Figure 4.

Gene structure and genomic Southern blot analysis of J3-crystallin. (a) The J3-crystallin gene was cloned as follows. Five identical 2.2-kbp genomic fragments (G₁) were obtained from an EcoRI genomic library by using J3-crystallin cDNA as a probe. Next, a 4.7-kbp clone (G₂) was obtained from a XbaI genomic library by screening with G₁. Partial sequencing and PCR with primers derived from the J3-crystallin cDNA were used to identify part of exon 2 and all of exon 3 in G₁ and exons 2 and 3 in G₂. Exons 4–7 were identified on a 7.4-kbp clone (G₃) that was obtained from the XbaI library by screening with three deoxyoligonucleotides (5′-gagtgcgagaacatagtgcga-3′; 5′-ctgtaattattcgaaggaaag-3′; 5′ctgtgcagtgctgggctact-3′) derived further downstream on the J3-crystallin cDNA. The 5′ end of the J3-crystallin gene was determined by cloning a PCR product using primers with EcoRI linkers on their ends. The 5′ primer was derived from the beginning of the cDNA and the 3′ primer was derived from the 5′ end of G₂. The resulting clone (G₄) contained exon 1 at its 5′ end. (b) Southern blot of genomic J3-crystallin sequences. Lane 1, J3-crystallin cDNA probe; lanes 2 and 4, G₁ probe; lane 3, G₄ probe.