Behal A, Prakash K, D'Eustachio P, Adesnik M, Sabatini D D, Kreibich G
Department of Cell Biology, New York University School of Medicine, New York 10016.
J Biol Chem. 1990 May 15;265(14):8252-8.
Ribophorin I is a type I transmembrane glycoprotein characteristic of the rough portions of the endoplasmic reticulum where it is thought to play a role in the cotranslational insertion of nascent polypeptides. A rat ribophorin I cDNA was used to isolate four overlapping genomic clones from a rat EMBL3 genomic library. Restriction mapping, Southern blotting, and DNA sequencing showed that these clones, spanning approximately 21 kilobases of chromosomal DNA, include the entire ribophorin I gene, as well as 15 kilobases (kb) of upstream sequences. Southern blotting analysis of DNA from a panel of mouse-Chinese hamster cell hybrids demonstrated that the ribophorin I gene is located on mouse chromosome six. The ribophorin I gene contains 10 exons, seven of which encode the luminal domain of the polypeptide. Exon 8 encodes the trans-membrane domain and small portions of the flanking luminal and cytoplasmic domains. Exons 9 and 10 encode the remainder of the cytoplasmic domain, and the latter includes the 3'-untranslated portion of the mRNA. Six closely spaced transcription start sites located 3 to 24 base pairs upstream from the initiation codon were identified by primer extension analysis and S1 mapping. The sequence of a 1.3-kb region upstream of the cap sites was determined and found to contain three GC-rich potential Sp1-binding sites beginning at -14, -24, and -91 base pairs (bp), two octamer-like sequences at -233 and -1248 bp, and a CAAT-like box at -41 bp. The possible roles of these elements in regulating expression of the ribophorin gene in all cells and in differentiated cell types characterized by a well developed rough endoplasmic reticulum is discussed.
核糖体结合蛋白I是一种I型跨膜糖蛋白，在内质网的粗糙部分具有特征性，人们认为它在新生多肽的共翻译插入过程中发挥作用。利用大鼠核糖体结合蛋白I的cDNA从大鼠EMBL3基因组文库中分离出四个重叠的基因组克隆。限制性图谱分析、Southern印迹分析和DNA测序表明，这些克隆跨越约21千碱基对的染色体DNA，包括整个核糖体结合蛋白I基因以及15千碱基对的上游序列。对一组小鼠-中国仓鼠细胞杂种的DNA进行Southern印迹分析表明，核糖体结合蛋白I基因位于小鼠6号染色体上。核糖体结合蛋白I基因包含10个外显子，其中7个编码多肽的腔内结构域。外显子8编码跨膜结构域以及侧翼腔内和细胞质结构域的小部分。外显子9和10编码细胞质结构域的其余部分，后者包括mRNA的3'非翻译部分。通过引物延伸分析和S1图谱鉴定出位于起始密码子上游3至24个碱基对处的六个紧密间隔的转录起始位点。确定了帽位点上游1.3千碱基对区域的序列，发现其包含三个富含GC的潜在Sp1结合位点，分别位于-14、-24和-91碱基对处，两个八聚体样序列位于-233和-1248碱基对处，以及一个CAAT样框位于-41碱基对处。讨论了这些元件在调节核糖体结合蛋白基因在所有细胞以及以发育良好的粗糙内质网为特征的分化细胞类型中的表达方面可能发挥的作用。
