Coffman J A, Kirchhamer C V, Harrington M G, Davidson E H
Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA.
Dev Biol. 1996 Feb 25;174(1):43-54. doi: 10.1006/dbio.1996.0050.
In this paper we present a structural and functional characterization of a new sea urchin embryo transcription factor, SpRunt-1. This factor was isolated by means of its specific interaction with a cis-regulatory target site of the CyIIIa gene. Here we show that this target site, the P7I site, is required for normal embryonic activation of CyIIIa x CAT reporter gene constructs. An oligonucleotide affinity column bearing the P7I target site purifies a 21-kDa polypeptide from blastula-stage nuclear extracts, and the amino acid sequence obtained from this polypeptide was used to generate a nucleic acid probe with which the corresponding cDNA was cloned. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a "runt domain" that is closely homologous to those of Drosophila and mammalian runt domain transcription factors. RNA and genomic blots show that SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. By RNA probe protection we found that transcripts of SpRunt-1 increase in concentration dramatically after the blastula stage of development, suggesting that the up-regulation of CyIIIa that occurs after blastula stage is a function of zygotically transcribed SpRunt-1. These results are discussed with reference to known features of the runt domain family of transcription factors.
在本文中,我们展示了一种新的海胆胚胎转录因子SpRunt-1的结构和功能特征。该因子是通过其与CyIIIa基因的顺式调控靶位点的特异性相互作用而分离得到的。在此我们表明,这个靶位点,即P7I位点,对于CyIIIa x CAT报告基因构建体的正常胚胎激活是必需的。带有P7I靶位点的寡核苷酸亲和柱从囊胚期核提取物中纯化出一种21 kDa的多肽,从该多肽获得的氨基酸序列被用于生成一种核酸探针,并用其克隆相应的cDNA。该cDNA编码一种约60 kDa的蛋白质SpRunt-1,它包含一个“runt结构域”,该结构域与果蝇和哺乳动物的runt结构域转录因子的结构域密切同源。RNA和基因组印迹显示,SpRunt-1由单个胚胎转录本代表,由可能两个含runt结构域的基因之一编码。通过RNA探针保护实验,我们发现SpRunt-1的转录本在发育的囊胚期之后浓度急剧增加,这表明囊胚期后发生的CyIIIa上调是合子转录的SpRunt-1的功能。我们将结合转录因子runt结构域家族的已知特征对这些结果进行讨论。