EXON DETECTION IN VERTEBRATE DNA

人类基因组的定义将需要识别个体 基因.从理论上讲，编码序列可以通过 剪接共有序列，所述共有序列界定并限定它们的组成性 外显子不幸的是，外显子非常短， 脊椎动物的内含子。此外，剪接共有序列是 短的;并且在3'剪接位点不很保守的情况下， 脊椎动物，使外显子检测序列比较困难。的 然而，剪接机器有效且正确地识别外显子。 该提案描述了一种实验策略， 脊椎动物剪接系统检测和克隆人类外显子。的方法 利用了脊椎动物外显子选择的规则， 我们在脊椎动物RNA剪接和多聚腺苷酸化方面的研究成果。最终 期望的产物将是外显子文库（具有和不具有相邻的 内含子序列）。因为这是一项基于DNA的技术， （外显子）预计将以等量表示;无论 它们在任何给定细胞类型中的表达水平。最初的目标将是 分离含有3'末端（含poly（A）位点）的外显子文库 外显子每个基因应代表一次（如果不是交替的话 聚腺苷酸化）。脊椎动物3'末端外显子的平均大小为635 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.