Moormann R J, Jongbloed R, Schoenmakers J G
Gene. 1984 Jul-Aug;29(1-2):1-9. doi: 10.1016/0378-1119(84)90159-8.
Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.
通过将大鼠基因组DNA的Sau3A部分酶切产生的不同大小片段克隆到黏粒载体pTM中，构建了两个文库，共包含约10⁶个菌落。用两个cDNA克隆进行筛选，一个包含αA2-晶状体蛋白序列，另一个包含βB1-晶状体蛋白序列，分离出14个黏粒克隆，它们是βB1-晶状体蛋白特异性的；未发现包含αA2-晶状体蛋白序列的克隆。βB1克隆的插入片段长度在35至45kb之间，包含覆盖超过60kb大鼠基因组DNA的重叠DNA片段。该区域的复合BamHI限制性图谱显示有一个单一的βB1-晶状体蛋白基因，被几个内含子序列中断。还从这些文库中分离出了五个与两个不同的大鼠晶状体γ-晶状体蛋白cDNA克隆杂交的重组体。其中四个包含31至41kb的插入片段，而第五个重组体包含一个12.2kb的插入片段。用5'和3'特异性cDNA片段进行杂交分析表明，这些插入片段总共包含六个γ-晶状体蛋白基因，其中三个位于仅31kb的一个插入片段上。
