STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

一、德系犹太人Tay-Sachs携带者和 患者是14号外显子HEXA基因第11外显子上的4个碱基对(Bp)插入 导致过早的终止密码子。携带4个碱基对的六个等位基因 插入表达的mRNA被一种原因不明的基因迅速降解 机制。我们已经开发了一个系统来调查4个 BP对HEXA基因表达的影响。构建了一个8kb的“mini-HEXA基因”。 将由外显子1至7指定的编码区作为单个块融合到 包含剩余外显子和内含子的基因组片段。这一构造 并在稳定表达的L细胞和HEXA细胞中进行了表达 Northern分析检测基因表达水平。我们发现细胞 用含有容易检测到的正常构建体 正确拼接的HEXA mRNA。转微基因的细胞 然而，4个碱基的突变产生了检测不到的HEXA mRNA。在基因水平上 当1个碱基被修复时，含有4个碱基突变的微型基因被恢复 删除了上游以更正读数框。 溶酶体酶基因表现出溶酶体酶的表达 增强版。为了深入了解溶酶体的调节 酶基因我们已经确定了HEXB基因的启动子区域。 我们发现，小鼠和人类的HEXB基因都含有一个同源的50 功能启动子活性在各自的40个碱基内的碱基区 RNA起始点。该区域包含几个结合位点 转录因子。

I. The major mutation among Ashkenazi Jewish Tay-Sachs carriers and patients is a 4 base pair(bp) insertion in exon 11 of the 14 exon HEXA gene resulting in a premature stop codon. HEXA alleles carrying the 4 bp insertion express mRNA that is rapidly degraded by an unexplained mechanism. We have developed a system to investigate the effect of the 4 bp on HEXA mRNA expression. A 8 kb "mini-HEXA gene" was constructed by fusing the coding region specified by exons 1 to 7 as a single block to a genomic segment containing the remaining exons and introns. This construct and derivatives were expressed in stably transfected L-cells and the HEXA mRNA levels were assessed by Northern analysis. We found that cells transfected with the normal construct contained readily detectable and properly spliced HEXA mRNA. Cells transfected with a mini-gene containing the 4 bp mutation, however, yielded undetectable HEXA mRNA. The mRNA level was restored with a mini-gene containing the 4 bp mutation when 1 bp was deleted upstream to correct the reading frame. II. Lysosomal enzyme genes exhibit an expression of lysosomal enzymes is enhanced. In order to gain insight into the regulation of the lysosomal enzyme genes we have characterized the promotor region of the HEXB gene. We found that both the mouse and human HEXB genes contain a homologous 50 bp region with functional promotor activity within 40 bp of the respective RNA start sites. The region contains several binding sites for transcription factors.