domain. Transcripts retaining the intron would encode a truncated form of DNA-PKcs with a predicted mass of ~441kD (as opposed to ~469kD) which would theoretically lack protein kinase activity. This transcript is expressed in all species examined to date and is strongly conserved. We have preliminary evidence that this transcript is translated. Furthermore, analyses of DNA-PK activity in cells, which express high levels of this transcript, encodes a regulatory from of the kinase. Finally, expression of these DNA-PKcs splice variants is markedly higher in resting cells than in cycling cells suggesting a link between DNA-PKcs and cell cycle progression. The hypothesis of the proposed research is that since each DNA-PKcs transcript encodes a structurally distinct protein, each from will have distinct functions. The overall goal of the proposed research is to determine the function of each DNA-PKcs using both biochemical and genetic approaches.