STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

溶酶体酶的结构-功能关系

• 批准号: 3840463
• 负责人: R L PROIA
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3840463
• 关键词: L cell; RNA splicing; Tay Sachs disease; alleles; enzyme mechanism; enzyme structure; fusion gene; gene deletion mutation; gene expression; genetic promoter element; genetic regulation; human genetic material tag; laboratory mouse; lysosomes; messenger RNA; northern blottings; nucleic acid sequence; protein structure function; transcription factor; transfection

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.

I. 德系犹太人泰-萨克斯病携带者的主要突变， 患者是在14外显子HEXA基因的外显子11中插入4个碱基对（bp） 导致提前终止密码子。 携带4 bp的HEXA等位基因 插入表达的mRNA被一种无法解释的 机制 我们已经开发了一个系统来研究4 bp对HEXA mRNA表达的影响。 构建了8 kb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。 该构建体 和衍生物在稳定转染的L细胞中表达，HEXA 通过北方分析评估mRNA水平。 我们发现细胞 用正常构建体转染的细胞中含有容易检测到的 正确剪接的HEXA mRNA。 转染了含有微基因的细胞 然而，4 bp突变产生不可检测的HEXA mRNA。 mrna水平 用含有4 bp突变的迷你基因恢复，当1bp被 删除上游以校正阅读帧。 二. 溶酶体酶基因表现出溶酶体酶的表达， 增强 为了深入了解溶酶体的调节， 酶基因，我们已经表征了HEXB基因的启动子区。 我们发现小鼠和人的HEXB基因都含有一个同源的50 具有功能性启动子活性的40 bp区域， RNA起始位点。 该区域含有几个结合位点， 转录因子