Comparative Analysis of the &Crystallin / Quinone Reductase Gene in

Guinea Pig and Mouse

Pedro Gonzalez, * Carlos Herndndez-Calzadilla,? P. Vasantha Rao, *

Ignacio R. Rodriguez, * J. Samuel Zigler, Jr., * and Teresa Borrds$
*Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health;
TDepartamento de Bioquimica y Biologia Molecular, Universidad de La Laguna; and SLaboratorios Cusi

c-Crystallin is a novel nicotinamide adenine dinucleotide phosphate : quinone reductase, present at enzymatic levels
in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and
camelids. We report here the complementary DNA (cDNA) cloning of c-crystallin from liver libraries in guinea
pig ( Cavia porcellus), where I;-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus),
where expression in the lens occurs only at enzymatic levels. A 5’ untranslated sequence different from the one
previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic
clones including the complete guinea pig c-crystallin gene and the 5’ region of this gene in mouse. These results
show the presence of two promoters in the guinea pig L-crystallin gene, one responsible for expression at enzymatic
levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in
the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained
by the acquisition of an alternative lens-specific promoter.

Introduction
Crystallins are water-soluble proteins present in the recruitment of enzymes as lens crystallins represents
high concentrations in the ocular lens. These include a selective process in which the expression in the lens
the a-, p- and y-crystallins, which are ubiquitously ex- of particular proteins confers some selective advantage
pressed in all vertebrate lenses, and the taxon-specific or whether it is a more neutral process in which the
crystallins, which have limited phylogenetic distribu- functional or structural properties of the protein are not
tions. This latter group is also called “enzyme/crystal- so critical (de Jong et al. 1989).
lins” because it has been demonstrated that they are, in <-Crystallin is a nicotinamide adenine dinucle-
fact, enzymes which are highly expressed in lens where otide phosphate ( NADPH ) : quinone reductase ( Rao
they presumably serve as structural proteins (crystallins) et al. 1992) present at enzymatic levels in various
(for reviews, see de Jong et al. 1989; Piatigorsky and nonlenticular tissues of different species (Rao and
Wistow 1989 ) . Since only one gene is generally respon- Zigler 1992b), which is also a lens crystallin in some
sible for the normal expression at enzymatic levels as hystricomorph rodents (Huang et al. 1987) and cam-
well as for the expression at high levels in the lens, the elids (Garland et al. 199 1)) where it constitutes 6 10%
recruitment of an enzyme as a lens protein requires the of total protein. As in the case of other enzyme/crys-
modification of the pattern of gene expression in the tallins, the acquisition of a new function occurred
lens, without having an important effect on gene expres- without gene duplication (Borras et al. 1990). This
sion in other tissues. This process, whereby the product protein is particularly interesting because a hereditary
of a single gene has two distinct functions in different nuclear cataract in guinea pigs (Cavia porcellus) is
tissues, has been called “gene sharing’: ( Wistow and Pia- associated with a genomic mutation causing a deletion
tigorsky 1987). The mechanism by which such modi- in c-crystallin (Rodriguez et al. 1992), the only re-
fication of expression occurs has not been fully elucidated ported case of a cataract resulting from a mutation in
for any enzyme/crystallin gene. It is uncertain whether an enzyme / crystallin. Moreover, the high expression
of c-crystallin in the lens occurs in two groups of
Key words: guinea pig, mouse, alternative promoter, ocular lens, mammals: hystricomorphs and camelids, which had
molecular evolution. already diverged before the radiation of mammals <75
Address for correspondence and reprints: J. Samuel Zigler, Jr., Mya (Novacek 1992). This peculiar distribution could
National Institutes of Health, Building 6, Room 237, Bethesda, Mary- be explained either by independent recruitment in
land 20892.
each group or by an early recruitment in the evolution
Mol. Biol. Evol. 11(2):305-315. 1994.
of mammals, with subsequent disappearance from the
0 1994 by The University of Chicago. All rights reserved.
0737-4038/94/l 102~0013$02.00 lens in many taxons.

305
306 Gonzalez et al.

Lens cDNA Exon 1 Exon 2 Met

AAGTCTGAACTCTTGCAGCACAGCTCCTCGCTCCTGCAACTGTTTCTGACACTTACACTGCTTGTGTTTGTATCTGCATCACCATG
IIIIIIIIIIIIIIIII
TCTTGCCAAAGTTGAAAACAGCCTGTCCTCTCCCTCTTGCAGTCC~GGGAGCCATAGCAGGTTGTGATCCC~CTCTGGAGGCGTATCTGCATCACCATG
Liver cDNA pCH18 *Liver cDNA pPG103

FIG. l .-5 ’Untranslated sequences of the lens cDNA and liver cDNA clones pCH 18 and pPG 103. The sequences corresponding to exon
2 (common to lens and liver cDNA sequences) are underlined, and the first amino acid of the coding region is shown above its respective
codon. The position of the antisense oligonucleotide used for the primer extension analysis (see Material and Methods) is indicated with an
arrow. The asterisk (* ) denotes the first nucleotide in clone pPG 163.

Here we report the analysis of the structure of the
<-crystallin gene in a species exhibiting high expression
in the lens (guinea pig) and in a second, related species
(laboratory mouse, Mus musculus) with a low level of
lens expression. The gene in guinea pig is regulated by
two promoters, one of which is responsible for the high
expression in the lens. This lens-preferred promoter is
not present in the mouse gene. c-Crystallin is the first
instance in which the recruitment of an enzyme as a
lens crystallin can be attributed to the acquisition by the
gene of a second, lens promoter.
used as full-length probe. Fragments corresponding to
nucleotides 142-446, 553-690, and 1368-1729 of the
insert of clone pTB 100 and to the first 220 nt of clone
pCH 18 (fig. 1) were generated by the PCR and used in
mapping the guinea pig clones.
The inserts of the positive clones were subcloned
in the vector pBluescript II KS+ (Stratagene). The re-
combinant plasmids were sequenced with ( 35S)dATP
by using the Sequenase II system (USB). Computer
analysis was carried out with the GCG package of pro-
grams in the NIH Convex unit.

Material and Methods Primer Extension Analysis of Guinea Pig Liver RNA
Library Screening, Subcloning, and Sequencing For the primer extension analysis, one 36-mer syn-
A hgt 10, oligo dT-primed guinea pig adult liver thetic oligonucleotide ( 5 ‘-CGCCTCCAGAGCAGG-
complementary DNA (cDNA) library was obtained GATCACAACCTGCTATGGCTC-3’) complementary
from Clontech (GL1007a). The probe used for the to the liver cDNA untranslated region was end labeled
screening was the insert of the guinea pig lens c-crystallin with ( 32P)ATP and hybridized with 50 kg of total RNA
cDNA clone pTB 100 (Rodokanaki et al. 1989). A from guinea pig liver previously extracted by the method
hEMBL3 guinea pig genomic library (Clontech; of Chomczynsky and Sacchi ( 1987). The primer exten-
GL1002d) was screened with the insert of the guinea sion was carried out as described by Sambrook et al.
pig lens cDNA clone pTB 100 and with a fragment, gen- ( 1989, vol. 1, pp. 7.79-7.85) using AMV reverse tran-
erated by polymerase chain reaction (PCR), corre- scriptase ( Promega) .
sponding to the first 120 nt in the 5 ’region of pTB 100
PCRs
insert.
The mouse liver cDNA library hgt 11 (~207 1; Pro- In order to amplify fragments of the sizes expected
mega) was screened with the insert of clone pTB100. for some introns, we used the buffer described by Ponce
Two mouse genomic libraries from Clontech ( ML 103Oj and Micol ( 1992). This buffer has been especially for-
and ML1044j ), constructed in hEMBL3, were screened mulated to get amplifications of products ~6 kb in
with a nested PCR fragment corresponding to the first length. The Taq polymerase and deoxynucleotides were
2 10 nt in the 5’ end of mouse liver cDNA clone purchased from Perkin-Elmer.
pMLR107 (fig. 4).
All the probes were labeled by the random primer Rapid Amplification of cDNA Ends (RACE)
method with ( 32P)dCTP. Screenings were performed For the RACE, total RNA from the mouse lens-
using nitrocellulose filters ( Schleicher and Schuell ), with derived cell line aTN4 was used. The first-strand cDNA
hybridizations at 68°C in 6 X saline-sodium citrate synthesis was performed by priming the total RNA with
(SSC), 0.5% sodium dodecyl sulfate (SDS), 0.5% Den- a <-crystallin antisense oligonucleotide ( 5 ‘-CTGATG-
hardt’s with 100 pg Escherichia coli or salmon-sperm GCTCTGTGGAACAG-3 ‘, nucleotides 2 lo- 190 of
DNA ( Sigma)/ml and washes at 68°C in 2 X SSC, clone pMLR 107 ) by using MLV reverse transcriptase
0.1% SDS. (Promega). After dA tailing with TdT enzyme from
The positive clones were analyzed by Southern blot BRL, a run of amplification was performed using a
analysis as described by Sambrook et al. ( 1989, vol. 2, polyT oligo with an EcoRI restriction site ( 5 ‘-GGAC-
pp. 9.3 l-9.57 ) . Hybridizations were carried out under TCGAATTCGACTGCTTTTTTTTTTTTTTT-
the same conditions as for the library screenings. The TT-3’) and an internal c-crystallin antisense oligonucle-
insert of the guinea pig lens cDNA clone pTB 100 was otide ( 5 ‘-AAACTCAAACACTCGAATAG-3 ‘, nucleo-
308 Gonzalez et al.

ATG TGA
A

Liver Lens 2 3 4 5 6 78 9
exon exon

B hPG- 1

hPG-2

UG-7

5 kb
I I

FIG. 3.-A, Genomic organization of the guinea pig t;-crystallin gene. Exons are shown as boxes numerated below. The unblackened boxe
represent untranslated regions, and the blackened boxes represent translated regions. The location of the start codon (ATG) and stop coda
(TGA) are indicated with arrows. B,Extent of the guinea pig genomic clones used for the sequence analysis.

isolated (fig. 3). The sequence corresponding to the 5’
end of the liver cDNA was found in clone 3LPG7 -3 kb
upstream of the lens exon. This exon is followed by a
consensus donor splicing sequence at the 3’ end, but no
evident acceptor splicing sequence is present at the 5’
region. The first translated exon is located - 1 kb down-
stream of the lens first exon. The methionine codon is
preceded by 13 untranslated nucleotides, and the exon
encodes for 36 additional amino acids. This exon is fol-
lowed by seven other exons ranging in size from 52 nt
(exon 5) to 933 nt (exon 9). The last exon, exon 9,
encodes 53 amino acids of the C-terminal region of tb
protein as well as the entire 772 bp of the 3’ noncodin:
region of the cDNA. All of the exon-intron junction
display the minimal consensus sequences GT and AC
(Breathnach and Chambon 1981; Mount 1982) at thl
5’ and 3’ boundaries of every intron (table 1) . The exor
sequences obtained from the genomic clones were iden
tical to the cDNA sequence reported elsewhere (Rodo
kanaki et al. 1989), except for one extra A at positior
2 ( 5 ’untranslated region).
The sequence upstream of the liver exon does no

Table 1
Intron-Exon Junctions of the t;-Crystallin Gene in Guinea Pig

Exon
Length lntron
Exon (bp) 5’Splice Donora Length 3’Splice Acceptora Codon Phas

I (liver) >88 GCGgtaagtggttttaaa 4.3 kb atttcttttccttagTAT . *.

I (lens) . 70 TTGzgagtatgattatt 1.1 kb atttcttttccttGTAT ...
Gin- -Val
II 124 CAGgtggaaatgattact - 1.2kb atgttgtcattcgagGTT 0
Lys- _Lys
III 153 MGgtattacacttttaa -3.8 kb tgatctcttccctagAM 0
Se- -r
IV 164 CAGgtaaaggcacacagg -4.2 kb tttttttttacgcagTGC II
Gly- -Val
v 52 GGAgtaagtatttattta -3.8 kb atatttattttttagGTT 0
Lys- _Lys
VI 150 AAGgtaaaattttctata -2.5 kb ttttttactaaacagAAA 0
Ile- -1le
VII 102 ATCgtaagtaaagttttc 108 bp ttgtttccatgatagATT 0
Lys- -Glu
VIII . 96 AAGgtgagaaaagaaggt 441 bp ttttctttctcttagGAA
- 0
IX . 933 AAAmCTACAGAGC end

a Minimal consensus splice sequences are underlined.

 <-Crystallin Gene in Guinea Pig and Mouse 309

contain TATA or CAAT boxes. The main characteristic 8.10, respectively. The similarity with the guinea pig se-
of this region is its high content of GpC (G+C=6 1% in quence is 80.3% and with the human 80.6%. A detailed
472 nt). The upstream region of the lens exon includes comparative analysis of the amino acid sequences of c-
TATA box, Sp 1, and some putative regulatory elements crystallins, including the mouse sequence reported here,
present in the promoters of other crystallins, including has recently been published (Jiirnvall et al. 1993).
C/EBP (Johnson et al. 1987), aCE2 (Matsuo and Ya-
suda 1992), aCRYBP1 (Nakamura et al. 1990), and a 5 ’Region of c-Crystallin Gene in Mouse
string of T (McDermott et al. 1992). Functional analysis
of this region has demonstrated that it contains a very Two mouse genomic libraries were screened with
active promoter which is virtually lens specific (Lee et a probe corresponding to the 5’ region of the cDNA.
al. 1992; D. C. Lee, P. Gonzalez, and G. J. Wistow, Three independent clones were isolated: AMG 1 included
personal communication ). only -300 nt upstream of the first translated exon.
Clones hMG75 and hMG78 extended > 10 kb to the
Characterization of c-Crystallin cDNA in Mouse upstream region of the gene. The sequence correspond-
As a first step in comparing the regulatory regions ing to the liver untranslated exon was found 8 kb up-
of the c-crystallin gene in guinea pig with that of species stream of the second exon, and the complete intron was
lacking high expression in the lens, a cDNA library from sequenced. If we assume a genetic distance between
mouse liver was screened and three independent clones guinea pig and mouse ~75 Myr (Novacek 1992) and a
(pMLRlO5, pMLR106, and pMLR107; fig. 4) were ob- ratio of 4.6 X 10 -9 substitutions/ nucleotide site/year
tained. The 5 ’untranslated sequence of clone pMLR 106 (Li et al. 198 1 ), the similarity expected in intron se-
extended only 12 nt upstream of the ATG start codon, quences without a functional role is -65%. It is therefore
and clones pMLR 105 and pMLR107 extended 85 and possible to distinguish sequences which were originally
99 nt upstream of the start codon, respectively. This in the gene before the divergence of guinea pig and
sequence shows similarity with the 5’ untranslated region mouse from those that resulted from recombinatory
found in cDNA clones from guinea pig liver (fig. 5 ) . In events after the divergence of the two species. The region
order to determine whether the 5’ untranslated sequence upstream of the liver exon showed 67% similarity with
in mouse lens cells was identical to that found in the that in guinea pig. As in guinea pig, this sequence is GpC
liver cDNA clones or whether it was similar to the one rich ( 65.4% in 358 nt ) and lacks TATA or CAAT boxes
in guinea pig lens cDNA, a RACE experiment was per- (fig. 5). Similarity with the guinea pig gene ends just
formed with RNA from the mouse lens cell line oTN4. after the splicing donor consensus sequence, and it reap-
This cell line was selected because the lens-specific pro- pears in the region corresponding to the guinea pig lens
moter of guinea pig shows high activity in transfection exon. The complete region responsible for the lens pro-
experiments using these particular cells (D. C. Lee, P. moter activity in guinea pig (D. C. Lee, P. Gonzalez,
Gonzalez, and G. J. Wistow, personal communication). and G. J. Winstow, personal communication) is absent
The results showed no difference between the lens and in the mouse gene (fig. 6). The fragment of 6.5 kb not
liver sequences, suggesting that a lens promoter similar homologous to the guinea pig intron sequence includes
to the one in the guinea pig gene was not present in two GT repeats at positions 2875-2935 and 5958-6009,
mouse. counting from the first nucleotide in intron 1, and a
All the clones had a long polyA tail in the 3’ end truncated LlMdA2 repetitive sequence (Loeb et al.
15 nt downstream of the motif AGTAAA which is prob- 1986) at position 3153-4166. The similarity between
ably the functional polyadenylation signal. An additional guinea pig and mouse intron sequences from this point
consensus polyadenylation signal ( AATAAA) was lo- to exon 2 is -74%, with the exception of a fragment of
cated in the same relative position ( 1479-1484) as in 235 nt in guinea pig and of 400 nt in mouse, located
guinea pig (Rodokanaki et al. 1989) and human (Gon- 480 nt upstream of exon 2. The similarity between the
zalez et al. 1993). two species ends again after the consensus splicing donor
The sequence surrounding the putative initiation sequence of intron 2. A graphic representation of the
ATG ( Met ) codon ( 5 ‘-GTCTCCCATGG-3 ‘) matches conserved and nonconserved sequences between the two
the consensus for the translation initiation region of ver- species, in the 5 ’ region of the gene, is shown in fig-
tebrate genes (Kozak 199 1). The termination codon ure 7.
TGA was located at position 1093- 1095. The open
reading frame was 990 nt in length and coded for a pro- Discussion
tein with 330 amino acids, two residues longer than in
guinea pig and human. The two new residues are Asp The analysis of the guinea pig and mouse <-crys-
and Lys located at positions 2 16 and 2 17. The calculated tallin genes presented here indicates clearly that the dif-
molecular mass and isoelectric point are 35,137 D and ference in the level of expression in the lens in these two
*mm07 *MLR105 *RACE-l *RACE-2
CTAGCGAAGTTAGGGAAACC CAGCCCACCTCTTX!CCCACAGACCTTCTGGAGCTTAGGAGG TTTXXTTCCTACTCTAGA 81

*RACE-3 hfLR106 ATGQKLMRAIRVFEFGGPEV 20

~CTCTAGGTCGCCATGGCMCTGGGCAGAAGTTGATGAGGGCTATTC~~~G~~~C~~ 162

LKLQSDVVVPVPQSHQVLIKVHACGVN 47
CTGAAGC'PCCAGTCGGATGIY;GTCGTGCCTGTTCCACA~CA~A~~CTM~~~CAC~C~~~~MC 243

PVETYIRSGAYSRKPALPYTPGSDVAG 74
CCZ?GTAGAGACATACATTCGCTC %GGGCCTACAGTCGAAAACC!IGCCCTACCCTACAC!lCCAGGCTCTGATGTGGCTGGG 324

IIESVGDKVSAFKKGDRVFCYSTVSGG 101
ATCATAGAATCCG~~~~~~~M~~~C~~TM3TGCTACAGCACCGTCTCTCGTGGC 405

YAEFALAADDTIYPLPETLNFRQGAAL 128
TACGCGGAATTTGCTCTTGCAGC!CGATGACACTATCTACCCCCTGCCCGAGACACTGAACTTCAGGCAGGGGGCTGCCCTG 486

GIPYFTACRALFHSARARAGESVLVHG 155
~~CCGTAC~ACGGCCTGCCGAGCTC~CACAL3n;CCCGTGCTCGAGCTGGCGAAAGCG TTTTGGTTCATGGG 567

ASGGVGLATCQIARAHGLKVLGTAGSE 182
GCCAGn;G1AGGAGTIIGGATTAGCTACATGCCAGATTGC TTTTGGGCACAGCTGGTTCTGAG 648

EGKKLVLQNGAHEVFNHKEANYIDKIK 209
~~TTGTTCTGCAAAACGGAGCCCACGAAGTGTTTM~~~~~~CTATATTGATAAAATCAAG 729

MSVGDKDKGVDVIIEMLANENLSNDLK 236
ATGTCTGTIY;GTGALMGGACM~ACGGGGTTGATGTGATTATT~GAATGAGAACCTTAGCMTGACCTGAAG 810

LLSHGGRVVVVGCRGPIEINPRDTMAK 263
CT'ICTGTCCCACGGAGGACGGGTCGTCGTCGTTGGCTGTCGAGG~CCATTGAGATAAACCCMGGGACACCATGGCAAM 891

ETSIIGVSLSSSTKEEFQQFAGLLQAG 290
GAAACAAGCATCA !lTGGGGTCTCTCTCTCTTCTTCC!ACC
AAGGACGAATTTCAACMTTCGCAGGCCTTCl'CCAAGCAGGA 972

IEKGWVKPVIGSEYPLEKAAQAHEDII 317
ATAGAMAAGGTTGGGTGAAAC C'JGTGATAGGT!tCTGAGTATCCATB AGCCCAGGCCCACGATT 1053

HGSGKTGKMILLL 330
CACGGCAGTGGGAAGAC BTGATTCTCCTCTTA j?GJGACCTGTGTCACTGGGCTCCTTCTTCCTCAAGTCBX'GC 1134

ACCACCATCTTTAAACAG!tGTTATTTGAlTGAGGTTCACGTATT AAAAACAGGACTTTAGGGAGTAGACACAGTGAGTGAC1215

CTCTTTAGGGAGTCACCTGCCCCCTCAGCCATGGCTGTCT TGAAAAATATCCTTTTM 1620

AACTMTGTCATC!LCAGTCTGTAAGCCTC%GAAAGC TTCGGCAAAAATGAGCMTACT~ CTCCCAGTCAGCTT 1701

TCCTAACATTTTTGAGAATATCAGAATACTGCA'lGC!CGGTCTGTAGTC TGGGGGGltAGCAGCCACCTGTA 1782

GTCTGGGAGGTCACCAGCTACCTGTAGTC ~~AGCMGCCTTA~CTGGGTCn;TGGTC~AGTATCGTCACC 1863

ATCAAATGCTTCA!BXXCA13TATAGTTTGCTGATATGTGACATGAGTCTCATAGTTTCACTTAAMTTGTMGTTCACAAA 1944

TTTAACATGTTMGAC!CTGTCTGCTTlGGTTTl'CATAGTATCTTAACCTGGAGTAAATTCTGTAGMC!AC~ 2025
3 14 Gonzalez et al.

ATG
MOUSE
Ll Md-A2 t
Put
live
pro

GUINEA PIG

Liver exon Lens exon exon 2

I II
FIG. 7.-Comparison between the 5’ region of guinea pig and mouse <-crystallin genes. The regions conserved (similarity -70%) betwee
the two species are represented as gray boxes. The sequences specific to the mouse and guinea pig genes are represented as unblackened an
blackened boxes, respectively. The mouse Ll MdA2 repetitive element and the GT repeats are also shown. The region labeled as lens promott
in the guinea pig gene is based on the functional analysis by D. C. Lee, P. Gonzalez, and G. J. Winstow (personal communication). Th
sequences corresponding to the areas indicated as liver exon (I) and guinea pig lens exon (II) correspond to those sequences shown in figs.
and 6, respectively.

Since the studies carried out on other enzyme/ Acknowledgments

crystallins have not always involved the analysis of the
5 ’untranslated sequences of the cDNA in nonlenticular
tissues, it seems possible that the presence of alternative
promoters may not be limited to c-crystallin. A recent
analysis of two enzymes recruited as lens crystallins (ar-
gininosuccinate lyase/ &crystallin and lactate dehydro-
genase-B / a-crystallin ) indicated that in those cases there
is apparently one promoter responsible for expression
in both lens and liver ( Hodin and Wistow 1993 ). More
recently, it has been reported that the high expression
of lactate dehydrogenase-B /a-crystallin in the lens in
duck may be mediated by the presence of a promoter-
specific enhancer(s) present in the first intron of the
gene. Taken together, these studies suggest that the
mechanism of recruitment of enzymes as lens crystallins
does not have a single common basis but occurs by dif-
ferent means ( Kraft et al. 1993 ) .
In conclusion, the analysis of the c-crystallin gene
reported here provides the first example of an enzyme/
crystallin in which the double role as enzyme and lens
crystallin can be explained by the acquisition of an ad-
ditional lens-specific promoter. This finding raises fur-
ther interesting questions about the evolution of the gene.
One such question is whether the lens-specific promoter
We thank Dr. S. Tumminia for providing tota
RNA from aTN4 cells. P.G. was supported in part b
a grant from the Ministerio de Education y Cienci,
(Spain) : programa sectorial de betas de formation d
profesorado y personal investigador en el extranjero.
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Received August 10, 1993
Accepted October 27, 1993