Moormann R J, den Dunnen J T, Mulleners L, Andreoli P, Bloemendal H, Schoenmakers J G
J Mol Biol. 1983 Dec 25;171(4):353-68. doi: 10.1016/0022-2836(83)90034-7.
Two complementary DNA clones pRL gamma-2 and pRL gamma-3 of different rat lens gamma-crystallin messenger RNAs have been used to identify gamma-crystallin gene sequences in rat genomic DNA. Subsequently, the DNA present in the 18,000 to 20,000 bases region of the EcoRI digest, giving rise to a strong doublet hybridization signal, was cloned in lambda phage Charon-4A. One of the clones, lambda RCH gamma-3, carrying an insert of 17,500 bases has been characterized in detail. From analysis at the restriction enzyme level with 5'-, "middle" and 3'-specific subprobes of pRL gamma-3 it could be deduced that lambda RCH gamma-3 contains only one gamma-crystallin gene. The coding sequences of this gene are interrupted by intronic DNA. The primary structure of this gene and its flanking regions have been established by sequencing the relevant regions of a subclone of lambda RCH gamma-3, designated pRCH gamma-3 . 1. The sequence data show that the gamma-crystallin gene extends over 2700 bases of rat genomic DNA. The gene is split by two introns, one of 87 base-pairs after the third translation codon and a large one of 1880 base-pairs after codon 84. The mosaic structure of the gene is strictly co-linear with the structure of the gamma-crystallin polypeptide in that the large intron is positioned in a region which specifies the so-called "connecting peptide" and which links the two highly symmetrical and homologous protein domains. Although expected from the cDNA and protein sequence no introns were observed between the coding regions in the DNA specifying the two homologous folding motifs present in each protein domain. The relevance of this phenomenon in terms of the evolution of the mature gamma-crystallin gene is discussed.
两个不同的大鼠晶状体γ-晶状体蛋白信使核糖核酸的互补DNA克隆pRLγ-2和pRLγ-3已被用于鉴定大鼠基因组DNA中的γ-晶状体蛋白基因序列。随后，在EcoRI酶切片段18,000至20,000碱基区域中出现强烈双峰杂交信号的DNA被克隆到λ噬菌体Charon-4A中。其中一个克隆λRCHγ-3，携带一个17,500碱基的插入片段，已被详细表征。通过使用pRLγ-3的5'-、“中间”和3'-特异性亚探针在限制性内切酶水平上进行分析，可以推断出λRCHγ-3仅包含一个γ-晶状体蛋白基因。该基因的编码序列被内含子DNA打断。通过对λRCHγ-3的一个亚克隆（命名为pRCHγ-3.1）的相关区域进行测序，确定了该基因及其侧翼区域的一级结构。序列数据表明，γ-晶状体蛋白基因在大鼠基因组DNA中延伸超过2700个碱基。该基因被两个内含子隔开，一个在第三个翻译密码子后87个碱基对，另一个大的在密码子84后1880个碱基对。该基因的镶嵌结构与γ-晶状体蛋白多肽的结构严格共线，因为大内含子位于一个指定所谓“连接肽”的区域，该区域连接两个高度对称且同源的蛋白质结构域。尽管从cDNA和蛋白质序列可以预期，但在指定每个蛋白质结构域中存在的两个同源折叠基序的DNA编码区域之间未观察到内含子。讨论了这种现象在成熟γ-晶状体蛋白基因进化方面的相关性。
