U.S. patent application number 09/880427 was filed with the patent office on 2002-04-11 for mutations in nucleic acid molecules encoding 11-cis retinol dehydrogenase, the mutated proteins, and uses thereof.
Invention is credited to Berson, Eliot L., Dryja, Thaddeus P., Eriksson, Ulf, Simon, Andras, Yamamoto, Hioyuji.
Application Number | 20020042116 09/880427 |
Document ID | / |
Family ID | 23185741 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020042116 |
Kind Code |
A1 |
Simon, Andras ; et
al. |
April 11, 2002 |
Mutations in nucleic acid molecules encoding 11-CIS retinol
dehydrogenase, the mutated proteins, and uses thereof
Abstract
The invention relates to mutations in the gene encoding 11-cis
retinal dehyrogenase. The mutations in the gene and in the
resulting encoded protein are correlated to ocular disorders, such
as fundus albipunctatus.
Inventors: |
Simon, Andras; (Stockholm,
SE) ; Eriksson, Ulf; (Stockholm, SE) ; Dryja,
Thaddeus P.; (Boston, MA) ; Berson, Eliot L.;
(Boston, MA) ; Yamamoto, Hioyuji; (Boston,
MA) |
Correspondence
Address: |
Fulbright & Jaworski LLP
666 Fifth Avenue
New York
NY
10103
US
|
Family ID: |
23185741 |
Appl. No.: |
09/880427 |
Filed: |
June 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09880427 |
Jun 13, 2001 |
|
|
|
09306538 |
May 6, 1999 |
|
|
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Current U.S.
Class: |
435/190 ;
435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 2799/021 20130101;
C12Q 1/6883 20130101; C12Y 101/01105 20130101; C12N 9/0006
20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
435/190 ;
435/69.1; 536/23.2; 435/325 |
International
Class: |
C12N 009/04; C07H
021/04; C12N 005/06; C12P 021/02 |
Claims
We claim:
1. An isolated protein comprising the amino acid sequence of wild
type retinol dehydrogenase as set forth in SEQ ID NO: 1, with the
proviso that (i) amino acid 238 is not Gly or (ii) amino acid 73 is
not Ser, or (iii) amino acid 33 is not Ile.
2. The isolated protein of claim 1, wherein amino acid 238 is Trp
rather than Gly.
3. The isolated protein of claim 1, wherein amino acid 73 is Phe
rather than Ser.
4. The isolated protein of claim 1, wherein amino acid 33 is Val
rather than Ile.
5. An isolated nucleic acid molecule which encodes the protein of
claim 1.
6. An isolated nucleic acid molecule which encodes the protein of
claim 2.
7. An isolated nucleic acid molecule which encodes the protein of
claim 3.
8. An isolated nucleic acid molecule which encodes the protein of
claim 4.
9. Expression vector comprising the isolated nucleic acid molecule
of claim 5, operably linked to a promoter.
10. Recombinant cell comprising the isolated nucleic acid molecule
of claim 1.
11. Recombinant cell comprising the expression vector of claim
9.
12. A method for determining possible presence of a disorder of the
eye, comprising assaying a sample taken from a subject believed to
have an eye disorder for a mutation in the nucleic acid molecule
which encodes retinol dehydrogenase, presence of said mutation
being indicative of possible presence of said disorder.
13. The method of claim 12, said method comprising polymerase chain
reaction.
14. The method of claim 12, wherein said mutation is a mutation at
the codon which encodes amino acid 73 of retinol dehydrogenase.
15. The method of claim 12, wherein said mutation is a mutation at
the codon which encodes amino acid 238 of retinol
dehydrogenase.
16. The method of claim 15, wherein said disorder is fundus
albipunctatus.
17. The method of claim 16, wherein said mutation occurs in both
alleles of said subject's gene which encodes retinol
dehydrogenase.
18. The method of claim 17, wherein both of said alleles carry the
same mutation.
19. The method of claim 17, wherein each of said alleles carries a
different mutation
20. The method of claim 18, wherein said mutation results in a
change from glycine to tryptophan at the codon for amino acid
238.
21. The method of claim 19, wherein one of said alleles carries a
mutation resulting in a change from glycine to tryptophan at the
codon for amino acid 238, and the other allele carries a mutation
resulting in a change at the codon for amino acid 73, resulting in
a change from serine to phenylalanine.
Description
FIELD OF THE INVENTION
[0001] This invention relates to mutations in nucleic acid
molecules encoding the protein 11-cis retinol dehydrogenase, or
"RDH5," and the resulting mutated protein. These mutations are
implicated in ocular disorders, such as fundus albipunctatus. The
diagnostic and therapeutic ramifications of these mutations are
also discussed and are features of the invention.
BACKGROUND AND PRIOR ART
[0002] Retinoids (vitamin A-derivatives) have important
physiological functions in a variety of biological processes.
During embryonic growth and development, as well as during growth
and differentiation of adult organisms, retinoids act as hormones
and participate in the regulation of gene expression in a number of
cell types. See Lied et al. Trends Genet., 17:427-433 (1992). It is
believed that these effects are medicated through two classes of
nuclear ligand-controlled transcription factors, the retinoic acid
receptors (RARs) and the retinoid X receptors (RXRs), Benbrook et
al., Nature, 333:669-672 (1988); Brand et al., Nature, 332:850-853
(1988); Giguere et al., Nature, 330:624-629 (1987); Mangelsdorf et
al., Nature, 345:224-229 (1990); Mangelsdorf, et al. Genes Dev.
6:329-344 (1992); Petkovich et al. Nature 330:440-450 (1987); and
Zelent et al., Nature 339:714-717 (1989).
[0003] Apart from their function as hormones in cellular growth and
differentiation, retinoids are also involved in the visual process,
as the stereo isomer 11-cis retinaldehyde is the chromophore of the
visual pigments. See, e.g. Bridges, The Retinoids, Vol. 2, pp
125-176, Academic Press, Orlando, Fla., (1984).
[0004] Under normal physiological conditions most cells, both
ocular and non- ocular, obtain all-trans retinol as their major
source of retinoids. Despite the many different metabolic events
taking place in different tissues, it is known that a common
extracellular transport machinery for retinol has evolved.
Specifically, in plasma, retinol is transported by plasma retinol
binding protein (RBP). See Goodman et al., The Retinoids, Academic
Press, Orlando, Fla., Volume 2, pp. 41-88 (1984). The active
derivatives of retinol, retinoic acid in non-ocular tissues and
mostly 11-cis retinaldehyde for ocular tissues, are then generated
by cellular conversion using specific mechanisms. To date, none of
these mechanisms have been fully defined at the molecular level and
several of the enzymes involved have only been identified by
enzymatic activities. See Lion et al., Biochem. Biophys. Acta.
384:283-292 (1975); Zimmermann et al., Exp. Eye Res. 21:325-332
(1975); Zimmerman, Exp. Eye Res. 23:159-164 (1976) and Posch et
al., Biochemistry 30:6224-6230 (1991).
[0005] Polarized retinal pigment epithelial cells (RPE) are unique
with regard to retinoid uptake since all-trans retinol enters these
cells via two different mechanisms. Retinol accumulated from RBP is
taken up through the basolateral plasma membrane, while all-trans
retinol, presumably taken up from the interstitial retinol-binding
protein (IRBP) following bleaching of the visual pigments, may
enter through the apical plasma membrane. See Bok et al., Exp. Eye
Res. 22:395-402 (1976); Alder et al., Biochem. Biophys. Res.
Commun. 108:1601-1608 (1982); Lai et al., Nature 298:848-849
(1982); and Inu et al., Vision Res. 22:1457-1468 (1982).
[0006] The transfer of retinol from RBP to cells is a subject under
investigation. In a number of cell types, including RPE, specific
membrane receptors for RBP have been identified, which is
consistent with a receptor-mediated uptake mechanism for retinol.
For example, isolated retinol binding protein receptors, nucleic
acid molecule coding for these receptors and antibodies binding to
the receptor are known.. These teachings relate to the first of the
two mechanisms. See Bavik et al., J. Biol. Chem. 266:14978-14985
(1991); Bavik, et al. J. Biol. Chem. 267:23035-23042 1992; Bavik et
al., J. Biol. Chem. 267:20540-20546 (1993); and U.S. Pat. Nos.
5,573,939 and 5,679,772, all of which are incorporated by reference
. See also Heller, J. Biol. Chem. 250:3613-3619 (1975); and Bok et
al., Exp. Eye Res. 22:395-402 (1976).
[0007] Retinol uptake on the apical side of the RPE for the
regeneration of 11-cis retinaldehyde ("11-cis retinal" hereafter)
is less well characterized. However, regardless of the origin of
all - trans retinol, the synthesis and apical secretion of 11-cis
retinal seems to be the major pathway for accumulated retinol in
the RPE. At present, it is not known whether similar mechanisms are
used with regard to cellular retinol uptake through the basolateral
and the apical plasma membranes. However, available data show that
functional receptors for RBP are exclusively expressed on the
basolateral plasma membrane of RPE-cells. Bok et al., Exp. Eye Res.
22:395-402 (1976).
[0008] It is also known that retinal pigment epithelial cells (RPE)
express a 63 kDa protein (p63). It has also been shown by chemical
cross-linking that this protein may be part of an oligomeric
protein complex which functions as a membrane receptor for plasma
retinol-binding protein (RBP) in RPE-cells, or a component of the
retinoid uptake machinery in RPE cells. See Bavik et al., J. Biol.
Chem. 266:14978-14875 (1991); Bavik et al., J. Biol, Chem.
267:23035-23042 (1992), and U.S. Pat. Nos. 5,573,939 and 5,679,772
The p63 protein has been isolated and the corresponding cDNA
cloned. See Bavik et al., J. Biol. Chem. 267:20540-20546 (1993)and
the '939 and "772 patents referred to supra. All of these
references are incorporated by reference.
[0009] 11-cis retinal, referred to supra is important in vision,
because it is the light sensing chromophore found in cone opsins
and rod opsins (i.e., "rhodopsin"), in both cone and rod
photoreceptor cells. Deficiencies in vitamin A result in reduction
in concentrations of rhodopsin in the retina, which is followed by
night blindness. In turn, if night blindness is left untreated it
is followed by degeneration of rod photoreceptors, and then cone
photoreceptors.In fact, vitamin A supplementation has been reported
to slow the course of retinal degeneration in diseases such as
retinitis pigmentosa (Berson et al, Arch. Ophthalmol
111:761-772(1993), and to reverse the night blindness found in
Sorsby fundus dystrophy, at least temporarily. See Jaconson, et al,
Nature Genet. 11:27-32(1995).
[0010] Deficiencies in vitamin A can be attributed to one or more
causes, including poor diet, or a deficiency in one or more of the
proteins involved in transport of vitamin A through the
bloodstream. See, e.g., Wetterau, et al., Science 258:999-1001
(1992), and Narcisi, et al., Am. J. Hum. Genet 57:1298-1310 (1995),
discussing an inherited deficiency in microsomal triglyceride
transfer protein, and Seeliger, et al., Invest. Ophthal Vis. Sci.
40:3-11 (1999), discussing an inherited deficiency in serum retinol
binding protein.
[0011] Physiological abnormalities and visual symptoms also arise
from defects in the storage or metabolism of vitamin A within the
retina. With respect to the storage of the vitamin, a number of
proteins are thought to bind 11-cis and all-trans vitamin A
alcohols and aldehydes in the retina and the retinal pigment
epithelium. These proteins include "CRALBP", or cellular
relinaldehyde binding protein, "IRBP", or "inter-photoreceptor
retinoid binding protein", and "CRBP", or cellular retinol-binding
protein. CRALBP and IRBP are known to be essential to photoreceptor
physiology, since null mutations in the genes encoding these
proteins cause photoreceptor degeneration in mammals. See Mau, et
al., Nature Genet 17:198-200 (1997); Morimura, et al., Invest.
Ophthalmal. Vis. Sci. 40:1000-1004 (1999); Burstedt, et al.,
Invest. Ophthal. Vis. Sci. 40:995-1000 (1999); Liou, et al., J.
Neurasci 18:4511-4520 (1998).
[0012] In contrast to the understanding of the pathways and
mechanisms discussed supra, abnormalities in the metabolic pathways
which convert all-trans retinol from the bloodstream into 11-cis
retinal, and that reconvert the all-trans retinal produced after
cone and rod photopigments absorb photons of light back to 11-cis
retinol are not well understood. Various enzymes are involved in
this pathway, and are found in photoreceptor cells, as well as
neighboring retinal pigment epithelium, and Muller cells of the
retina. Several of these enzymes have been purified only recently.
For example, see Simon et al., J. Biol. Chem 270:1107-1112 (1995),
and U.S. Pat. No. 5,731,195, both of which are incorporated by
reference for teachings relating to purified 11-cis retinal
dehydrogenase, and molecules encoding this enzyme, Haeseleer, et
al., J. Biol. Chem 273:21790-21799 (1998), for teachings related to
all-trans retinal oxido reductase, and Ruiz, et al., J. Biol. Chem
274:3834-3841 (1999), for teachings relating to lecithin: retinal
acyltransferase.
[0013] It has now been found that ocular disorders are associated
with mutations in the nucleic acid molecules encoding 11-cis
retinal dehydrogenase (RDH5), and the resulting mutated proteins.
These are features of the invention which are set out in the
disclosure which follows.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows gel analysis of DNA taken from patients,
showing mutation at codon 238 of the RDH5 gene.
[0015] FIG. 2 shows gel analysis of DNA taken from patients,
showing mutations at codons 238 and 73
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
[0016] Given the role of 11-cis retinol dehydrogenase (SEQ ID NO:
1) in vision, it was assumed that patients suffering from
hereditary retinal degeneration or malfunction might provide a
source of mutated RDH5 genes. Patients with diseases featuring
subretinal white or pale yellow spots were chosen for study,
because these spots can arise from a lack of vitamin A, and are
also found in patients with hereditary retinal degeneration caused
by a lack of serum retinol binding protein (Seeliger et al.,
Invest. Ophthalmol Vis. Sci. 40:3-11 (1999)), or CRALBP (Morimura,
et al., Invest. Ophthalmol Vis. Sci. 40:1000-10004 (1999));
Burstedt, et al., Invest. Ophthalmol Vis. Sci. 40:995-1000 (1999)).
Twenty nine unrelated patients were evaluated who exhibited retinal
degeneration with subretinal spots (retinitis punctata albescens,
or albipunctate dystrophy), two patients were evaluated who
suffered from night blindness and subretinal spots (fundus
albipunctatus), as were 94 normal controls, 71 patients with
recessive retinitis pigmentosa, and 73 with dominant retinitis
pigmentosa. The latter two groups were used because there were no
assurances that the assumption underlying the study, i.e., that
mutations would be found in the RDH5 gene of patients who exhibited
subretinal spots, would be correct.
[0017] The RDH5 gene contains 4 translated exons. Exon 1 is not
translated. Single strand confirmation analysis was used to screen
the 4 translated exons, as well as flanking intron sequences.
[0018] Genomic DNA was analyzed via polymerase chain reaction.
Primers were based upon the sequence of RDH5 published by Simon et
al., Genomics 36:424-430 (1996), and Gu, et al., (GenBank Acc. No.
AF037062), both of which are incorporated by reference and SEQ ID
NO: 2. The primer pairs employed were:
[0019] 5'- GGCCACAGTA AACTGGACAA-3'
[0020] (nucleotides 2301-2320 of SEQ ID NO:2)(sense)
[0021] 5'- AGCCGGTGAT GAAGACAAAG-3'
[0022] (Nucelotides 2458-2477 of SEQ ID NO:2)(antisense)
[0023] which amplify exon 2a of the RDH5 gene;
[0024] 5'- TTACTCTGGG CAGTGCTGTG-3'
[0025] (nucleotides 2399-2418 of SEQ ID NO:2)(sense)
[0026] 5'- AGGACTCGGA AGCCTCTCTG-3'
[0027] (nucleotides 2519-2538 of SEQ ID NO:2)(antisense)
[0028] which amplify exon 2b;
[0029] 5'- TTCTGGCACT GCAGCTGGAC-3'
[0030] (nucleotides 2499-2518 of SEQ ID NO:2)(sense)
[0031] 5'- TTCCTGGTGG TCTACCATAC-3'
[0032] (nucleotides 2686-2705 of SEQ ID NO:2)(antisense)
[0033] which amplify exon 2c;
[0034] 5'- CCCCAGCATC CTTTTCATCT-3'
[0035] (nucleotides 2848-2867 of SEQ ID NO:2)(sense)
[0036] 5'- GACGCTGGTG ATGTTGATCA-3'
[0037] (nucleotides 3038-3057 of SEQ ID NO:2))(antisense)
[0038] which amplify exon 3a;
[0039] 5'- TGAACACMT GGGTCCCATC-3'
[0040] (nucleotides 2969-2988 of SEQ I D NO:2)(sense)
[0041] 5'- TGTTAGTCCT GGAACCCAGG-3'
[0042] (nucleotides 3151-3170 of SEQ ID NO:2)(antisense)
[0043] which amplify exon 3b;
[0044] 5'- AAGAACCCAG CAACTTCGCT-3'
[0045] (nucleotides 4030-4049 of SEQ ID NO:2)(sense)
[0046] 5'- TTCCCTTCAT GTGCCCCTGT-3'
[0047] (nucleotides 5247-5266 of SEQ ID NO:2)(antisense)
[0048] which amplify exon 4;
[0049] 5'- CTGATTGCM CCACCTATGG-3'
[0050] (nucleotides 5473-5492 of SEQ ID NO:2)(sense)
[0051] 5'- AGAGCAGCTT GGCATCCCAA-3'
[0052] (nucleotides5619-5639 of SEQ ID NO:2)(antisense)
[0053] which amplify exon 5a; and
[0054] 5'- TAACCAAGGT GAGCCGATGC-3'
[0055] (nucleotides 5549-5568 of SEQ ID NO:2))(sense)
[0056] 5'- CAATCTCTTG CTGGAAGGCT-3'
[0057] (nucleotides 5731-5748 of SEQ ID NO:2)(antisense)
[0058] which amplify exon 5b.
[0059] Amplification of exon fragments was carried out by isolating
DNA from leukocytes of subjects, and then adding 20-100 ng of the
DNA to 20 .mu.l of a solution containing 20 pM of each pair of the
primers described supra, 20 mM Tris-HCI (pH 8.4), from 0.5 to 1.5
mM MgCl.sub.2 (explained infra), 50 mM KCl, 0.02 mM of each of
dATP, dTTP, and dGTP, and 0.002 mM of dCTP, 0.6 .mu.Ci
[.alpha.-.sup.32P] dCTP (3000 Ci/mmol), 0.1 mg/ml bovine serum
albumin, 10% DMSO (except for assays on exons 4 and 5a), and 0.25
units of Taq polymerase.
[0060] The amount of MgCl.sub.2 varied, depending on the primer
pair being used, to obtain optimal amplification. In the assays
where exons 2a, 2c and 3b were amplified, 0.5 mM was used. When
amplifying exons 2b, 3a, and 5a, 1.0 mM was used, and 1.5 mM was
used when amplifying exons 4 and 5b. Samples were heated to
94.degree. C. for 5 minutes to denature the double stranded DNA,
and then from 22-30 cycles of amplification were carried out, with
a cycle being defined as 30 seconds of denaturing at 94.degree. C.,
30 seconds at 50-60.degree. C. for primer annealing, and 30 seconds
of extension at 71.degree. C. More specifically, the annealing
temperature for exons 2a,3a,and 5a was 58.degree. C., it was
50.degree. C. for exons 2c and 3b, and 60.degree. C. for 2b and 5b
The final extension involved heating at 71.degree. C. for 5
minutes. In all cases, the pH of the reaction was 8.4.
[0061] Amplified DNA was heat denatured, using standard methods,
and aliquots of the resulting single stranded fragments were
separated through two sets of 6% polyacrylamide gels. One set
contained 10% glycerol, and the other did not. Amplification
products of the assay for exon 2c were also evaluated by
electrophoresis through MDE (mutation detection enhancement) gels.
The electrophoresis was carried out for 5-18 hours, at room
temperature, and at 8-12 W, before drying and autoradiography.
Variant bands (defined as bands migrating at a faster or slower
than normal speed through any one of the gels) were evaluated by
sequencing the corresponding PCR amplified segments, using standard
methods.
[0062] The two patients with fundus albipunctatus were found to
have missense changes. Specifically, one patient was homozygous for
a change in exon 4 at codon 238 (GGG to TGG),(nucleotides 5207-5209
of SEQ ID NO:2) leading to a change in the amino acid encoded by
the gene (a change from Gly to Trp), while a second patient was
heterozygous for the same change, and for a second change in exon 2
at codon 73 (TCC to TTC)(nucleotides 2589-2591 of SEQ ID NO:2),
leading to a change in the amino acid encoded (Ser to Phe). One
patient with dominant retinitis pigmentosa exhibited a missense
change in exon 2 at codon 33 (ATC to GTC)(nucleotides 2468-2470 of
SEQ ID NO:2), leading to an amino acid change (Ile to Val). Silent
polymorphisms were also found, at codon 141 (ATC to
ATT)(nucleotides 2986-2988 of SEQ ID NO:2) and codon 200 (GTC to
GTG)(nucleotides 5093-5095 of SEQ ID NO:2).
[0063] The missense changes at 238 and 73 were studied further via
familial analysis. For both subjects, the missense change
segregated as would be expected if they caused the disease. For
example, a sibling of the patient homozygous for the 238 missense
change was also homozygous for the change, and was afflicted with
the disease, while the patient's mother and two unafflicted
siblings were heterozygous. With respect to the patient who was
heterozygous for the missense changes at 238 and 73, there were no
affected relatives; however, an unaffected brother was heterozygous
for the 238 missense change, and his mother was heterozygous for
the 73 missense change. These data are set forth at FIGS. 1 and 2.
It can be seen in FIG. 1, for example, afflicted individuals are
homozygous for a change at position 238, where "G" has been
replaced by "T" in both alleles. In contrast, non afflicted
familial individuals are heterozygous for "G" and "T" at this
position. The data in FIG. 2 show that individuals can be "compound
heterozygotes" in that there are single mutations at both positions
238 and 73. What this indicates is that compound heterozygosity can
lead to the condition.
[0064] The results show that a homozygous mutation at position 238
is indicative of the condition, i.e., fundus albipunctatus.
Heterozygous mutations, i.e., situations where only one allele
carried the mutation, did not result in the mutation. On the other
hand, the data also evidence compound heterozygosity, in that more
than one, heterozygous mutation, results in an abnormal
condition.
EXAMPLE 2
[0065] These experiments describe studies to determine the effect
of the missense mutations described supra, i.e., Gly238Trp and
Ser73Phe on the activity of 11-cis retinol dehydrogenase.
[0066] The first set of experiments was designed to generate mutant
forms of the enzyme in vitro.
[0067] Human cDNA encoding 11-cis retinol dehydrogenase (RDH5)a is
known, as per Simon, et al., supra, and allowed U.S. patent
application Ser. No. 08/258,418, filed Jun. 10, 1994, and PCT
W095/34580 published May 29, 1997, as SEQ ID NO: 14, all of which
are incorporated by reference. See SEQ ID NO: 5 as well. The human
cDNA molecule described in these references was subcloned into
eukaryotic expression vector pSG5, described by Green, et al.,
Nucl. Acids Res. 16:369 (1988), incorporated by reference.
Expression vectors which encoded mutant forms of the enzyme were
then generated using single strand mutagenesis, in accordance with
Kunkel, et al., Methods Enzymol. 154:367-382 (1987), and Viera, et
al., Methods Enzymol. 154:3-11 (1987), both of which are
incorporated by reference. The following primers were used to
generate the Ser73Phe and Gly238Trp mutants, respectively:
[0068] 5'- CTGCAGCGGG TGGCCTTCTC CCGCCTCCAC ACC- 3'
[0069] (SEQ ID NO: 3)
[0070] 5'- ACACAGGCCC ACTATTGGGG GGCCTTCCTC ACC- 3'
[0071] (SEQ ID NO: 4)
[0072] In SEQ ID NOS: 3 and 4, codons for the missense mutations
are underlined.
[0073] Following verification that plasmids carried the introduced
mutations, these were used to separately transfect COS-1 cells. As
controls, expression vectors expressing wild type 11-cis-retinol
dehydrogenase (RDH5), or an empty vector were used. All
transfections were carried out using the well known DEAE-dextran
methodology, as described by Simon, et al., J. Biol. Chem.
270:1107-1112 (1995), incorporated by reference. An expression
vector which expressed .beta.-galactosidase (pSV.beta. gal), which
is commercially available, was cotransfected in each experiment.
The transfectants were cultured for 48 hours, under standard
conditions, after which they were harvested. Microsomes were
prepared from these cells, via gentle homogenization and
centrifugation at 7000.times.g for 10 minutes, in order to remove
debris, and unbroken cells.
[0074] Microsomes were collected from supernatant via
centrifugation at 100,000.times.g for 60 minutes. The microsomes
were suspended in PBS, protein concentrations were determined using
standard methods, and then aliquots were stored at -80.degree. C.,
until used. Equal efficiencies of the transfections were verified
by measuring .beta.-galactosidase activity using
o-nitrophenyl-.beta.-D-galactopyranoside at 405 nm.
[0075] Expression levels of wild type and mutant enzymes were
determined via immunoblotting, using pre-existing polyclonal rabbit
antiserum, in accordance with the ECL methodology as described by
Simon, et al., J. Cell Sci. 112:549-558 (1999).
[0076] It was ascertained that both mutants were expressed at
levels 8-12 times lower than wild type enzyme.
[0077] To monitor the ability of the wild-type and mutant enzymes
to catalyze the oxidation of 11-cis retinol to 11-cis retinal,
11-cis retinol, which had been synthesized by reduction of 11-cis
retinal with NaBH4 and stored under argon at -80.degree. C., was
added to reaction mixtures containing microsomes from the
transfected cells. Microsomes containing the wild-type enzyme
exhibited the expected ability to catalyze the oxidation of 11-cis
retinol to 11-cis retinal in the presence of an excess of the
cofactor NAD.
[0078] The enzyme reactions, in a total volume of 100 .mu.I were
carried out in 50 mM Tris-HCl buffer pH7.5 containing 5 mM NAD or
NADH, 50 M 11-cis retinol, or 11-cis retinal, and microsomes at 37
.degree. C. All manipulations of retinoids were performed in dim
light. The reactions were stopped by puffing the tubes on ice, and
the retinoids were immediately extracted with 200 .mu.l n-hexane.
Subsequently, 75 .mu.l aliquots were analyzed by reverse-phase HPLC
using a C18 column. The mobile phase was acetonitrile/water (85/15,
v/v), and the column was eluted under isocratic conditions at a
flow rate of 1ml/min. 11-cis retinol and 11-cis retinal eluted at
12.0-12.2 and 14.0-14.3 minutes, respectively. Retinoids were
quantified at 320 and 370 nm. To measure the Km and Vmax values of
wild type 11-cis retinol dehydrogenase, reactions were carried out
as above, using a concentration of 11-cis retinol between 0.5 .mu.M
and 50 M. In these assays, 2 .mu.g of microsomal protein containing
the wild-type enzyme were used in 8-minute incubations. Formed
11-cis retinal was quantified in the HPLC analyses, and the Km and
Vmax values were calculated from Lineweaver-Burk plots. The values
were compensated for the extraction efficiency (60-70%, as
determined experimentally).
[0079] The Km and Vmax values for 11-cis retinol in this reaction
were determined to be 6.7 M (average of duplicates 5.5 and 7.8),
and 8.4 nmol/mg protein/minute (average of duplicates 7.1 and
9.6)., respectively. These results are consistent with a report by
Wang, et al., Biochem. J. 338:23(1999). As expected, the wild-type
enzyme was also able to catalyze the reverse reaction, i.e., the
reduction of 11-cis retinal to 11-cis retinol, in the presence of
an excess of NADH.
[0080] The relative activities of the wild type and mutant enzymes
were then determined. To do this, microsomes containing a mutant
enzyme (20 .mu.g),or wild type enzyme(2 .mu.g) together with 18
.mu.g of microsomal protein from mock-transfected cells, to
standardize the experimental conditions, or no enzyme were combined
with 50 mM Tris-HCI buffer (pH 7.5), containing 5 mM of NAD, and 50
.mu.M 11-cis-retinol (forward, oxidation reaction) or 5 mM NADH and
50 .mu.M 11-cis-retinal (reverse, reduction reaction) in a total
volume of 100 .mu.l. 5 .mu.M 11-cis retinol is approximately a
seven-fold higher substrate concentration than the estimated Km
value for the wild-type enzyme. The mixtures were incubated for 10
minutes at 37.degree. C., after which the reactions were stopped by
putting the mixtures on ice. Retinoids were extracted immediately
with 200 .mu.l of n-hexane. Subsequently, 75 .mu.l aliquots were
analyzed via reversed phase HPLC using a C.sub.18 column. The
mobile phase was acetonitrile/water (85:15 v/v), and the column was
eluted under isocratic conditions at a flow rate of 1 ml/min. The
11-cis-retinol eluted at 12.0-12.2 minutes, and 11-cis-retinal at
14.0-14.3 minutes. Retinoids were quantified at 320 (retinol) and
370 (retinal) nm. Relative activities of wild-type and mutant
enzymes were calculated as the ratio of formed 11-cis retinal by
transfected versus mock-transfected microsomes. The results were n
ormalized for the total extracted retinoid content from each
reaction
[0081] The wild type enzyme exhibited the expected ability to
catalyze the oxidation of 11-cis-retinol to 11-cis-retinal in the
presence of an excess of NAD. Similarly, it catalyzed the reverse
reaction, i.e., reduction of 11-cis-retinal to 11-cis-retinol, in
the presence of excess NADH.
[0082] Both mutants exhibited dramatic reductions in their ability
to catalyze the relevant reactions, even though they were used at
10 fold higher concentrations in view of their lower expression
levels. The Gly238Trp mutant showed no activity above background
level, while the Ser73Phe mutant showed residual activity. Similar
results were obtained when catalyzing the reverse reaction
[0083] The Gly238Trp mutant was tested further, to determine if it
had any activity, by incubating for 20 and 40 minute periods before
extracting retinoids. Wild type and Ser73Phe mutant were used as
controls. The Gly238Trp mutant showed 2.2.+-.0.14 fold activity
above background activity (using mock transformants), as compared
to 42.9.+-.4.5 fold (wild type), and 9.1.+-.1 fold (Ser273Phe),
after 40 minutes. This indicates that the Ser273Phe mutant has
approximately 5 fold less activity, and the Gly238Trp mutant
approximately 19 fold less activity than wild type enzyme.
[0084] The foregoing examples describe the invention, which relates
to mutations in the nucleic acid molecule which encodes 11-cis
retinol dehydrogenase, as well as the resulting mutated protein.
Specifically, mutations at the codon which encodes amino acid 238
and/or the codon which encodes amino acid 73 are a feature of the
invention. In particular, with respect to codon 238 Trp, rather
than Gly, is encoded, and with respect to amino acid 73Phe, rather
than Ser, is encoded. Also a feature of the invention are molecules
where the mutation is at position 33, especially those where Val,
rather than lie is encoded. In addition to the specific mutations
described supra, it will be understood by the skilled artisan that,
in view of codon degeneracy, more than one type of mutation may
result in the specific changes.
[0085] As was explained, supra, the mutated forms of the encoding
molecules and the proteins are involved in ocular disorders. Hence,
a further feature of this invention is the determination of the
possible presence of an ocular disorder, via determining presence
of a mutated form of either the nucleic acid molecule, the protein,
or both. Such methods include, e.g., hybridization assays such as
the polymerase chain reaction, antibody assays involving antibodies
specific for an epitope defined by the mutation, and so forth. One
specific type of disorder which may be determined via these
methodologies is fundus albipunctatus.
[0086] Fundus albipunctatus is a form of night blindness wherein
patients who suffer from rod photoreceptor malfunction recover
after prolonged exposure to darkness. This condition is
characterized further by delays in recovery of both rod and cone
function after exposure to light; however, fundus albipunctatus is
characterized as a form of night blindness, because cone recovery
from exposure to light is generally not sufficiently impaired to be
subjectively important.
[0087] The experiments set forth supra show that the abnormally
slow rates of regeneration of code and rod photopigmentfound in
fundus albipunctatus resultfrom slower than normal rates of
production of 11-cis retinal, due to reduced 11-cis retinol
dehydrogenase activity. Further, decreased, steady state levels of
mutant enzymes were seen, suggesting folding or stability problems.
Indeed, these two phenomena point to a net capacity of the enzyme
to generate 11-cis retinal at one order of magnitude lower than
normal. These observations suggest a further application of the
invention, which is the treatment of conditions characterized by
mutations in the gene encoding 11-cis retinal dehydrogenase, or
mutated forms of the protein, by administration of an amount of
normal, or "wild type" 11-cis retinol dehydrogenase sufficient to
alleviate the disorder. Similarly, since the enzyme is involved in
the production of 11-cis retinal, the therapy can also take the
form of administration of 11-cis-retinal, in any of the standard
forms of ocular administration. Similarly, principles of gene
therapy, such as homologous recombination, can be employed to
correct the mutation, upon its detection
[0088] As the mutated forms of the enzyme are useful in the ways
discussed supra, it is desirable to have a ready source of the
materials. Hence, another feature of this invention is the
recombinant production of the enzyme, via the use of transformed or
transected cells, where the resulting recombinant cells produce the
mutant enzyme, as well as multiple copies of the desired nucleic
acid molecule.
[0089] Mutations in the nucleic acid molecule, in addition to those
described above, i.e., at codons for amino acids 238, or 73, or 33,
which result in inactive or less active forms of the enzyme, are
also a part of this invention. These mutations can include other
missense changes, as well as deletions, insertions, frame shift
mutations, mutations affecting intron splice donor or acceptor
sites, mutations in the promotor region, and so forth.
Identification of such mutations via, e.g., PCR, or other forms of
assays, are indicative of possible presence of an optical
disorder.
[0090] Other features of the invention will be clear to the skilled
artisan, and are not set forth herein.
[0091] The terms and expressions which have been employed are used
as terms of description and not of limitation, and there is no
intention in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, it being recognized that various modifications are
possible within the scope of the invention.
Sequence CWU 1
1
5 1 318 PRT Homo sapiens 1 Met Trp Leu Pro Leu Leu Leu Gly Ala Leu
Leu Trp Ala Val Leu Trp 1 5 10 15 Leu Leu Arg Asp Arg Gln Ser Leu
Pro Ala Ser Asn Ala Phe Val Phe 20 25 30 Ile Thr Gly Cys Asp Ser
Gly Phe Gly Arg Leu Leu Ala Leu Gln Leu 35 40 45 Asp Gln Arg Gly
Phe Arg Val Leu Ala Ser Cys Leu Thr Pro Ser Gly 50 55 60 Ala Glu
Asp Leu Gln Arg Val Ala Ser Ser Arg Leu His Thr Thr Leu 65 70 75 80
Leu Asp Ile Thr Asp Pro Gln Ser Val Gln Gln Ala Ala Lys Trp Val 85
90 95 Glu Met His Val Lys Glu Ala Gly Leu Phe Gly Leu Val Asn Asn
Ala 100 105 110 Gly Val Ala Gly Ile Ile Gly Pro Thr Pro Trp Leu Thr
Arg Asp Asp 115 120 125 Phe Gln Arg Val Leu Asn Val Asn Thr Met Gly
Pro Ile Gly Val Thr 130 135 140 Leu Ala Leu Leu Pro Leu Leu Gln Gln
Ala Arg Gly Arg Val Ile Asn 145 150 155 160 Ile Thr Ser Val Leu Gly
Arg Leu Ala Ala Asn Gly Gly Gly Tyr Cys 165 170 175 Val Ser Lys Phe
Gly Leu Glu Ala Phe Ser Asp Ser Leu Arg Arg Asp 180 185 190 Val Ala
His Phe Gly Ile Arg Val Ser Ile Val Glu Pro Gly Phe Phe 195 200 205
Arg Thr Pro Val Thr Asn Leu Glu Ser Leu Glu Lys Thr Leu Gln Ala 210
215 220 Cys Trp Ala Arg Leu Pro Pro Ala Thr Gln Ala His Tyr Gly Gly
Ala 225 230 235 240 Phe Leu Thr Lys Tyr Leu Lys Met Gln Gln Arg Ile
Met Asn Leu Ile 245 250 255 Cys Asp Pro Asp Leu Thr Lys Val Ser Arg
Cys Leu Glu His Ala Leu 260 265 270 Thr Ala Arg His Pro Arg Thr Arg
Tyr Ser Pro Gly Trp Asp Ala Lys 275 280 285 Leu Leu Trp Leu Pro Ala
Ser Tyr Leu Pro Ala Ser Leu Val Asp Ala 290 295 300 Val Leu Thr Trp
Val Leu Pro Lys Pro Ala Gln Ala Val Tyr 305 310 315 2 6330 DNA Homo
sapiens unsure 5357, 5448 nucleotide not determined 2 ccaggttttc
cctcccttcc cccactcagc tgcaggaact cctttttggg gtttggatct 60
ggtatttttc tattcagctc cgagcttggc tctcctgggg aatcctggga gtgaaaggaa
120 ggagctgggt ttatttgcat gtactggtag tcatttgcat cacatccaaa
aatggccaaa 180 attataaccc ctgattcttg gctgaactgg gactgctgca
atggaatatt attcccggaa 240 accaccccca actagctgga gctaatctcc
tccctcctcc aaccccccat tttggcccag 300 gcctacataa accaaaaaaa
gctggaccat aaggtgaaaa ccctacaggt ccaggctgcc 360 caatttgcca
agcaaacagg ccattggatc gaaatggtga aaaacttcaa ccaggcactc 420
aaggtgggcc atactcccta cctcaccacc ccaatcctgg gcccccattg gctgcctcca
480 gtcaggttac ctcaggttta ggttaaggag gaagtagggt ggtcccagaa
accccatcta 540 tagccccagt gtcagaaaag gttgagaaag aaagaaaagc
agttggtggg tccaagttaa 600 agccttttcc aggagatgaa taaaacttat
tccccaatgg aagccatagt ctacccattc 660 tgattcctgg gtcccaactc
ctctccccct ttccaggaaa ttggggatgt ggagaatggg 720 cttggagcat
tgagctggaa atgcgcacca ttgccaatgc aatggaatat gtttacaaag 780
ggcagctgca gtttgcccct tcctagcccc tgttccctcc cccaacccta tccctcctac
840 ctcacccgca gggggaagga gggaggctga caagccttga ataaaaaaca
agcctccgtt 900 tttttgtggt gtgtttcaga gaggtaatag ctccagtgtc
gggggtggga gtggaaggtt 960 caaaggtggt ttccctgagg gacaggtacc
ttttggggag agggtggaaa tagcttcctt 1020 ttactatccc aaattttttt
tcctccatgg cccttgtgca ggtgtttgtt aggcaagcag 1080 agggtgggag
ttcccatccc tcctgagaga aggtcctagt agccctgccc caagcttcct 1140
aattcaggaa ttgtttccta cagaagagaa acaaggcaag tacacctggt ccccagctct
1200 ggctttctgc ctctccacgt gctcatggcc tctccccagg ctaactctaa
gcagtgtcat 1260 gagtctgagc caggtgggag attaattcct gggggcactt
cagggctgag aagggggagg 1320 aatgacaggt ccagtaaccg ttaccaacag
agcagtgcag ctgccatcct tgacagctcc 1380 ctcctccttg gagaccatga
catagatggt caggaaccca ggctgagaaa gacagccaag 1440 gggtgggggg
agcctaggca aatctggcct ctgccaagtc ctggcttcag ccaggcaagc 1500
tccagcctcc ctggctcctc ctcctcctca gtcctatccc caccctgtca cacatacact
1560 taatacgcct ggcatccaag tccacccact ccggactttg gccttagcag
tagttagtgt 1620 gggaggctgg gaagactggg agcagtctct taaacaaaag
caaaagaata agcttcgggc 1680 gctgtagtac ctgccagctt tcgccacagg
aggtaagtgg atctgggagc tgggggaact 1740 gagaagacta gccagatatt
acatgtattg ccaactcaaa actttcagct tttaacatgc 1800 ttcctcacac
attatcccct ttgatcctcc acaactctga ggtggacctg gtgggtctta 1860
gccccacttg gtagatgaga aaataggttg agagagacag tgagatgctc agtatcacac
1920 agcaaacctc ttggccctat acatcattcc aaacacaaga cccaggttgc
atatagaagg 1980 ttcagtgtcc ctggtttaga aggagaggtg gtgtgaggca
agcaagaaga tgcctctgct 2040 gcactccagc ctgggcgaca gagtgagact
ccatctcaaa aaaaaaaaaa aacgatgcct 2100 ctgctccata cagcaggtct
gtacacagga tctggctcat gtggttttag ttaagttagc 2160 cacaaataca
gggtctgccc acatctttgc tttgaacaga tgagccatgg ttggccaatt 2220
atctgccaac cagataattt ctcaatatgc tcacaccaga tgcttccagc tagggagggt
2280 attaggggaa agggcttgag ggccacagta aactggacaa gtttttctgc
ccagcctagg 2340 ctgccacctg taggtcactt gggctccagc tatgtggctg
cctcttctgc tgggtgcctt 2400 actctgggca gtgctgtggt tgctcaggga
ccggcagagc ctgcccgcca gcaatgcctt 2460 tgtcttcatc accggctgtg
actcaggctt tgggcgcctt ctggcactgc agctggacca 2520 gagaggcttc
cgagtcctgg ccagctgcct gaccccctcc ggggccgagg acctgcagcg 2580
ggtggcctcc tcccgcctcc acaccaccct gttggatatc actgatcccc agagcgtcca
2640 gcaggcagcc aagtgggtgg agatgcacgt taaggaagca ggtaagtatg
gtagaccacc 2700 aggaatatgg tgtggggtgt cctgatcccc acagtcaccc
caggagtcac ctgcaagggc 2760 tgtggtaagc taaagggaca atttgaggag
aagcagtttt cagatgctcc caggaagaag 2820 agggagctgt gggagtgcct
cacctacccc cagcatcctt ttcatctccc cacagggctt 2880 tttggtctgg
tgaataatgc tggtgtggct ggtatcatcg gacccacacc atggctgacc 2940
cgggacgatt tccagcgggt gctgaatgtg aacacaatgg gtcccatcgg ggtcaccctt
3000 gccctgctgc ctctgctgca gcaagcccgg ggccgggtga tcaacatcac
cagcgtcctg 3060 ggtcgcctgg cagccaatgg tgggggctac tgtgtctcca
aatttggcct ggaggccttc 3120 tctgacagcc tgaggtgagg ggtacagggc
tctgggttcc aggactaaca gcagcccact 3180 caacaaacgt gggccagcag
aggtggttaa aatacagcac attggaatag ttaaaaagag 3240 acagtttagg
gctaaacttc atgggttcaa tgaagtctac ccttatgtaa gctttgtgac 3300
cataagtaga ttacttctct ttacccattt ttaacgtgtt tgttttttgt tttttgagat
3360 ggagtcttgc tctgtcgcca ggctggagtg cagtggcgcg atcttggctc
accacaattt 3420 ccacccccgg ggttcaagcg attctcctgc ctcagcctcc
cgagtagctg ggactacagg 3480 catgcgccac catgcctggc taatttttgt
atttttagta gagacagggt ttcactatgt 3540 tggccaggtt ggtctcaaac
tcctgacctc gtgatccgcc cacctcagcc tcccaaagtg 3600 ctgggattac
aggtgtgagc caccacgccc ggccttgcct ctcgtcttta aacaataagg 3660
ttcaaagttc cgtgggagca caaaggagac atgatgagga caacgggagt tagggcctga
3720 gtttttttgg tttttttttt ttaagcgttt tgctcttgtt gcctaggctg
gagtgcaatg 3780 gcgagatctc agctcacagc aacccctgcc tctcaggttc
atgtgattct cctgcctcag 3840 cctcccgatt agctgggctt acaggcacgt
gccaccactc ccagctaaat ttttaggtag 3900 agatggagtt tataccatgt
ggccagggtg ggtttgaatt cctgacctca cctgatccac 3960 cggaccggcc
ttcccaaagt gctgggatta caggcatgag ccaccacaca cggcccaagg 4020
cctgagttct tagcaggagt ataaggcgcc taagcttagt ctaccttcta aggaagcctg
4080 cgtttgtcac catcactcag caaataaccg gaattgtctc ctgtctctca
gccttaattt 4140 ttcaggcagc atcatgggac acatactttt agttttgaga
caaggccttg ctctcaccca 4200 gggtggagtg cagtggtgca gtcacggccc
actgaacttc aaactcctag gctcaagcag 4260 ctcaagcgat atccgcctca
gcctcctgag tagctgagac cacaggcgcg tgccagcatg 4320 cctggctagt
atttttttac agatggggtc ttgctgtggt gaccagactt gtctccaact 4380
cccggcctca agcgatgctt ccgcctgggc ctcccaaagt gttgggatta taggtgtgag
4440 ccactgcata ctggaacaca tactttatac ttgaattttt ttttatcccc
ttccttcgtg 4500 ctcctaacct atacttggat ttctacatct gtgccagggc
agtgggatgt atccccactt 4560 tccccatcag cttaccctcc agcaaatacg
agactatacc cttcaatatc cagcactcag 4620 ggctcaacca tgtgttttgg
gagcaaggga atggggttcc tctaggtcag gaatcggcaa 4680 actcagtact
caagccagat ttggccagct gcctacaagc tgataatggt tttttttatt 4740
tttaaatggt tacattgtaa actgttatat aagtacctga taatatcatt aattttgttt
4800 cttggcctgc catgcttaaa atattaactc tctggccctt taagaaaaaa
acgtgctgac 4860 ccctgctcta gatcaaagaa aacaaacctc aaaaatactt
tcctccctct accccacttg 4920 acccttgtcc cggggcagta ggcatctccg
tcaaaactct tgtccctggt ctgtggtaac 4980 tttctcagct ccccaaccca
tgtccctcaa agtcccctcc ctatagggca agaacccagc 5040 aacttcgctc
tgccccgact ctaggcggga tgtagctcat tttgggatac gagtctccat 5100
cgtggagcct ggcttcttcc gaacccctgt gaccaacctg gagagtctgg agaaaaccct
5160 gcaggcctgc tgggcacggc tgcctcctgc cacacaggcc cactatgggg
gggccttcct 5220 caccaagtgt gagtagccag gcccacacag gggcacatga
agggaaacaa gtaccagaaa 5280 ggccagtcct gcataagcct gctaggaggt
gggtggggca cccagggcag ggttgagggt 5340 gaacaggatg ttacaanagt
gcccaggcca tgtggaacct gcccactccc cacactgagg 5400 aggggactga
gggtgacaag cccagggccc caaaaaacag tacctaanat gggctggagt 5460
gaggaaggga aactgattgc aaccacctat ggggctgcag acctgaaaat gcaacagcgc
5520 atcatgaacc tgatctgtga cccggaccta accaaggtga gccgatgcct
ggagcatgcc 5580 ctgactgctc gacacccccg aacccgctac agcccaggtt
gggatgccaa gctgctctgg 5640 ctgcctgcct cctacctgcc agccagcctg
gtggatgctg tgctcacctg ggtccttccc 5700 aagcctgccc aagcagtcta
ctgaatccag ccttccagca agagattgtt tttcaaggac 5760 aaggactttg
atttatttct gcccccaccc tggtactgcc tggtgcctgc cacaaaataa 5820
gcactaacaa aagtgtattg tttaaaaaat aaaaagaagg tgggcagaaa tgtgcccagt
5880 ggaaggctga ccccatttaa gtgccaacta ctccaaaccg acatgctcac
ggtctctggc 5940 ctgttcagtc cctgcaaaac agctagcacc cacagtgggg
cgccagggaa ctgcctcaca 6000 tctacagctg cacgtcgggg agtggccatc
aaagggcact ttaatacatt tcccttattt 6060 tctgaagggg agtaaggttg
caattcagtg tctgtactgg gaatggtctt catatttctt 6120 gggggagaag
agcaggtgat gagggttctg ggccaggctg ggtggcttcc atggaagaaa 6180
aggcaatatt cacataaatt ctcctgctaa ggacactgac cacacaggtg tgcaaggcaa
6240 cttatcatac ttcgaaagga gctggatccc ttgaggattg gccaggaagg
gaggtgctgg 6300 gcccttagcg gtgcacagaa ggccaggaag 6330 3 33 DNA Homo
sapiens 3 ctgcagcggg tggccttctc ccgcctccac acc 33 4 33 DNA Homo
sapiens 4 acacaggccc actattgggg ggccttcctc acc 33 5 1128 DNA Homo
sapiens 5 taagcttcgg gcgctgtagt acctgccagc tttcgccaca ggaggctgcc
acctgtaggt 60 cacttgggct ccagctatgt ggctgcctct tctgctgggt
gccttactct gggcagtgct 120 gtggttgctc agggaccggc agagcctgcc
cgccagcaat gcctttgtct tcatcaccgg 180 ctgtgactca ggctttgggc
gccttctggc actgcagctg gaccagagag gcttccgagt 240 cctggccagc
tgcctgaccc cctccggggc cgaggacctg cagcgggtgg cctcctcccg 300
cctccacacc accctgttgg atatcactga tccccagagc gtccagcagg cagccaagtg
360 ggtggagatg cacgttaagg aagcagggct ttttggtctg gtgaataatg
ctggtgtggc 420 tggtatcatc ggacccacac catggctgac ccgggacgat
ttccagcggg tgctgaatgt 480 gaacacaatg ggtcccatcg gggtcaccct
tgccctgctg cctctgctgc agcaagcccg 540 gggccgggtg atcaacatca
ccagcgtcct gggtcgcctg gcagccaatg gtgggggcta 600 ctgtgtctcc
aaatttggcc tggaggcctt ctctgacagc ctgaggcggg atgtagctca 660
ttttgggata cgagtctcca tcgtggagcc tggcttcttc cgaacccctg tgaccaacct
720 ggagagtctg gagaaaaccc tgcaggcctg ctgggcacgg ctgcctcctg
ccacacaggc 780 ccactatggg ggggccttcc tcaccaagta cctgaaaatg
caacagcgca tcatgaacct 840 gatctgtgac ccggacctaa ccaaggtgag
ccgatgcctg gagcatgccc tgactgctcg 900 acacccccga acccgctaca
gcccaggttg ggatgccaag ctgctctggc tgcctgcctc 960 ctacctgcca
gccagcctgg tggatgctgt gctcacctgg gtccttccca agcctgccca 1020
agcagtctac tgaatccagc cttccagcaa gagattgttt ttcaaggaca aggactttga
1080 tttatttctg cccccaccct ggtactgcct ggtgcctgcc acaaaata 1128
* * * * *