Extensive Intragenic Sequence Homology in Two Distinct Rat Lens Y

Proc. NatL Acad. Sci. USA Vol. 79, pp. 6876-6880, November 1982 Biochemistry

Extensive intragenic sequence homology in two distinct rat lens y-crystallin cDNAs suggests duplications of a primordial gene (cDNA cloning/DNA sequence determination/gene generation) ROB J. M. MOORMANN*, JOHAN T. DEN DUNNEN*, HANS BLOEMENDALt, AND JOHN G. G. SCHOENMAKERS* Departments of *Molecular Biology and tBiochemistry, University of Nijmegen, Nijmegen, 6525 ED, The Netherlands Communicated by L. N. M. Duysens, August 17, 1982 ABSTRACT The nucleotide sequences of two different rat formation and intermolecular interactions of the crystallins in lens y-crystallin cDNA clones, pRLy2 and pRLy3, have been the lens fiber cells in situ. However, it has been established that determined. pRLy3 contains the complete coding information for increased light scattering ofsenile nuclear cataract is associated a y-crystallin of 173 amino acids whereas pRLy2 is incomplete in with denaturation, chemical crosslinking including disulfide that it lacks the codons for the first three amino acids ofa separate bridges, and aggregation of crystallins (15). but very homologous y-crystallin of identical length. Both rat y- Recently, the three-dimensional structure of a lens protein, crystallins are homologous to the known amino acid sequence of bovine y-crystallin II, has been determined by Blundell et aL bovine y-crystallin II which is only a single amino acid longer. The (16). It turned out that the sulfhydryl groups in the highly sym- length of the region downstream the coding sequence to the A-A- 3 T-A-A-A polyadenylylation signal sequence is 40 nucleotides in metric two-domain structure of this protein were found in each clone. In pRLy3 the poly(A) signal sequence is followed at positions that would allow both intra- and intermolecular cross- 14 nucleotides by a remnant ofthe poly(A) tail which indicates that linking. Disulfide bridges can be formed through some sulfhy- this clone contains a complete 3' noncoding region. pRLy2 has dryls without destabilization or denaturation ofthe protein, but only seven nucleotides following this signal sequence and no denaturation would certainly result if the sulfhydryls that are poly(A) tail, suggesting an incomplete 3' end. The cDNA clones buried in hydrophobic regions were covalently crosslinked. show an overall nucleotide sequence homology of85%. The mutual Moreover, a large decrease in the y-crystallin content of the homology at the amino acid level is 73% whereas their amino acid lens has been demonstrated in hereditary cataracts (see ref. 7 homology with bovine y-crystallin H is about 70%. The nucleotide and citations therein). sequence ofeach clone also reveals a high intragenic homology and These findings and the observations that the y-crystallins are seems to be duplicated in itself. We suggest that the y-crystallin expressed exclusively in the cortical fibers (17) in which they genes have arisen by multiple duplications of a primordial gene are synthesized at a remarkably decreased rate upon aging (18, which consisted of about 120 nucleotides. 19) prompted us to a more detailed analysis of the y-crystallin genes as a first step toward a better understanding of their The transparency and high refractive index of the normal eye expression in normal and disordered lens. Few studies have lens necessary for focusing visible light on the retina is achieved dealtwith crystallin genes (20-22). We previously described the not only by a regular arrangement ofthe lens fiber cells during cloning and characterization ofdouble-stranded DNA copies of growth of the lenticular body but also by the extremely high various a-, ,(, and y-crystallin-specific rat lens mRNAs (11) and concentration and the supramolecular organization of the lens- determined the nucleotide sequence of most ofthe mRNA en- specific proteins, the crystallins, within each fiber cell. In the coding the aA2-crystallin chain (23). In this study we describe mammalian lens, three major classes of crystallins are distin- the nucleotide sequence of two mRNAs coding for two highly guished: a, B, and 'y. The largest oligomeric protein, a-crys- homologous rat y-crystallins. tallin, is composed of two primary gene products aA2 and aB2 of Mr 20,000 which, together with their deamidation products MATERIALS AND METHODS aA1 and aB1, associate to aggregates with an average Mr of Enzymes and Chemicals. All restriction endonucleases used 800,000 (1-3). The ,B-crystallins are an even more heteroge- were purchased from either Boehringer Mannheim or Bethesda neous class of oligomeric proteins; at least five distinct poly- Research Laboratories. Polynucleotide kinase was from P-L peptides are involved in their association and the Mrs range Biochemicals. Calf intestine alkaline phosphatase and Esche- from 20,000 to 32,000 (4, 5). The y-crystallins, which contribute richia coli DNA polymerase I (large fragment) were from Boeh- about 40% of the total protein mass in rat and mouse lens (6, ringer Mannheim. [y-32P]ATP (3,000 Ci/mmol; 1 Ci = 3.7 X 7), are monomeric and have a Mr of 20,000 (8-10). At least 101 becquerels) and [a-32P]dCTP (3,000 Ci/mmol) were from five closely related y-crystallins have been identified in the Amersham. The synthetic oligonucleotide primer 5' d(G-T-A- bovine (8) and rat lens (11). A-A-A-C-G-A-C-G-G-C-C-A-G-T-G)3' which is complemen- The y-crystallins are rich in cysteine compared with the other tary to 18 bases directly preceding the region containing mul- water-soluble lens proteins a- and -crystallins (10, 12). The tiple restriction sites in M13mp7 (24) was constructed by J. van most prominent species in the bovine lens, 'y-crystallin II, has Boom and co-workers (Department ofOrganic Chemistry, Lei- six free sulfhydryl groups (9). Although it was shown in the ro- den University). dent lens that, with aging, there is an almost complete oxidation DNA Sequence Analysis. The isolation ofrecombinant DNA (SH to S-S) oflens protein (13, 14), little is known of the con- and the determination of nucleotide sequences by the method of Maxam and Gilbert (25) were carried out essentially as de- The publication costs ofthis article were defrayed in part by page charge scribed (23). In some cases, ambiguities in the nucleotide se- payment. This article must therefore be hereby marked "advertise- quence ofpRLy3 could not clearly be solved by this way ofse- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. quence determination. Therefore, Pst I fragments ofthis clone 6876 Biochemistry: Moormann et al. Proc. Natl. Acad. Sci. USA 79 (1982) 6877 were inserted in Pst I-linearized, alkaline phosphatase-treated that, compared with pRLy3, pRLy2 lacks three amino acid M13mp7 (24) and transfected in E. coli JM103 (24). Single- residues at its NH2 terminus. pRLy3 most probably comprises stranded DNA from colorless plaques was subsequently ana- the complete coding information contained in the y-crystallin lyzed by the chain-termination method of DNA sequence de- mRNA. This is deduced from comparison with the known amino termination as described by Sanger et al. (26) with the 18-base acid sequence of y-crystallin II from bovine origin (9, 16). The oligonucleotide mentioned above as primer. sequence of this protein starts with a glycine and is completely homologous with the first 13 amino acids of pRLy3 (Fig. 2). In RESULTS contrast to earlier findings by Zigman et al. (27) who reported alanine as the NH2 terminus, Wagner and Fu (12) showed by After restriction enzyme analysis and positive hybridization se- Edman degradation analysis that rat crystallins only have gly- lection, the cDNA clones pRLy2 and pRLy3 and another one cine at their NH2 terminus. In line with this is our finding of designated pRLyl were selected as appropriate candidates for T-G preceding the NH2-terminal glycine codon in pRLy3. full-length transcripts (650-750 base pairs) of the 10S mRNA From extended analysis of genomic clones, which will be pre- class in which the y-crystallin mRNAs are dominantly present sented elsewhere, it turned out that in pRLy3 the A of the in- (11). Refinement of the physical maps and determination ofthe itiator ATG codon is missing. nucleotide sequences indicated that pRLyl and pRLy2 were Comparison of the two rat clones at the amino acid level in- identical except for the 5' and 3' termini which were slightly dicates that 60% of the changes in amino acid sequences were shorter in pRLyl (data not shown). due to two or more nucleotide changes in a single codon. More Fig. 1 shows the physical maps of pRLy2 and pRLy3. Al- or less nonconservative changes are apparent at amino acid po- though these clones had several restriction cleavage sites in sition 15 (Ser-His), 22 (Cys-His), 23 (Pro-Ser), 27 (Thr-Pro), 53 common at identical positions (only those used in determining (His-Cys), 100 (Val-Gln), 109 (Ser-Pro), 10 (Cys-His), 148 (Gln- the DNA sequence are indicated), pRLy3 was distinctly dif- Gly), and 168 (Ser-Arg). The mutual homology at the amino acid ferent from pRLy2 in that the former had one extra Pst I site level between the two clones is 73%. The amino acid homology and two extra Hinfl sites. with bovine y-crystallin II is 66% for pRLy2 and 70% for It should be noted that a positive hybridization assay cannot pRLy3. The bovine y-crystallin II has one extra amino acid discriminate between these clones because the same set of y- compared to the two rat y-crystallins. crystallins as translation products show up with each clone (11). Another interesting feature is the high content of cysteine From this we inferred that pRLy2 and pRLy3 are different but residues in the y-crystallins. Although there is no evidence for highly homologous y-crystallin-specific cDNA clones. This as- disulfide bridges in the mature protein (10), it is known that the sumption is clearly sustained by the nucleotide sequences of location of these residues is often highly conserved in protein. the cDNAs (Fig. 2). At this level the clones show an overall Ifthe NH2-terminal glycine ofpRLy3 is taken as the first amino homology of85%. However, the homology varies in subregions; acid, Cys residues 18, 32, and 108 are at the same positions in up to nucleotide 250 it is 90%, from nucleotide 250 to 340 it pRLy2, pRLy3, and bovine crystallin II as well and Cys-41 is diminishes to 60%, and from nucleotide 340 to 580 it increases the same only in the two rat clones. pRLy2 has additional non- again to 80%. shared cysteine residues at positions 22, 110, and 129 whereas The nucleotide sequences of pRLy2 and pRLy3 contain pRLy3 has an extra cysteine at position 53. Bovine crystallin open reading frames encoding polypeptides of 170 and 173 II has two additional cysteine residues at positions 15 and 23. amino acids, respectively. The amino acid sequences indicate It has been shown (13, 14) that, during the process of aging of the rat lens, almost all soluble protein SH is oxidized to S-S. Because the y-crystallins are rich in cysteines compared to a-

9 C a > a - t- > and ,B-crystallin (10, 12, 28), it is mainly these proteins that Is < a<< I I I should undergo inter- and intramolecular crosslinking during I T0- .3 this process. In the 3' noncoding regions in the rat cDNA clones we find a stretch of 14 A residues at 22 nucleotides after the A-A-T-A- pRLy-2 631 3362 A-A polyadenylylation signal sequence (29) in pRLy3. The poly(A) stretch being a remnant of the oligonucleotide primer used in the synthesis ofthe cDNA indicates that this clone contains the complete 3' end ofthe mRNA. No poly(A) stretch was found in pRLy2. In this clone the poly(dC) tail used in cloning started 11 nucleotides after the polyadenylylation signal sequence. In view of this and the high degree of homology between pRLy2 and pRLy3, we anticipate that approximately 10-15 nucleotides ofthe mRNA are missing at the 3' end ofthis clone. The homology between the short (65 nucleotides for pRLy3) 3' noncoding regions is 75%, which is only slightly 0.1 kb lower than the overall homology between these clones. This feature is in agreement with the retained homology in the short FIG. 1. Restriction map of pRLy2 and pRLy3, showing the restriction sites used for sequence analysis. The thin horizontal arrows 3' noncoding regions of 52 and 53 nucleotides, respectively, of indicate the orientation and approximate length of sequences obtained two rat preproinsulin genes (30) but contrasts with findings in by the chemical degradation method (25); the thick arrows indicate other families ofclosely related genes like the human leukocyte orientation and length of sequences deduced by the dideoxynucleotide interferons (31) and several human (32) and mouse P-globin-like method (26). The cleavage coordinates of the Hinfl sites flanking the genes (33) in which the 3' noncoding regions diverge more rap- Pst I site of pBR322 are indicated. Open boxes, y-crystallin mRNA idly than the protein coding regions. sequences; hatched boxes, poly(dG-dC) tracks; black box in pRLy3, remnant of the poly(A) track of the mRNA template used in cDNA Experiments involving in vitro translation of mRNAs with synthesis; asterisk, a methylated Ava II site. large 3' deletions suggest that intact 3' noncoding regions are 6878 Biochemistry: Moormann et aL Proc. Natd. Acad. Sci. USA 79 (1982)

20 4I0 60 a0 pRL-y2 C ACC TTC TAC GAG GAC CGA GGC TTC CAG GGC CGC AGC TAT GAG TGC AGC AGC GAC TGC CCC MAC CTG CAG ACC ** ** ** * * pRL-y3 TG GGG MG ATC ACC TTC TAT GAG GAC CGC GGC TTC CAG GGC CGC CAC TAT GAG TGC AGC ACA GAC CAC TCC AAC CTG CAG CCC rat y2 Thr Phe Tyr Giu Asp Arg Giy Phe Gin Giy Arg Ser Tyr Giu Cys Ser Ser Asp Cys Pro Asn Leu GinLThr rat y3 Gly Lys Ile Thr Phe yr .Giu. Asp Arg Gly Phe Gin Giy Arg His Tyr Giu Cys Ser ThAsp His Ser Asn Leu Gin Pro calf yII IGiy Lys Ile Thr Phe Tyr Giu Asp Arg Gly Phe Gin Giy s Cys TAsn Asn Cys Leu GI 20

100 120 140 160 TAC TTC AGC CGC TGC AAC TCC.GTC CGC GTG GAC AGT GGC TGC TGG ATG CTC TAT GAG CGC CCC MC TAC CAG GGC CAC CAG TAC TTC CTG * * ** * *** ** * * TAC TTC AGC CGC TGC AAC TCT GTG CGC GTG GAC AGT GGC TGC TGG ATG CTC TAT GAG CAG CCC MC TTC ACA GGC TGC CAG TAT TTC CTT Tyr Phe Ser Arg Cys Asn Ser Vai Arg Vai Asp Ser Giy Cys Trp Met Leu Iyr Giu Ar Pro Asn YrGin Giy His Gin Tyr Phe Leu Tyr Phe Ser Arg Cys Asn Ser Vai Arg Val Asp Ser Giy Cys Trp Met Leu Tyr Giu [GjnPro Asn Phe Thr Giy C Gn Tyr Phe Leu Tyr Phe Ser Arg Cys Asn SerlieeArg Vai Vai His Ser arAspT Arg y His Gin TyrFMetLLeu 4 0

180 200 . 220 240 260 CGA CGC GGG GAC TAC CCT GAC TAC CAG CAG TGG ATG GGT TTC AGC GAC TCC ATT CGC TCC TGC CGC CTC ATC CCC CGT ACA GGT TCC * * ** *** * ** * CGT CGC GGG GAC TAC CCT GAC TAC CAG CAG TGG ATG GGT TTC AGC GAC TCT GTC CGC TCC TGC CGC CTC ATC CCC CAC TCC AGC TCT Arg Arg Giy Asp Tyr Pro Asp Tyr Gln Gin Trp Met Giy Phe Ser Asp Ser Ile Arg Ser Cys Arg Leu Ile Pro Arg[FT| Gly Ser Arg Arg Giy Asp Tyr Pro Asp Tyr Gin Gin Trp Met Giy Phe Ser Asp SerrVa-iArg Ser Cys Arg Leu Ile Pro His Ser Ser &r Met G As ArCLeu le Pro Gin His Thr 6 0 a80

280 300 320 340 CAG AGG ATG CGT CTG TAT GAG AAA G.M GAT CAC AAA GGC GTC ATG ATG GAG CTG AGT G.M GAC TGC TCC TGC ATC CAG GAC CGC TTC CAC * * ** * ** * ** * * * * *** * * * **. * * ** * * CAC AGA ATC AGG ATC TAC GAG CGA GAG GAC TAC AGA GGC CAG ATG GTG GAG ATC ACA GAC GAC TGC CCC CAC CTG CAG GAC CGC TTC CAC Gin Arg Met ArgLLeu Tyr Giu L Gu AspHis MseVat Met G GiuAsp Cys Ser C~ys Ile Gin Asp Arg Phe His His Arg eArg Ile Tyr Giu Arg Giu Asp Tyr G Met Vai Giu Ie Thr Asp Asp Cys Pro His Leu Gin Asp Arg Phe His Phe Arg Met Arg Ile Tyr Giu ArgspAsp PheArg Gin Met Ser Glu Ile Thr Asp Asp Cys Pro Ser Leu Gin Asp Arg Phe His

360 380 400 420 440 CTC AGT GAG GTG CGC TCG CTG CAC GTG CTA GAG GGC TGC TGG GTC CTC TAT GAG ATG.CCT MC TAC CGA GGC CGG CAG TAT CTG CTG AGG * * *.* * * ** * * *. * * * * TTC AGT GAC TTC CAC TCT TTC CAC GTG ATG GAG GGC TAC TGG GTC CTC TAT GAG ATG CCC MC TAC CGG GGG CGG CAG TAC CTG CTG AGG Leu Ser Glu VaZ|Arg Ser Leu His Val Leu Giu Giy Cys Trp Vai Leu Tyr Giu Met Pro Asn Tyr Arg GZy Arg Gin Tyr Leu Leu Arg Phe Ser Asp Phe His Ser Phe His Vai Met Giu Giy Tyr Trp Vai Leu Tyr Giu Met Pro Asn Tyr Arg Giy Arg Gin Tyr Leu Leu Arg Leu fShr;GlualAsn Ser Vai Arg Vai Leu Giu Giy Ser Trp VaIIle Tyr Giu Met Pro SerTyr Arg Giy Arg Gin Tyr Leu Leu Arg 120 140

460 480 500 520 CCT CAA GAG.TAC CGG CGC TAC CAC GAC TGG GGC GCT GTA GAT GCT MAG GCA GGC TCT TTG CGG AGC GC6 GTA GAT TTA TAC:TAA MAT AGG ** * * * * ** * * * * * **** * * * *:.*.* ** CCT GGA GAM TAC AGG'CGC TAC CAC GAC TGG GGC GCC ATG MT GCC AGG GTA GGC TCT CTG AGG AGA ATC ATG GAT TTC TAT:TGAj MAT ATT Pro Gin Giu Tyr Arq Arg Tyr His Asp Trp GI' AiagVai AsryAarLs Aa Giy Ser Leu Arg Ser Aia Vai AspLe Tyr Pro GZy Giu Tyr Arg Arg Tyr His Asp Trp Giy Aia Met Asn Aia7 Val Giy Ser Leu Arg Arg. Ile Met Asp Phe Tyr Pro Giy Giu Tyr Arg Arg TyreAsp Trp Giy Aia Met Asn Aia Lys Vai Giy Ser Leu Arg Arg Vai Met Asp Phe Tyr 1 6 0 173

540 560 580 603 TGA ACG CTA CCA TTT TCT CAT TTT GGA ACC AGT ATT TAC GAIC 1'_- ** * ** * *** TMTAX:...... :.:::.:.:::* * **l 25i TTT ACT CTA CCA TTT TCT CCA TCT GGA CAT T.AA,,,..AA,,kAT ATT TCC TGT GTG TTT CAT GCAAAAAAAAAAAAAAIC7

FIG. 2. Nucleotide sequence of the rat lens y-crystallin cDNA inserts in pRLy2 and pRLy3 andthe deduced amino acid sequence. For comparison, the amino acid sequence of bovine y-crystallin II (9) as modified by Blundell et al. (16) is also given. Stippled area, nucleotide sequence involved in poly(A) addition; asterisks, differences in the nucleotide sequence between the rat y-crystallin cDNAs; boxes, regions with identical amino acid sequences in both rat y-crystallins and bovine y-crystallin II. not needed for efficient translation of the protein encoding re- DISCUSSION gions in the mRNA (34, 35). Furthermore, we found that the Close analysis of the two. y-crystallin cDNA sequences shows aA2-crystallin mRNA has a 3' noncoding region of 520 nucleo- that they have an axis of symmetry which divides them into tides which almost equals the length ofthe coding region ofthis NH2- and COOH-terminal halves. A two-by-two arrangement mRNA (23). However, no specific function could be assigned of these halves (Fig. 3) yields regions a-a' and b-b'; the latter to this large noncoding region. These findings suggest that the reveals the higher homology. In order to get a higher degree length and the precise sequence of much of the 3' noncoding of homology between the a and a' regions of pRLy2 and regions of these eukaryotic mRNAs are not essential for func- pRLy3, a six-nucleotide deletion was introduced following the tion. It remains to be seen, however, whether the 3' noncoding C at position 83 in the a region of both cDNA sequences. For regions in the other not yet analyzed y-crystallin genes are as the same reason, a trinucleotide was deleted at position 311 in conserved as those in pRLy2 and pRLy3. If so, this might re- the a' regions of pRLy2 and pRLy3. However, this needs to flect a functional importance of these sequences in the y-crys- be compensated for six nucleotides upstream in thecorrespond- tallin multigene family. ing a region by deletion ofa trinucleotide behind the nucleotide Biochemistry: Moormann et al. Proc. Natl. Acad. Sci. USA 79 (1982) 6879

a ACC TTC TAC GAG GAC CGA GGC TTC CAG GGC CGC AGC TAT TGC AGC AGC GAC TGC CC CTG CAG ACC TAC TTC AGC CGC TGC AAC TCC GTC CGC GTG GAC AGT C * ** *** * * * * * *** * * GAG** ** ***** MC**** *** ***** ***** pRL-y2 a CAG AGG ATG CGT CTG TAT GAG AAA GAA GAT CAC AAA GGC GTC ATG ATG GAG CTG AGT GAA GAC TGC TCC TGC ATC CAG GAC CGC TTC CAC CTC AGT GAG GTG CGC TCG CTG CAC GTG CTA GAG I...... Z1...... Thr Phe Tyr Giu Asp Arg GCy Phe Gtn GZy Arg Sir Tyr Gb Cys Ser-Ser Amp Cvi Pro Amn Liu GtIn Thr TyrPPhe Ser Arg Cys Asn Ser Vi Arg Val Asp Ser GIn Arg Met Arg Lois Pis Gtu Lys Giu Asp His Lys GZy Val Met MitGlu LiS Sw Gbu Asp Cys Sir Li Cn Asp Arg Phe HisL| GCu Val A Lou Hi Val Leu Glu 87 ...... 107 . 127 Gt Arg Gin Asp Tyr A GI Gtn Aret Vi GI Ile Ths Asp As Ps Hi Gtn Asp As-pg Phe His Ph Asp P:e H Ph Lys Ile Thr Phe G Asp GZy Phe G His Tyr Ser A Sw Pro Arg Cys Asn Va Asp Sw GCy Arg G Gb Cys Thr H i nLm u Ph Ser Ser 1Pr _tu tn ...... ty A SJSP r uCn Pyr s 39~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... t a' CAC AGA ATC AGG ATC TAC GAG CGA GAG GAC TAC AGA GGC CAG ATG GTG GAG ATC ACA GAC GAC TGC CCC CAC CTG CAG GAC CGC TTC CAC TTC AGT GAC TTC CAC TCT TTC CAC GTG ATG GAG * *** *** * * * * * *** * *** * * *** *** * ** ** *** *** * ** *** ** * * * ** *** * * * *** PRL-Y3 a GGG MG ATC ACC TIC TAT GAG GAC CGC GGC TTC CAG GGC CGC CAC TAT GAG TGC AGC ACA GAC CAC TCC MC CTG CAG CCC TAC TTC AGC CGC TGCGMC TCT GTG CGC GTG GAC AGT

b GGC TGC TGG ATG CTC TAT GAG CGC CCC MC TAC CAG GGC CAC CAG TAC TTC CTG CGA CGC GGG GAC TAC CCT GAC TAC CAG CAG TGG ATG GGT TTC AGC GAC TCC ATT CGC TCC TGC CGC CTC ATC CCC b GGC TGC TGG GIC CGC TAT GAG ATG CCT MC TAC CGA GGC CGG CAG TAT CTG CTG AGG CCT CAA GAG TAC CGG CGC TAC CAC GAC TGG GGC GCT GTA GAT GCT MG GCA GGC TCT TTG CGG AGC GCG GTA

40 40 CS r W Lo .. d Pro Ai r Gin His Gln Ph* Le r As Pro Am Gin G~n t PeS s e l r e Tyr':::::::::::. Arg Ty ...... Tyr ArgGP 61]82Tyr _~ Tp Gly ...... ,. Gty Cyi Trp Vat Liu Tyr Gtu Met Pro Asn TyPr Asrg Gy Arg Gsn syr Leu Leu Arg Pro Gin Gtu Pyir Arg Arg Pr His Asp TSp Gty Ata Vii ASp Ata Lys Ab Gty Sep Leu Args Sr Ab Vol

40 ...... s . L 61 ;; L 82

GG TA pRL-y3 b' TG GG GIGCT TAT GAG ATG CCC MG TAG CGG GGG CGG GAG TAG GIG GIG AGG CCI GGA GM TAG AGG CGGC TAG GAG GAG TGG GGC GGC AIG MT GCC AGG GTA GGC TCT GIG AGG AGA ATC ATG Yb GGC TGG TGG A;G TAT GAG GAG CCC MC TIC ACA GGC TGC GAG TAT TIC CTT CGT CGC GGG GAG TAG CCI GAG TAG GAG GAG ;GG AIG GGT TIC AGC GAG ICT GIG CGC ICC TGC CGC A;C CCC

4 120 249 261 384 513 603 4 I LEW41 I I F9 I...I ~lLXL - a b a'

FIG. 3. Intragenic nucleotide sequence homology in the rat lens y-crystallin genes. The nucleotide sequence of pRLy2 and pRLy3 has been divided into subregions (a, a', b, and b') as indicated below the sequence. Arabic numerals represent the nucleotide positions as given in Fig. 2. Region 249-261 represents the "connecting peptide" (see text). The subregions and their corresponding amino acid sequences have been arranged in such a way that a mutual comparison can be made between a and a' (or b and b') of either pRLy2 or pRL-y3 and also between these subregions in both y-crystallin sequences. Nucleotide sequence homology in each subregion is indicated by asterisks. Amino acids in each rat y-crystallin sequence that differ from the bovine y-crystallin II amino acid sequence (cf. Fig. 2) are given in boldface. Boxed areas, amino acid residues that are identical in a and a' (or b and b') of both rat y-crystallins; stippled areas, amino acid residues located at topologically equivalent positions in all subregions of rat y-crystallin and bovine y-crystallin II as well. at position 62. It is possible to increase homology by one base and b' as indicated in Fig. 3. The two domains are formed by in this small region of pRLy3 by placing the deletion in the a a + b and a' + b' and are connected at b and a' by a 12-nu- stretch six nucleotides upstream from where it is placed now. cleotide region, nucleotide positions 249-260, which codes for We prefer the former sequence alignment because the other the so-called connecting peptide. way of matching would introduce a deleted amino acid in the The backbone of the intramotif structure in the protein is a strain of pRLy3 which would be two positions further than mainly formed by the four residues mentioned above. The chain the deletion in pRLy2. This, in turn, should imply that the tri- folds between Glu-7 and Gly-13. This allows Tyr-6 to pack nucleotide deletions at these positions in the two genes would against Phe-il which is only nonconservatively varied to His have occurred after duplication of the ancestral 'y-crystallin in subregion a' of pRLy2. The other conserved residue is Ser- gene. We favor, however, the idea that the deletions occurred 34 which stabilizes the structure by hydrogen bonding to Phe- as indicated and before duplication of the ancestral gene. 11 (or to a topologically equivalent residue). The fifth residue The overall degree of homology, as deduced from Fig. 3, which participates in this unusual foldover feature is Arg-36 between regions a and a' is 44% for pRLy2 and 52% for pRLy3. which, however, is rather conservatively varied to His in subre- This is lower than the overall homology between regions b and gion a' of pRLy2 and pRLy3. The deletion of six nucleotides b', 55% for pRLy2 and 56% for pRLy3. Because the deletions introduced in subregion a of pRLy2 and pRL'y3 corresponds introduced in regions a and a' only enhance the mutual ho- with hypothetical amino acids located at a position in the protein mology between these regions, the actual homology difference where Blundell et al (16) deleted three amino acids in bovine between a/a' and b/b' is even higher and mainly due to the y-crystallin II. The amino acid deletions at position 18 and 21 extremely high homology between the first 13 codons in regions are located just before and at the end ofthe second turn in motif b and b'. I and motif III. It is conceivable that two deletions have been The four residues tyrosine, glutamate, glycine, and serine are introduced here because ofthe fact that deletion ofonly a single conserved at equivalent positions in all the subregions of the amino acid residue would have had such an impact on the struc- two cDNA clones (shaded boxed areas in Fig. 3). From x-ray ture in this region that a second nearby deletion was necessary. analysis at 2.6- resolution, Blundell et al. (16) deduced that Furthermore, motif II and motif IV (subregions b and b') in bovine y-crystallin II shows the highest degree of intrachain both rat y-crystallins are four residues larger than motif I symmetry ofany protein whose three-dimensional structure has (subregion a) which is exactly the same as in bovine y-crystallin been elucidated so far. The protein has a two-domain 3-struc- II. The contacts between the two domains are largely hydro- ture folded into four remarkably similar Greek key motifs. phobic. The residues involved in packing the two domains to- These motifs correspond exactly with the subregions a, b, a', gether are methionine and valine at positions 43 and 131, re- 6880 Biochemistry: Moormann et al. Proc. Natl. Acad. Sci. USA 79 (1982)

spectively, and phenylalanine and leucine at positions 56 and 5. Siezen, R. J., Bindels, J. G. & Hoenders, H. J. (1978) Eur. J. 144. It is interesting to note that methionine and phenylalanine Biochem. 91, 387-396. are in the reversed orientation in bovine y-crystallin II (cf. Fig. 6. Ochen, P. R., Fu, S. C. J., Hart, R., White, J. H., Wagner, B. J. & Lewis, K. F. (1977) Exp. Eye Res. 24, 355-367. 2). 7. Wada, E., Sugiura, T., Nakamura, H. & Tsumita, T. (1981) The connecting peptide which forms the covalent linkage Biochim. Biophys. Acta 667, 251.-259. between the two domains consists offour residues (positions 83- 8. Bj6rk, I. (1970) Exp. Eye Res. 9, 152-157. 86) which are conservatively varied among rat and calf. How- 9. Croft, L. R. (1972) Biochem. J. 128, 961-970. ever, there is a clear difference when the residues, directly 10. Slingsby, C. & Croft, L. R. (1978) Exp. Eye Res. 26, 291-304. flanking the connecting peptide are considered. The glutamine 11. Dodemont, H. J., Andreoli, P. M., Moormann, R. J. M., Ra- maekers, F. C. S., Schoenmakers, J. G. G. & Bloemendal, H. preceding the connecting peptide in bovine y-crystallin II has (1981) Proc. Natl. Acad. Sci. USA 78, 5320-5324. been deleted in both rat y-crystallins whereas the phenylala- 12. Wagner, B. J. & Fu, S. C. J. (1978) Exp. Eye Res. 26, 255-265. nine following the connecting peptide is rather nonconserva- 13. East, E. J., Chang, R. C., Yu, N. T. & Kuck, J. F. R. (1978) J. tively varied to histidine or glutamine in pRLy3 and pRLy2, Biol Chem. 253, 1436-1441. respectively. How far this feature interferes with interdomain 14. Askren, C. C., Yu, N. T. & Kuck, J. F. R. (1979) Exp. Eye Res. linkage is hard to say. Because these amino acids are next to the 29, 647-654. 15. Harding, J. (1981) in Molecular and Cellular Biology of the Eye connecting peptide which is at the surface of the molecule, it Lens, ed. Bloemendal, H. (Wiley, New York), pp. 327-365. is feasible that there should be some space for amino acid vari- 16. Blundell, T., Lindley, P., Miller, L., Moss, D., Slingsby, C., ation without dramatic effects on tertiary structure. It is also Tickle, I., Turnell, B. & Wistow, G. (1981) Nature (London) 289, conceivable that the reversion ofphenylalanine and methionine 771-777. has taken place to accommodate small three-dimensional struc- 17. Papaconstantinou, J. (1965) Biochim. Biophys. Acta 107, 81-90. tural alterations that could have taken place. 18. Slingsby, C. & Croft, L. R. (1973) Exp. Eye Res. 17, 369-376. 19. Bours, J., Doepfmer, K. & Hockwin, O. (1976) Doc. Ophthalmol. From the results presented in this study it is clear that the Proc. Ser. 8, 75. y-crystallins are well conserved among calfand rat at the struc- 20. Bhat, S. P., Jones, R. E., Sullivan, M. A. & Piatigorsky, J. (1980) tural level. Moreover, the structure of the cDNA sequences Nature (London) 284, 234-238. suggests that the primordial y-crystallin gene consisted ofonly 21. Tomarev, M., Dolgilevich, S. M., Kozlov, K. A., Zinovieva, R, one subdomain-say a. Duplication ofthis subregion then gen- D., Dzhumagaliev, E. B., Kogan, G. L., Skobelev, N. A., Mik- erated a domain-like gene which, by tandem duplication, hailov, A. T., Frovola, L. & Gause, G. G. (1982) Gene 17, 131- 138. evolved into the mature y-crystallin gene described above. In 22. King, C. R., Shinohara, T. & Piatigorsky, J. (1982) Science 215, this respect, y-crystallin is not unique and seems to resemble 985-987. well-characterized genes-for instance, the chicken ovomucoid 23. Moormann, R. J. M., van der Velden, H. M. W., Dodemont, H. gene, which has three domains showing 40-60% of homology J., Andreoli, P. M., Bloemendal, H. & Schoenmakers, J. G. G. at the nucleotide level (36), or the chicken collagen gene for (1981) Nucleic Acids Res. 9, 4813-4822. which it has been shown that the ancestral gene might have 24. Messing, J. M., Crea, R. & Seeburg, P. H. (1981) Nucleic Acids Res. 9, 309-321. arisen by multiple duplication events of a single genetic unit 25. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499- consisting of only 54 base pairs (37). 560. The occurrence ofdifferent nonallelic y-crystallin genes was 26. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. suggested in 1978 (10). Our recent data on restricted genomic Sci. USA 74, 5463-5467. DNAand subsequent molecular cloning demonstrate that there 27. Zigman, S., Schultz, J. & Yulo, T. (1970) Biochim. Biophys. Acta are at least five genes in rat. This indicates 221, 576-582. y-crystallin that, 28. Croft, L. R. (1980) Proc. Int. Soc. Eye Res. 1, 118 (abstr.). besides the intragenic type of duplication, single or tandem 29. Proudfoot, N. J. & Brownlee, G. G. (1976) Nature (London) 263, duplications of mature y-crystallin sequences also have taken 211-214. place more recently to generate a multigene family. 30. Lomecido, P., Rosenthal, N., Efstratiadis, A., Gilbert, W., Ko- A considerable increase in the resolution to 1.9 A has mean- lodner, R. & Tizard, R. (1979) Cell 18, 545-558. while been achieved in x-ray studies (T. Blundell and C. 31. Goeddel, D. V., Leung, D. W., Dull, T. J., Gross, M., Lawn, Slingsby, personal communication). This enabled them to refine R. M., McCandliss, R., Seeburg, P. H., Ulrich, A., Yelverton, E. & Gray, P. W. (1981) Nature (London) 290, 20-26. further the previously reported (9, 16) calf yII-crystallin amino 32. Efstratiadis, A., Posakony, J. W., Maniatis, T., Lawn, R. M., acid sequence. Therefore, the degree of homology between the O'Connell, C., Spritz, A. R., DeRiel, J. K., Forget, B. G., calfand rat y-crystallins as presented in this paper must be con- Weissman, S. M., Slightom, J. R., Blechl, A. E., Smithies, O., sidered as a minimum. Baralle, F. E., Shoulders, C. C. & Proudfoot, N. J. (1980) Cell 21, 653-668. This work was supported by The Netherlands Foundation for Chem- 33. Konkel, D. A., Maizel, J. V. & Leder, P. (1979) Cell 18, 865-873. ical Research (SON) with financial aid from the Netherlands Organi- 34. Kronenberg, H. M., Roberts, B. E. & Efstratiadis, A. (1979) Nu- zation for the Advancement of Pure Research (ZWO). cleic Acids Res. 6, 153-166. 1. Van der Ouderaa, F. J., de Jong, W. W. & Bloemendal, H. 35. Nunberg, J. H., Kaufman, R. J., Chang, A. C. Y., Cohen, S. N. (1973) Eur. J. Biochem. 39, 207-222. & Schminke, R. T. (1980) Cell 19, 355-364. 2. Van der Ouderaa, F. J., de Jong, W. W., Hilderink, A. & Bloe- 36. Stein, J. P., Catterall, J. F., Kristo, P., Means, A. R. & O'Malley, mendal, H. (1974) Eur. J. Biochem. 49, 157-168. B. W. (1980) Cell 21, 681-687. 3. Spector, A. & Katz, E. (1965) J. BioL Chem. 240, 1979 1985. 37. Yamada, Y., Anvedimento, V. 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