Identification of the sequences controlling cyclic AMP regulation and cell-type-specific expression of a prestalk-specific gene in Dictyostelium discoideum.
Identification of the sequences controlling cyclic AMP regulation and cell-type-specific expression of a prestalk-specific gene in Dictyostelium discoideum.
复制标题
盘基网柄菌中控制环 AMP 调节和细胞类型特异性表达的前柄特异性基因的序列的鉴定。
DOI：
10.1128/mcb.7.1.149-159.1987
复制
发表时间：
1987
影响因子：
5.3
通讯作者：
Firtel,RA
中科院分区：
文献类型：
--
作者：
Datta,S;Firtel,RA
我们克隆并分析了一个发育和空间调控的盘基网柄藻前柄细胞特异基因。该基因编码与溶酶体半胱氨酸蛋白酶组织蛋白酶H和组织蛋白酶B高度同源的蛋白质。这些酶之间的氨基酸比较表明，活性位点的氨基酸是保守的，因为是已知的是重要的催化和残基，形成分子内半胱氨酸桥的氨基酸。我们在帽位点5′端300个碱基对区域内构建了一系列内部缺失、重复和连接扫描突变。对转化体中突变表达的分析鉴定了一个~ 35碱基对的GC富集区，该区域包含一个dAdC/dGdT回文重复序列和一个与回文重复序列的3′ GT半同源的G富集盒。G盒的缺失或破坏导致在单细胞培养中响应于环AMP的转化体中基因融合的表达水平下降约50倍，但不影响由环AMP调节或控制的时间模式。在正常的多细胞分化过程中，这种构建体的表达与内源基因的表达相同;然而，来自构建体的RNA水平仅比含有G盒的构建体的RNA水平低约10倍。缺失回文序列的3′端和G盒区域导致转录水平显著降低，尽管构建体仍显示出适当的时间表达。这些结果表明，这35个碱基对的区域作为一个重要的部分，调节区域的细胞类型和环AMP的调节。
We have cloned and analyzed a developmentally and spatially regulated prestalk cell-specific gene fromDictyostelium discoideum. The gene encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. Amino acid comparisons between these enzymes showed that the active-site amino acids were conserved, as were amino acids known to be important for catalysis and residues which form the intramolecular cysteine bridges. We have constructed a series of internal deletions, duplications, and linker scanner mutations within the region 300 base pairs 5′ to the cap site. Analysis of expression of the mutations in transformants identified a ~ 35-base pair GC-rich region containing a dAdC/dGdT palindromic repeat and a G-rich box which is homologous to the 3′ GT half of the palindromic repeat. Deletion or disruption of the G box resulted in a ~ 50-fold drop in the level of expression of the gene fusion in transformants in response to cyclic AMP in single-cell culture but did not affect the temporal pattern of regulation or control by cyclic AMP. The expression of such constructs during normal multicellular differentiation paralleled that of the endogenous gene; however, the level of RNA from the constructs was only ~ 10-fold lower than that of constructs containing the G box. Deletion of the 3′ half of the palindromic sequence and the G box region resulted in a dramatic decrease in the level of transcription, although the constructs still showed proper temporal expression. These results suggest that this 35-base-pair region acts as an important part of the regulatory region for cell type and cyclic AMP regulation.