EXON DETECTION IN VERTEBRATE DNA

对人类基因组的定义将需要对个体进行识别 基因。从理论上讲，编码序列可以通过 拼接共同序列，界定它们的构成 外显子。不幸的是，外显子很短，相隔很长 脊椎动物的内含子。此外，拼接一致序列是 短；在3‘剪接位点没有很好保存的情况下 脊椎动物，这使得通过序列比较来检测外显子变得困难。这个 然而，剪接机器可以有效和正确地识别外显子。 这项建议描述了一种实验策略，利用天然的 用于检测和克隆人类外显子的脊椎动物剪接系统。该方法 利用已出现的脊椎动物外显子选择规则 来自我们在脊椎动物RNA剪接和聚腺苷酸化方面的工作。决赛 所需产品将是外显子文库(包括相邻的和不相邻的 内含子序列)。因为这是一项基于DNA的技术，所有的基因 (外显子)将以相同的数量表达； 它们在任何给定细胞类型中的表达水平。最初的目标是 分离含有3‘末端的外显子文库(含Poly(A)位点) 外显子。每个基因应该代表一次(如果不是交替的话 多聚腺苷)。脊椎动物3‘端外显子的平均大小为635 核苷酸；因此，3‘外显子文库将是一个方便的来源 用于原位染色体定位方法的基因特异性探针。

Definition of the human genome will require identification of individual genes. Theoretically, coding sequences could be located by virtue of the splicing consensus sequences that border and define their constitutive exons. Unfortunately, exons are very short and are separated by very long introns in vertebrates. Furthermore, splicing consensus sequences are short; and in the case of 3' splice sites not well conserved in vertebrates, making exon detection by sequence comparison difficult. The splicing machinery, however, recognizes exons efficiently and correctly. This proposal describes an experimental strategy to utilize the natural vertebrate splicing system to detect and clone human exons. The approach takes advantage of rules for exon selection in vertebrates that have arisen from our work on vertebrate RNA splicing and polyadenylation. The final desired product will be exon libraries (both with and without adjacent intron sequences). Because this is a DNA-based technology, all genes (exons) will be expected to be represented in equal amounts; regardless of their level of expression in any given cell type. The initial goal will be to isolate exon libraries containing 3' terminal (poly(A) site-containing) exons. Each gene should be represented once (if not alternatively polyadenylated). The average size of vertebrate 3' terminal exons is 635 nucleotides; therefore, a library of 3' exons will be a convenient source for gene-specific probes for in situ chromosome location approaches.