CHARACTERIZATION OF A HIGHLY CONSERVED 23 KDA BASIC PROTEIN

在分离和鉴定基因克隆的过程中 ADP-核糖化因子，约20 kDa的GTP结合蛋白，人脑 包含一个编码48个氨基酸的部分开放阅读框的 其中10株是碱性的，是分离出来的。这种多肽是不寻常的，因为它的 预测等电点为11.7。该序列特异的寡核苷酸探针 在HL-60细胞中与两个丰富的mRNA杂交，其中一个似乎 与ARF3mRNA配对。因为这种多肽的不同寻常的性质 除了mRNAs的大小和丰度，研究还进行了 确定并研究这些mRNAs之间的关系。CDNA3 利用片段从二丁酰基中分离重叠克隆 CAMP分化的HL-60和牛视网膜cDNA文库。推定的 确定了编码23.6 kDa蛋白质(p23,203)的开放阅读框 氨基酸)，预测等电点为11.6。推导出的氨基酸序列 在人和牛之间高度保守，显示出97%的同源性；它们 如果包括保守的替代，则99%相同。人类和 牛核苷酸序列在整个编码区表现出91%的同源性。 这两个物种的克隆都含有一个多聚腺苷信号(AATAAA)4 终止密码子下游的核苷酸(3‘)以及 Poly(A)+Tail。然而，其他克隆超出了PolyA+的范围 加法。在一个牛克隆中，3‘非翻译区延伸了442 核苷酸超出多聚腺苷酸化信号，以聚(A)+尾巴结尾； 在Poly(A)+之前发现了21个核苷酸的第二多聚腺苷信号 尾巴。牛不同组织和脑中Poly(A)+RNA的杂交 几种哺乳动物物种具有特异性的cDNAs和寡核苷酸 该编码区鉴定出两个丰富的mRNA物种，分别为1.2-和0.8kb。 针对延伸的3‘-非翻译区的寡核苷酸探针 与1.2-kb的mRNA杂交，但不与0.8-kb的mRNA杂交。这两个mRNA都是 在所有哺乳动物组织和物种中都能观察到。这些研究表明， 一种普遍存在的碱性23.6 kDa蛋白在哺乳动物和 由两种不同的信使核糖核酸编码，这两种信使核糖核酸在 多聚腺苷酸化。

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.