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U-Crystallin Is a Mammalian Homologue of Agrobacterium

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U-Crystallin Is a Mammalian Homologue of Agrobacterium Proc. Natl. Acad. Sci. USA Vol. 89, pp. 9292-92%, October 1992 Evolution ,u-Crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina (crystallin recritme t/molecular evolution/amino add metabolism) ROBERT Y. KIM*, ROBIN GASSERt, AND GRAEME J. WISTOW*t *Section on Molecular Structure and Function, Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892; and tThe University of Melbourne, School of Veterinary Science, Werribee, Victoria 3030, Australia Communicated by Charles Sibley, July 6, 1992 (received for review June 4, 1992) ABSTRACT I-Crystallin is the major component of the MATERIALS AND METHODS in Australian marsupials. The complete se- eye lens several RNA Extraction and cDNA Cloning. Total RNA was ex- of kangaroo -crystalln has now been obtained by quence tracted from gray kangaroo lenses by the guanidinium thio- cDNA cloning. The predicted amino acid sequence shows with RNazol reagents (Tel-Test, Friends- tu- cyanate method (7) similary with ornithine cyclodeaminases encoded by the wood, TX). Five micrograms of poly(A)+ RNA was then mor-inducing (Ti) plasmids of Agrobacterium tumefaciens. purified by using Dynabeads (Dynal, Great Neck, NY). Until now, neither ornithine cyclodeaminase nor any structur- cDNA was synthesized using the Lambda Zap kit from ally related enzymes have been observed in eukaryotes. RNA Stratagene, following manufacturer's specifications. A cDNA analysis ofkangaroo tissues shows that It-crystallin is expressed probe for j.-crystallin was produced by PCR of 1 pug of at high abundance in lens, but outside the lens #-crystallin is kangaroo lens total RNA with oligo primers GARGGGGTG- preferentially expressed in neural tissues, retina, and brain. An GTGCARCC and GATCACATCCCCAGAYTC, designed almost full-length cDNA for p-crystallin was cloned from from previously determined wallaby IA-crystallin peptides (5), human retina. In human tissues, pt-crystallin mRNA is present with a Perkin-Elmer reverse transcription PCR kit. The in neural tissue, muscle, and kidney. This pattern ofexpression probe was labeled by random-priming (United States Bio- and relationship to an enzyme involved in unusual amino acid chemical) and used to screen 200,000 plaques of the cDNA metabolism suggests the interesting possibility that mam- library. Approximately 5% of the plaques gave positive malian p-crystallins could be enzymes participating in pro- hybridization; plaques were purified by standard methods cesses such as osmoregulation or the metabolism of excitatory (8). A full-length clone was sequenced by using Sequenase amino acids. reagents and protocols from United States Biochemical. This clone was used as a probe to screen a human fetal retina Crystallins, the major structural components ofthe lens, have cDNA library (Stratagene). arisen by the direct recruitment of enzymes and stress Primer-Extension Analysis. Two different primers were proteins (1-4). In many, perhaps all, cases the acquisition of used to extend on a template of kangaroo lens total RNA. A another role occurred without prior gene duplication. Prob- single initiation site was identified by both primers. Oligo ably because of the evolutionary plasticity of the lens, many primers (5936, CCGCCCGCTGTCCGCAGCCT; 5937, different taxon-specific crystallins with recent, independent AAAGCCGGACTCCAACTCAT) were labeled with [(y32p]J It dATP (Amersham). Labeled primer (106 cpm) was hybridized recruitment events have been found in different lineages. to 10 pZg of lens total RNA and extended by using a cDNA has been suggested that all the recruited proteins represent synthesis kit from Boehringer Mannheim. molecules expressed in the developing lens for other func- Southern Analysis. Ten micrograms of liver DNA was tional reasons, such as metabolic control or response to digested with BamHI, EcoRI, and HindIII restriction en- stresses such as the osmotic swelling oflens fiber cells (5, 6). zymes (BRL). Gels were run according to standard methods The genes for such proteins are already active in the lens and (8) and blotted onto nylon membranes. A cDNA probe was may need only minor modification to increase expression to generated by PCR of the pu-crystallin clone shown in Fig. 1 structurally useful levels. Recent recruitment events may with T3 and T7 primers used to amplify the entire insert (9). have selected enzymes to dilute the lens-hardening effects of Probe was labeled with 32P by using the United States the more specialized y-crystallins. Biochemical random priming kit. Hybridization was in Nyl- ,u-Crystallin accounts for about a quarter of total lens ohybe (Oncor, Gaithersburg, MD) at 420C overnight. The blot protein in several Australian marsupials (5), although it is not was washed in 0.1 x standard saline citrate (SSC)/1% SDS at detectable in the lenses of an American marsupial or of other 650C. A commercial "zoo blot" (Evoblot, Bios, New Haven, vertebrates. Partial peptide sequences of u-crystallins from a CT) hybridized with the same probe. Hybridization was in macropod and a dasyurid suggested distant similarities with Nylohybe at 420C overnight, and subsequent washing was in certain dehydrogenases (5), but no obvious superfamily re- 0.1x SSC/1% SDS at room temperature. lationship was indicated. To obtain complete identification of Northern (RNA) Analysis. Total RNA was extracted from ,I-crystallin a gray kangaroo (Macropus fuliginosus) lens kangaroo lens, cornea, retina, brain, liver, heart, and kidney cDNA library has been constructed and screened with probes as for Fig. 1. Five micrograms was loaded in each lane; an derived from known peptide sequences.§ Results suggest that additional lens lane contained 0.5 t&g. Northern blots were ,u-crystallin is a eukaryotic enzyme related only to bacterial ornithine cyclodeaminases (OCDs), enzymes of unusual Abbreviation: OCD, ornithine cyclodeaminase. amino acid metabolism. *To whom reprint requests should be addressed at: Section on Molecular Structure and Function, Laboratory of Molecular and Developmental Biology, National Eye Institute, Room 222, Build- The publication costs of this article were defrayed in part by page charge ing 6, National Institutes of Health, Bethesda, MD 20892. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M90841). 9292 Evolution: Kim et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9293 A B 1 2 3 4 1 CTGGCAGCGAACAGCCGGCAGCAGGCTGCGGACAGCGGGCGGCGGACAGCCGGCAGCG - 1 MetSerTrpSerProAlaPheLeuArgSerGluAspValGluArgTyrLeuGly 58 GGCGCCATGAGTTGGAGTCCGGCTTTCCTGAGGTCCGAGGATGTGGAGCGGTACCTGGGC _ amp-a 19 SerSerSerIleLeuLeuProAlaLeuGluLy laLeuAlaAsnPheSerSerGlySer _::. 118 AGCTCCAGCATCCTGCTACCAGCCCTGGAAAAGGCCCTGGCCAACTTTTCTAGCGGCTCG HE -a -127 ,.s- 39 GluGlyGlyValValGlnProValAr ¢ThrValIleProValAlaLy +iisGlnGlyPhe A. 178 GAGGGAGGAGTCGTGCAGCCAGTTCGCACCGTGATACCTGTGGCGAAACACCAAGGTTTC _. seuValThr A..... 59 LeuGlyIleMetProValTyrSerAlaSerGluAspAlaLeuThahrLy .,gW, . 238 CTGGGTATCATGCCTGTCTACAGCGCTAGTGAAGATGCACTTACCACCAAGCTAGTAACC A.: 4.:X...,.:Be;... 79 PheTyrGluGlyMetSerProThrSerThrAlaProSerHisGlnThrThrValLeuPhe .. .:......... 298 TTCTATGAGGGAATGAGCCCCACTTCCACTGCTCCCTCACATCAGACCACAGTGCTTTTC w. a_ ..95,,,,. -J,.S ..: ':::.:' *: ..: 99 PheAspPr erAsnGlySerLeuLeuSerIleMetAspGlyAsnIleIleThrAlaLys 358 TTTGACCCCAGCAATGGCTCTCTACTCTCGATCATGGATGGAAACATAATTACTGCAAAG A_ 119 ArgThrAlaAlaValSerAlaIleAlaThrLysPheLeuLysProProSerSerGluVal 418 AGGACAGCTGCTGTGTCAGCTATTGCAACCAAGTTTCTAAAACCCCCTTCAAGTGAAGTT nt42 nt84 139 LeuCysIleLeuGlyAlaGlyValGlnAlaTyrSerHisTyrGluIlePheLysGluGln 478 TTGTGCATACTTGGTGCTGGTGTCCAGGCCTACAGTCATTATGAAATCTTCAAAGAGCAA jcDNA 159 PheSerPheLysGluValArgIleTrpAsnArgThrLysLysAsnAlaGluLysPheAla 538 TTCTCTTTTAAAGAGGTGCGGATATGGAATCGAACCAAGAAGAATGCTGAGAAGTTTGCT 179 GlnThrValLysGlyAspValArgValCysSerSerValGlnGluAlaValThrGlyAla 598 CAGACAGTGAAGGGGGATGTGCGAGTCTGCTCATCTGTCCAGGAGGCAGTGACAGGCGCA 199 AspValIleIleThrValThrMetAlaThrLysProIleLeuPheGlyGluTrpValLys 658 GATGTGATCATCACTGTCACCATGGCAACAAAACCCATTTTATTTGGAGAATGGGTGAAG ots FIGURE: SIZE 219 ProGlyAlaHisIleAsnAlaValGlyAlaSerArgProAspTrpArgGluLeuAspAsp 718 CCAGGAGCCCATATCAATGCTGTTGGAGCCAGCAGACCTGACTGGAGAGAACTAGATGAT 239 GluIleMetLysAsnCysValLeuTyrValAspSerArgGluAlaAlaLeuLy{uSer 778 GAAATTATGAAGAATTGTGTGCTATACGTAGATTCTCGTGAGGCAGCCCTCAAGGAGTCT 1. of gray 259 GlyAspValIleLeuSerGlyAlaGluIlePheAlaGluLeuGlyluValVaL ly FIG. (A) Sequence kangaroo 838 GGAGATGTCATACTGTCTGGGGCTGAGATCTTTGCAGAGCTGGGAGAAGTGGTGAAGGGA A-crystallin. Deduced amino acid se- quence is shown above cDNA sequence. 279 ValLysProAlaHisArgGluLysThrThrValPheLysSerLeuGlyMetAlaValGlu Peptides sequenced previously (5) are were 898 GTGAAGCCAGCCCACCGTGAGAAAACTACAGTGTTCAAATCCTTAGGAATGGCGGTGGAA boxed. Some peptides that sequenced as mixtures are slightly reinterpreted. (B) Primer-extension analysis. Two different 299 AspAlaValAlaAlaLy4euValTyrAspSerTrpSerSerGlyLy*fnd primers were used to extend on a template 958 GATGCAGTTGCTGCCAAACTGGTCTATGATTCTTGGTCATCTGGTAAATGAAGCCAAGGA of kangaroo lens total RNA. A single ini- 1018 AGTAGCATTGAAATGCTTCAACATTAAGACCTATTATTGCTAGTCATCTCCCATAGTTTC tiation site was identified by both primers. 1078 TCAGGGGAGAATACTCTTTTGTACTTAGTATCATCTGCCTCAAAAACTAAACTGTTTGTG Lanes: 1 and 4, sequence ladders; 2 and 3, 1138 GTTTAAAAAAAAAAAAATGACAGTATTGTGTATTTCCTTTCCTCTATCTTATCATAGTAT extended products.
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