The human myristoylated alanine-rich C kinase substrate (MARCKS) gene (MACS). Analysis of its gene product, promoter, and chromosomal localization.
The human myristoylated alanine-rich C kinase substrate (MARCKS) gene (MACS). Analysis of its gene product, promoter, and chromosomal localization.
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DOI：
10.1016/s0021-9258(18)98698-9
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发表时间：
1991-08
期刊：
影响因子：
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通讯作者：
D. Harlan;J. Graff;D. Stumpo;D. Stumpo;R. Eddy;T. Shows;J. Boyle;P. Blackshear
中科院分区：
文献类型：
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作者：
D. Harlan;J. Graff;D. Stumpo;D. Stumpo;R. Eddy;T. Shows;J. Boyle;P. Blackshear
蛋白激酶C的主要细胞底物MARCKS蛋白的表达以细胞、组织和发育阶段特异性的方式进行调节;此外，这种表达可以在某些细胞类型中受到各种细胞因子的急性刺激。我们已经开始描述人类基因的特征，以阐明这种高度调控表达的遗传因素。我们首先克隆了人MARCKS cDNA，其编码332个氨基酸（Mr 31，600）的预测蛋白质，分别与牛、小鼠和鸡的蛋白质约89%、74%和59%相同。在氨基酸水平上保守的区域包括氨基末端豆蔻酰化共有序列、内含子剪接位点和磷酸化位点结构域。人cDNA用于证明肿瘤坏死因子-α可以快速刺激人早幼粒细胞白血病细胞系HL 60中MARCKS基因的转录。然后分离基因组克隆;序列分析鉴定了一个假定的启动子区域，该区域没有TATA盒，并且在跨越57个碱基对（bp）的区域中含有多个转录起始位点。随后是约400 bp的5'-非翻译区，其显示出复杂的预测二级结构，Δ G为-73.4 kcal/mol。然后将含有52 - 1453 bp与人生长激素基因连接的人MARCKS启动子的质粒构建体用于瞬时表达实验。含有52和110 bp的MARCKS启动子的构建体没有表现出启动子功能，而较大的构建体都表现出启动子功能; MARCKS启动子的248 bp片段在刺激完整细胞中人生长激素表达方面与人铁蛋白启动子的有效性相同，为80%。使用来自人类基因组克隆的插入片段作为探针，我们确定人类6号染色体q21-qter为MARCKS基因的位置;这已被指定为基因符号MACS。
The expression of a major cellular substrate for protein kinase C, the MARCKS protein, is regulated in a cell-, tissue-, and developmental stage-specific fashion; in addition, this expression can be stimulated acutely by various cytokines in certain cell types. We have begun to characterize the human gene in order to elucidate the genetic elements responsible for this highly regulated expression. We first cloned a human MARCKS cDNA, which encoded a predicted protein of 332 amino acids (Mr 31,600) that was approximately 89, 74, and 59% identical to the bovine, mouse, and chicken proteins, respectively. Regions conserved at the amino acid level included the amino-terminal myristoylation consensus sequence, the site of intron splicing, and the phosphorylation site domain. The human cDNA was used to demonstrate that tumor necrosis factor-alpha could rapidly stimulate MARCKS gene transcription in the human promyelocytic leukemia cell line HL60. Genomic clones were then isolated; sequence analysis identified a putative promoter region that had no TATA box and contained multiple transcription initiation sites in a region spanning 57 base pairs (bp). This was followed by a 5'-untranslated region of approximately 400 bp, which displayed a complex predicted secondary structure with a delta G of -73.4 kcal/mol. Plasmid constructions containing between 52 and 1453 bp of the human MARCKS promoter linked to the human growth hormone gene were then used in transient expression experiments. Constructions containing 52 and 110 bp of the MARCKS promoter did not exhibit promoter function while the larger constructions all exhibited promotor function; the 248-bp fragment of the MARCKS promoter was 80% as effective as the human ferritin promoter in stimulating expression of human growth hormone in intact cells. Using an insert from the human genomic clone as a probe, we identified human chromosome 6, q21-qter, as the location of the MARCKS gene; this has been assigned the gene symbol MACS.