Borst D E, Nickerson J M
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, Bethesda, MD 20892.
Exp Eye Res. 1988 Dec;47(6):825-38. doi: 10.1016/0014-4835(88)90066-8.
The gene locus for bovine IRBP, as well as several kilobases of sequence flanking the gene on either end, has been cloned. Two of the several clones seem to contain full-length copies of the protein encoding portion of the gene. Using these clones and cDNA clones, we have determined that there are one or perhaps two copies of the IRBP gene per haploid genome in several species. The gene is compact considering the large size of the protein (145,000 daltons) and its large mRNA (about 6.5 kb). Surprisingly, the gene is no more than 14 kb, being fully contained on lambda clones of maximum packaging size 20 kb. Small parts of the gene were sequenced for the purpose of proving the identity of the genomic clones. DNA sequencing of one of the IRBP gene clones demonstrates the existence of an intron in the gene. The sequence analysis of another fragment identified the N-terminus which has been sequenced at the protein level. The DNA sequence analysis showed the existence of a putative signal sequence and the potential existence of a short five amino acid sequence between the signal sequence and the authentic N-terminus of the secreted extracellular IRBP. This confirms and validates the finding of the extra five amino acid sequence that is present on 40-50% of the polypeptides in monkey and human IRBP which have been isolated from the subretinal space. The presence of the appropriate gene sequence for the pentapeptide but its absence in bovine IRBP indicates differences in processing among the vertebrate IRBPs.
牛视黄醇结合蛋白（IRBP）的基因位点以及该基因两端侧翼的几千个碱基对序列已被克隆。几个克隆中有两个似乎包含该基因蛋白质编码部分的全长拷贝。利用这些克隆和cDNA克隆，我们已确定在几个物种中，每个单倍体基因组有一个或可能两个IRBP基因拷贝。考虑到该蛋白质的大尺寸（145,000道尔顿）及其大mRNA（约6.5 kb），该基因结构紧凑。令人惊讶的是，该基因不超过14 kb，完全包含在最大包装尺寸为20 kb的λ克隆上。为了证实基因组克隆的身份，对该基因的小部分进行了测序。对其中一个IRBP基因克隆的DNA测序表明该基因中存在一个内含子。对另一个片段的序列分析确定了已在蛋白质水平进行测序的N端。DNA序列分析表明存在一个假定的信号序列，并且在分泌的细胞外IRBP的信号序列与真实N端之间可能存在一个短的五氨基酸序列。这证实并验证了在从视网膜下腔分离的猴子和人类IRBP中40 - 50%的多肽上存在额外五个氨基酸序列的发现。五肽的适当基因序列的存在但在牛IRBP中不存在，表明脊椎动物IRBP之间在加工过程上存在差异。
