CHARACTERIZATION OF A HIGHLY CONSERVED 23 KDA BASIC PROTEIN

高度保守的 23 KDA 碱性蛋白的表征

• 批准号: 3878906
• 负责人: S R PRICE
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/3878906
• 关键词: ADP ribosylation; clone cells; cow; guanine nucleotide binding protein; human tissue; hybrid cells; molecular cloning; nucleic acid probes; protein sequence; protein signal sequence; protein structure function; species difference

During the isolation and characterization of cDNA clones for ADP-ribosylation factors, about 20 kDa GTP-binding proteins, a human brain cDNA containing a partial open reading frame encoding 48 amino acids, of which 10 were basic, was isolated. This peptide was unusual based on its predicted pI of 11.7. Oligonucleotide probes specific for this sequence hybridized with two abundant mRNAs in HL-60 cells, one of which appeared to comigrate with an ARF3 mRNA. Because of the unusual nature of this peptide as well as the size and abundance of the mRNAs, studies were performed to identify and investigate the relationship between these mRNAs. The cDNA fragment was used to isolate overlapping clones from dibutyryl cAMP-differentiated HL-60 and bovine retinal cDNA libraries. A putative open reading frame was identified that encodes a 23.6 kDa protein (p23, 203 amino acids) with a predicted pI of 11.6. The deduced amino acid sequence is highly conserved between human and bovine, exhibiting 97% identity; they are 99% identical if conservative substitutions are included. The human and bovine nucleotide sequences exhibit 91% identity across the coding region. Clones from both species contain a poly-adenylation signal (AATAAA) 4 nucleotides downstream (3') from the termination codon as well as a poly(A)+ tail. Other clones, however, extended beyond the point of poly(A)+ addition. In one bovine clone, the 3'-untranslated region extended 442 nucleotides beyond the polyadenylation signal to end in a poly(A)+ tail; a second polyadenylation signal was found 21 nucleotides before the poly (A)+ tail. Hybridization of poly(A)+ RNA from various bovine tissues and brains of several mammalian species with cDNA and oligonucleotides specific for the coding region identified two abundant mRNA species of 1.2- and 0.8-kb. Oligonucleotide probes specific for the extended 3'-untranslated region hybridized with the 1.2-kb, but not the 0.8-kb, mRNA. Both mRNAs were observed in all mammalian tissues and species. These studies indicate that a ubiquitous, basic 23.6 kDa protein is highly conserved among mammals and is encoded by two mRNA species that appear to differ in the site of polyadenylation.

在 cDNA 克隆的分离和表征过程中 ADP-核糖基化因子，约 20 kDa GTP 结合蛋白，人脑 含有编码48个氨基酸的部分开放阅读框的cDNA， 其中10个是基本的，是孤立的。这种肽的不同寻常之处在于它 预测 pI 为 11.7。该序列特异的寡核苷酸探针 与 HL-60 细胞中两种丰富的 mRNA 杂交，其中一种似乎 与 ARF3 mRNA 共迁移。由于这种肽的不寻常性质 以及 mRNA 的大小和丰度，进行了研究 识别并研究这些 mRNA 之间的关系。 cDNA 片段用于从二丁酰中分离重叠克隆 cAMP 分化的 HL-60 和牛视网膜 cDNA 文库。一个推定的 已鉴定出编码 23.6 kDa 蛋白质（p23、203 氨基酸），预测 pI 为 11.6。推导的氨基酸序列 在人和牛之间高度保守，表现出 97% 的同一性；他们 如果包括保守替换，则 99% 相同。人类和 牛核苷酸序列在整个编码区表现出 91% 的同一性。 两个物种的克隆均含有聚腺苷酸化信号 (AATAAA) 4 终止密码子下游（3'）的核苷酸以及 聚（A）+尾。然而，其他克隆的范围超出了 Poly(A)+ 的范围 添加。在一个牛克隆中，3'-非翻译区延伸了第442章 超出聚腺苷酸化信号的核苷酸以聚（A）+尾结束；一个 在多聚 (A)+ 之前的 21 个核苷酸处发现了第二个多聚腺苷酸化信号 尾巴。来自各种牛组织和大脑的 Poly(A)+ RNA 的杂交 几种哺乳动物物种的 cDNA 和寡核苷酸特异性 编码区鉴定出两种丰富的 1.2-kb 和 0.8-kb mRNA 种类。 特异于延伸的 3'-非翻译区的寡核苷酸探针 与 1.2-kb mRNA 杂交，但不与 0.8-kb mRNA 杂交。两种 mRNA 均 在所有哺乳动物组织和物种中均观察到。这些研究表明 一种普遍存在的基本 23.6 kDa 蛋白质在哺乳动物中高度保守 由两种 mRNA 编码，这两种 mRNA 的位点似乎有所不同 聚腺苷酸化。