Exp. Eye Res. (1998), 66, 267–269

LETTER TO THE EDITORS

Extralenticular Expression of the Rodent βB2-Crystallin Gene

It has long been thought that the crystallins, the

abundant lenticular structural proteins, were lens-

specific proteins. However, it is now known that some

of the crystallin genes are used elsewhere as house-

keeping or stress genes ; the expression of these genes

is merely upregulated in the lens (for reviews, see

Wistow and Piatigorsky, 1987; De Jong et al., 1989;

Piatigorsky and Wistow, 1989, 1991; Bloemendal

and de Jong, 1991; De Jong, Lubsen and Kraft, 1994).

Examples of such genes are the α-crystallin genes and

the taxon-specific crystallin genes. There is accumu-

lating evidence that the β- and γ-crystallin genes are

also expressed at low levels outside the lens. Expression

of β-crystallins has been detected in the mouse, cat

and chicken retina (Head, Peter and Clayton, 1991;

Head, Sedowofia and Clayton, 1995) and extra-

lenticular expression of β- and γ-crystallin mRNA was

detected in early embryonic stages of Xenopus de-

velopment (Smolich et al., 1994; Brunekreef et al.,

1997).

To determine if rodent β-crystallin genes are

expressed in tissues other than retina and lens, we

focussed on the βB2-crystallin gene as the protein

product of this gene was detected in the mouse retina

(Head et al., 1995). Since we failed to detect βB2-

crystallin mRNA in the retina by Northern blot

analysis, we used the highly sensitive reverse trans-

criptase polymerase chain reaction (RT-PCR) assay to

test for βB2-crystallin RNA in the retina and other

tissues.

RNA was isolated from retina and from several

other tissues of newborn rats and reverse transcribed

into cDNA. Following 25 cycles of amplification, βB2-

crystallin PCR products were visualized by Southern

blot analysis [Fig. 1(A)]. As expected, a 500 bp βB2-

crystallin PCR product is visible in retina. A PCR

product is also present in intestine, spleen, liver and

kidney, at a lower level in heart, but not in brain [see

also Fig. 1(C)]. The identity of the RT-PCR fragment

was confirmed by restriction enzyme analysis (data

not shown). Several control experiments were per-

formed to rule out the possibility that the PCR signal

results from contamination of RNA samples or

reagents with RNA from the lens (data not shown). As

the extralenticular expression of βB2-crystallin was

originally detected in the retina of the adult mouse, we

repeated the RT-PCR analysis on RNA derived from

several adult mouse organs [Fig. 1(B)]. A 500 bp PCR

product is detectable at approximately equally high

levels in muscle and liver, at a lower level in the retina,

but not in heart and in skin. To estimate the amount

of βB2-crystallin mRNA present in the extralenticular

tissues, the RT-PCR signal from newborn rat intestine

RNA was compared with the signal obtained from

serial dilutions of lens RNA [Fig. 1(C)]. The βB2

F. 1. (A) βB2-crystallin mRNA is present at low levels inseveral non-lens tissues of the newborn rat. RNA isolatedfrom the indicated rat tissues was reverse transcribed withAMV reverse transcriptase using oligo(dT) as a primer andthe cDNA was amplified in 25 PCR cycles using Vent DNApolymerase (Biolabs). The rat βB2 exon 2-specific oligo-nucleotide 5«-(CCA CCA TGG CCT CAG ACC ACC AGACAC)-3« was used as the sense primer and the exon 6-specificprimer 5«-GTC CTT GTA ATC CCC CTT CTC CAG CAG)-3« asthe intisense primer (sequence according to Aarts, Lubsenand Schoenmakers, 1989). Denaturation was for 1 min at94°C, annealing for 2 min at 50°C and polymerization for3 min at 72°C. RT-PCR samples were fractionated onagarose gels (Sambrook, Fritsch and Maniatis, 1989),Southern blotted and hybridized with a $#P-labelled rat βB2-crystallin cDNA probe according to standard protocols(Sambrook et al., 1989). (B) βB2-crystallin mRNA in non-lens tissues of the adult mouse. RNA from the indicatedmouse tissues was analysed by RT-PCR as described in (A).(C) Expression level of the rat βB2-crystallin gene inextralenticular tissues. The indicated amounts of RNAderived from newborn rat intestine, brain and lens wereanalysed by RT-PCR as described in (A).

0014–4835}98}020267­03 $25.00}0}ey970439 # 1998 Academic Press Limited

268 LETTER TO THE EDITORS

F. 2. βB2-crystallin protein in extralenticular tissues of the newborn rat. Organs were dissected from newborn rats and 2-months-old mice and washed in phosphate buffered saline. Crude lysates were prepared by homogenization in water. Aftercentrifugation (20 min, 4°C, eppendorf centrifuge), the supernatants, containing the water-soluble protein, were used forwestern blot analysis. Protein concentration was determined by colorimetric staining (Bio-Rad). Aliquots of water solubleprotein were boiled in SDS containing sample buffer for 5 min and electrophoresed in an SDS}polyacrylamide gel according tostandard protocols (Sambrook et al., 1989). The protein was transferred to nitrocellulose by electroblotting (Sambrook et al.,1989) and assayed for βB2-crystallin content. The first antibody was a rabbit polyclonal antibody raised against recombinantrat βB2-crystallin. The second antibody was an alkalic phosphatase-conjugated goat anti-rabbit-IgG antibody (Boehringer),which was subsequently detected by chemoluminescence using AMPPD (Tropix) as a substrate according to the manufacturer’sprotocol. (A) Crude lysates from rat brain (50 µg), lens (250 ng), intestine and retina (100 µg). Pure recombinant βB2-crystallin was used as an internal size marker. (B) As in (A), except that the anti-βB2-crystallin was used as an internal sizemarker. (B) As in (A), except that the anti-βB2-antibody had been preincubated with 200-fold excess of recombinant βB2-crystallin.

expression level in intestine is approximately 0±1–0±2%

of the level expressed in the newborn rat lens.

Our data indicate that βB2-crystallin mRNA is

expressed at very low levels outside the lens. To

determine whether the extralenticular expression of

βB2-crystallin could also be detected at the protein

level, crude lysates from newborn rat brain, intestine,

retina and lens were analysed by western blot analysis

using an anti-βB2-crystallin antibody [Fig. 2(A)]. A

27 kDa protein that comigrates with recombinant

βB2-crystallin and interacts with the anti-βB2-

crystallin antibody is present in lens and retina,

whereas the antibody detects a slightly smaller protein

in brain and intestine. Preincubation of the antibody

with excess recombinant βB2-crystallin greatly

reduces the signal of the 27 kDa protein in lens and

retina, but does not affect the signal of the slightly

smaller protein in brain and intestine [Fig. 2(B)]. The

βB2-crystallin is a highly stable protein and resistent

to heating for 10 min at 100°C. However, when the

lysate from intestine was boiled for 10 min prior to

western blot analysis, the 27 kDa protein could no

longer be detected (data not shown). We also

considered the possibility that βB2-crystallin is part of

the water insoluble fraction of the intestine, but

western blot analysis of the water insoluble protein

fraction or of whole cell lysates from intestine did not

reveal any βB2-crystallin (data not shown).

Efforts to detect βB2-crystallin at the protein level in

other tissues failed as well, although others have

reported the presence of βB2-crystallin in adult mouse

brain and testes (Kantorow et al., 1997). Hence, even

though the βB2-crystallin mRNA level in retina is not

significantly higher than that in some of the other

non-lens tissues tested, only retina expresses detectable

levels of βB2-crystallin protein. This may indicate that

expression of the βB2-crystallin gene in non-ocular

tissues is inhibited at the translational level. It has

been previously suggested that, even in lens, βB2-

crystallin mRNA is poorly translated (Aarts, Lubsen

and Schoenmakers, 1989). Alternatively, the βB2-

crystallin gene could have alternative transcription

initiation sites, as reported for the αB-crystallin gene

(Iwaki et al., 1990). The longer transcript of this gene

is expressed at low levels in virtually all tissues and

contains several open reading frames upstream of the

code for αB-crystallin, which may negatively affect the

translation of the mRNA. However, the 5« non-coding

region of full length βB2-crystallin cDNA clones

isolated from intestine or lens RNA did not differ in

length or sequence (data not shown).

At this moment, we can only speculate about the

putative function of βB2-crystallin in extralenticular

tissues, assuming that it is translated under certain

conditions. Recently, β- and γ-crystallin mRNA was

found to be present in early embryonic stages of

Xenopus laevis (Smolich et al., 1994; Brunekreef et al.,

1997) and the members of the β}γ-crystallin super-

family could play some unknown role during early

development. Whether βB2-crystallin plays a vital role

in the mouse or man is questionable, given the fact that

the only visible phenotypical effect of a mutation in the

βB2-crystallin gene is the development of cataract

(Chambers and Russell, 1991; Litt et al., 1997).

LETTER TO THE EDITORS 269

Acknowledgements

This work has been carried out under the auspices of theNetherlands Foundation for Chemical Research (SON) andwith financial aid from the Netherlands Organization for theAdvancement of Pure Research (NWO) and was alsosupported by grant 1R01 EY09187 from the NationalInstitutes of Health, and a twinning grant from the EC.

RON P. H. DIRKS,

SIEBE T. VAN GENESEN, JACQUELINE

J. C. M. KRU> SE,

LEJA JORISSEN

NICOLETTE H. LUBSEN*

Department of Molecular Biology,

University of Nijmegen,

Toernooiveld 1,

6525 ED, Nijmegen, The Netherlands

* Author to whom all correspondence should be addressed.

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