Aey2, a New Mutation in the ␤B2-Crystallin–Encoding Gene of the Mouse Jochen Graw,1 Jana Lo¨ster,1 Dian Soewarto,2 Helmut Fuchs,2 Andre´ Reis,3,4 Eckhard Wolf,5 Rudi Balling,1,6 and Martin Hrabe´ de Angelis2 PURPOSE. During an ethylnitrosourea (ENU) mutagenesis he ␤- and ␥-crystallins form the major part of the water- screen, mice were tested for the occurrence of dominant Tsoluble proteins of the eye lens. The ␤- and ␥-crystallins are cataracts. One particular mutant was found that caused pro- recognized as members of a superfamily and have been con- gressive opacity and was referred to as Aey2. The purpose of sidered for a long time to be present only in the eye and mainly the study was to provide a morphologic description, to map in the ocular lens.1,2 However, just recently, expression of the the mutant gene, and to characterize the underlying molecular ␤B2-crystallin mRNA and protein was reported also in brain lesion. and testis.3 The common characteristic of all ␤- and ␥-crystal- METHODS. Isolated lenses were photographed, and histologic lins is the so-called Greek key motif. Crystallography of bovine sections of the eye were analyzed according to standard pro- ␤B2- and ␥B-crystallins has shown that each of the ␤- and cedures. Linkage analysis was performed using a set of micro- ␥-crystallins is composed of two domains, each of them built satellite markers covering all autosomal chromosomes. cDNA up by two Greek key motifs.4,5 from candidate genes was amplified after reverse transcription It is widely accepted that ␤- and ␥-crystallins evolved in two of lens mRNA. duplication steps from an ancestral protein folded like a Greek 6 RESULTS. The cortical opacification visible at eye opening pro- key. Greek key motifs also have been reported for a few other gressed to an anterior suture cataract and reached its final proteins, such as a yeast killer toxin from Williopsis mrakii 7 phenotype as total opacity at 8 weeks of age. There was no (WmKT; 1 Greek key motif) the protein S from Myxococcus obvious difference between heterozygous and homozygous xanthus (4 Greek key motifs), spherulin 3c from Physarum mutants. The mutation was mapped to chromosome 5 proxi- polycephalon (2 Greek key motifs), the epidermis-specific pro- mal to the marker D5Mit138 (8.7 Ϯ 4.2 centimorgan [cM]) and tein from Cynops pyrrhogaster (EDSP or ED37; 4 Greek key distal to D5Mit15 (12.8 Ϯ 5.4 cM). No recombinations were motifs),6 and the putative human tumor suppressor protein observed to the markers D5Mit10 and D5Mit25. This position AIM1 (12 Greek key motifs).8 makes the genes within the ␤A4/␤B-crystallin gene cluster The function of the Greek key motifs has not been elabo- excellent candidate genes. Sequence analysis revealed a muta- rated in detail; however, computer-based analysis suggests that tion of T3A at position 553 in the Crybb2 gene, leading to an it may be responsible for particular protein–protein interac- exchange of Val for Glu. It affects the same region of the tions similar to those seen with immunoglobulins.9 Similar to Crybb2 gene as in the Philly mouse. Correspondingly, the loss the immunoglobulins, the ␤- and ␥-crystallins are folded in an of the fourth Greek key motif is to be expected. all-␤ structure. Undoubtedly, the accumulation of hydrogen CONCLUSIONS. The Aey2 mutant represents the second allele of bonds in the symmetrical, twisted, antiparallel, ␤-sheet struc- Crybb2 in mice. Because an increasing number of ␤- and ture of each domain, and the hydrophobic interactions be- ␥-crystallin mutations have been reported, a detailed pheno- tween them, contribute significantly to their stability. This type–genotype correlation will allow a clearer functional un- overall stability can be understood as the sum of the contribu- derstanding of ␤- and ␥-crystallins. (Invest Ophthalmol Vis Sci. tion of independent folding units.10,11 2001;42:1574–1580) Mutations in the ␤- and ␥-crystallin–encoding genes (gene symbols are Cryb and Cryg, respectively) have been demon- strated to lead to lens opacification in mice12–17 and hu- mans.18–24 In most cases, the formation of at least one of the 1 From Forschungszentrum fu¨r Umwelt und Gesundheit (GSF; Na- Greek key motifs is affected by the mutation. In total, 13 genes tional Research Center for Environment and Health), Institute of Mam- belong to this superfamily in mammals. malian Genetics, Neuherberg; the 2Institute of Experimental Genetics, 3 In the ␤-crystallins, individual Greek key motifs are encoded Neuherberg; the Institute of Molecular Genetics, Max Delbru¨ck Cen- 25 ter for Molecular Medicine, Berlin; and the 5Lehrstuhl fu¨r Molekulare by separate exons. The Cryb genes consist of six exons: The Tierzucht und Haustiergenetik, Ludwig Maximilians Universita¨t, Mu- first exon is not translated, the second exon encodes the nich, Germany. N-terminal extension, and the subsequent four exons are re- 4Present affiliation: Institute of Human Genetics, University of sponsible for one Greek key motif each. Biochemically, the Erlangen-Nu¨rnberg, Germany. 6 ␤-crystallins are characterized as oligomers (the molecular Present affiliation: German Research Center for Biotechnology masses of the monomers ranges between 22 and 28 kDa) with (GBF), Braunschweig, Germany. native molecular masses ranging up to 200 kDa for octomeric Supported by Grant 01KW9610/1 from the German Human Ge- 6,26 nome Project (RB, EW, MHdA). forms. The N termini are blocked by acetylation. Submitted for publication November 28, 2000; revised January 29, The family of ␤-crystallins can be divided into more acidic 2001; accepted February 7, 2001. (␤A-) and more basic (␤B-) crystallins. Each subgroup is en- Commercial relationships policy: I, C (RB, MHdA); N (all others). coded by three genes (respectively, Cryba1,-2, and -4; and The publication costs of this article were defrayed in part by page Crybb1,-2, and -3), however, Cryba1 encodes two proteins: charge payment. This article must therefore be marked “advertise- ␤A1- and ␤A3-crystallins. This feature is conserved among all ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Jochen Graw, GSF–National Research Cen- vertebrates. In mammals, the Cryb genes are distributed among ter for Environment and Health, Institute of Mammalian Genetics, three chromosomes (mouse: 1, 5, and 11; man: 2, 17, and 22; Ingolsta¨dter Landstrasse 1, D-85764 Neuherberg, Germany. for recent review see Ref. 15 and references therein). Among [email protected] the Cryb genes, the nucleotide sequence of Crybb2 was char- Investigative Ophthalmology & Visual Science, June 2001, Vol. 42, No. 7 1574 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 10/02/2021 IOVS, June 2001, Vol. 42, No. 7 Novel ␤B2-Crystallin Mutation in the Mouse 1575 TABLE 1. PCR Primers with Annealing Temperatures and Length of the Products Accession Annealing Temperature (Designation Lab Number Sequence (5؅ 3 3؅) Number (°C); Fragment Size (bp Cryba4-L1 25026 GGC CCT TCT TGG ACC GTG C AJ272228 53–60; 747 Cryba4-R2 26526 TGG CGG GGC ATG GGC AJ272228 Crybb1-L1 25028 CCA ATA CAG CAC CAG GAA CCA TGT C AF106853 60; 517 Crybb1-R1 25029 CTC TTG GAT CTC CAT GGT GTT GCC AF106853 Crybb2-L1 24468 CTC GAC GCC AGA GAG TCC ACC M60559 55; 728 Crybb2-R1 24469 GGC ACG AGC CAC ACT TTA TTC TTC M60559 Crybb2-L2 27037 GGC TAC CGT GGG CTG CAG TAC M60559 58; 215 Crybb2-R1 24469 GGC ACG AGC CAC ACT TTA TTC TTC M60559 Crybb3-L1 25030 CTG GTT CTC CTC CAG TTT GAG GC AJ272229 53–60; 738 Crybb3-R2 25031 GGC TGG GCA CAT CGT CAT CTA C AJ272229 acterized at first because of the high abundance of this basic, PCR and Sequencing principal ␤-crystallin.27 In the course of analysis of mouse mutants obtained by a For the molecular analysis, RNA was isolated from lenses of C3HeB/FeJ, large-scale ethylnitrosourea (ENU) mutagenesis program,28,29 T-stock, 129, JF-1, and C57BL/6J wild-type mice and from homozygous we identified several cataract mutations. Herein, we report one mutant mice at the age of 4 weeks. RNA from lens, brain, and testis of them, which was mapped to mouse chromosome 5 and were transcribed to cDNA by using a kit (Ready-to-Go; Pharmacia identified as the second allele in the mouse Crybb2 gene. Biotech, Freiburg, Germany). Genomic DNA was isolated from tail tips or spleen of wild-type C3HeB/FeJ and C57BL/6J mice or homozygous mutants, according to standard procedures. For amplification of cDNA MATERIALS AND METHODS from Cryba4 and the three Crybb genes, primers were selected from the EMBL GenBank databases (provided in the public domain by the Mice European Bioinformatics Institute at Hinxton, Cambridge, UK, at Mice were kept under specific pathogen-free conditions at the National http//:www.ebi.ac.uk/ and the National Center for Biotechnology In- Research Center for Environment and Health (GSF) and monitored formation at http//:www.ncbi.nlm.nih.gov/, respectively; Table 1). within the ENU mouse mutagenesis screen project.28,29 The use of PCR was performed using a thermocycler (Hybaid OmniGene; animals was in accordance with the ARVO Statement for the Use of MWG BioTech, Ebersberg, Germany). The annealing temperatures are Animals in Ophthalmic and Vision Research and with the German Law included in Table 1. PCR products were analyzed on a 1% agarose gel. on Animal Protection. Sequencing was performed commercially (SequiServe, Vaterstetten, Male C3HeB/FeJ mice were treated with ENU (160 mg/kg) at the Germany) immediately after isolation of the DNA from the gel using an age of 10 weeks according to Ehling et al.30 and mated to untreated extraction kit (Qia-Quick; Qiagen, Hilden, Germany).