U.S. patent application number 13/151375 was filed with the patent office on 2012-02-16 for methods of gene therapy using nucleic acid sequences for atp-binding cassette transporter.
This patent application is currently assigned to Baylor College of Medicine. Invention is credited to Rando Allikmets, Kent L. Anderson, Michael Dean, Mark Leppert, Richard A. Lewis, Yixin Li, James R. Lupski, Jeremy Nathans, Amir Rattner, Noah F. Shroyer, Nanda Singh, Philip Smallwood, Hui Sun.
Application Number | 20120040456 13/151375 |
Document ID | / |
Family ID | 27759870 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120040456 |
Kind Code |
A1 |
Allikmets; Rando ; et
al. |
February 16, 2012 |
METHODS OF GENE THERAPY USING NUCLEIC ACID SEQUENCES FOR
ATP-BINDING CASSETTE TRANSPORTER
Abstract
The present invention provides nucleic acid and amino acid
sequences of an ATP binding cassette transporter and mutated
sequences thereof associated with macular degeneration. Methods of
detecting agents that modify ATP-binding cassette transporter
comprising combining purified ATP binding cassette transporter and
at least one agent suspected of modifying the ATP binding cassette
transporter an observing a change in at least one characteristic
associated with ATP binding cassette transporter. Methods of
detecting macular degeneration is also embodied by the present
invention.
Inventors: |
Allikmets; Rando;
(Frederick, MD) ; Anderson; Kent L.; (Houston,
TX) ; Dean; Michael; (Frederick, MD) ;
Leppert; Mark; (Salt Lake City, UT) ; Lewis; Richard
A.; (Houston, TX) ; Li; Yixin; (Houston,
TX) ; Lupski; James R.; (Houston, TX) ;
Nathans; Jeremy; (Baltimore, MD) ; Rattner; Amir;
(Baltimore, MD) ; Shroyer; Noah F.; (Houston,
TX) ; Singh; Nanda; (Salt Lake City, UT) ;
Smallwood; Philip; (Woodbine, MD) ; Sun; Hui;
(Baltimore, MD) |
Assignee: |
Baylor College of Medicine
Houston
TX
|
Family ID: |
27759870 |
Appl. No.: |
13/151375 |
Filed: |
June 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11636909 |
Dec 11, 2006 |
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13151375 |
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10336219 |
Jan 3, 2003 |
7192579 |
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11636909 |
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09032438 |
Feb 27, 1998 |
6713300 |
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10336219 |
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60039388 |
Feb 27, 1997 |
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Current U.S.
Class: |
435/348 ;
435/252.3; 435/252.31; 435/252.33; 435/252.34; 435/254.2;
435/254.21; 435/320.1; 435/349; 435/358; 435/363; 435/367;
536/23.1 |
Current CPC
Class: |
C07K 14/705 20130101;
A01K 2217/05 20130101 |
Class at
Publication: |
435/348 ;
435/349; 435/358; 435/363; 435/367; 435/252.3; 435/252.31;
435/252.33; 435/252.34; 435/254.2; 435/254.21; 435/320.1;
536/23.1 |
International
Class: |
C12N 1/21 20060101
C12N001/21; C12N 15/11 20060101 C12N015/11; C12N 15/63 20060101
C12N015/63; C12N 5/10 20060101 C12N005/10; C12N 1/19 20060101
C12N001/19 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This work was supported in part by research grants from the
Department of Health and Human Services, grant numbers DHHS #2
T32GM07330-19 and #3 T32EY07102-0553, the National Institutes of
Health, grant number M01-RR00064. The United States Government may
have certain rights in this invention.
Claims
1.-6. (canceled)
7. A purified nucleic acid molecule comprising a nucleotide
sequence encoding an ABCR amino acid sequence of SEQ ID NO:11.
8. The nucleic acid molecule of claim 7, wherein the nucleotide
sequence encodes wild-type ABCR.
9. A vector comprising a nucleotide sequence encoding an ABCR amino
acid sequence according to SEQ ID NO: 11.
10. The vector of claim 9, wherein the vector is a viral
vector.
11. The vector of claim 9, wherein the nucleotide sequence encodes
wild-type ABCR.
12. An isolated cell comprising a nucleotide sequence encoding an
ABCR amino acid sequence of SEQ ID NO:11.
13. The cell of claim 12, wherein the nucleotide sequence encodes
wild-type ABCR.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. application Ser. No.
10/336,219, filed Jan. 3, 2003, which is a divisional of U.S. Pat.
No. 6,713,300, filed Feb. 27, 1998, which claims benefit of U.S.
Provisional Application No. 60/039,388, filed Feb. 27, 1997. Each
of these applications and patents is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0003] Macular degeneration affects approximately 1.7 million
individuals in the U.S. and is the most common cause of acquired
visual impairment in those over the age of 65. Stargardt disease
(STGD; McKusick Mendelian Inheritance (MIM) #248200) is arguably
the most common hereditary recessive macular dystrophy and is
characterized by juvenile to young adult onset, central visual
impairment, progressive bilateral atrophy of the macular retinal
pigment epithelium (RPE) and neuroepithelium, and the frequent
appearance of orange-yellow flecks distributed around the macula
and/or the midretinal periphery (Stargardt, 1909; Anderson et al.,
1995). A clinically similar retinal disorder (Fundus
Flavimaculatus, FFM, Franceschetti, 1963) often displays later age
of onset and slower progression (Fishman, 1976; Noble and Carr,
1979). From linkage analysis, it has been concluded that STGD and
FFM are most likely allelic autosomal recessive disorders with
slightly different clinical manifestations caused by mutation(s) of
a gene at chromosome 1p13-p21 (Gerber et al., 1995; Anderson et
al., 1995). The STGD gene has been localized to a 4 cM region
flanked by the recombinant markers D1S435 and D1S236 and a complete
yeast artificial chromosome (YAC) contig of the region has been
constructed (Anderson et al., 1995). Recently, the location of the
STGD/FFM locus on human chromosome 1p has been refined to a 2 cM
interval between polymorphic markers D1S406 and D1S236 by genetic
linkage analysis in an independent set of STGD families (Hoyng et
al., 1996). Autosomal dominant disorders with somewhat similar
clinical phenotypes to STGD, identified in single large North
American pedigrees, have been mapped to chromosome 13q34 (STGD2;
MIM #153900; Zhang et al., 1994) and to chromosome 6q11-q14 (STGD3;
MIM #600110; Stone et al., 1994) although these conditions are not
characterized by the pathognomonic dark choroid observed by
fluorescein angiography (Gass, 1987).
[0004] Members of the superfamily of mammalian ATP binding cassette
(ABC) transporters are being considered as possible candidates for
human disease phenotypes. The ABC superfamily includes genes whose
products are transmembrane proteins involved in energy-dependent
transport of a wide spectrum of substrates across membranes (Childs
and Ling, 1994; Dean and Allikmets, 1995). Many disease-causing
members of this superfamily result in defects in the transport of
specific substrates (CFTR, Riordan et al, 1989; ALD, Mosser et al.,
1993; SUR, Thomas et al., 1995; PMP70, Shimozawa et al., 1992;
TAP2, de la Salle et al., 1994). In eukaryotes, ABC genes encode
typically four domains that include two conserved ATP-binding
domains (ATP) and two domains with multiple transmembrane (TM)
segments (Hyde et al., 1990). The ATP-binding domains of ABC genes
contain motifs of characteristic conserved residues (Walker A and B
motifs) spaced by 90-120 amino acids. Both this conserved spacing
and the "Signature" or "C" motif just upstream of the Walker B site
distinguish members of the ABC superfamily from other ATP-binding
proteins (Hyde et al., 1990; Michaelis and Berkower, 1995). These
features have allowed the isolation of new ABC genes by
hybridization, degenerate PCR, and inspection of DNA sequence
databases (Allikmets et al., 1993, 1995; Dean et al., 1994; Luciani
et al., 1994).
[0005] The characterization of twenty-one new members of the ABC
superfamily may permit characterization and functions assigned to
these genes by determining their map locations and their patterns
of expression (Allikmets et al., 1996). That many known ABC genes
are involved in inherited human diseases suggests that some of
these new loci will also encode proteins mutated in specific
genetic disorders. Despite regionally localizing a gene by mapping,
the determination of the precise localization and sequence of one
gene nonetheless requires choosing the certain gene from about 250
genes, four to about five million base pairs, from within the
regionally localized chromosomal site.
[0006] While advancements have been made as described above,
mutations in retina-specific ABC transporter (ABCR) in patients
with recessive macular dystrophy STGD/FFM have not yet been
identified to Applicants' knowledge. That ABCR expression is
limited to photoreceptors, as determined by the present invention,
provides evidence as to why ABCR has not yet been sequenced.
Further, the ABC1 subfamily of ABC transporters is not represented
by any homolog in yeast (Michaelis and Berkower, 1995), suggesting
that these genes evolved to perform specialized functions in
multicellular organisms, which also lends support to why the ABCR
gene has been difficult to identify. Unlike ABC genes in bacteria,
the homologous genes in higher eukaryotes are much less well
studied. The fact that prokaryotes contain a large number of ABC
genes suggests that many mammalian members of the superfamily
remain uncharacterized. The task of studying eukaryote ABC genes is
more difficult because of the significantly higher complexity of
eukaryotic systems and the apparent difference in function of even
highly homologous genes. While ABC proteins are the principal
transporters of a number of diverse compounds in bacterial cells,
in contrast, eukaryotes have evolved other mechanisms for the
transport of many amino acids and sugars. Eukaryotes have other
reasons to diversify the role of ABC genes, for example, performing
such functions as ion transport, toxin elimination, and secretion
of signaling molecules.
[0007] Accordingly, there remains a need for the identification of
the sequence of the gene, which in mutated forms is associated with
retinal and/or macular degenerative diseases, including Stargardt
Disease and Fundus Flavimaculatus, for example, in order to provide
enhanced diagnoses and improved prognoses and interventional
therapies for individuals affected with such diseases.
SUMMARY OF THE INVENTION
[0008] The present invention provides sequences encoding an ATP
binding cassette transporter. Nucleic acid sequences, including SEQ
ID NO: 1 which is a genomic sequence, and SEQ ID NOS: 2 and 5 which
are cDNA sequences, are sequences to which the present invention is
directed.
[0009] A further aspect of the present invention provides ATP
binding cassette transporter polypeptides and/or proteins. SEQ ID
NOS: 3 and 6 are novel polypeptides of the invention produced from
nucleotide sequences encoding the ATP binding cassette transporter.
Also within the scope of the present invention is a purified ATP
binding cassette transporter.
[0010] The present invention also provides an expression vector
comprising a nucleic acid sequence encoding an ATP binding cassette
transporter, a transformed host cell capable of expressing a
nucleic acid sequence encoding an ATP binding cassette transporter,
a cell culture capable of expressing an ATP binding cassette
transporter, and a protein preparation comprising an ATP binding
cassette transporter.
[0011] The present invention is also directed to a method of
screening for an agent that modifies ATP binding cassette
transporter comprising combining purified ATP binding cassette
transporter with an agent suspected of modifying ATP binding
cassette transporter and observing a change in at least one
characteristic associated with ATP binding cassette transporter.
The present invention provides methods of identifying an agent that
inhibits macular degeneration comprising combining purified ATP
binding cassette transporter from a patient suspected of having
macular degeneration and an agent suspected interacting with the
ATP binding cassette transporter and observing an inhibition in at
least one of the characteristics of diseases associated with the
ATP binding cassette transporter. In addition, the present
invention provides for methods of identifying an agent that induces
onset of at least one characteristic associated with ATP binding
cassette transporter comprising combining purified wild-type ATP
binding cassette transporter with an agent suspected of inducing a
macular degenerative disease and observing the onset of a
characteristic associated with macular degeneration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1A and 1B display the ABCR gene and amplification
products. FIG. 1A displays a physical map of the ABCR gene.
Mega-YAC clones from the CEPH mega-YAC genomic library
(Bellane-Chantelot et al., 1992) encompassing the 4 cM critical
region for STGD are represented by horizontal bars with shaded
circles indicating confirmed positives for STSs by landmark
mapping. The individual STS markers and their physical order are
shown below the YACs with arrows indicating the centromeric (cen)
and telomeric (1pter) direction (Anderson et al., 1995). The
horizontal double head arrow labeled STGD indicates the refined
genetic interval delineated by historical recombinants (Anderson et
al., 1995). FIG. 1B displays the results of agarose gel
electrophoresis of PCR amplification products with primers from the
5' (GGTCTTCGTGTGTGGTCATT, SEQ ID NO: 114, GGTCCAGTTCTTCCAGAG, SEQ
ID NO: 115, labeled 5' ABCR) or 3' (ATCCTCTGACTCAGCAATCACA, SEQ ID
NO:116, TTGCAATTACAAATGCAATGG, SEQ ID NO: 117, labeled 3' ABCR)
regions of ABCR on the 13 different YAC DNA templates indicated as
diagonals above the gel. The asterisk denotes that YAC
680_b.sub.--5 was positive for the 5' ABCR PCR but negative for the
3' ABCR PCR. These data suggest the ABCR gene maps within the
interval delineated by markers D1S3361-D1S236 and is transcribed
toward the telomere, as depicted by the open horizontal box.
[0013] FIG. 2 exhibits the size and tissue distribution of ABCR
transcripts in the adult rat. A blot of total RNA from the
indicated tissues was hybridized with a 1.6 kb mouse Abcr probe
(top) and a ribosomal protein S26 probe (bottom; Kuwano et al.,
1985). The ABCR probe revealed a predominant transcript of
approximately 8 kb that is found in retina only. The mobility of
the 28S and 18S ribosomal RNAs are indicated at the right. B,
brain; H, heart; K, kidney; Li, liver; Lu, lung; R, retina; S,
spleen.
[0014] FIGS. 3A-H show the sequence of the ABCR coding region
within the genomic ABCR sequence, SEQ ID NO: 1. The sequence of the
ABCR cDNA, SEQ ID NO: 2, is shown with the predicted protein
sequence, SEQ ID NO: 3, in one-letter amino acid code below. The
location of splice sites is shown by the symbol |.
[0015] FIGS. 4A-D display the alignment of the ABCR protein, SEQ ID
NO:3, with other members of the ABC1 subfamily. The deduced amino
acid sequence of ABCR is shown aligned to known human and mouse
proteins that are members of the same subfamily. Mouse Abc1 (SEQ ID
NO:118); Abc2; mouse Abc2 (SEQ ID NO:119); and ABCC, human ABC gene
(SEQ ID NO:120). The Walker A and B motifs and the Signature C
motif are designated by underlining and the letters A, B, and C,
respectively.
[0016] FIG. 5 exhibits the location of Abcr from a Jackson BSS
Backcross showing a portion of mouse chromosome 3. The map is
depicted with the centromere toward the top. A 3 cM scale bar is
also shown. Loci mapping to the same position are listed in
alphabetical order.
[0017] FIG. 6 shows the segregation of SSCP variants in exon 49 of
the ABCR gene in kindred AR293. Sequence analysis of SSCP bands
revealed the existence of wild-type sequence (bands 1 and 3) and
mutant sequence (bands 2 and 4). DNA sequencing revealed a 15 base
pair deletion, while the affected children (lanes 2 and 3) are
homozygous. Haplotype analysis demonstrated homozygosity at the
STGD locus in the two affected individuals.
[0018] FIGS. 7A-H show the localization of ABCR transcripts to
photoreceptor cells. In situ hybridization was performed with
digoxygenin-labeled riboprobes and visualized using an alkaline
phosphatase conjugated anti-digoxygenin antibody. FIGS. 7A-D
display hybridization results of retina and choroid from a
pigmented mouse (C57/B16); FIGS. 7E and 7F show hybridization
results of retina and choroid from an albino rat; and FIGS. 7G and
7H exhibit hybridization results of retina from a macaque monkey.
FIGS. 7A, 7E, and 7G display results from a mouse abcr antisense
probe; FIG. 7B exhibits results from a mouse abcr sense probe; FIG.
7C shows results from a macaque rhodopsin antisense probe; and
FIGS. 7D, 7F, and 7H display results from a mouse blue cone pigment
antisense probe. ABCR transcripts are localized to the inner
segments of the photoreceptor cell layer, a pattern that matches
the distribution of rhodopsin transcripts but is distinct from the
distribution of cone visual pigment transcripts. Hybridization is
not observed in the RPE or choroid, as seen most clearly in the
albino rat eye (arrowhead in FIG. 7E). The retinal layers indicated
in FIG. 7B are: OS, outer segments; IS, inner segments; ONL, outer
nuclear layer; OPL, outer plexiform layer; INL, inner nuclear
layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
[0019] FIG. 8 provides a pGEM..RTM.-T Vector map.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0020] The present invention is directed to the nucleic acid and
protein sequences encoding ATP binding cassette transporter. The
ATP binding cassette transporter of the present invention is retina
specific ATP binding cassette transporter (ABCR); more
particularly, ABCR may be isolated from retinal cells, preferably
photoreceptor cells. The present invention provides nucleotide
sequences of ABCR including genomic sequences, SEQ ID NO: 1, and
cDNA sequences SEQ ID NO: 2 and 5. Novel polypeptide sequences, SEQ
ID NOS: 3 and 6, for ABCR, and the translated products of SEQ ID
NOS: 2 and 5, respectively, and are also included in the present
invention.
[0021] SEQ ID NO: 1 provides the human genomic DNA sequence of
ABCR. SEQ ID NOS: 2 and 5 provide wild-type cDNA sequences of human
ABCR, which result in translated products SEQ ID NOS: 3 and 6,
respectively. While not intending to be bound by any particular
theory or theories of operation, it is believed that SEQ ID NOS: 2
and 5 are isoforms of ABCR cDNA. The difference between SEQ ID NOS:
2 and 5 may be accounted for by an additional sequence in SEQ ID
NO: 2 which is added between bases 4352 and 4353 of SEQ ID NO: 5.
This difference is thought to arise from alternative splicing of
the nascent transcript of ABCR, in which an alternative exon 30,
SEQ ID NO: 4, is excluded. This alternative exon encodes an
additional 38 amino acids, SEQ ID NO: 11.
[0022] Nucleic acids within the scope of the present invention
include cDNA, RNA, genomic DNA, fragments or portions within the
sequences, antisense oligonucleotides. Sequences encoding the ABCR
also include amino acid, polypeptide, and protein sequences.
Variations in the nucleic acid and polypeptide sequences of the
present invention are within the scope of the present invention and
include N terminal and C terminal extensions, transcription and
translation modifications, and modifications in the cDNA sequence
to facilitate and improve transcription and translation efficiency.
In addition, changes within the wild-type sequences identified
herein which changed sequence retains substantially the same
wild-type activity, such that the changed sequences are
substantially similar to the ABCR sequences identified, are also
considered within the scope of the present invention. Mismatches,
insertions, and deletions which permit substantial similarity to
the ABCR sequences, such as similarity in residues in
hydrophobicity, hydrophilicity, basicity, and acidity, will be
known to those of skill in the art once armed with the present
disclosure. In addition, the isolated, or purified, sequences of
the present invention may be natural, recombinant, synthetic, or a
combination thereof. Wild-type activity associated with the ABCR
sequences of the present invention include, inter alia, all or part
of a sequence, or a sequence substantially similar thereto, that
codes for ATP binding cassette transporter.
[0023] The genomic, SEQ ID NO: 1, and cDNA, SEQ ID NOS: 2 and 5,
sequences are identified in FIG. 3 and encode ABCR, certain
mutations of which are responsible for the class of retinal
disorders known as retinal or macular degenerations. Macular
degeneration is characterized by macular dystrophy, various
alterations of the peripheral retina, central visual impairment,
progressive bilateral atrophy of the macular retinal pigment
epithelium (RPE) and neuroepithelium, frequent appearance of
orange-yellow flecks distributed around the macula and/or the
midretinal periphery, and subretinal deposition of lipofuscin-like
material. Retinal and macular degenerative diseases include and are
not limited to Stargardt Disease, Fundus Flavimaculatus,
age-related macular degeneration, and may include disorders
variously called retinitis pigmentosa, combined rod and cone
dystrophies, cone dystrophies and degenerations, pattern dystrophy,
bull's eye maculopathies, and various other retinal degenerative
disorders, some induced by drugs, toxins, environmental influences,
and the like. Stargardt Disease is an autosomal recessive retinal
disorder characterized by juvenile to adult-onset macular and
retinal dystrophy. Fundus Flavimaculatus often displays later age
of onset and slower progression. Some environmental insults and
drug toxicities may create similar retinal degenerations. Linkage
analysis reveals that Stargardt Disease and Fundus Flavimaculatus
may be allelic autosomal recessive disorders with slightly
different clinical manifestations. The identification of the ABCR
gene suggests that different mutations within ABCR may be
responsible for these clinical phenomena.
[0024] The present invention is also directed to a method of
screening for an agent that modifies ATP binding cassette
transporter comprising combining purified ATP binding cassette
transporter with an agent of modifying ATP binding cassette
transporter and observing a change in at least one characteristic
associated with ATP binding cassette transporter.
[0025] "Modify" and variations thereof include changes such as and
not limited to inhibit, suppress, delay, retard, slow, suspend,
obstruct, and restrict, as well as induce, encourage, provoke, and
cause. Modified may also be defined as complete inhibition such
that macular degeneration is arrested, stopped, or blocked.
Modifications may, directly or indirectly, inhibit or substantially
inhibit, macular degeneration or induce, or substantially induce,
macular degeneration, under certain circumstances.
[0026] Methods of identifying an agent that inhibits macular
degeneration are embodied by the present invention and comprise
combining purified ATP binding cassette transporter from a patient
suspected of having macular degeneration and an agent suspected of
interacting with the ATP binding cassette transporter and observing
an inhibition in at least one of the characteristics of diseases
associated with the ATP binding cassette transporter. Accordingly,
such methods serve to reduce or prevent macular degeneration, such
as in human patients. In addition, the present invention provides
for methods of identifying an agent that induces onset of at least
one characteristic associated with ATP binding cassette transporter
comprising combining purified wild-type ATP binding cassette
transporter with an agent suspected of inducing a macular
degenerative disease and observing the onset of a characteristic
associated with macular degenerative. Thus, such methods provide
methods of using laboratory animals to determine causative agents
of macular degeneration. The ATP binding cassette transporter may
be provided for in the methods identified herein in the form of
nucleic acids, such as and not limited to SEQ ID NOS: 1, 2, and 5
or as an amino acid, SEQ ID NOS: 3 and 6, for example. Accordingly,
transcription and translation inhibitors may be separately
identified. Characteristics associated with macular degeneration
include and are not limited to central visual impairment,
progressive bilateral atrophy of the macular retinal pigment
epithelium (RPE) and neuroepithelium, and the frequent appearance
of orange-yellow flecks distributed around the macula and/or the
midretinal periphery. Accordingly, observing one or more of the
characteristics set forth above results in identification of an
agent that induces macular degeneration, whereas reduction or
inhibition of at least one of the characteristics results in
identification of an agent that inhibits macular degeneration.
[0027] Mutational analysis of ABCR in Stargardt Disease families
revealed thus far seventy four mutations including fifty four
single amino acid substitutions, five nonsense mutations resulting
in early truncation of the protein, six frame shift mutations
resulting in early truncation of the protein, three in-frame
deletions resulting in loss of amino acid residues from the
protein, and six splice site mutations resulting in incorrect
processing of the nascent RNA transcript, see Table 2. Compound
heterozygotes for mutations in ABCR were found in forty two
families. Homozygous mutations were identified in three families
with consanguineous parentage. Accordingly, mutations in wild-type
ABCR which result in activities that are not associated with
wild-type ABCR are herein referred to as sequences which are
associated with macular degeneration. Such mutations include
missense mutations, deletions, insertions, substantial differences
in hydrophobicity, hydrophilicity, acidity, and basicity.
Characteristics which are associated with retinal or macular
degeneration include and are not limited to those characteristics
set forth above.
[0028] Mutations in wild-type ABCR provide a method of detecting
macular degeneration. Retinal or macular degeneration may be
detected by obtaining a sample comprising patient nucleic acids
from a patient tissue sample; amplifying retina-specific ATP
binding cassette receptor specific nucleic acids from the patient
nucleic acids to produce a test fragment; obtaining a sample
comprising control nucleic acids from a control-tissue sample;
amplifying control nucleic acids encoding wild-type retina-specific
ATP binding cassette receptor to produce a control fragment;
comparing the test fragment with the control fragment to detect the
presence of a sequence difference in the test fragment, wherein a
difference in the test fragment indicates macular degeneration.
Mutations in the test fragment, including and not limited to each
of the mutations identified above, may provide evidence of macular
degeneration.
[0029] A purified ABCR protein is also provided by the present
invention. The purified ABCR protein may have an amino acid
sequence as provided by SEQ ID NOS: 3 and 6.
[0030] The present invention is directed to ABCR sequences obtained
from mammals from the Order Rodentia, including and not limited to
hamsters, rats, and mice; Order Logomorpha, such as rabbits; more
particularly the Order Carnivora, including Felines (cats) and
Canines (dogs); even more particularly the Order Artiodactyla,
Bovines (cows) and Suines (pigs); and the Order Perissodactyla,
including Equines (horses); and most particularly the Order
Primates, Ceboids and Simoids (monkeys) and Anthropoids (humans and
apes). The mammals of most preferred embodiments are humans.
[0031] Generally, the sequences of the invention may be produced in
host cells transformed with an expression vector comprising a
nucleic acid sequence encoding ABCR. The transformed cells are
cultured under conditions whereby the nucleic acid sequence coding
for ABCR is expressed. After a suitable amount of time for the
protein to accumulate, the protein may be purified from the
transformed cells.
[0032] A gene coding for ABCR may be obtained from cDNA library.
Suitable libraries can be obtained from commercial sources such as
Clontech, Palo Alto, Calif. Libraries may also be prepared using
the following non-limiting examples: hamster insulin-secreting
tumor (HIT), mouse .alpha.TC-6, and rat insulinoma (RIN) cells.
Positive clones are then subjected to DNA sequencing to determine
the presence of a DNA sequence coding for ABCR. DNA sequencing is
accomplished using the chain termination method of Sanger et al.,
Proc. Nat'l. Acad. Sci. U.S.A., 1977, 74, 5463. The DNA sequence
encoding ABCR is then inserted into an expression vector for later
expression in a host cell.
[0033] Expression vectors and host cells are selected to form an
expression system capable of synthesizing ABCR. Vectors including
and not limited to baculovirus vectors may be used in the present
invention. Host cells suitable for use in the invention include
prokaryotic and eukaryotic cells that can be transformed to stably
contain and express ABCR. For example, nucleic acids coding for the
recombinant protein may be expressed in prokaryotic or eukaryotic
host cells, including the most commonly used bacterial host cell
for the production of recombinant proteins, E. coli. Other
microbial strains may also be used, however, such as Bacillus
subtilis, and other enterobacteriaceae such as Salmonella
typhimurium or Serratia marcescens, various species of Pseudomonas,
or other bacterial strains.
[0034] The preferable eukaryotic system is yeast, such as
Saccharomyces cerevisiae. Yeast artificial chromosome (YAC) systems
are able to accommodate the large size of ABCR gene sequence or
genomic clone. The principle of the YAC system is similar to that
used in conventional cloning of DNA. Large fragments of cDNA are
ligated into two "arms" of a YAC vector, and the ligation mixture
is then introduced into the yeast by transformation. Each of the
arms of the YAC vector carries a selectable marker as well as
appropriately oriented sequences that function as telomeres in
yeast. In addition, one of the two arms carries two small fragments
that function as a centromere and as an origin of replication (also
called an ARS element-autonomously replicating sequences). Yeast
transformants that have taken up and stably maintained an
artificial chromosome are identified as colonies on agar plates
containing the components necessary for selection of one or both
YAC arms. YAC vectors are designed to allow rapid identification of
transformants that carry inserts of genomic DNA. Insertion of
genomic DNA into the cloning site interrupts a suppressor tRNA gene
and results in the formation of red rather than white colonies by
yeast strains that carry an amber ade2 gene.
[0035] To clone in YAC vectors, genomic DNA from the test organism
is prepared under conditions that result in relatively little
shearing such that its average size is several million base pairs.
The cDNA is then ligated to the arms of the YAC vector, which has
been appropriately prepared to prevent self-ligation. As an
alternative to partial digestion with EcoRI, YAC vectors may be
used that will accept genomic DNA that has been digested to
completion with rarely cutting restriction enzymes such as NotI or
MluI.
[0036] In addition, insect cells, such as Spodoptera frugiperda;
chicken cells, such as E3C/O and SL-29; mammalian cells, such as
HeLa, Chinese hamster ovary cells (CHO), COS-7 or MDCK cells and
the like may also be used. The foregoing list is illustrative only
and is not intended in any way to limit the types of host cells
suitable for expression of the nucleic acid sequences of the
invention.
[0037] As used herein, expression vectors refer to any type of
vector that can be manipulated to contain a nucleic acid sequence
coding for ABCR, such as plasmid expression vectors, viral vectors,
and yeast expression vectors. The selection of the expression
vector is based on compatibility with the desired host cell such
that expression of the nucleic acid encoding ABCR results. Plasmid
expression vectors comprise a nucleic acid sequence of the
invention operably linked with at least one expression control
element such as a promoter. In general, plasmid vectors contain
replicon and control sequences derived from species compatible with
the host cell. To facilitate selection of plasmids containing
nucleic acid sequences of the invention, plasmid vectors may also
contain a selectable marker such as a gene coding for antibiotic
resistance. Suitable examples include the genes coding for
ampicillin, tetracycline, chloramphenicol, or kanamycin
resistance.
[0038] Suitable expression vectors, promoters, enhancers, and other
expression control elements are known in the art and may be found
in Sambrook et al., Molecular Cloning: A Laboratory Manual, second
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989), incorporated herein by reference in its entirety.
[0039] Transformed host cells containing a DNA sequence encoding
ABCR may then be grown in an appropriate medium for the host. The
cells are then grown until product accumulation reaches desired
levels at which time the cells are then harvested and the protein
product purified in accordance with conventional techniques.
Suitable purification methods include, but are not limited to, SDS
PAGE electrophoresis, phenylboronate-agarose, reactive green
19-agarose, concanavalin A sepharose, ion exchange chromatography,
affinity chromatography, electrophoresis, dialysis and other
methods of purification known in the art.
[0040] Protein preparations, of purified or unpurified ABCR by host
cells, are accordingly produced which comprise ABCR and other
material such as host cell components and/or cell medium, depending
on the degree of purification of the protein.
[0041] The invention also includes a transgenic non-human animal,
including and not limited to mammals, such as and not limited to a
mouse, rat, or hamster, comprising a sequence encoding ABCR, or
fragment thereof that substantially retains ABCR activity,
introduced into the animal or an ancestor of the animal. The
sequence may be wild-type or mutant and may be introduced into the
animal at the embryonic or adult stage. The sequence is
incorporated into the genome of an animal such that it is
chromosomally incorporated into an activated state. A transgenic
non-human animal has germ cells and somatic cells that contain an
ABCR sequence. Embryo cells may be transfected with the gene as it
occurs naturally, and transgenic animals are selected in which the
gene has integrated into the chromosome at a locus which results in
activation. Other activation methods include modifying the gene or
its control sequences prior to introduction into the embryo. The
embryo may be transfected using a vector containing the gene.
[0042] In addition, a transgenic non-human animal may be engineered
wherein ABCR is suppressed. For purposes of the present invention,
suppression of ABCR includes, and is not limited to strategies
which cause ABCR not to be expressed. Such strategies may include
and are not limited to inhibition of protein synthesis, pre-mRNA
processing, or DNA replication. Each of the above strategies may be
accomplished by antisense inhibition of ABCR gene expression. Many
techniques for transferring antisense sequences into cells are
known to those of skill, including and not limited to
microinjection, viral-mediated transfer, somatic cell
transformation, transgene integration, and the like, as set forth
in Pinkert, Carl, Transgenic Animal Technology, 1994, Academic
Press, Inc., San Diego, Calif., incorporated herein by reference in
its entirety.
[0043] Further, a transgenic non-human animal may be prepared such
that ABCR is knocked out. For purposes of the present invention, a
knock-out includes and is not limited to disruption or rendering
null the ABCR gene. A knock-out may be accomplished, for example,
with antisense sequences for ABCR. The ABCR gene may be knocked out
by injection of an antisense sequence for all or part of the ABCR
sequence such as an antisense sequence for all or part of SEQ ID
NO: 2. Once ABCR has been rendered null, correlation of the ABCR to
macular degeneration may be tested. Sequences encoding mutations
affecting the ABCR may be inserted to test for alterations in
various retinal and macular degenerations exhibited by changes in
the characteristics associated with retinal and macular
degeneration.
[0044] An ABCR knock-out may be engineered by inserting synthetic
DNA into the animal chromosome by homologous recombination. In this
method, sequences flanking the target and insert DNA are identical,
allowing strand exchange and crossing over to occur between the
target and insert DNA. Sequences to be inserted typically include a
gene for a selectable marker, such as drug resistance. Sequences to
be targeted are typically coding regions of the genome, in this
case part of the ABCR gene. In this process of homologous
recombination, targeted sequences are replaced with insert
sequences thus disrupting the targeted gene and rendering it
nonfunctional. This nonfunctional gene is called a null allele of
the gene.
[0045] To create the knockout mouse, a DNA construct containing the
insert DNA and flanking sequences is made. This DNA construct is
transfected into pluripotent embryonic stem cells competent for
recombination. The identical flanking sequences align with one
another, and chromosomal recombination occurs in which the targeted
sequence is replaced with the insert sequence, as described in
Bradley, A., Production and Analysis of Chimeric Mice, in
Tetracarcinomas and Embryonic Stem Cells--A Practical Approach,
1987, E. Roberson, Editor, IRC Press, pages 113-151. The stem cells
are injected into an embryo, which is then implanted into a female
animal and allowed to be born. The animals may contain germ cells
derived from the injected stem cells, and subsequent matings may
produce animals heterozygous and homozygous for the disrupted
gene.
[0046] Transgenic non-human animals may also be useful for testing
nucleic acid changes to identify additional mutations responsible
for macular degeneration. A transgenic non-human animal may
comprise a recombinant ABCR.
[0047] The present invention is also directed to gene therapy. For
purposes of the present invention, gene therapy refers to the
transfer and stable insertion of new genetic information into cells
for the therapeutic treatment of diseases or disorders. A foreign
sequence or gene is transferred into a cell that proliferates to
spread the new sequence or gene throughout the cell population.
Sequences include antisense sequence of all or part of ABCR, such
as an antisense sequence to all or part of the sequences identified
as SEQ ID NO: 1, 2, and 5. Known methods of gene transfer include
microinjection, electroporation, liposomes, chromosome transfer,
transfection techniques, calcium-precipitation transfection
techniques, and the like. In the instant case, macular degeneration
may result from a loss of gene function, as a result of a mutation
for example, or a gain of gene function, as a result of an extra
copy of a gene, such as three copies of a wild-type gene, or a gene
over expressed as a result of a mutation in a promoter, for
example. Expression may be altered by activating or deactivating
regulatory elements, such as a promoter. A mutation may be
corrected by replacing the mutated sequence with a wild-type
sequence or inserting an antisense sequence to bind to an over
expressed sequence or to a regulatory sequence.
[0048] Numerous techniques are known in the art for the
introduction of foreign genes into cells and may be used to
construct the recombinant cells for purposes of gene therapy, in
accordance with this embodiment of the invention. The technique
used should provide for the stable transfer of the heterologous
gene sequence to the stem cell, so that the heterologous gene
sequence is heritable and expressible by stem cell progeny, and so
that the necessary development and physiological functions of the
recipient cells are not disrupted. Techniques which may be used
include but are not limited to chromosome transfer (e.g., cell
fusion, chromosome-mediated gene transfer, micro cell-mediated gene
transfer), physical methods (e.g., transfection, spheroplast
fusion, microinjection, electroporation, liposome carrier), viral
vector transfer (e.g., recombinant DNA viruses, recombinant RNA
viruses) and the like (described in Cline, M. J., 1985, Pharmac.
Ther. 29:69-92, incorporated herein by reference in its
entirety).
[0049] The term "purified", when used to describe the state of
nucleic acid sequences of the invention, refers to nucleic acid
sequences substantially free of nucleic acid not coding for ABCR or
other materials normally associated with nucleic acid in
non-recombinant cells, i.e., in its "native state."
[0050] The term "purified" or "in purified form" when used to
describe the state of an ABCR nucleic acid, protein, polypeptide,
or amino acid sequence, refers to sequences substantially free, to
at least some degree, of cellular material or other material
normally associated with it in its native state. Preferably the
sequence has a purity (homogeneity) of at least about 25% to about
100%. More preferably the purity is at least about 50%, when
purified in accordance with standard techniques known in the
art.
[0051] In accordance with methods of the present invention, methods
of detecting retinal or macular degenerations in a patient are
provided comprising obtaining a patient tissue sample for testing.
The tissue sample may be solid or liquid, a body fluid sample such
as and not limited to blood, skin, serum, saliva, sputum, mucus,
bone narrow, urine, lymph, and a tear; and feces. In addition, a
tissue sample from amniotic fluid or chorion may be provided for
the detection of retinal or macular degeneration in utero in
accordance with the present invention.
[0052] A test fragment is defined herein as an amplified sample
comprising ABCR-specific nucleic acids from a patient suspected of
having retinal or macular degeneration. A control fragment is an
amplified sample comprising normal or wild-type ABCR-specific
nucleic acids from an individual not suspected of having retinal or
macular degeneration.
[0053] The method of amplifying nucleic acids may be the polymerase
chain reaction using a pair of primers wherein at least one primer
within the pair is selected from the group consisting of SEQ ID
NOS: 12-113. When the polymerase chain reaction is the
amplification method of choice, a pair of primers may be used such
that one primer of the pair is selected from the group consisting
of SEQ ID NOS: 12-113.
[0054] Nucleic acids, such as DNA (such as and not limited to,
genomic DNA and cDNA) and/or RNA (such as, and not limited to,
mRNA), are obtained from the patient sample. Preferably RNA is
obtained.
[0055] Nucleic acid extraction is followed by amplification of the
same by any technique known in the art. The amplification step
includes the use of at least one primer sequence which is
complementary to a portion of ABCR-specific expressed nucleic acids
or sequences on flanking intronic genomic sequences in order to
amplify exon or coding sequences. Primer sequences useful in the
amplification methods include and are not limited to SEQ ID NOS:
12-113, which may be used in the amplification methods. Any primer
sequence of about 10 nucleotides to about 35 nucleotides, more
preferably about 15 nucleotides to about 30 nucleotides, even more
preferably about 17 nucleotides to about 25 nucleotides may be
useful in the amplification step of the methods of the present
invention. In addition, mismatches within the sequences identified
above, which achieve the methods of the invention, such that the
mismatched sequences are substantially complementary and thus
hybridizable to the sequence sought to be identified, are also
considered within the scope of the disclosure. Mismatches which
permit substantial similarity to SEQ ID NOS: 12-113, such as and
not limited to sequences with similar hydrophobicity,
hydrophilicity, basicity, and acidity, will be known to those of
skill in the art once armed with the present disclosure. The
primers may also be unmodified or modified. Primers may be prepared
by any method known in the art such as by standard phosphoramidite
chemistry. See Sambrook et al., supra.
[0056] The method of amplifying nucleic acids may be the polymerase
chain reaction using a pair of primers wherein at least one primer
within the pair is selected from the group consisting of SEQ ID
NOS: 12-113. When the polymerase chain reaction is the
amplification method of choice, a pair of primers may be used such
that one primer of the pair is selected from the group consisting
of SEQ ID NOS: 12-113.
[0057] When an amplification method includes the use of two
primers, a first primer and a second primer, such as in the
polymerase chain reaction, one of the first primer or second primer
may be selected from the group consisting of SEQ ID NOS: 12-113.
Any primer pairs which copy and amplify nucleic acids between the
pairs pointed toward each other and which are specific for ABCR may
be used in accordance with the methods of the present
invention.
[0058] A number of template dependent processes are available to
amplify the target sequences of interest present in a sample. One
of the best known amplification methods is the polymerase chain
reaction (PCR) which is described in detail in U.S. Pat. Nos.
4,683,195, 4,683,202 and 4,800,159, and in Innis et al., PCR
Protocols, Academic Press, Inc., San Diego Calif., 1990, each of
which is incorporated herein by reference in its entirety. Briefly,
in PCR, two primer sequences are prepared which are complementary
to regions on opposite complementary strands of the target
sequence. An excess of deoxynucleoside triphosphates are added to a
reaction mixture along with a DNA polymerase (e.g., Taq
polymerase). If the target sequence is present in a sample, the
primers will bind to the target and the polymerase will cause the
primers to be extended along the target sequence by adding on
nucleotides. By raising and lowering the temperature of the
reaction mixture, the extended primers will dissociate from the
target to form reaction products, excess primers will bind to the
target and to the reaction products and the process is repeated.
Alternatively, a reverse transcriptase PCR amplification procedure
may be performed in order to quantify the amount of mRNA amplified.
Polymerase chain reaction methodologies are well known in the
art.
[0059] Another method for amplification is the ligase chain
reaction (referred to as LCR), disclosed in EPA No. 320,308,
incorporated herein by reference in its entirety. In LCR, two
complementary probe pairs are prepared, and in the presence of the
target sequence, each pair will bind to opposite complementary
strands of the target such that they abut. In the presence of a
ligase, the two probe pairs will link to form a single unit. By
temperature cycling, as in PCR, bound ligated units dissociate from
the target and then serve as "target sequences" for ligation of
excess probe pairs. U.S. Pat. No. 4,883,750, incorporated herein by
reference in its entirety, describes an alternative method of
amplification similar to LCR for binding probe pairs to a target
sequence.
[0060] Qbeta Replicase, described in PCT Application No.
PCT/US87/00880, incorporated herein by reference in its entirety,
may also be used as still another amplification method in the
present invention. In this method, a replicative sequence of RNA
which has a region complementary to that of a target is added to a
sample in the presence of an RNA polymerase. The polymerase will
copy the replicative sequence which can then be detected.
[0061] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'-[alpha-thio]triphosphates in one strand of a restriction site
(Walker, G. T., et al., Proc. Natl. Acad, Sci. (U.S.A.) 1992,
89:392-396, incorporated herein by reference in its entirety), may
also be useful in the amplification of nucleic acids in the present
invention.
[0062] Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis, i.e.
nick translation. A similar method, called Repair Chain Reaction
(RCR) is another method of amplification which may be useful in the
present invention and which involves annealing several probes
throughout a region targeted for amplification, followed by a
repair reaction in which only two of the four bases are present.
The other two bases can be added as biotinylated derivatives for
easy detection. A similar approach is used in SDA.
[0063] ABCR-specific nucleic acids can also be detected using a
cyclic probe reaction (CPR). In CPR, a probe having a 3' and 5'
sequences of non-ABCR specific DNA and middle sequence of ABCR
specific RNA is hybridized to DNA which is present in a sample.
Upon hybridization, the reaction is treated with RNaseH, and the
products of the probe identified as distinctive products, generate
a signal which is released after digestion. The original template
is annealed to another cycling probe and the reaction is repeated.
Thus, CPR involves amplifying a signal generated by hybridization
of a probe to a ABCR-specific expressed nucleic acid.
[0064] Still other amplification methods described in GB
Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025, each of which is incorporated by reference in its
entirety, may be used in accordance with the present invention. In
the former application, "modified" primers are used in a PCR like,
template and enzyme dependent synthesis. The primers may be
modified by labeling with a capture moiety (e.g., biotin) and/or a
detector moiety (e.g., enzyme). In the latter application, an
excess of labeled probes are added to a sample. In the presence of
the target sequence, the probe binds and is cleaved catalytically.
After cleavage, the target sequence is released intact to be bound
by excess probe. Cleavage of the labeled probe signals the presence
of the target sequence.
[0065] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS) (Kwoh D., et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 1989, 86:1173, Gingeras T. R., et
al., PCT Application WO 88/10315, each of which is incorporated
herein by reference in its entirety), including nucleic acid
sequence based amplification (NASBA) and 3SR. In NASBA, the nucleic
acids can be prepared for amplification by standard
phenol/chloroform extraction, heat denaturation of a clinical
sample, treatment with lysis buffer and minispin columns for
isolation of DNA and RNA or guanidinium chloride extraction of RNA.
These amplification techniques involve annealing a primer which has
ABCR-specific sequences. Following polymerization, DNA/RNA hybrids
are digested with RNase H while double standard DNA molecules are
heat denatured again. In either case the single stranded DNA is
made fully double stranded by addition of second ABCR-specific
primer, followed by polymerization. The double standard DNA
molecules are then multiply transcribed by a polymerase such as T7
or SP6. In an isothermal cyclic reaction, the RNAs are reverse
transcribed into double stranded DNA, and transcribed once again
with a polymerase such as T7 or SP6. The resulting products,
whether truncated or complete, indicate ABCR-specific
sequences.
[0066] Davey, C., et al., European Patent Application Publication
No. 329,822, incorporated herein by reference in its entirety,
disclose a nucleic acid amplification process involving cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and
double-stranded DNA ("dsDNA") which may be used in accordance with
the present invention. The ssRNA is a first template for a first
primer oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from
resulting DNA:RNA duplex by the action of ribonuclease H(RNase H,
an RNase specific for RNA in a duplex with either DNA or RNA). The
resultant ssDNA is a second template for a second primer, which
also includes the sequences of an RNA polymerase promoter
(exemplified by T7 RNA polymerase) 5' to its homology to its
template. This primer is then extended by DNA polymerase
(exemplified by the large "Klenow" fragment of E. coli DNA
polymerase I), resulting as a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA
between the primers and having additionally, at one end, a promoter
sequence. This promoter sequence can be used by the appropriate RNA
polymerase to make many RNA copies of the DNA. These copies can
then re-enter the cycle leading to very swift amplification. With
proper choice of enzymes, this amplification can be done
isothermally without addition of enzymes at each cycle. Because of
the cyclical nature of this process, the starting sequence can be
chosen to be in the form of either DNA or RNA.
[0067] Miller, H. I., et al., PCT application WO 89/06700,
incorporated herein by reference in its entirety, disclose a
nucleic acid sequence amplification scheme based on the
hybridization of a promoter/primer sequence to a target
single-stranded DNA ("ssDNA") followed by transcription of many RNA
copies of the sequence. This scheme is not cyclic; i.e. new
templates are not produced from the resultant RNA transcripts.
Other amplification methods include "race" disclosed by Frohman, M.
A. In: PCR Protocols: A Guide to Methods and Applications 1990,
Academic Press, N.Y.) and "one-sided PCR" (Ohara, O., et al., Proc.
Natl. Acad. Sci. (U.S.A.) 1989, 86:5673-5677), all references
herein incorporated by reference in their entirety.
[0068] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide (Wu, D. Y. et al., Genomics 1989, 4:560,
incorporated herein by reference in its entirety), may also be used
in the amplification step of the present invention.
[0069] Test fragment and control fragment may be amplified by any
amplification methods known to those of skill in the art, including
and not limited to the amplification methods set forth above. For
purposes of the present invention, amplification of sequences
encoding patient and wild-type ABCR includes amplification of a
portion of a sequence such as and not limited to a portion of an
ABCR sequence of SEQ ID NO:1, such as sequence of a length of about
10 nucleotides to about 1,000 nucleotides, more preferably about 10
nucleotides to about 100 nucleotides, or having at least 10
nucleotides occurring anywhere within the SEQ ID NO:1, where
sequence differences are known to occur within ABCR test fragments.
Thus, for example, a portion of the sequence encoding ABCR of a
patient sample and a control sample may be amplified to detect
sequence differences between these two sequences.
[0070] Following amplification of the test fragment and control
fragment, comparison between the amplification products of the test
fragment and control fragment is carried out. Sequence changes such
as and not limited to nucleic acid transition, transversion, and
restriction digest pattern alterations may be detected by
comparison of the test fragment with the control fragment.
[0071] Alternatively, the presence or absence of the amplification
product may be detected. The nucleic acids are fragmented into
varying sizes of discrete fragments. For example, DNA fragments may
be separated according to molecular weight by methods such as and
not limited to electrophoresis through an agarose gel matrix. The
gels are then analyzed by Southern hybridization. Briefly, DNA in
the gel is transferred to a hybridization substrate or matrix such
as and not limited to a nitrocellulose sheet and a nylon membrane.
A labeled probe encoding an ABCR mutation is applied to the matrix
under selected hybridization conditions so as to hybridize with
complementary DNA localized on the matrix. The probe may be of a
length capable of forming a stable duplex. The probe may have a
size range of about 200 to about 10,000 nucleotides in length,
preferably about 500 nucleotides in length, and more preferably
about 2,454 nucleotides in length. Mismatches which permit
substantial similarity to the probe, such as and not limited to
sequences with similar hydrophobicity, hydrophilicity, basicity,
and acidity, will be known to those of skill in the art once armed
with the present disclosure. Various labels for visualization or
detection are known to those of skill in the art, such as and not
limited to fluorescent staining, ethidium bromide staining for
example, avidin/biotin, radioactive labeling such as .sup.32P
labeling, and the like. Preferably, the product such as the PCR
product, may be run on an agarose gel and visualized using a stain
such as ethidium bromide. See Sambrook et al., supra. The matrix
may then be analyzed by autoradiography to locate particular
fragments which hybridize to the probe. Yet another alternative is
the sequencing of the test fragment and the control fragment to
identify sequence differences. Methods of nucleic acid sequencing
are known to those of skill in the art, including and not limited
to the methods of Maxam and Gilbert, Proc. Natl. Acad. Sci. USA
1977, 74, 560-564 and Sanger, Proc. Natl. Acad. Sci., USA 1977, 74,
5463-5467.
[0072] A pharmaceutical composition comprising all or part of a
sequence for ABCR may be delivered to a patient suspected of having
retinal or macular degeneration. The sequence may be an antisense
sequence. The composition of the present invention may be
administered alone or may generally be administered in admixture
with a pharmaceutical carrier. The pharmaceutically-acceptable
carrier may be selected with regard to the intended route of
administration and the standard pharmaceutical practice. The dosage
will be about that of the sequence alone and will be set with
regard to weight, and clinical condition of the patient. The
proportional ratio of active ingredient to carrier will naturally
depend, inter alia, on the chemical nature, solubility, and
stability of the sequence, as well as the dosage contemplated.
[0073] The sequences of the invention may be employed in the method
of the invention singly or in combination with other compounds,
including and not limited to other sequences set forth in the
present invention. The method of the invention may also be used in
conjunction with other treatments such as and not limited to
antibodies, for example. For in vivo applications the amount to be
administered will also depend on such factors as the age, weight,
and clinical condition of the patient. The composition of the
present invention may be administered by any suitable route,
including as an eye drop, inoculation and injection, for example,
intravenous, intraocular, oral, intraperitoneal, intramuscular,
subcutaneous, topically, and by absorption through epithelial or
mucocutaneous linings, for example, conjunctival, nasal, oral,
vaginal, rectal and gastrointestinal.
[0074] The mode of administration of the composition may determine
the sites in the organism to which the compound will be delivered.
For instance, topical application may be administered in creams,
ointments, gels, oils, emulsions, pastes, lotions, and the like.
For parenteral administration, the composition may be used in the
form of sterile aqueous or non-aqueous solution which may contain
another solute, for example, sufficient salts, glucose or dextrose
to make the solution isotonic. A non-aqueous solution may comprise
an oil, for example. For oral mode of administration, the present
invention may be used in the form of tablets, capsules, lozenges,
troches, powders, syrups, elixirs, aqueous solutions and
suspension, and the like. Various disintegrants, such as starch,
and lubricating agents may be used. For oral administration in
capsule form, useful diluents are lactose and high molecular weight
polyethylene glycols. When aqueous suspensions are required for
oral use, certain sweetening and/or flavoring agents may be
added.
[0075] A diagnostic kit for detecting retinal or macular
degeneration comprising in one or more containers at least one
primer which is complementary to an ABCR sequence and a means for
visualizing amplified DNA is also within the scope of the present
invention. Alternatively, the kit may comprise two primers. In
either case, the primers may be selected from the group consisting
of SEQ ID NOS:12-113, for example. The diagnostic kit may comprise
a pair of primers wherein one primer within said pair is
complementary to a region of the ABCR gene, wherein one of said
pair of primers is selected from the group consisting of SEQ ID NO:
12-113, a probe specific to the amplified product, and a means for
visualizing amplified DNA, and optionally including one or more
size markers, and positive and negative controls. The diagnostic
kit of the present invention may comprise one or more of a
fluorescent dye such as ethidium bromide stain, .sup.32P, and
biotin, as a means for visualizing or detecting amplified DNA.
Optionally the kit may include one or more size markers, positive
and negative controls, restriction enzymes, and/or a probe specific
to the amplified product.
[0076] The following examples are illustrative but are not meant to
be limiting of the invention.
Examples
Identification of the ABCR as a Candidate Gene for STGD
[0077] One of the 21 new human genes from the ABC superfamily,
hereafter called ABCR (retina-specific ABC transporter), was
identified (Allikmets et al. 1996) among expressed sequence tags
(ESTs) obtained from 5,000 human retina cDNA clones (Wang, Y.,
Macke, J. P., Abella, B. S., Andreasson, K., Worley, P., Gilbert,
D. J., Copeland, N. G., Jenkins, N. A., and Nathans, J. (1996)) and
among ESTs obtained from human retina cDNA clones by the I.M.A.G.E.
consortium (Lennon et al., 1996). ABCR is closely related to the
previously described mouse and human ABC1 and ABC2 genes (Luciani
et al., 1994; Allikmets et al., 1995). To determine whether ABCR
might cause a disease, the gene was mapped with a whole genome
radiation hybrid panel (GeneBridge 4; Research Genetics,
Huntsville, Ala.). ABCR mapped to the human chromosome 1p13-p21
region, close to microsatellite markers D1S236 and D1S188. To
define further the location of the gene, PCR primers, 3'UTR-For
5'ATCCTCTGACTCAGCAATCACA, SEQ ID NO: 7, and 3'UTR-Rev
5'TTGCAATTACAAATGCAATGG, SEQ ID NO:8, from the putative 3'
untranslated region were used to screen YACs from the previously
described contig between these anonymous markers (Anderson et al.,
1995). At least 12 YACs contain the 3' end of the ABCR gene,
including 924_e.sub.--9, 759_d.sub.--7, 775_c..sub.--2,
782_b.sub.--4, 982_g.sub.--5, 775_b.sub.--2, 765_a.sub.--3,
751_f.sub.--2, 848_e.sub.--3, 943_h.sub.--8, 934_g.sub.--7, and
944_b.sub.--12 (FIG. 1). These YACs delineate a region containing
the STGD gene between markers D1S3361 and D1S236 (Anderson et al.,
1995).
Expression of the ABCR Gene
[0078] Additional support suggesting that ABCR is a candidate STGD
gene came from expression studies and inspection of the EST
databases.
[0079] Searches of the dbEST (Boguski et al., 1993) database were
performed with BLAST on the NCBI file server (Altschul et al.,
1990). Amino acid alignments were generated with PILEUP (Feng and
Doolittle, 1987). Sequences were analyzed with programs of the
Genetics Computer Group package (Devereaux et al., 1984) on a VAX
computer.
[0080] Clones corresponding to the mouse ortholog of the human ABCR
gene were isolated from the mouse retina cDNA library and
end-sequenced. The chromosomal location of the mouse ABCR gene was
determined on The Jackson Laboratory (Bar Harbor, Me.)
interspecific backcross mapping panel
(C57BU6JEi.times.SPRET/Ei)F1.times.SPRET/Ei (Rowe et al., 1994)
known as Jackson BSS. Mapping was performed by SSCP analysis with
the primers MABCR1F 5'ATC CAT ACC CTT CCC ACT CC, SEQ ID NO:9, and
MABCR1R 5'GCA GCA GAA GAT AAG CAC ACC, SEQ ID NO. 10. The allele
pattern of the ABCR was compared to the 250 other loci mapped
previously in the Jackson BSS cross (http://www.jax.org).
[0081] DNA fragments used as probes were purified on a 1%
low-melting temperature agarose gel. The probe sequences are set
forth within the genomic sequence of SEQ ID NO: 1 and FIG. 3. DNA
was labeled directly in agarose with the Random Primed DNA Labeling
Kit (Boehringer Mannheim, Indianapolis, Ind.) and hybridized to
multiple tissue Northern blot and a Master blot (Clontech, Palo
Alto, Calif.), according to the manufacturer's instructions. Each
blot contained 2 .mu.g of poly A.sup.+ RNA from various human
tissues. Total RNA was isolated from adult rat tissues using the
guanidinium thiocyanate method (Chomczynski and Saachi, 1987 and
resolved by agarose gel electrophoresis in the presence of
formaldehyde (Sambrook et al., 1989). Hybridization with the mouse
ABCR probe was performed in 50% formamide, 5.times.SSC at
42.degree. C., and filters were washed in 0.1.times.SSC at
68.degree. C.
[0082] Hybridization of a 3' ABCR cDNA probe to a multiple tissue
Northern blot and a MasterBlot (Clontech, Palo Alto, Calif.)
indicated that the gene was not expressed detectably in any of the
50 non-retinal fetal and adult tissues examined, consistent with
the observation that all 12 of the ABCR clones in the EST database
originated from retinal cDNA libraries. Furthermore, screening cDNA
libraries from both developing mouse eye and adult human retina
with ABCR probes revealed an estimated at 0.1%-1% frequency of ABCR
clones of all cDNA clones in the library. Hybridization of the ABCR
probe to a Northern blot containing total RNA from rat retina and
other tissues showed that the expression of this gene is uniquely
retina-specific (FIG. 2). The transcript size is estimated to be 8
kb.
Sequence and Exon/Intron Structure of the ABCR cDNA
[0083] Several ESTs that were derived from retina cDNA libraries
and had high similarity to the mouse Abel gene were used to
facilitate the assembly of most of the ABCR cDNA sequence. Retina
cDNA clones were linked by RT-PCR, and repetitive screening of a
human retina cDNA library with 3' and 5' PCR probes together with
5' RACE were used to characterize the terminal sequences of the
gene.
[0084] cDNA clones containing ABCR sequences were obtained from a
human retina cDNA library (Nathans et al., 1986) and sequenced
fully. Primers were designed from the sequences of cDNA clones from
5' and 3' regions of the gene and used to link the identified cDNA
clones by RT-PCR with retina QUICK-Clone cDNA (Clontech, Palo Alto,
Calif.) as a template. PCR products were cloned into pGEM.RTM.-T
vector (Promega, Madison, Wis.). Mouse ABCR cDNA clones were
obtained from screening a developing mouse eye cDNA library (H.
Sun, A. Lanahan, and J. Nathans, unpublished). The pGEM.RTM.-T
Vector is prepared by cutting pGEM.RTM.-5Zf(+) DNA with EcoR V and
adding to a 3' terminal thymidine to both ends. These single 3'-T
overhangs at the insertion site greatly improve the efficiency of
ligation of PCR products because of the nontemplate-dependent
addition of a single deoxyadenosine (A) to the 3'-ends of PCR
products by many thermostable polymerases. The pGEM.RTM. 5Zf(+)
Vector contains the origin of replication of the filamentous phage
f1 and can be used to produce ssDNA. The plasmid also contains T7
and SP6 RNA polymerase promoters flanking a multiple cloning region
within the .alpha.-peptide coding region for the enzyme
beta-galactosidase. Insertional inactivation of the alpha-peptide
allows recombinant clones to be identified directly by color
screening on indicator plates. cDNA clones from various regions of
the ABCR gene were used as probes to screen a human genomic library
in Lambda FIX II (#946203, Stratagene, LaJolla, Calif.).
Overlapping phase clones were mapped by EcoRI and BamHI digestion.
A total of 6.9 kb of the ABCR sequence was assembled, (FIG. 3)
resulting in a 6540 bp (2180 amino acid) open reading frame.
[0085] Screening of a bacteriophage lambda human genomic library
with cDNA probes yielded a contig that spans approximately 100 kb
and contains the majority of the ABCR coding region. The
exon/intron structure of all fifty one exons of the gene were
characterized by direct sequencing of genomic and cDNA clones.
Intron sizes were estimated from the sizes of PCR products using
primers from adjacent exons with genomic phage clones as
templates.
[0086] Primers for the cDNA sequences of the ABCR were designed
with the PRIMER program (Lincoln et al., 1991). Both ABCR cDNA
clones and genomic clones became templates for sequencing.
Sequencing was performed with the Taq Dyedeoxy Terminator Cycle
Sequencing kit (Appliedn Biosystems, Foster City, Calif.),
according to the manufacturer's instructions. Sequencing reactions
were resolved on an ABI 373A automated sequencer. Positions of
introns were determined by comparison between genomic and cDNA
sequences. Primers for amplification of individual exons were
designed from adjacent intron sequences 20-50 bp from the splice
site and are set forth in Table 1.
TABLE-US-00001 TABLE 1 Exon/intron Primers for ABCR PRIMER SEQUENCE
SEQ ID NO ABCR.EXON1: F ACCCTCTGCTAAGCTCAGAG 12 ABCR.EXON1 R
ACCCCACACTTCCAACCTG 13 ABCR.EXON2: F AAGTCCTACTGCACACATGG 14
ABCR.EXON2: R ACACTCCCACCCCAAGATC 15 ABCR.EXON3: F
TTCCCAAAAAGGCCAACTC 16 ABCR.EXON3: R CACGCACGTGTGCATTCAG 17
ABCR.EXON4: F GCTATTTCCTTATTAATGAGGC 18 ABCR.EXON4: R
CCAACTCTCCCTGTTCTTTC 19 ABCR.EXON5: F TGTTTCCAATCGACTCTGGC 20
ABCR.EXON5: R TTCTTGCCTTTCTCAGGCTGG 21 ABCR.EXON6: F
GTATTCCCAGGTTCTGTGG 22 ABCR.EXON6: R TACCCCAGGAATCACCTTG 23
ABCR.EXON7: F AGCATATAGGAGATCAGACTG 24 ABCR.EXON7: R
TGACATAAGTGGGGTAAATGG 25 ABCR.EXON8: F GAGCATTGGCCTCACAGCAG 26
ABCR.EXON8: R CCCCAGGTTTGTTTCACC 27 ABCR.EXON9: F
AGACATGTGATGTGGATACAC 28 ABCR.EXON9: R GTGGGAGGTCCAGGGTACAC 29
ABCR.EXON10: F AGGGGCAGAAAAGACACAC 30 ABCR.EXON10: R
TAGCGATTAACTCTTTCCTGG 31 ABCR.EXON11: F CTCTTCAGGGAGCCTTAGC 32
ABCR.EXON11: R TTCAAGACCACTTGACTTGC 33 ABCR.EXON12: F
TGGGACAGCAGCCTTATC 34 ABCR.EXON12: R CCAAATGTAATTTCCCACTGAC 35
ABCR.EXON13: F AATGAGTTCCGAGTCACCCTG 36 ABCR.EXON13: R
CCCATTCGCGTGTCATGG 37 ABCR.EXON14: F TCCATCTGGGCTTTGTTCTC 38
ABCR.EXON14: R AATCCAGGCACATGAACAGG 39 ABCR.EXON15: F
AGGCTGGTGGGAGAGAGC 40 ABCR.EXON15: R AGTGGACCCCCTCAGAGG 41
ABCR.EXON16: F CTGTTGCATTGGATAAAAGGC 42 ABCR.EXONI6: R
GATGAATGGAGAGGGCTGG 43 ABCR.EXON17: F CTGCGGTAAGGTAGGATAGGG 44
ABCR.EXON17: R CACACCGTTTACATAGAGGGC 45 ABCR.EXON18: F
CCTCTCCCCTCCTTTCCTG 46 ABCR.EXON18: R GTCAGTTTCCGTAGGCTTC 47
ABCR.EXON19: F TGGGGCCATGTAATTAGGC 48 ABCR.EXON19: R
TGGGAAAGAGTAGACAGCCG 49 ABCR.EXON20: F ACTGAACCTGGTGTGGGG 50
ABCR.EXON20: R TATCTCTGCCTGTGCCCAG 51 ABCR.EXON21: F
GTAAGATCAGCTGCTGGAAG 52 ABCR.EXON2I: R GAAGCTCTCCTGCACCAAGC 53
ABCR.EXON22: F AGGTACCCCCACAATGCC 54 ABCR.EXON22: R
TCATTGTGGTTCCAGTACTCAG 55 ABCR.EXON23: F TTTTTGCAACTATATAGCCAGG 56
ABCR.EXON23: R AGCCTGTGTGAGTAGCCATG 57 ABCR.EXON24: F
GCATCAGGGCGAGGCTGTC 58 ABCR.EXON24: R CCCAGCAATACTGGGAGATG 59
ABCR.EXON25: F GGTAACCTCACAGTCTTCC 60 ABCR.EXON25: R
GGGAACGATGGCTTTTTGC 61 ABCR.EXON26: F TCCCATTATGAAGCAATACC 62
ABCR.EXON26: R CCTTAGACTTTCGAGATGG 63 ABCR.EXON27: F
GCTACCAGCCTGGTATTTCATTG 64 ABCR.EXON27: R GTTATAACCCATGCCTGAAG 65
ABCR.EXON28: F TGCACGCGCACGTGTGAC 66 ABCR.EXON28: R
TGAAGGTCCCAGTGAAGTGGG 67 ABCR.EXON29: F CAGCAGCTATCCAGTAAAGG 68
ABCR.EXON29: R AACGCCTGCCATCTTGAAC 69 ABCR.EXON30: F
GTTGGGCACAATTCTTATGC 70 ABCR.EXON30: R GTTGTTTGGAGGTCAGGTAC 71
ABCR.EXON31: F AACATCACCCAGCTGTTCCAG 72 ABCR.EXON31: R
ACTCAGGAGATACCAGGGAC 73 ABCR.EXON32: F GGAAGACAACAAGCAGTTTCAC 74
ABCR.EXON32: R ATCTACTGCCCTGATCATAC 75 ABCR.EXON33: F
AAGACTGAGACTTCAGTCTTC 76 ABCR.EXON33: R GGTGTGCCTTTTAAAAGTGTGC 77
ABCR.EXON34: F TTCATGTTTCCCTACAAAACCC 78 ABCR.EXON34: R
CATGAGAGTTTCTCATTCATGG 79 ABCR.EXON35: F TGTTTACATGGTTTTTAGGGCC 80
ABCR.EXON35: R TTCAGCAGGAGGAGGGATG 81 ABCR.EXON36: F
CCTTTCCTTCACTGATTTCTGC 82 ABCR.EXON36: R AATCAGCACTTCGCGGTG 83
ABCR.EXON37: F TGTAAGGCCTTCCCAAAGC 84 ABCR.EXON37: R
TGGTCCTTCAGCGCACACAC 85 ABCR.EXON38: F CATTTTGCAGAGCTGGCAGC 86
ABCR.EXON38: R CTTCTGTCAGGAGATGATCC 87 ABCR.EXON39: F
GGAGTGCATTATATCCAGACG 88 ABCR.EXON39: R CCTGGCTCTGCTTGACCAAC 89
ABCR.EXON40: F TGCTGTCCTGTGAGAGCATC 90 ABCR.EXON40: R
GTAACCCTCCCAGCTTTGG 91 ABCR.EXON41: F CAGTTCCCACATAAGGCCTG 92
ABCR.EXON41: R CAGTTCTGGATGCCCTGAG 93 ABCR.EXON42: F
GAAGAGAGGTCCCATGGAAAGG 94 ABCR.EXON42: R GCTTGCATAAGCATATCAATTG 95
ABCR.EXON43: F CTCCTAAACCATCCTTTGCTC 96 ABCR.EXON43: R
AGGCAGGCACAAGAGCTG 97 ABCR.EXON44: F CTTACCCTGGGGCCTGAC 98
ABCR.EXON44: R CTCAGAGCCACCCTACTATAG 99 ABCR.EXON45: F
GAAGCTTCTCCAGCCCTAGC 100 ABCR.EXON45: R TGCACTCTCATGAAACAGGC 101
ABCR.EXON46: F GTTTGGGGTGTTTGCTTGTC 102 ABCR.EXON46: R
ACCTCTTTCCCCAACCCAGAG 103 ABCR.EXON47: F GAAGCAGTAATCAGAAGGGC 104
A13CR.EXON47: R GCCTCACATTCTTCCATGCTG 105 ABCR.EXON48: F
TCACATCCCACAGGCAAGAG 106 ABCR.EXON48: R TTCCAAGTGTCAATGGAGAAC 107
ABCR.EXON49: F ATTACCTTAGGCCCAACCAC 108 ABCR.EXON49: R
ACACTGGGTGTTCTGGACC 109 ABCR.EXON50: F GTGTAGGGTGGTGTTTTCC 110
ABCR.EXON50: R AAGCCCAGTGAACCAGCTGG 111 ABCR.EXON51: F
TCAGCTGAGTGCCCTTCAG 112 ABCR.EXON51: R AGGTGAGCAAGTCAGTTTCGG
113
[0087] In Table 1, "F" indicates forward, i.e., 5' to 3', "R"
indicates reverse, i.e., 3' to 5'. PCR conditions were 95.degree.
C. for 8 minutes; 5 cycles at 62.degree. C. for 20 seconds,
72.degree. C. for 30 seconds; 35 cycles at 60.degree. C. for 20
seconds, 72.degree. C. for 30 seconds; 72.degree. C. for 5 minutes
(except that .sup.a was performed at 94.degree. C. for 5 minutes);
5 cycles at 94.degree. C. for 40 seconds; 60.degree. C. for 30
seconds; 72.degree. C. for 20 seconds; 35 cycles at 94.degree. C.
for 40 seconds; 56.degree. C. for 30 seconds; 72.degree. C. for 20
seconds, and 72.degree. C. for 5 minutes.
[0088] Amplification of exons was performed with AmpliTaq Gold
polymerase in a 25 microliter volume in 1.times.PCR buffer supplied
by the manufacturer (Perkin Elmer, Foster City, Calif.). Samples
were heated to 95.degree. C. for 10 minutes and amplified for 35-40
cycles at 96.degree. C. for 20 seconds; 58.degree. C. for 30
seconds; and 72.degree. C. for 30 seconds. PCR products were
analyzed on 1-1.5% agarose gels and in some cases digested with an
appropriate restriction enzyme(s) to verify their sequence. Primer
sequences and specific reaction conditions are set forth in Table
1. The sequence of the ABCR cDNA has been deposited with GenBank
under accession #U88667.
Homology to ABC Superfamily Members
[0089] A BLAST search revealed that ABCR is most closely related to
the previously characterized mouse Abc1 and Abc2 genes (Luciani et
al., 1994) and to another human gene (ABCC) which maps to
chromosome 16p13.3 (Klugbauer and Hofmann, 1996). These genes,
together with ABCR and a gene from C. elegans (GenBank #Z29117),
form a subfamily of genes specific to multicellular organisms and
not represented in yeast (Michaelis and Berkower, 1995; Allikmets
et al., 1996). Alignment of the cDNA sequence of ABCR with the
Abc1, ABc2, and ABCC genes revealed, as expected, the highest
degree of homology within the ATP-binding cassettes. The predicted
amino acid identity of the ABCR gene to mouse Abel was 70% within
the ATP-binding domains; even within hydrophobic membrane-spanning
segments, homology ranged between 55 and 85% (FIG. 4). The putative
ABCR initiator methionine shown in FIGS. 3 and 4 corresponds to a
methionine codon at the 5' end of Abc1 (Luciani et al., 1994).
[0090] ABCR shows the composition of a typical full-length ABC
transporter that consists of two transmembrane domains (TM), each
with six membrane spanning hydrophobic segments, as predicted by a
hydropathy plot (data not shown), and two highly conserved
ATP-binding domains (FIGS. 3 and 4). In addition, the HH1
hydrophobic domain, located between the first ATP and second TM
domain and specific to this subfamily (Luciani et al., 1994),
showed a predicted 57% amino acid identity (24 of 42 amino acids)
with the mouse Abc1 gene.
[0091] To characterize the mouse ortholog of ABCR, cDNA clones from
a developing mouse eye library were isolated. A partial sequence of
the mouse cDNA was utilized to design PCR primers to map the mouse
ABCR gene in an interspecific backcross mapping panel (Jackson
BSS). The allele pattern of ABCR was compared to 2450 other loci
mapped previously in the Jackson BSS cross; linkage was found to
the distal end of chromosome 3 (FIG. 5). No recombinants were
observed between ABCR and D13Mit13. This region of the mouse genome
is syntenic with human chromosome 1p13-p21. Thus far, no eye
disease phenotype has been mapped to this region of mouse
chromosome 3.
Compound Heterozygous and Homozygous Mutations in STGD Patients
[0092] One hundred forty-five North American and three Saudi
Arabian families with STGD/FFM were examined. Among these, at least
four were consanguineous families in which the parents were first
cousins. Entry criteria for the characterization of the clinical
and angiographic diagnosis of Stargardt disease, ascertainment of
the families, and methodology for their collection, including the
consanguineous families from Saudi Arabia, were as provided in
Anderson et al., 1995; and Anderson, 1996.
[0093] Mutational analysis of the ABCR gene was pursued in the
above identified one hundred forty-eight STGD families previously
ascertained by strict definitional criteria and shown to be linked
to chromosome 1 p (Anderson et al., 1995; Anderson, 1996). To date,
all 51 exons have been used for mutation analysis.
[0094] Mutations were detected by a combined SSCP (Orita et al.,
1989) and heteroduplex analysis (White et al., 1992) under
optimized conditions (Glavac and Dean, 1993). Genomic DNA samples
(50 ng) were amplified with AmpliTaq Gold polymerase in 1.times.PCR
buffer supplied by the manufacturer (Perkin Elmer, Foster City,
Calif.) containing [.alpha.-.sup.32P] dCTP. Samples were heated to
95.degree. C. for 10 minutes and amplified for 35-40 cycles at
96.degree. C. for 20 seconds; 58.degree. C. for 30 seconds; and
72.degree. C. for 30 seconds. Products were diluted in 1:3 stop
solution, denatured at 95.degree. C. for 5 minutes, chilled in ice
for 5 minutes, and loaded on gels. Gel formulations include 6%
acrylamide:Bis (2.6% cross-linking), 10% glycerol at room
temperature, 12W; and 10% acrylamide:Bis (1.5% cross-linking), at
4.degree. C., 70 W. Gels were run for 2-16 hours (3000 Vh/100 bp),
dried, and exposed to X-ray film for 2-12 hours. Some exons were
analyzed by SSCP with MDE acrylamide (FMC Bioproducts, Rockland,
Me.) with and without 10% glycerol for 18 hours, 4 watts at room
temperature with .alpha .alpha..-P.sup.32-dCTP labeled DNA.
Heteroduplexes were identified from the double-stranded DNA at the
bottom of the gels, and SSCPs were identified from the
single-stranded region. Samples showing variation were compared
with other family members to assess segregation of the alleles and
with at least 40 unrelated control samples, from either Caucasian
or Saudi Arabian populations, to distinguish mutations from
polymorphisms unrelated to STGD. PCR products with SSCP or
heteroduplex variants were obtained in a 25 .mu.l volume, separated
on a 1% agarose gel, and isolated by a DNA purification kit (PGC
Scientific, Frederick, Md.). Sequencing was performed on an ABI
sequencer with both dye primer and dye terminator chemistry.
[0095] Some mutations were identified with a heteroduplex analysis
protocol (Roa et al., 1993). Equimolar amounts of control and
patient PCR products were mixed in 0.2 ml tubes. Two volumes of PCR
products from a normal individual served as a negative control, and
MPZ exon 3 from patient BAB731 as a positive control (Roa et al.,
1996). Samples were denatured at 95.degree. C. for 2 minutes and
cooled to 35.degree. C. at a rate of 1.degree. C./minute. Samples
were loaded onto 1.0 mm thick, 40 cm MDE gels (FMC Bioproducts,
Rockland, Me.), electrophoresed at 600-800 V for 15-20 hours, and
visualized with ethidium bromide. Samples showing a variant band
were reamplified with biotinylated forward and reverse primers and
immobilized on streptavidin-conjugated beads (Warner et al., 1996).
The resulting single strands were sequenced by the
dideoxy-sequencing method with Sequenase 2.0 (Amersham, Arlington
Heights Ill.).
[0096] A total of seventy five mutations were identified, the
majority representing missense mutations in conserved amino acid
positions. However, several insertions and deletions representing
frameshifts were also found (Table 2). Two missense alterations
(D847H, R943Q) were found in at least one control individual,
suggesting that they are neutral polymorphisms. The remaining
mutations were found in patients having macular degeneration and
were not found in at least 220 unrelated normal controls (440
chromosomes), consistent with the interpretation that these
alterations represent disease-causing mutations, not polymorphisms.
One of the mutations, 5892+1 G.fwdarw.T, occurs in family AR144 in
which one of the affected children is recombinant for the flanking
marker D1S236 (Anderson et al., 1995). This mutation, however, is
present in the father as well as in both affected children.
Therefore, the ABCR gene is non-recombinant with respect to the
Stargardt disease locus.
[0097] The mutations are scattered throughout the coding sequence
of the ABCR gene (see Table 2 and FIG. 3), although clustering
within the conserved regions of the ATP-binding domains is
noticeable. Homozygous mutations were detected in three likely
consanguineous families, two Saudi Arabian and one North American
(Anderson et al., 1995), in each of which only the affected
individuals inherited the identical disease allele (Table 2; FIG.
6). Forty two compound heterozygous families were identified in
which the two disease alleles were transmitted from different
parents to only the affected offspring (Table 2).
TABLE-US-00002 TABLE 2 Mutations in the ABCR gene in STGD Families
Nucleotide Amino Acid #Families Exon 0223T->G C75G 1 3
0634C->T R212C 1 6 0664de113 fs 1 6 0746A->G D249G 1 6
1018T->G Y340D 2 8 1411 G->A E471 K 1 11 1569T->G D523E 1
12 1715G->A R572Q 2 12 1715G->C R572P 1 12 1804C->T R602W
1 13 1822T->A F608I 1 13 1917C->A Y639X 1 13 2453G->A
G818E 1 16 2461T->A W821R 1 16 2536G->C D846H 1 16
2588G->C G863A 11 17 2791G->A V931M 1 19 2827C->T R943W 1
19 2884de1C fs 1 19 2894A->G N965S 3 19 3083C->T A1028V 14 21
3211de1GT fs 1 22 3212C->T S1071L 1 22 3215T->C V1072A 1 22
3259G->A E1087K 1 22 3322C->T R1108C 6 22 3364G->A El122K
1 23 3385G->T R1129C 1 23 3386G->T R1129L 1 23 3602T->G
L1201R 1 24 3610G->A D1204N 1 25 4139C->T P1380L 2 28
4195G->A E1399K 1 28 4222T->C W1408R 3 28 4232insTATG fs 1 28
4253 + 5G->T splice 1 28 4297G->A V1433I 1 29 4316G->A
G1439D 1 29 4319T->C F1440S 1 29 4346G->A W1449X 1 29
4462T->C C1488R 1 30 4469G->A C1490Y 1 31 4577C->T T1526M
6 32 4594G->A D1532N 2 32 4947de1C fs 1 36 5041 de115
VVAIC1681del 1 37 5196 + 2T->C splice 1 37 5281de19 PAL1761de1 1
38 5459G->C R1820P 1 39 5512C->T H1838Y 1 40 5527C->T
R1843W 1 40 5585 + 1G->A splice 1 41 5657G->A G1886E 1 41
5693G->A R1898H 4 41 5714 + 5G->A splice 8 41 5882G->A
G1961E 16 43 5898 + 1G->A splice 3 43 5908C->T L1970F 1 44
5929G->A G1977S 1 44 6005 + 1 G->T splice 1 44 6079C->T
L2027F 11 45 6088C->T R2030X 1 45 6089G->A R2030Q 1 45
6112C->T R2038W 1 45 6148G->C V2050L 2 46 6166A->T K2056X
1 46 6229C->T R2077W 1 46 6286G->A E2096K 1 47 6316C->T
R2106C 1 47 6391G->A E2131K 1 48 6415C->T R2139W 1 48
6445C->T R2149X 1 48 6543de136 1181dell2 1 49 6709de1G fs 1
49
[0098] Mutations are named according to standard nomenclature. The
column headed "Exon" denotes which of the 51 exons of ABCR contain
the mutation. The column headed "# Families" denotes the number of
Stargardt families which displayed the mutation. The column headed
"Nucleotide" gives the base number starting from the A in the
initiator ATG, followed by the wild type sequence and an arrow
indicating the base it is changed to: del indicates a deletion of
selected bases at the given position in the ABCR gene; ins
indicates an insertion of selected bases at the given position;
splice donor site mutations are indicated by the number of the last
base of the given exon, followed by a plus sign and the number of
bases into the intron where the mutation occurs. The column headed
"Amino Acid" denotes the amino acid change a given mutation causes;
fs indicates a frameshift mutation leading to a truncated protein;
splice indicates a splice donor site mutation; del indicates an
in-frame deletion of the given amino acids.
[0099] Mutations are named according to standard nomenclature. Exon
numbering according to the nucleotide position starting from the A
in the initiator ATG.
In Situ Hybridization
[0100] STGD is characterized histologically by a massive
accumulation of a lipofuscin-like substance in the retinal pigment
epithelium (RPE). This characteristic has led to the suggestion
that STGD represents an RPE storage disorder (Blacharski et al.,
1988). It was therefore of interest that ABCR transcripts were
found to be abundant in the retina. To identify the site(s) of ABCR
gene expression at higher resolution and to determine whether the
gene is also expressed in the RPE, the distribution of ABCR
transcripts was visualized by in situ hybridization to mouse, rat,
bovine, and macaque ocular tissues.
[0101] In situ hybridization with digoxigenin-labeled riboprobes
was performed as described by Schaeren-Wiemers and Gerfin-Moser,
1993. For mouse and rat, unfixed whole eyes were frozen and
sectioned; macaque retinas were obtained following cardiac
perfusion with paraformaldehyde as described (Thou et al., 1996).
An extra incubation of 30 min in 1% Triton X-100, 1.times.PBS was
applied to the fixed monkey retina sections immediately after the
acetylation step. The templates for probe synthesis were: (1) a 1.6
kb fragment encompassing the 3' end of the mouse ABCR coding
region, (2) a full length cDNA clone encoding the mouse blue cone
pigment (Chiu et al., 1994), and (3) a macaque rhodopsin coding
region segment encoding residues 133 to 254 (Nickells, R. W.,
Burgoyne, C. F., Quigley, H. A., and Zack, D. J. (1995)).
[0102] This analysis showed that ABCR transcripts are present
exclusively within photoreceptor cells (FIG. 7). ABCR transcripts
are localized principally to the rod inner segments, a distribution
that closely matches that of rhodopsin gene transcripts.
Interestingly, ABCR hybridization was not observed at detectable
levels in cone photoreceptors, as judged by comparisons with the
hybridization patterns obtained with a blue cone pigment probe
(compare FIG. 7A and FIG. 7D, FIG. 7E with FIG. 7F and FIG. 7G with
FIG. 7H). Because melanin granules might obscure a weak
hybridization signal in the RPE of a pigmented animal, the
distribution of ABCR transcripts was also examined in both albino
rats and albino mice. In these experiments, the ABCR hybridization
signal was seen in the photoreceptor inner segments and was
unequivocally absent from the RPE (FIG. 7E). Given that ABCR
transcripts in each of these mammals, including a primate, are
photoreceptor-specific, it is highly likely that the distribution
of ABCR transcripts conforms to this pattern as well in the human
retina.
[0103] The disclosures of each patent, patent application and
publication cited or described in this document are hereby
incorporated herein by reference, in their entirety.
[0104] Various modifications of the invention in addition to those
shown and described herein will be apparent to those skilled in the
art from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
REFERENCES
[0105] Allikmets, R., Singh, N., Sun, H., Shroyer, N. F.,
Hutchinson, A., Chidambaram, A., Gerrard, B., Baird, L., Stauffer,
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L., Lewis, R. A., Nathans, J., Leppert, M., Dean, M., Lupski, J.
R., (1997) A photoreceptor cell-specific ATP-binding transporter
gene (ABCR) is mutated in recessive Stargardt macular dystrophy.
Nature Genetics. 15(3):236-46. [0106] Allikmets, R., Shroyer, N.
F., Singh, N., Seddon, J. M., Lewis, R. A., Bernsteinm P. S.,
Peiffer, A., Zabriskie, N. A., Li, Y., Hutchinson, A., Dean, M.,
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Sequence CWU 1
1
12017783DNAHomo sapiens 1cccctacccc tctgctaagc tcagggataa
cccaactagc tgaccataat gacttcagtc 60attacggagc aagatgaaag actaaaagag
ggagggatca cttcagatct gccgagtgag 120tcgattggac ttaaagggcc
agtcaaaccc tgactgccgg ctcatggcag gctcttgccg 180aggacaaatg
cccagcctat atttatgcaa agagattttg ttccaaactt aaggtcaaag
240atacctaaag acatccccct caggaacccc tctcatggag gagagtgcct
gagggtcttg 300gtttcccatt gcatccccca cctcaatttc cctggtgccc
agccacttgt gtctttaggg 360ttctctttct ctccataaaa gggagccaac
acagtgtcgg cctcctctcc ccaactaagg 420gcttatgtgt aattaaaagg
gattatgctt tgaaggggaa aagtagcctt taatcaccag 480gagaaggaca
cagcgtccgg agccagaggc gctcttaacg gcgtttatgt cctttgctgt
540ctgaggggcc tcagctctga ccaatctggt cttcgtgtgg tcattagcat
gggcttcgtg 600agacagatac agcttttgct ctggaagaac tggaccctgc
ggaaaaggca aaagattcgc 660tttgtggtgg aactcgtgtg gcctttatct
ttatttctgg tcttgatctg gttaaggaat 720gccaacccgc tctacagcca
tcatgaatgc catttcccca acaaggcgat gccctcagca 780ggaatgctgc
cgtggctcca ggggatcttc tgcaatgtga acaatccctg ttttcaaagc
840cccaccccag gagaatctcc tggaattgtg tcaaactata acaactccat
cttggcaagg 900gtatatcgag attttcaaga actcctcatg aatgcaccag
agagccagca ccttggccgt 960atttggacag agctacacat cttgtcccaa
ttcatggaca ccctccggac tcacccggag 1020agaattgcag gaagaggaat
acgaataagg gatatcttga aagatgaaga aacactgaca 1080ctatttctca
ttaaaaacat cggcctgtct gactcagtgg tctaccttct gatcaactct
1140caagtccgtc cagagcagtt cgctcatgga gtcccggacc tggcgctgaa
ggacatcgcc 1200tgcagcgagg ccctcctgga gcgcttcatc atcttcagcc
agagacgcgg ggcaaagacg 1260gtgcgctatg ccctgtgctc cctctcccag
ggcaccctac agtggataga agacactctg 1320tatgccaacg tggacttctt
caagctcttc cgtgtgcttc ccacactcct agacagccgt 1380tctcaaggta
tcaatctgag atcttgggga ggaatattat ctgatatgtc accaagaatt
1440caagagttta tccatcggcc gagtatgcag gacttgctgt gggtgaccag
gcccctcatg 1500cagaatggtg gtccagagac ctttacaaag ctgatgggca
tcctgtctga cctcctgtgt 1560ggctaccccg agggaggtgg ctctcgggtg
ctctccttca actggtatga agacaataac 1620tataaggcct ttctggggat
tgactccaca aggaaggatc ctatctattc ttatgacaga 1680agaacaacat
ccttttgtaa tgcattgatc cagagcctgg agtcaaatcc tttaaccaaa
1740atcgcttgga gggcggcaaa gcctttgctg atgggaaaaa tcctgtacac
tcctgattca 1800cctgcagcac gaaggatact gaagaatgcc aactcaactt
ttgaagaact ggaacacgtt 1860aggaagttgg tcaaagcctg ggaagaagta
gggccccaga tctggtactt ctttgacaac 1920agcacacaga tgaacatgat
cagagatacc ctggggaacc caacagtaaa agactttttg 1980aataggcagc
ttggtgaaga aggtattact gctgaagcca tcctaaactt cctctacaag
2040ggccctcggg aaagccaggc tgacgacatg gccaacttcg actggaggga
catatttaac 2100atcactgatc gcaccctccg cctggtcaat caatacctgg
agtgcttggt cctggataag 2160tttgaaagct acaatgatga aactcagctc
acccaacgtg ccctctctct actggaggaa 2220aacatgttct gggccggagt
ggtattccct gacatgtatc cctggaccag ctctctacca 2280ccccacgtga
agtataagat ccgaatggac atagacgtgg tggagaaaac caataagatt
2340aaagacaggt attgggattc tggtcccaga gctgatcccg tggaagattt
ccggtacatc 2400tggggcgggt ttgcctatct gcaggacatg gttgaacagg
ggatcacaag gagccaggtg 2460caggcggagg ctccagttgg aatctacctc
cagcagatgc cctacccctg cttcgtggac 2520gattctttca tgatcatcct
gaaccgctgt ttccctatct tcatggtgct ggcatggatc 2580tactctgtct
ccatgactgt gaagagcatc gtcttggaga aggagttgcg actgaaggag
2640accttgaaaa atcagggtgt ctccaatgca gtgatttggt gtacctggtt
cctggacagc 2700ttctccatca tgtcgatgag catcttcctc ctgacgatat
tcatcatgca tggaagaatc 2760ctacattaca gcgacccatt catcctcttc
ctgttcttgt tggctttctc cactgccacc 2820atcatgctgt gctttctgct
cagcaccttc ttctccaagg ccagtctggc agcagcctgt 2880agtggtgtca
tctatttcac cctctacctg ccacacatcc tgtgcttcgc ctggcaggac
2940cgcatgaccg ctgagctgaa gaaggctgtg agcttactgt ctccggtggc
atttggattt 3000ggcactgagt acctggttcg ctttgaagag caaggcctgg
ggctgcagtg gagcaacatc 3060gggaacagtc ccacggaagg ggacgaattc
agcttcctgc tgtccatgca gatgatgctc 3120cttgatgctg cgtgctatgg
cttactcgct tggtaccttg atcaggtgtt tccaggagac 3180tatggaaccc
cacttccttg gtactttctt ctacaagagt cgtattggct tagcggtgaa
3240gggtgttcaa ccagagaaga aagagccctg gaaaagaccg agcccctaac
agaggaaacg 3300gaggatccag agcacccaga aggaatacac gactccttct
ttgaacgtga gcatccaggg 3360tgggttcctg gggtatgcgt gaagaatctg
gtaaagattt ttgagccctg tggccggcca 3420gctgtggacc gtctgaacat
caccttctac gagaaccaga tcaccgcatt cctgggccac 3480aatggagctg
ggaaaaccac caccttgtcc atcctgacgg gtctgttgcc accaacctct
3540gggactgtgc tcgttggggg aagggacatt gaaaccagcc tggatgcagt
ccggcagagc 3600cttggcatgt gtccacagca caacatcctg ttccaccacc
tcacggtggc tgagcacatg 3660ctgttctatg cccagctgaa aggaaagtcc
caggaggagg cccagctgga gatggaagcc 3720atgttggagg acacaggcct
ccaccacaag cggaatgaag aggctcagga cctatcaggt 3780ggcatgcaga
gaaagctgtc ggttgccatt gcctttgtgg gagatgccaa ggtggtgatt
3840ctggacgaac ccacctctgg ggtggaccct tactcgagac gctcaatctg
ggatctgctc 3900ctgaagtatc gctcaggcag aaccatcatc atgcccactc
accacatgga cgaggccgac 3960caccaagggg accgcattgc catcattgcc
cagggaaggc tctactgctc aggcacccca 4020ctcttcctga agaactgctt
tggcacaggc ttgtacttaa ccttggtgcg caagatgaaa 4080aacatccaga
gccaaaggaa aggcagtgag gggacctgca gctgctcgtc taagggtttc
4140tccaccacgt gtccagccca cgtcgatgac ctaactccag aacaagtcct
ggatggggat 4200gtaaatgagc tgatggatgt agttctccac catgttccag
aggcaaagct ggtggagtgc 4260attggtcaag aacttatctt ccttcttcca
aataagaact tcaagcacag agcatatgcc 4320agccttttca gagagctgga
ggagacgctg gctgaccttg gtctcagcag ttttggaatt 4380tctgacactc
ccctggaaga gatttttctg aaggtcacgg aggattctga ttcaggacct
4440ctgtttgcgg gtggcgctca gcagaaaaga gaaaacgtca acccccgaca
cccctgcttg 4500ggtcccagag agaaggctgg acagacaccc caggactcca
atgtctgctc cccaggggcg 4560ccggctgctc acccagaggg ccagcctccc
ccagagccag agtgcccagg cccgcagctc 4620aacacgggga cacagctggt
cctccagcat gtgcaggcgc tgctggtcaa gagattccaa 4680cacaccatcc
gcagccacaa ggacttcctg gcgcagatcg tgctcccggc tacctttgtg
4740tttttggctc tgatgctttc tattgttatc cttccttttg gcgaataccc
cgctttgacc 4800cttcacccct ggatatatgg gcagcagtac accttcttca
gcatggatga accaggcagt 4860gagcagttca cggtacttgc agacgtcctc
ctgaataagc caggctttgg caaccgctgc 4920ctgaaggaag ggtggcttcc
ggagtacccc tgtggcaact caacaccctg gaagactcct 4980tctgtgtccc
caaacatcac ccagctgttc cagaagcaga aatggacaca ggtcaaccct
5040tcaccatcct gcaggtgcag caccagggag aagctcacca tgctgccaga
gtgccccgag 5100ggtgccgggg gcctcccgcc cccccagaga acacagcgca
gcacggaaat tctacaagac 5160ctgacggaca ggaacatctc cgacttcttg
gtaaaaacgt atcctgctct tataagaagc 5220agcttaaaga gcaaattctg
ggtcaatgaa cagaggtatg gaggaatttc cattggagga 5280aagctcccag
tcgtccccat cacgggggaa gcacttgttg ggtttttaag cgaccttggc
5340cggatcatga atgtgagcgg gggccctatc actagagagg cctctaaaga
aatacctgat 5400ttccttaaac atctagaaac tgaagacaac attaaggtgt
ggtttaataa caaaggctgg 5460catgccctgg tcagctttct caatgtggcc
cacaacgcca tcttacgggc cagcctgcct 5520aaggacagga gccccgagga
gtatggaatc accgtcatta gccaacccct gaacctgacc 5580aaggagcagc
tctcagagat tacagtgctg accacttcag tggatgctgt ggttgccatc
5640tgcgtgattt tctccatgtc cttcgtccca gccagctttg tcctttattt
gatccaggag 5700cgggtgaaca aatccaagca cctccagttt atcagtggag
tgagccccac cacctactgg 5760gtgaccaact tcctctggga catcatgaat
tattccgtga gtgctgggct ggtggtgggc 5820atcttcatcg ggtttcagaa
gaaagcctac acttctccag aaaaccttcc tgcccttgtg 5880gcactgctcc
tgctgtatgg atgggcggtc attcccatga tgtacccagc atccttcctg
5940tttgatgtcc ccagcacagc ctatgtggct ttatcttgtg ctaatctgtt
catcggcatc 6000aacagcagtg ctattacctt catcttggaa ttatttgata
ataaccggac gctgctcagg 6060ttcaacgccg tgctgaggaa gctgctcatt
gtcttccccc acttctgcct gggccggggc 6120ctcattgacc ttgcactgag
ccaggctgtg acagatgtct atgcccggtt tggtgaggag 6180cactctgcaa
atccgttcca ctgggacctg attgggaaga acctgtttgc catggtggtg
6240gaaggggtgg tgtacttcct cctgaccctg ctggtccagc gccacttctt
cctctcccaa 6300tggattgccg agcccactaa ggagcccatt gttgatgaag
atgatgatgt ggctgaagaa 6360agacaaagaa ttattactgg tggaaataaa
actgacatct taaggctaca tgaactaacc 6420aagatttatc tgggcacctc
cagcccagca gtggacaggc tgtgtgtcgg agttcgccct 6480ggagagtgct
ttggcctcct gggagtgaat ggtgccggca aaacaaccac attcaagatg
6540ctcactgggg acaccacagt gacctcaggg gatgccaccg tagcaggcaa
gagtatttta 6600accaatattt ctgaagtcca tcaaaatatg ggctactgtc
ctcagtttga tgcaatcgat 6660gagctgctca caggacgaga acatctttac
ctttatgccc ggcttcgagg tgtaccagca 6720gaagaaatcg aaaaggttgc
aaactggagt attaagagcc tgggcctgac tgtctacgcc 6780gactgcctgg
ctggcacgta cagtgggggc aacaagcgga aactctccac agccatcgca
6840ctcattggct gcccaccgct ggtgctgctg gatgagccca ccacagggat
ggacccccag 6900gcacgccgca tgctgtggaa cgtcatcgtg agcatcatca
gaaaagggag ggctgtggtc 6960ctcacatccc acagcatgga agaatgtgag
gcactgtgta cccggctggc catcatggta 7020aagggcgcct ttcgatgtat
gggcaccatt cagcatctca agtccaaatt tggagatggc 7080tatatcgtca
caatgaagat caaatccccg aaggacgacc tgcttcctga cctgaaccct
7140gtggagcagt tcttccaggg gaacttccca ggcagtgtgc agagggagag
gcactacaac 7200atgctccagt tccaggtctc ctcctcctcc ctggcgagga
tcttccagct cctcctctcc 7260cacaaggaca gcctgctcat cgaggagtac
tcagtcacac agaccacact ggaccaggtg 7320tttgtaaatt ttgctaaaca
gcagactgaa agtcatgacc tccctctgca ccctcgagct 7380gctggagcca
gtcgacaagc ccaggactga tctttcacac cgctcgttcc tgcagccaga
7440aaggaactct gggcagctgg aggcgcagga gcctgtgccc atatggtcat
ccaaatggac 7500tggcccagcg taaatgaccc cactgcagca gaaaacaaac
acacgaggag catgcagcga 7560attcagaaag aggtctttca gaaggaaacc
gaaactgact tgctcacctg gaacacctga 7620tggtgaaacc aaacaaatac
aaaatccttc tccagacccc agaactagaa accccgggcc 7680atcccactag
cagctttggc ctccatattg ctctcatttc aagcagatct gcttttctgc
7740atgtttgtct gtgtgtctgc gttgtgtgtg attttcatgg aaa
778326819DNAHomo sapiens 2atgggcttcg tgagacagat acagcttttg
ctctggaaga actggaccct gcggaaaagg 60caaaagattc gctttgtggt ggaactcgtg
tggcctttat ctttatttct ggtcttgatc 120tggttaagga atgccaaccc
gctctacagc catcatgaat gccatttccc caacaaggcg 180atgccctcag
caggaatgct gccgtggctc caggggatct tctgcaatgt gaacaatccc
240tgttttcaaa gccccacccc aggagaatct cctggaattg tgtcaaacta
taacaactcc 300atcttggcaa gggtatatcg agattttcaa gaactcctca
tgaatgcacc agagagccag 360caccttggcc gtatttggac agagctacac
atcttgtccc aattcatgga caccctccgg 420actcacccgg agagaattgc
aggaagagga atacgaataa gggatatctt gaaagatgaa 480gaaacactga
cactatttct cattaaaaac atcggcctgt ctgactcagt ggtctacctt
540ctgatcaact ctcaagtccg tccagagcag ttcgctcatg gagtcccgga
cctggcgctg 600aaggacatcg cctgcagcga ggccctcctg gagcgcttca
tcatcttcag ccagagacgc 660ggggcaaaga cggtgcgcta tgccctgtgc
tccctctccc agggcaccct acagtggata 720gaagacactc tgtatgccaa
cgtggacttc ttcaagctct tccgtgtgct tcccacactc 780ctagacagcc
gttctcaagg tatcaatctg agatcttggg gaggaatatt atctgatatg
840tcaccaagaa ttcaagagtt tatccatcgg ccgagtatgc aggacttgct
gtgggtgacc 900aggcccctca tgcagaatgg tggtccagag acctttacaa
agctgatggg catcctgtct 960gacctcctgt gtggctaccc cgagggaggt
ggctctcggg tgctctcctt caactggtat 1020gaagacaata actataaggc
ctttctgggg attgactcca caaggaagga tcctatctat 1080tcttatgaca
gaagaacaac atccttttgt aatgcattga tccagagcct ggagtcaaat
1140cctttaacca aaatcgcttg gagggcggca aagcctttgc tgatgggaaa
aatcctgtac 1200actcctgatt cacctgcagc acgaaggata ctgaagaatg
ccaactcaac ttttgaagaa 1260ctggaacacg ttaggaagtt ggtcaaagcc
tgggaagaag tagggcccca gatctggtac 1320ttctttgaca acagcacaca
gatgaacatg atcagagata ccctggggaa cccaacagta 1380aaagactttt
tgaataggca gcttggtgaa gaaggtatta ctgctgaagc catcctaaac
1440ttcctctaca agggccctcg ggaaagccag gctgacgaca tggccaactt
cgactggagg 1500gacatattta acatcactga tcgcaccctc cgcctggtca
atcaatacct ggagtgcttg 1560gtcctggata agtttgaaag ctacaatgat
gaaactcagc tcacccaacg tgccctctct 1620ctactggagg aaaacatgtt
ctgggccgga gtggtattcc ctgacatgta tccctggacc 1680agctctctac
caccccacgt gaagtataag atccgaatgg acatagacgt ggtggagaaa
1740accaataaga ttaaagacag gtattgggat tctggtccca gagctgatcc
cgtggaagat 1800ttccggtaca tctggggcgg gtttgcctat ctgcaggaca
tggttgaaca ggggatcaca 1860aggagccagg tgcaggcgga ggctccagtt
ggaatctacc tccagcagat gccctacccc 1920tgcttcgtgg acgattcttt
catgatcatc ctgaaccgct gtttccctat cttcatggtg 1980ctggcatgga
tctactctgt ctccatgact gtgaagagca tcgtcttgga gaaggagttg
2040cgactgaagg agaccttgaa aaatcagggt gtctccaatg cagtgatttg
gtgtacctgg 2100ttcctggaca gcttctccat catgtcgatg agcatcttcc
tcctgacgat attcatcatg 2160catggaagaa tcctacatta cagcgaccca
ttcatcctct tcctgttctt gttggctttc 2220tccactgcca ccatcatgct
gtgctttctg ctcagcacct tcttctccaa ggccagtctg 2280gcagcagcct
gtagtggtgt catctatttc accctctacc tgccacacat cctgtgcttc
2340gcctggcagg accgcatgac cgctgagctg aagaaggctg tgagcttact
gtctccggtg 2400gcatttggat ttggcactga gtacctggtt cgctttgaag
agcaaggcct ggggctgcag 2460tggagcaaca tcgggaacag tcccacggaa
ggggacgaat tcagcttcct gctgtccatg 2520cagatgatgc tccttgatgc
tgcgtgctat ggcttactcg cttggtacct tgatcaggtg 2580tttccaggag
actatggaac cccacttcct tggtactttc ttctacaaga gtcgtattgg
2640cttagcggtg aagggtgttc aaccagagaa gaaagagccc tggaaaagac
cgagccccta 2700acagaggaaa cggaggatcc agagcaccca gaaggaatac
acgactcctt ctttgaacgt 2760gagcatccag ggtgggttcc tggggtatgc
gtgaagaatc tggtaaagat ttttgagccc 2820tgtggccggc cagctgtgga
ccgtctgaac atcaccttct acgagaacca gatcaccgca 2880ttcctgggcc
acaatggagc tgggaaaacc accaccttgt ccatcctgac gggtctgttg
2940ccaccaacct ctgggactgt gctcgttggg ggaagggaca ttgaaaccag
cctggatgca 3000gtccggcaga gccttggcat gtgtccacag cacaacatcc
tgttccacca cctcacggtg 3060gctgagcaca tgctgttcta tgcccagctg
aaaggaaagt cccaggagga ggcccagctg 3120gagatggaag ccatgttgga
ggacacaggc ctccaccaca agcggaatga agaggctcag 3180gacctatcag
gtggcatgca gagaaagctg tcggttgcca ttgcctttgt gggagatgcc
3240aaggtggtga ttctggacga acccacctct ggggtggacc cttactcgag
acgctcaatc 3300tgggatctgc tcctgaagta tcgctcaggc agaaccatca
tcatgcccac tcaccacatg 3360gacgaggccg accaccaagg ggaccgcatt
gccatcattg cccagggaag gctctactgc 3420tcaggcaccc cactcttcct
gaagaactgc tttggcacag gcttgtactt aaccttggtg 3480cgcaagatga
aaaacatcca gagccaaagg aaaggcagtg aggggacctg cagctgctcg
3540tctaagggtt tctccaccac gtgtccagcc cacgtcgatg acctaactcc
agaacaagtc 3600ctggatgggg atgtaaatga gctgatggat gtagttctcc
accatgttcc agaggcaaag 3660ctggtggagt gcattggtca agaacttatc
ttccttcttc caaataagaa cttcaagcac 3720agagcatatg ccagcctttt
cagagagctg gaggagacgc tggctgacct tggtctcagc 3780agttttggaa
tttctgacac tcccctggaa gagatttttc tgaaggtcac ggaggattct
3840gattcaggac ctctgtttgc gggtggcgct cagcagaaaa gagaaaacgt
caacccccga 3900cacccctgct tgggtcccag agagaaggct ggacagacac
cccaggactc caatgtctgc 3960tccccagggg cgccggctgc tcacccagag
ggccagcctc ccccagagcc agagtgccca 4020ggcccgcagc tcaacacggg
gacacagctg gtcctccagc atgtgcaggc gctgctggtc 4080aagagattcc
aacacaccat ccgcagccac aaggacttcc tggcgcagat cgtgctcccg
4140gctacctttg tgtttttggc tctgatgctt tctattgtta tccttccttt
tggcgaatac 4200cccgctttga cccttcaccc ctggatatat gggcagcagt
acaccttctt cagcatggat 4260gaaccaggca gtgagcagtt cacggtactt
gcagacgtcc tcctgaataa gccaggcttt 4320ggcaaccgct gcctgaagga
agggtggctt ccggagtacc cctgtggcaa ctcaacaccc 4380tggaagactc
cttctgtgtc cccaaacatc acccagctgt tccagaagca gaaatggaca
4440caggtcaacc cttcaccatc ctgcaggtgc agcaccaggg agaagctcac
catgctgcca 4500gagtgccccg agggtgccgg gggcctcccg cccccccaga
gaacacagcg cagcacggaa 4560attctacaag acctgacgga caggaacatc
tccgacttct tggtaaaaac gtatcctgct 4620cttataagaa gcagcttaaa
gagcaaattc tgggtcaatg aacagaggta tggaggaatt 4680tccattggag
gaaagctccc agtcgtcccc atcacggggg aagcacttgt tgggttttta
4740agcgaccttg gccggatcat gaatgtgagc gggggcccta tcactagaga
ggcctctaaa 4800gaaatacctg atttccttaa acatctagaa actgaagaca
acattaaggt gtggtttaat 4860aacaaaggct ggcatgccct ggtcagcttt
ctcaatgtgg cccacaacgc catcttacgg 4920gccagcctgc ctaaggacag
gagccccgag gagtatggaa tcaccgtcat tagccaaccc 4980ctgaacctga
ccaaggagca gctctcagag attacagtgc tgaccacttc agtggatgct
5040gtggttgcca tctgcgtgat tttctccatg tccttcgtcc cagccagctt
tgtcctttat 5100ttgatccagg agcgggtgaa caaatccaag cacctccagt
ttatcagtgg agtgagcccc 5160accacctact gggtgaccaa cttcctctgg
gacatcatga attattccgt gagtgctggg 5220ctggtggtgg gcatcttcat
cgggtttcag aagaaagcct acacttctcc agaaaacctt 5280cctgcccttg
tggcactgct cctgctgtat ggatgggcgg tcattcccat gatgtaccca
5340gcatccttcc tgtttgatgt ccccagcaca gcctatgtgg ctttatcttg
tgctaatctg 5400ttcatcggca tcaacagcag tgctattacc ttcatcttgg
aattatttga taataaccgg 5460acgctgctca ggttcaacgc cgtgctgagg
aagctgctca ttgtcttccc ccacttctgc 5520ctgggccggg gcctcattga
ccttgcactg agccaggctg tgacagatgt ctatgcccgg 5580tttggtgagg
agcactctgc aaatccgttc cactgggacc tgattgggaa gaacctgttt
5640gccatggtgg tggaaggggt ggtgtacttc ctcctgaccc tgctggtcca
gcgccacttc 5700ttcctctccc aatggattgc cgagcccact aaggagccca
ttgttgatga agatgatgat 5760gtggctgaag aaagacaaag aattattact
ggtggaaata aaactgacat cttaaggcta 5820catgaactaa ccaagattta
tctgggcacc tccagcccag cagtggacag gctgtgtgtc 5880ggagttcgcc
ctggagagtg ctttggcctc ctgggagtga atggtgccgg caaaacaacc
5940acattcaaga tgctcactgg ggacaccaca gtgacctcag gggatgccac
cgtagcaggc 6000aagagtattt taaccaatat ttctgaagtc catcaaaata
tgggctactg tcctcagttt 6060gatgcaatcg atgagctgct cacaggacga
gaacatcttt acctttatgc ccggcttcga 6120ggtgtaccag cagaagaaat
cgaaaaggtt gcaaactgga gtattaagag cctgggcctg 6180actgtctacg
ccgactgcct ggctggcacg tacagtgggg gcaacaagcg gaaactctcc
6240acagccatcg cactcattgg ctgcccaccg ctggtgctgc tggatgagcc
caccacaggg 6300atggaccccc aggcacgccg catgctgtgg aacgtcatcg
tgagcatcat cagaaaaggg 6360agggctgtgg tcctcacatc ccacagcatg
gaagaatgtg aggcactgtg tacccggctg 6420gccatcatgg taaagggcgc
ctttcgatgt atgggcacca ttcagcatct caagtccaaa 6480tttggagatg
gctatatcgt cacaatgaag atcaaatccc cgaaggacga cctgcttcct
6540gacctgaacc ctgtggagca gttcttccag gggaacttcc caggcagtgt
gcagagggag 6600aggcactaca acatgctcca gttccaggtc tcctcctcct
ccctggcgag gatcttccag 6660ctcctcctct cccacaagga cagcctgctc
atcgaggagt actcagtcac acagaccaca 6720ctggaccagg tgtttgtaaa
ttttgctaaa cagcagactg aaagtcatga cctccctctg 6780caccctcgag
ctgctggagc cagtcgacaa gcccaggac 681932273PRTHomo sapiens 3Met Gly
Phe Val Arg Gln Ile Gln Leu Leu Leu Trp Lys Asn Trp Thr1 5 10 15Leu
Arg Lys Arg Gln Lys Ile Arg Phe Val Val Glu Leu Val Trp Pro 20 25
30Leu Ser Leu Phe Leu Val Leu Ile Trp Leu Arg Asn Ala Asn Pro Leu
35 40 45Tyr Ser His His Glu Cys His Phe Pro Asn Lys Ala Met Pro Ser
Ala 50
55 60Gly Met Leu Pro Trp Leu Gln Gly Ile Phe Cys Asn Val Asn Asn
Pro65 70 75 80Cys Phe Gln Ser Pro Thr Pro Gly Glu Ser Pro Gly Ile
Val Ser Asn 85 90 95Tyr Asn Asn Ser Ile Leu Ala Arg Val Tyr Arg Asp
Phe Gln Glu Leu 100 105 110Leu Met Asn Ala Pro Glu Ser Gln His Leu
Gly Arg Ile Trp Thr Glu 115 120 125Leu His Ile Leu Ser Gln Phe Met
Asp Thr Leu Arg Thr His Pro Glu 130 135 140Arg Ile Ala Gly Arg Gly
Ile Arg Ile Arg Asp Ile Leu Lys Asp Glu145 150 155 160Glu Thr Leu
Thr Leu Phe Leu Ile Lys Asn Ile Gly Leu Ser Asp Ser 165 170 175Val
Val Tyr Leu Leu Ile Asn Ser Gln Val Arg Pro Glu Gln Phe Ala 180 185
190His Gly Val Pro Asp Leu Ala Leu Lys Asp Ile Ala Cys Ser Glu Ala
195 200 205Leu Leu Glu Arg Phe Ile Ile Phe Ser Gln Arg Arg Gly Ala
Lys Thr 210 215 220Val Arg Tyr Ala Leu Cys Ser Leu Ser Gln Gly Thr
Leu Gln Trp Ile225 230 235 240Glu Asp Thr Leu Tyr Ala Asn Val Asp
Phe Phe Lys Leu Phe Arg Val 245 250 255Leu Pro Thr Leu Leu Asp Ser
Arg Ser Gln Gly Ile Asn Leu Arg Ser 260 265 270Trp Gly Gly Ile Leu
Ser Asp Met Ser Pro Arg Ile Gln Glu Phe Ile 275 280 285His Arg Pro
Ser Met Gln Asp Leu Leu Trp Val Thr Arg Pro Leu Met 290 295 300Gln
Asn Gly Gly Pro Glu Thr Phe Thr Lys Leu Met Gly Ile Leu Ser305 310
315 320Asp Leu Leu Cys Gly Tyr Pro Glu Gly Gly Gly Ser Arg Val Leu
Ser 325 330 335Phe Asn Trp Tyr Glu Asp Asn Asn Tyr Lys Ala Phe Leu
Gly Ile Asp 340 345 350Ser Thr Arg Lys Asp Pro Ile Tyr Ser Tyr Asp
Arg Arg Thr Thr Ser 355 360 365Phe Cys Asn Ala Leu Ile Gln Ser Leu
Glu Ser Asn Pro Leu Thr Lys 370 375 380Ile Ala Trp Arg Ala Ala Lys
Pro Leu Leu Met Gly Lys Ile Leu Tyr385 390 395 400Thr Pro Asp Ser
Pro Ala Ala Arg Arg Ile Leu Lys Asn Ala Asn Ser 405 410 415Thr Phe
Glu Glu Leu Glu His Val Arg Lys Leu Val Lys Ala Trp Glu 420 425
430Glu Val Gly Pro Gln Ile Trp Tyr Phe Phe Asp Asn Ser Thr Gln Met
435 440 445Asn Met Ile Arg Asp Thr Leu Gly Asn Pro Thr Val Lys Asp
Phe Leu 450 455 460Asn Arg Gln Leu Gly Glu Glu Gly Ile Thr Ala Glu
Ala Ile Leu Asn465 470 475 480Phe Leu Tyr Lys Gly Pro Arg Glu Ser
Gln Ala Asp Asp Met Ala Asn 485 490 495Phe Asp Trp Arg Asp Ile Phe
Asn Ile Thr Asp Arg Thr Leu Arg Leu 500 505 510Val Asn Gln Tyr Leu
Glu Cys Leu Val Leu Asp Lys Phe Glu Ser Tyr 515 520 525Asn Asp Glu
Thr Gln Leu Thr Gln Arg Ala Leu Ser Leu Leu Glu Glu 530 535 540Asn
Met Phe Trp Ala Gly Val Val Phe Pro Asp Met Tyr Pro Trp Thr545 550
555 560Ser Ser Leu Pro Pro His Val Lys Tyr Lys Ile Arg Met Asp Ile
Asp 565 570 575Val Val Glu Lys Thr Asn Lys Ile Lys Asp Arg Tyr Trp
Asp Ser Gly 580 585 590Pro Arg Ala Asp Pro Val Glu Asp Phe Arg Tyr
Ile Trp Gly Gly Phe 595 600 605Ala Tyr Leu Gln Asp Met Val Glu Gln
Gly Ile Thr Arg Ser Gln Val 610 615 620Gln Ala Glu Ala Pro Val Gly
Ile Tyr Leu Gln Gln Met Pro Tyr Pro625 630 635 640Cys Phe Val Asp
Asp Ser Phe Met Ile Ile Leu Asn Arg Cys Phe Pro 645 650 655Ile Phe
Met Val Leu Ala Trp Ile Tyr Ser Val Ser Met Thr Val Lys 660 665
670Ser Ile Val Leu Glu Lys Glu Leu Arg Leu Lys Glu Thr Leu Lys Asn
675 680 685Gln Gly Val Ser Asn Ala Val Ile Trp Cys Thr Trp Phe Leu
Asp Ser 690 695 700Phe Ser Ile Met Ser Met Ser Ile Phe Leu Leu Thr
Ile Phe Ile Met705 710 715 720His Gly Arg Ile Leu His Tyr Ser Asp
Pro Phe Ile Leu Phe Leu Phe 725 730 735Leu Leu Ala Phe Ser Thr Ala
Thr Ile Met Leu Cys Phe Leu Leu Ser 740 745 750Thr Phe Phe Ser Lys
Ala Ser Leu Ala Ala Ala Cys Ser Gly Val Ile 755 760 765Tyr Phe Thr
Leu Tyr Leu Pro His Ile Leu Cys Phe Ala Trp Gln Asp 770 775 780Arg
Met Thr Ala Glu Leu Lys Lys Ala Val Ser Leu Leu Ser Pro Val785 790
795 800Ala Phe Gly Phe Gly Thr Glu Tyr Leu Val Arg Phe Glu Glu Gln
Gly 805 810 815Leu Gly Leu Gln Trp Ser Asn Ile Gly Asn Ser Pro Thr
Glu Gly Asp 820 825 830Glu Phe Ser Phe Leu Leu Ser Met Gln Met Met
Leu Leu Asp Ala Ala 835 840 845Cys Tyr Gly Leu Leu Ala Trp Tyr Leu
Asp Gln Val Phe Pro Gly Asp 850 855 860Tyr Gly Thr Pro Leu Pro Trp
Tyr Phe Leu Leu Gln Glu Ser Tyr Trp865 870 875 880Leu Ser Gly Glu
Gly Cys Ser Thr Arg Glu Glu Arg Ala Leu Glu Lys 885 890 895Thr Glu
Pro Leu Thr Glu Glu Thr Glu Asp Pro Glu His Pro Glu Gly 900 905
910Ile His Asp Ser Phe Phe Glu Arg Glu His Pro Gly Trp Val Pro Gly
915 920 925Val Cys Val Lys Asn Leu Val Lys Ile Phe Glu Pro Cys Gly
Arg Pro 930 935 940Ala Val Asp Arg Leu Asn Ile Thr Phe Tyr Glu Asn
Gln Ile Thr Ala945 950 955 960Phe Leu Gly His Asn Gly Ala Gly Lys
Thr Thr Thr Leu Ser Ile Leu 965 970 975Thr Gly Leu Leu Pro Pro Thr
Ser Gly Thr Val Leu Val Gly Gly Arg 980 985 990Asp Ile Glu Thr Ser
Leu Asp Ala Val Arg Gln Ser Leu Gly Met Cys 995 1000 1005Pro Gln
His Asn Ile Leu Phe His His Leu Thr Val Ala Glu His 1010 1015
1020Met Leu Phe Tyr Ala Gln Leu Lys Gly Lys Ser Gln Glu Glu Ala
1025 1030 1035Gln Leu Glu Met Glu Ala Met Leu Glu Asp Thr Gly Leu
His His 1040 1045 1050Lys Arg Asn Glu Glu Ala Gln Asp Leu Ser Gly
Gly Met Gln Arg 1055 1060 1065Lys Leu Ser Val Ala Ile Ala Phe Val
Gly Asp Ala Lys Val Val 1070 1075 1080Ile Leu Asp Glu Pro Thr Ser
Gly Val Asp Pro Tyr Ser Arg Arg 1085 1090 1095Ser Ile Trp Asp Leu
Leu Leu Lys Tyr Arg Ser Gly Arg Thr Ile 1100 1105 1110Ile Met Pro
Thr His His Met Asp Glu Ala Asp His Gln Gly Asp 1115 1120 1125Arg
Ile Ala Ile Ile Ala Gln Gly Arg Leu Tyr Cys Ser Gly Thr 1130 1135
1140Pro Leu Phe Leu Lys Asn Cys Phe Gly Thr Gly Leu Tyr Leu Thr
1145 1150 1155Leu Val Arg Lys Met Lys Asn Ile Gln Ser Gln Arg Lys
Gly Ser 1160 1165 1170Glu Gly Thr Cys Ser Cys Ser Ser Lys Gly Phe
Ser Thr Thr Cys 1175 1180 1185Pro Ala His Val Asp Asp Leu Thr Pro
Glu Gln Val Leu Asp Gly 1190 1195 1200Asp Val Asn Glu Leu Met Asp
Val Val Leu His His Val Pro Glu 1205 1210 1215Ala Lys Leu Val Glu
Cys Ile Gly Gln Glu Leu Ile Phe Leu Leu 1220 1225 1230Pro Asn Lys
Asn Phe Lys His Arg Ala Tyr Ala Ser Leu Phe Arg 1235 1240 1245Glu
Leu Glu Glu Thr Leu Ala Asp Leu Gly Leu Ser Ser Phe Gly 1250 1255
1260Ile Ser Asp Thr Pro Leu Glu Glu Ile Phe Leu Lys Val Thr Glu
1265 1270 1275Asp Ser Asp Ser Gly Pro Leu Phe Ala Gly Gly Ala Gln
Gln Lys 1280 1285 1290Arg Glu Asn Val Asn Pro Arg His Pro Cys Leu
Gly Pro Arg Glu 1295 1300 1305Lys Ala Gly Gln Thr Pro Gln Asp Ser
Asn Val Cys Ser Pro Gly 1310 1315 1320Ala Pro Ala Ala His Pro Glu
Gly Gln Pro Pro Pro Glu Pro Glu 1325 1330 1335Cys Pro Gly Pro Gln
Leu Asn Thr Gly Thr Gln Leu Val Leu Gln 1340 1345 1350His Val Gln
Ala Leu Leu Val Lys Arg Phe Gln His Thr Ile Arg 1355 1360 1365Ser
His Lys Asp Phe Leu Ala Gln Ile Val Leu Pro Ala Thr Phe 1370 1375
1380Val Phe Leu Ala Leu Met Leu Ser Ile Val Ile Leu Pro Phe Gly
1385 1390 1395Glu Tyr Pro Ala Leu Thr Leu His Pro Trp Ile Tyr Gly
Gln Gln 1400 1405 1410Tyr Thr Phe Phe Ser Met Asp Glu Pro Gly Ser
Glu Gln Phe Thr 1415 1420 1425Val Leu Ala Asp Val Leu Leu Asn Lys
Pro Gly Phe Gly Asn Arg 1430 1435 1440Cys Leu Lys Glu Gly Trp Leu
Pro Glu Tyr Pro Cys Gly Asn Ser 1445 1450 1455Thr Pro Trp Lys Thr
Pro Ser Val Ser Pro Asn Ile Thr Gln Leu 1460 1465 1470Phe Gln Lys
Gln Lys Trp Thr Gln Val Asn Pro Ser Pro Ser Cys 1475 1480 1485Arg
Cys Ser Thr Arg Glu Lys Leu Thr Met Leu Pro Glu Cys Pro 1490 1495
1500Glu Gly Ala Gly Gly Leu Pro Pro Pro Gln Arg Thr Gln Arg Ser
1505 1510 1515Thr Glu Ile Leu Gln Asp Leu Thr Asp Arg Asn Ile Ser
Asp Phe 1520 1525 1530Leu Val Lys Thr Tyr Pro Ala Leu Ile Arg Ser
Ser Leu Lys Ser 1535 1540 1545Lys Phe Trp Val Asn Glu Gln Arg Tyr
Gly Gly Ile Ser Ile Gly 1550 1555 1560Gly Lys Leu Pro Val Val Pro
Ile Thr Gly Glu Ala Leu Val Gly 1565 1570 1575Phe Leu Ser Asp Leu
Gly Arg Ile Met Asn Val Ser Gly Gly Pro 1580 1585 1590Ile Thr Arg
Glu Ala Ser Lys Glu Ile Pro Asp Phe Leu Lys His 1595 1600 1605Leu
Glu Thr Glu Asp Asn Ile Lys Val Trp Phe Asn Asn Lys Gly 1610 1615
1620Trp His Ala Leu Val Ser Phe Leu Asn Val Ala His Asn Ala Ile
1625 1630 1635Leu Arg Ala Ser Leu Pro Lys Asp Arg Ser Pro Glu Glu
Tyr Gly 1640 1645 1650Ile Thr Val Ile Ser Gln Pro Leu Asn Leu Thr
Lys Glu Gln Leu 1655 1660 1665Ser Glu Ile Thr Val Leu Thr Thr Ser
Val Asp Ala Val Val Ala 1670 1675 1680Ile Cys Val Ile Phe Ser Met
Ser Phe Val Pro Ala Ser Phe Val 1685 1690 1695Leu Tyr Leu Ile Gln
Glu Arg Val Asn Lys Ser Lys His Leu Gln 1700 1705 1710Phe Ile Ser
Gly Val Ser Pro Thr Thr Tyr Trp Val Thr Asn Phe 1715 1720 1725Leu
Trp Asp Ile Met Asn Tyr Ser Val Ser Ala Gly Leu Val Val 1730 1735
1740Gly Ile Phe Ile Gly Phe Gln Lys Lys Ala Tyr Thr Ser Pro Glu
1745 1750 1755Asn Leu Pro Ala Leu Val Ala Leu Leu Leu Leu Tyr Gly
Trp Ala 1760 1765 1770Val Ile Pro Met Met Tyr Pro Ala Ser Phe Leu
Phe Asp Val Pro 1775 1780 1785Ser Thr Ala Tyr Val Ala Leu Ser Cys
Ala Asn Leu Phe Ile Gly 1790 1795 1800Ile Asn Ser Ser Ala Ile Thr
Phe Ile Leu Glu Leu Phe Asp Asn 1805 1810 1815Asn Arg Thr Leu Leu
Arg Phe Asn Ala Val Leu Arg Lys Leu Leu 1820 1825 1830Ile Val Phe
Pro His Phe Cys Leu Gly Arg Gly Leu Ile Asp Leu 1835 1840 1845Ala
Leu Ser Gln Ala Val Thr Asp Val Tyr Ala Arg Phe Gly Glu 1850 1855
1860Glu His Ser Ala Asn Pro Phe His Trp Asp Leu Ile Gly Lys Asn
1865 1870 1875Leu Phe Ala Met Val Val Glu Gly Val Val Tyr Phe Leu
Leu Thr 1880 1885 1890Leu Leu Val Gln Arg His Phe Phe Leu Ser Gln
Trp Ile Ala Glu 1895 1900 1905Pro Thr Lys Glu Pro Ile Val Asp Glu
Asp Asp Asp Val Ala Glu 1910 1915 1920Glu Arg Gln Arg Ile Ile Thr
Gly Gly Asn Lys Thr Asp Ile Leu 1925 1930 1935Arg Leu His Glu Leu
Thr Lys Ile Tyr Leu Gly Thr Ser Ser Pro 1940 1945 1950Ala Val Asp
Arg Leu Cys Val Gly Val Arg Pro Gly Glu Cys Phe 1955 1960 1965Gly
Leu Leu Gly Val Asn Gly Ala Gly Lys Thr Thr Thr Phe Lys 1970 1975
1980Met Leu Thr Gly Asp Thr Thr Val Thr Ser Gly Asp Ala Thr Val
1985 1990 1995Ala Gly Lys Ser Ile Leu Thr Asn Ile Ser Glu Val His
Gln Asn 2000 2005 2010Met Gly Tyr Cys Pro Gln Phe Asp Ala Ile Asp
Glu Leu Leu Thr 2015 2020 2025Gly Arg Glu His Leu Tyr Leu Tyr Ala
Arg Leu Arg Gly Val Pro 2030 2035 2040Ala Glu Glu Ile Glu Lys Val
Ala Asn Trp Ser Ile Lys Ser Leu 2045 2050 2055Gly Leu Thr Val Tyr
Ala Asp Cys Leu Ala Gly Thr Tyr Ser Gly 2060 2065 2070Gly Asn Lys
Arg Lys Leu Ser Thr Ala Ile Ala Leu Ile Gly Cys 2075 2080 2085Pro
Pro Leu Val Leu Leu Asp Glu Pro Thr Thr Gly Met Asp Pro 2090 2095
2100Gln Ala Arg Arg Met Leu Trp Asn Val Ile Val Ser Ile Ile Arg
2105 2110 2115Lys Gly Arg Ala Val Val Leu Thr Ser His Ser Met Glu
Glu Cys 2120 2125 2130Glu Ala Leu Cys Thr Arg Leu Ala Ile Met Val
Lys Gly Ala Phe 2135 2140 2145Arg Cys Met Gly Thr Ile Gln His Leu
Lys Ser Lys Phe Gly Asp 2150 2155 2160Gly Tyr Ile Val Thr Met Lys
Ile Lys Ser Pro Lys Asp Asp Leu 2165 2170 2175Leu Pro Asp Leu Asn
Pro Val Glu Gln Phe Phe Gln Gly Asn Phe 2180 2185 2190Pro Gly Ser
Val Gln Arg Glu Arg His Tyr Asn Met Leu Gln Phe 2195 2200 2205Gln
Val Ser Ser Ser Ser Leu Ala Arg Ile Phe Gln Leu Leu Leu 2210 2215
2220Ser His Lys Asp Ser Leu Leu Ile Glu Glu Tyr Ser Val Thr Gln
2225 2230 2235Thr Thr Leu Asp Gln Val Phe Val Asn Phe Ala Lys Gln
Gln Thr 2240 2245 2250Glu Ser His Asp Leu Pro Leu His Pro Arg Ala
Ala Gly Ala Ser 2255 2260 2265Arg Gln Ala Gln Asp 22704114DNAHomo
sapiens 4ggagtacccc tgtggcaact caacaccctg gaagactcct tctgtgtccc
caaacatcac 60ccagctgttc cagaagcaga aatggacaca ggtcaaccct tcaccatcct
gcag 11456705DNAHomo sapiens 5atgggcttcg tgagacagat acagcttttg
ctctggaaga actggaccct gcggaaaagg 60caaaagattc gctttgtggt ggaactcgtg
tggcctttat ctttatttct ggtcttgatc 120tggttaagga atgccaaccc
gctctacagc catcatgaat gccatttccc caacaaggcg 180atgccctcag
caggaatgct gccgtggctc caggggatct tctgcaatgt gaacaatccc
240tgttttcaaa gccccacccc aggagaatct cctggaattg tgtcaaacta
taacaactcc 300atcttggcaa gggtatatcg agattttcaa gaactcctca
tgaatgcacc agagagccag 360caccttggcc gtatttggac agagctacac
atcttgtccc aattcatgga caccctccgg 420actcacccgg agagaattgc
aggaagagga atacgaataa gggatatctt gaaagatgaa 480gaaacactga
cactatttct cattaaaaac atcggcctgt ctgactcagt ggtctacctt
540ctgatcaact ctcaagtccg tccagagcag ttcgctcatg gagtcccgga
cctggcgctg 600aaggacatcg cctgcagcga ggccctcctg gagcgcttca
tcatcttcag ccagagacgc 660ggggcaaaga cggtgcgcta tgccctgtgc
tccctctccc agggcaccct acagtggata 720gaagacactc tgtatgccaa
cgtggacttc ttcaagctct tccgtgtgct tcccacactc 780ctagacagcc
gttctcaagg tatcaatctg agatcttggg gaggaatatt atctgatatg
840tcaccaagaa ttcaagagtt tatccatcgg ccgagtatgc aggacttgct
gtgggtgacc 900aggcccctca tgcagaatgg tggtccagag acctttacaa
agctgatggg catcctgtct 960gacctcctgt gtggctaccc cgagggaggt
ggctctcggg tgctctcctt caactggtat 1020gaagacaata actataaggc
ctttctgggg attgactcca caaggaagga tcctatctat 1080tcttatgaca
gaagaacaac atccttttgt aatgcattga tccagagcct ggagtcaaat
1140cctttaacca aaatcgcttg gagggcggca aagcctttgc tgatgggaaa
aatcctgtac 1200actcctgatt cacctgcagc acgaaggata ctgaagaatg
ccaactcaac ttttgaagaa 1260ctggaacacg ttaggaagtt ggtcaaagcc
tgggaagaag tagggcccca
gatctggtac 1320ttctttgaca acagcacaca gatgaacatg atcagagata
ccctggggaa cccaacagta 1380aaagactttt tgaataggca gcttggtgaa
gaaggtatta ctgctgaagc catcctaaac 1440ttcctctaca agggccctcg
ggaaagccag gctgacgaca tggccaactt cgactggagg 1500gacatattta
acatcactga tcgcaccctc cgcctggtca atcaatacct ggagtgcttg
1560gtcctggata agtttgaaag ctacaatgat gaaactcagc tcacccaacg
tgccctctct 1620ctactggagg aaaacatgtt ctgggccgga gtggtattcc
ctgacatgta tccctggacc 1680agctctctac caccccacgt gaagtataag
atccgaatgg acatagacgt ggtggagaaa 1740accaataaga ttaaagacag
gtattgggat tctggtccca gagctgatcc cgtggaagat 1800ttccggtaca
tctggggcgg gtttgcctat ctgcaggaca tggttgaaca ggggatcaca
1860aggagccagg tgcaggcgga ggctccagtt ggaatctacc tccagcagat
gccctacccc 1920tgcttcgtgg acgattcttt catgatcatc ctgaaccgct
gtttccctat cttcatggtg 1980ctggcatgga tctactctgt ctccatgact
gtgaagagca tcgtcttgga gaaggagttg 2040cgactgaagg agaccttgaa
aaatcagggt gtctccaatg cagtgatttg gtgtacctgg 2100ttcctggaca
gcttctccat catgtcgatg agcatcttcc tcctgacgat attcatcatg
2160catggaagaa tcctacatta cagcgaccca ttcatcctct tcctgttctt
gttggctttc 2220tccactgcca ccatcatgct gtgctttctg ctcagcacct
tcttctccaa ggccagtctg 2280gcagcagcct gtagtggtgt catctatttc
accctctacc tgccacacat cctgtgcttc 2340gcctggcagg accgcatgac
cgctgagctg aagaaggctg tgagcttact gtctccggtg 2400gcatttggat
ttggcactga gtacctggtt cgctttgaag agcaaggcct ggggctgcag
2460tggagcaaca tcgggaacag tcccacggaa ggggacgaat tcagcttcct
gctgtccatg 2520cagatgatgc tccttgatgc tgcgtgctat ggcttactcg
cttggtacct tgatcaggtg 2580tttccaggag actatggaac cccacttcct
tggtactttc ttctacaaga gtcgtattgg 2640cttagcggtg aagggtgttc
aaccagagaa gaaagagccc tggaaaagac cgagccccta 2700acagaggaaa
cggaggatcc agagcaccca gaaggaatac acgactcctt ctttgaacgt
2760gagcatccag ggtgggttcc tggggtatgc gtgaagaatc tggtaaagat
ttttgagccc 2820tgtggccggc cagctgtgga ccgtctgaac atcaccttct
acgagaacca gatcaccgca 2880ttcctgggcc acaatggagc tgggaaaacc
accaccttgt ccatcctgac gggtctgttg 2940ccaccaacct ctgggactgt
gctcgttggg ggaagggaca ttgaaaccag cctggatgca 3000gtccggcaga
gccttggcat gtgtccacag cacaacatcc tgttccacca cctcacggtg
3060gctgagcaca tgctgttcta tgcccagctg aaaggaaagt cccaggagga
ggcccagctg 3120gagatggaag ccatgttgga ggacacaggc ctccaccaca
agcggaatga agaggctcag 3180gacctatcag gtggcatgca gagaaagctg
tcggttgcca ttgcctttgt gggagatgcc 3240aaggtggtga ttctggacga
acccacctct ggggtggacc cttactcgag acgctcaatc 3300tgggatctgc
tcctgaagta tcgctcaggc agaaccatca tcatgcccac tcaccacatg
3360gacgaggccg accaccaagg ggaccgcatt gccatcattg cccagggaag
gctctactgc 3420tcaggcaccc cactcttcct gaagaactgc tttggcacag
gcttgtactt aaccttggtg 3480cgcaagatga aaaacatcca gagccaaagg
aaaggcagtg aggggacctg cagctgctcg 3540tctaagggtt tctccaccac
gtgtccagcc cacgtcgatg acctaactcc agaacaagtc 3600ctggatgggg
atgtaaatga gctgatggat gtagttctcc accatgttcc agaggcaaag
3660ctggtggagt gcattggtca agaacttatc ttccttcttc caaataagaa
cttcaagcac 3720agagcatatg ccagcctttt cagagagctg gaggagacgc
tggctgacct tggtctcagc 3780agttttggaa tttctgacac tcccctggaa
gagatttttc tgaaggtcac ggaggattct 3840gattcaggac ctctgtttgc
gggtggcgct cagcagaaaa gagaaaacgt caacccccga 3900cacccctgct
tgggtcccag agagaaggct ggacagacac cccaggactc caatgtctgc
3960tccccagggg cgccggctgc tcacccagag ggccagcctc ccccagagcc
agagtgccca 4020ggcccgcagc tcaacacggg gacacagctg gtcctccagc
atgtgcaggc gctgctggtc 4080aagagattcc aacacaccat ccgcagccac
aaggacttcc tggcgcagat cgtgctcccg 4140gctacctttg tgtttttggc
tctgatgctt tctattgtta tccttccttt tggcgaatac 4200cccgctttga
cccttcaccc ctggatatat gggcagcagt acaccttctt cagcatggat
4260gaaccaggca gtgagcagtt cacggtactt gcagacgtcc tcctgaataa
gccaggcttt 4320ggcaaccgct gcctgaagga agggtggctt ccgtgcagca
ccagggagaa gctcaccatg 4380ctgccagagt gccccgaggg tgccgggggc
ctcccgcccc cccagagaac acagcgcagc 4440acggaaattc tacaagacct
gacggacagg aacatctccg acttcttggt aaaaacgtat 4500cctgctctta
taagaagcag cttaaagagc aaattctggg tcaatgaaca gaggtatgga
4560ggaatttcca ttggaggaaa gctcccagtc gtccccatca cgggggaagc
acttgttggg 4620tttttaagcg accttggccg gatcatgaat gtgagcgggg
gccctatcac tagagaggcc 4680tctaaagaaa tacctgattt ccttaaacat
ctagaaactg aagacaacat taaggtgtgg 4740tttaataaca aaggctggca
tgccctggtc agctttctca atgtggccca caacgccatc 4800ttacgggcca
gcctgcctaa ggacaggagc cccgaggagt atggaatcac cgtcattagc
4860caacccctga acctgaccaa ggagcagctc tcagagatta cagtgctgac
cacttcagtg 4920gatgctgtgg ttgccatctg cgtgattttc tccatgtcct
tcgtcccagc cagctttgtc 4980ctttatttga tccaggagcg ggtgaacaaa
tccaagcacc tccagtttat cagtggagtg 5040agccccacca cctactgggt
gaccaacttc ctctgggaca tcatgaatta ttccgtgagt 5100gctgggctgg
tggtgggcat cttcatcggg tttcagaaga aagcctacac ttctccagaa
5160aaccttcctg cccttgtggc actgctcctg ctgtatggat gggcggtcat
tcccatgatg 5220tacccagcat ccttcctgtt tgatgtcccc agcacagcct
atgtggcttt atcttgtgct 5280aatctgttca tcggcatcaa cagcagtgct
attaccttca tcttggaatt atttgataat 5340aaccggacgc tgctcaggtt
caacgccgtg ctgaggaagc tgctcattgt cttcccccac 5400ttctgcctgg
gccggggcct cattgacctt gcactgagcc aggctgtgac agatgtctat
5460gcccggtttg gtgaggagca ctctgcaaat ccgttccact gggacctgat
tgggaagaac 5520ctgtttgcca tggtggtgga aggggtggtg tacttcctcc
tgaccctgct ggtccagcgc 5580cacttcttcc tctcccaatg gattgccgag
cccactaagg agcccattgt tgatgaagat 5640gatgatgtgg ctgaagaaag
acaaagaatt attactggtg gaaataaaac tgacatctta 5700aggctacatg
aactaaccaa gatttatctg ggcacctcca gcccagcagt ggacaggctg
5760tgtgtcggag ttcgccctgg agagtgcttt ggcctcctgg gagtgaatgg
tgccggcaaa 5820acaaccacat tcaagatgct cactggggac accacagtga
cctcagggga tgccaccgta 5880gcaggcaaga gtattttaac caatatttct
gaagtccatc aaaatatggg ctactgtcct 5940cagtttgatg caatcgatga
gctgctcaca ggacgagaac atctttacct ttatgcccgg 6000cttcgaggtg
taccagcaga agaaatcgaa aaggttgcaa actggagtat taagagcctg
6060ggcctgactg tctacgccga ctgcctggct ggcacgtaca gtgggggcaa
caagcggaaa 6120ctctccacag ccatcgcact cattggctgc ccaccgctgg
tgctgctgga tgagcccacc 6180acagggatgg acccccaggc acgccgcatg
ctgtggaacg tcatcgtgag catcatcaga 6240aaagggaggg ctgtggtcct
cacatcccac agcatggaag aatgtgaggc actgtgtacc 6300cggctggcca
tcatggtaaa gggcgccttt cgatgtatgg gcaccattca gcatctcaag
6360tccaaatttg gagatggcta tatcgtcaca atgaagatca aatccccgaa
ggacgacctg 6420cttcctgacc tgaaccctgt ggagcagttc ttccagggga
acttcccagg cagtgtgcag 6480agggagaggc actacaacat gctccagttc
caggtctcct cctcctccct ggcgaggatc 6540ttccagctcc tcctctccca
caaggacagc ctgctcatcg aggagtactc agtcacacag 6600accacactgg
accaggtgtt tgtaaatttt gctaaacagc agactgaaag tcatgacctc
6660cctctgcacc ctcgagctgc tggagccagt cgacaagccc aggac
670562235PRTHomo sapiens 6Met Gly Phe Val Arg Gln Ile Gln Leu Leu
Leu Trp Lys Asn Trp Thr1 5 10 15Leu Arg Lys Arg Gln Lys Ile Arg Phe
Val Val Glu Leu Val Trp Pro 20 25 30Leu Ser Leu Phe Leu Val Leu Ile
Trp Leu Arg Asn Ala Asn Pro Leu 35 40 45Tyr Ser His His Glu Cys His
Phe Pro Asn Lys Ala Met Pro Ser Ala 50 55 60Gly Met Leu Pro Trp Leu
Gln Gly Ile Phe Cys Asn Val Asn Asn Pro65 70 75 80Cys Phe Gln Ser
Pro Thr Pro Gly Glu Ser Pro Gly Ile Val Ser Asn 85 90 95Tyr Asn Asn
Ser Ile Leu Ala Arg Val Tyr Arg Asp Phe Gln Glu Leu 100 105 110Leu
Met Asn Ala Pro Glu Ser Gln His Leu Gly Arg Ile Trp Thr Glu 115 120
125Leu His Ile Leu Ser Gln Phe Met Asp Thr Leu Arg Thr His Pro Glu
130 135 140Arg Ile Ala Gly Arg Gly Ile Arg Ile Arg Asp Ile Leu Lys
Asp Glu145 150 155 160Glu Thr Leu Thr Leu Phe Leu Ile Lys Asn Ile
Gly Leu Ser Asp Ser 165 170 175Val Val Tyr Leu Leu Ile Asn Ser Gln
Val Arg Pro Glu Gln Phe Ala 180 185 190His Gly Val Pro Asp Leu Ala
Leu Lys Asp Ile Ala Cys Ser Glu Ala 195 200 205Leu Leu Glu Arg Phe
Ile Ile Phe Ser Gln Arg Arg Gly Ala Lys Thr 210 215 220Val Arg Tyr
Ala Leu Cys Ser Leu Ser Gln Gly Thr Leu Gln Trp Ile225 230 235
240Glu Asp Thr Leu Tyr Ala Asn Val Asp Phe Phe Lys Leu Phe Arg Val
245 250 255Leu Pro Thr Leu Leu Asp Ser Arg Ser Gln Gly Ile Asn Leu
Arg Ser 260 265 270Trp Gly Gly Ile Leu Ser Asp Met Ser Pro Arg Ile
Gln Glu Phe Ile 275 280 285His Arg Pro Ser Met Gln Asp Leu Leu Trp
Val Thr Arg Pro Leu Met 290 295 300Gln Asn Gly Gly Pro Glu Thr Phe
Thr Lys Leu Met Gly Ile Leu Ser305 310 315 320Asp Leu Leu Cys Gly
Tyr Pro Glu Gly Gly Gly Ser Arg Val Leu Ser 325 330 335Phe Asn Trp
Tyr Glu Asp Asn Asn Tyr Lys Ala Phe Leu Gly Ile Asp 340 345 350Ser
Thr Arg Lys Asp Pro Ile Tyr Ser Tyr Asp Arg Arg Thr Thr Ser 355 360
365Phe Cys Asn Ala Leu Ile Gln Ser Leu Glu Ser Asn Pro Leu Thr Lys
370 375 380Ile Ala Trp Arg Ala Ala Lys Pro Leu Leu Met Gly Lys Ile
Leu Tyr385 390 395 400Thr Pro Asp Ser Pro Ala Ala Arg Arg Ile Leu
Lys Asn Ala Asn Ser 405 410 415Thr Phe Glu Glu Leu Glu His Val Arg
Lys Leu Val Lys Ala Trp Glu 420 425 430Glu Val Gly Pro Gln Ile Trp
Tyr Phe Phe Asp Asn Ser Thr Gln Met 435 440 445Asn Met Ile Arg Asp
Thr Leu Gly Asn Pro Thr Val Lys Asp Phe Leu 450 455 460Asn Arg Gln
Leu Gly Glu Glu Gly Ile Thr Ala Glu Ala Ile Leu Asn465 470 475
480Phe Leu Tyr Lys Gly Pro Arg Glu Ser Gln Ala Asp Asp Met Ala Asn
485 490 495Phe Asp Trp Arg Asp Ile Phe Asn Ile Thr Asp Arg Thr Leu
Arg Leu 500 505 510Val Asn Gln Tyr Leu Glu Cys Leu Val Leu Asp Lys
Phe Glu Ser Tyr 515 520 525Asn Asp Glu Thr Gln Leu Thr Gln Arg Ala
Leu Ser Leu Leu Glu Glu 530 535 540Asn Met Phe Trp Ala Gly Val Val
Phe Pro Asp Met Tyr Pro Trp Thr545 550 555 560Ser Ser Leu Pro Pro
His Val Lys Tyr Lys Ile Arg Met Asp Ile Asp 565 570 575Val Val Glu
Lys Thr Asn Lys Ile Lys Asp Arg Tyr Trp Asp Ser Gly 580 585 590Pro
Arg Ala Asp Pro Val Glu Asp Phe Arg Tyr Ile Trp Gly Gly Phe 595 600
605Ala Tyr Leu Gln Asp Met Val Glu Gln Gly Ile Thr Arg Ser Gln Val
610 615 620Gln Ala Glu Ala Pro Val Gly Ile Tyr Leu Gln Gln Met Pro
Tyr Pro625 630 635 640Cys Phe Val Asp Asp Ser Phe Met Ile Ile Leu
Asn Arg Cys Phe Pro 645 650 655Ile Phe Met Val Leu Ala Trp Ile Tyr
Ser Val Ser Met Thr Val Lys 660 665 670Ser Ile Val Leu Glu Lys Glu
Leu Arg Leu Lys Glu Thr Leu Lys Asn 675 680 685Gln Gly Val Ser Asn
Ala Val Ile Trp Cys Thr Trp Phe Leu Asp Ser 690 695 700Phe Ser Ile
Met Ser Met Ser Ile Phe Leu Leu Thr Ile Phe Ile Met705 710 715
720His Gly Arg Ile Leu His Tyr Ser Asp Pro Phe Ile Leu Phe Leu Phe
725 730 735Leu Leu Ala Phe Ser Thr Ala Thr Ile Met Leu Cys Phe Leu
Leu Ser 740 745 750Thr Phe Phe Ser Lys Ala Ser Leu Ala Ala Ala Cys
Ser Gly Val Ile 755 760 765Tyr Phe Thr Leu Tyr Leu Pro His Ile Leu
Cys Phe Ala Trp Gln Asp 770 775 780Arg Met Thr Ala Glu Leu Lys Lys
Ala Val Ser Leu Leu Ser Pro Val785 790 795 800Ala Phe Gly Phe Gly
Thr Glu Tyr Leu Val Arg Phe Glu Glu Gln Gly 805 810 815Leu Gly Leu
Gln Trp Ser Asn Ile Gly Asn Ser Pro Thr Glu Gly Asp 820 825 830Glu
Phe Ser Phe Leu Leu Ser Met Gln Met Met Leu Leu Asp Ala Ala 835 840
845Cys Tyr Gly Leu Leu Ala Trp Tyr Leu Asp Gln Val Phe Pro Gly Asp
850 855 860Tyr Gly Thr Pro Leu Pro Trp Tyr Phe Leu Leu Gln Glu Ser
Tyr Trp865 870 875 880Leu Ser Gly Glu Gly Cys Ser Thr Arg Glu Glu
Arg Ala Leu Glu Lys 885 890 895Thr Glu Pro Leu Thr Glu Glu Thr Glu
Asp Pro Glu His Pro Glu Gly 900 905 910Ile His Asp Ser Phe Phe Glu
Arg Glu His Pro Gly Trp Val Pro Gly 915 920 925Val Cys Val Lys Asn
Leu Val Lys Ile Phe Glu Pro Cys Gly Arg Pro 930 935 940Ala Val Asp
Arg Leu Asn Ile Thr Phe Tyr Glu Asn Gln Ile Thr Ala945 950 955
960Phe Leu Gly His Asn Gly Ala Gly Lys Thr Thr Thr Leu Ser Ile Leu
965 970 975Thr Gly Leu Leu Pro Pro Thr Ser Gly Thr Val Leu Val Gly
Gly Arg 980 985 990Asp Ile Glu Thr Ser Leu Asp Ala Val Arg Gln Ser
Leu Gly Met Cys 995 1000 1005Pro Gln His Asn Ile Leu Phe His His
Leu Thr Val Ala Glu His 1010 1015 1020Met Leu Phe Tyr Ala Gln Leu
Lys Gly Lys Ser Gln Glu Glu Ala 1025 1030 1035Gln Leu Glu Met Glu
Ala Met Leu Glu Asp Thr Gly Leu His His 1040 1045 1050Lys Arg Asn
Glu Glu Ala Gln Asp Leu Ser Gly Gly Met Gln Arg 1055 1060 1065Lys
Leu Ser Val Ala Ile Ala Phe Val Gly Asp Ala Lys Val Val 1070 1075
1080Ile Leu Asp Glu Pro Thr Ser Gly Val Asp Pro Tyr Ser Arg Arg
1085 1090 1095Ser Ile Trp Asp Leu Leu Leu Lys Tyr Arg Ser Gly Arg
Thr Ile 1100 1105 1110Ile Met Pro Thr His His Met Asp Glu Ala Asp
His Gln Gly Asp 1115 1120 1125Arg Ile Ala Ile Ile Ala Gln Gly Arg
Leu Tyr Cys Ser Gly Thr 1130 1135 1140Pro Leu Phe Leu Lys Asn Cys
Phe Gly Thr Gly Leu Tyr Leu Thr 1145 1150 1155Leu Val Arg Lys Met
Lys Asn Ile Gln Ser Gln Arg Lys Gly Ser 1160 1165 1170Glu Gly Thr
Cys Ser Cys Ser Ser Lys Gly Phe Ser Thr Thr Cys 1175 1180 1185Pro
Ala His Val Asp Asp Leu Thr Pro Glu Gln Val Leu Asp Gly 1190 1195
1200Asp Val Asn Glu Leu Met Asp Val Val Leu His His Val Pro Glu
1205 1210 1215Ala Lys Leu Val Glu Cys Ile Gly Gln Glu Leu Ile Phe
Leu Leu 1220 1225 1230Pro Asn Lys Asn Phe Lys His Arg Ala Tyr Ala
Ser Leu Phe Arg 1235 1240 1245Glu Leu Glu Glu Thr Leu Ala Asp Leu
Gly Leu Ser Ser Phe Gly 1250 1255 1260Ile Ser Asp Thr Pro Leu Glu
Glu Ile Phe Leu Lys Val Thr Glu 1265 1270 1275Asp Ser Asp Ser Gly
Pro Leu Phe Ala Gly Gly Ala Gln Gln Lys 1280 1285 1290Arg Glu Asn
Val Asn Pro Arg His Pro Cys Leu Gly Pro Arg Glu 1295 1300 1305Lys
Ala Gly Gln Thr Pro Gln Asp Ser Asn Val Cys Ser Pro Gly 1310 1315
1320Ala Pro Ala Ala His Pro Glu Gly Gln Pro Pro Pro Glu Pro Glu
1325 1330 1335Cys Pro Gly Pro Gln Leu Asn Thr Gly Thr Gln Leu Val
Leu Gln 1340 1345 1350His Val Gln Ala Leu Leu Val Lys Arg Phe Gln
His Thr Ile Arg 1355 1360 1365Ser His Lys Asp Phe Leu Ala Gln Ile
Val Leu Pro Ala Thr Phe 1370 1375 1380Val Phe Leu Ala Leu Met Leu
Ser Ile Val Ile Leu Pro Phe Gly 1385 1390 1395Glu Tyr Pro Ala Leu
Thr Leu His Pro Trp Ile Tyr Gly Gln Gln 1400 1405 1410Tyr Thr Phe
Phe Ser Met Asp Glu Pro Gly Ser Glu Gln Phe Thr 1415 1420 1425Val
Leu Ala Asp Val Leu Leu Asn Lys Pro Gly Phe Gly Asn Arg 1430 1435
1440Cys Leu Lys Glu Gly Trp Leu Pro Cys Ser Thr Arg Glu Lys Leu
1445 1450 1455Thr Met Leu Pro Glu Cys Pro Glu Gly Ala Gly Gly Leu
Pro Pro 1460 1465 1470Pro Gln Arg Thr Gln Arg Ser Thr Glu Ile Leu
Gln Asp Leu Thr 1475 1480 1485Asp Arg Asn Ile Ser Asp Phe Leu Val
Lys Thr Tyr Pro Ala Leu 1490 1495 1500Ile Arg Ser Ser Leu Lys Ser
Lys Phe Trp Val Asn Glu Gln Arg 1505 1510 1515Tyr Gly Gly Ile Ser
Ile Gly Gly Lys Leu Pro Val Val Pro Ile 1520 1525 1530Thr Gly Glu
Ala Leu Val Gly Phe Leu Ser Asp Leu Gly Arg Ile 1535 1540 1545Met
Asn Val Ser Gly Gly Pro Ile Thr Arg Glu Ala Ser Lys Glu 1550 1555
1560Ile Pro Asp Phe Leu Lys His Leu Glu Thr Glu Asp Asn Ile Lys
1565
1570 1575Val Trp Phe Asn Asn Lys Gly Trp His Ala Leu Val Ser Phe
Leu 1580 1585 1590Asn Val Ala His Asn Ala Ile Leu Arg Ala Ser Leu
Pro Lys Asp 1595 1600 1605Arg Ser Pro Glu Glu Tyr Gly Ile Thr Val
Ile Ser Gln Pro Leu 1610 1615 1620Asn Leu Thr Lys Glu Gln Leu Ser
Glu Ile Thr Val Leu Thr Thr 1625 1630 1635Ser Val Asp Ala Val Val
Ala Ile Cys Val Ile Phe Ser Met Ser 1640 1645 1650Phe Val Pro Ala
Ser Phe Val Leu Tyr Leu Ile Gln Glu Arg Val 1655 1660 1665Asn Lys
Ser Lys His Leu Gln Phe Ile Ser Gly Val Ser Pro Thr 1670 1675
1680Thr Tyr Trp Val Thr Asn Phe Leu Trp Asp Ile Met Asn Tyr Ser
1685 1690 1695Val Ser Ala Gly Leu Val Val Gly Ile Phe Ile Gly Phe
Gln Lys 1700 1705 1710Lys Ala Tyr Thr Ser Pro Glu Asn Leu Pro Ala
Leu Val Ala Leu 1715 1720 1725Leu Leu Leu Tyr Gly Trp Ala Val Ile
Pro Met Met Tyr Pro Ala 1730 1735 1740Ser Phe Leu Phe Asp Val Pro
Ser Thr Ala Tyr Val Ala Leu Ser 1745 1750 1755Cys Ala Asn Leu Phe
Ile Gly Ile Asn Ser Ser Ala Ile Thr Phe 1760 1765 1770Ile Leu Glu
Leu Phe Asp Asn Asn Arg Thr Leu Leu Arg Phe Asn 1775 1780 1785Ala
Val Leu Arg Lys Leu Leu Ile Val Phe Pro His Phe Cys Leu 1790 1795
1800Gly Arg Gly Leu Ile Asp Leu Ala Leu Ser Gln Ala Val Thr Asp
1805 1810 1815Val Tyr Ala Arg Phe Gly Glu Glu His Ser Ala Asn Pro
Phe His 1820 1825 1830Trp Asp Leu Ile Gly Lys Asn Leu Phe Ala Met
Val Val Glu Gly 1835 1840 1845Val Val Tyr Phe Leu Leu Thr Leu Leu
Val Gln Arg His Phe Phe 1850 1855 1860Leu Ser Gln Trp Ile Ala Glu
Pro Thr Lys Glu Pro Ile Val Asp 1865 1870 1875Glu Asp Asp Asp Val
Ala Glu Glu Arg Gln Arg Ile Ile Thr Gly 1880 1885 1890Gly Asn Lys
Thr Asp Ile Leu Arg Leu His Glu Leu Thr Lys Ile 1895 1900 1905Tyr
Leu Gly Thr Ser Ser Pro Ala Val Asp Arg Leu Cys Val Gly 1910 1915
1920Val Arg Pro Gly Glu Cys Phe Gly Leu Leu Gly Val Asn Gly Ala
1925 1930 1935Gly Lys Thr Thr Thr Phe Lys Met Leu Thr Gly Asp Thr
Thr Val 1940 1945 1950Thr Ser Gly Asp Ala Thr Val Ala Gly Lys Ser
Ile Leu Thr Asn 1955 1960 1965Ile Ser Glu Val His Gln Asn Met Gly
Tyr Cys Pro Gln Phe Asp 1970 1975 1980Ala Ile Asp Glu Leu Leu Thr
Gly Arg Glu His Leu Tyr Leu Tyr 1985 1990 1995Ala Arg Leu Arg Gly
Val Pro Ala Glu Glu Ile Glu Lys Val Ala 2000 2005 2010Asn Trp Ser
Ile Lys Ser Leu Gly Leu Thr Val Tyr Ala Asp Cys 2015 2020 2025Leu
Ala Gly Thr Tyr Ser Gly Gly Asn Lys Arg Lys Leu Ser Thr 2030 2035
2040Ala Ile Ala Leu Ile Gly Cys Pro Pro Leu Val Leu Leu Asp Glu
2045 2050 2055Pro Thr Thr Gly Met Asp Pro Gln Ala Arg Arg Met Leu
Trp Asn 2060 2065 2070Val Ile Val Ser Ile Ile Arg Lys Gly Arg Ala
Val Val Leu Thr 2075 2080 2085Ser His Ser Met Glu Glu Cys Glu Ala
Leu Cys Thr Arg Leu Ala 2090 2095 2100Ile Met Val Lys Gly Ala Phe
Arg Cys Met Gly Thr Ile Gln His 2105 2110 2115Leu Lys Ser Lys Phe
Gly Asp Gly Tyr Ile Val Thr Met Lys Ile 2120 2125 2130Lys Ser Pro
Lys Asp Asp Leu Leu Pro Asp Leu Asn Pro Val Glu 2135 2140 2145Gln
Phe Phe Gln Gly Asn Phe Pro Gly Ser Val Gln Arg Glu Arg 2150 2155
2160His Tyr Asn Met Leu Gln Phe Gln Val Ser Ser Ser Ser Leu Ala
2165 2170 2175Arg Ile Phe Gln Leu Leu Leu Ser His Lys Asp Ser Leu
Leu Ile 2180 2185 2190Glu Glu Tyr Ser Val Thr Gln Thr Thr Leu Asp
Gln Val Phe Val 2195 2200 2205Asn Phe Ala Lys Gln Gln Thr Glu Ser
His Asp Leu Pro Leu His 2210 2215 2220Pro Arg Ala Ala Gly Ala Ser
Arg Gln Ala Gln Asp 2225 2230 2235722DNAArtificial
SequenceDescription of Artificial Sequence Primer 7atcctctgac
tcagcaatca ca 22821DNAArtificial SequenceDescription of Artificial
Sequence Primer 8ttgcaattac aaatgcaatg g 21920DNAArtificial
SequenceDescription of Artificial Sequence Primer 9atccataccc
ttcccactcc 201021DNAArtificial SequenceDescription of Artificial
Sequence Primer 10gcagcagaag ataagcacac c 211138PRTHomo sapiens
11Glu Tyr Pro Cys Gly Asn Ser Thr Pro Trp Lys Thr Pro Ser Val Ser1
5 10 15Pro Asn Ile Thr Gln Leu Phe Gln Lys Gln Lys Trp Thr Gln Val
Asn 20 25 30Pro Ser Pro Ser Cys Arg 351220DNAArtificial
SequenceDescription of Artificial Sequence Primer 12accctctgct
aagctcagag 201319DNAArtificial SequenceDescription of Artificial
Sequence Primer 13accccacact tccaacctg 191420DNAArtificial
SequenceDescription of Artificial Sequence Primer 14aagtcctact
gcacacatgg 201519DNAArtificial SequenceDescription of Artificial
Sequence Primer 15acactcccac cccaagatc 191619DNAArtificial
SequenceDescription of Artificial Sequence Primer 16ttcccaaaaa
ggccaactc 191719DNAArtificial SequenceDescription of Artificial
Sequence Primer 17cacgcacgtg tgcattcag 191822DNAArtificial
SequenceDescription of Artificial Sequence Primer 18gctatttcct
tattaatgag gc 221920DNAArtificial SequenceDescription of Artificial
Sequence Primer 19ccaactctcc ctgttctttc 202020DNAArtificial
SequenceDescription of Artificial Sequence Primer 20tgtttccaat
cgactctggc 202121DNAArtificial SequenceDescription of Artificial
Sequence Primer 21ttcttgcctt tctcaggctg g 212219DNAArtificial
SequenceDescription of Artificial Sequence Primer 22gtattcccag
gttctgtgg 192319DNAArtificial SequenceDescription of Artificial
Sequence Primer 23taccccagga atcaccttg 192421DNAArtificial
SequenceDescription of Artificial Sequence Primer 24agcatatagg
agatcagact g 212521DNAArtificial SequenceDescription of Artificial
Sequence Primer 25tgacataagt ggggtaaatg g 212620DNAArtificial
SequenceDescription of Artificial Sequence Primer 26gagcattggc
ctcacagcag 202718DNAArtificial SequenceDescription of Artificial
Sequence Primer 27ccccaggttt gtttcacc 182821DNAArtificial
SequenceDescription of Artificial Sequence Primer 28agacatgtga
tgtggataca c 212920DNAArtificial SequenceDescription of Artificial
Sequence Primer 29gtgggaggtc cagggtacac 203019DNAArtificial
SequenceDescription of Artificial Sequence Primer 30aggggcagaa
aagacacac 193121DNAArtificial SequenceDescription of Artificial
Sequence Primer 31tagcgattaa ctctttcctg g 213219DNAArtificial
SequenceDescription of Artificial Sequence Primer 32ctcttcaggg
agccttagc 193320DNAArtificial SequenceDescription of Artificial
Sequence Primer 33ttcaagacca cttgacttgc 203418DNAArtificial
SequenceDescription of Artificial Sequence Primer 34tgggacagca
gccttatc 183522DNAArtificial SequenceDescription of Artificial
Sequence Primer 35ccaaatgtaa tttcccactg ac 223621DNAArtificial
SequenceDescription of Artificial Sequence Primer 36aatgagttcc
gagtcaccct g 213718DNAArtificial SequenceDescription of Artificial
Sequence Primer 37cccattcgcg tgtcatgg 183820DNAArtificial
SequenceDescription of Artificial Sequence Primer 38tccatctggg
ctttgttctc 203920DNAArtificial SequenceDescription of Artificial
Sequence Primer 39aatccaggca catgaacagg 204018DNAArtificial
SequenceDescription of Artificial Sequence Primer 40aggctggtgg
gagagagc 184118DNAArtificial SequenceDescription of Artificial
Sequence Primer 41agtggacccc ctcagagg 184221DNAArtificial
SequenceDescription of Artificial Sequence Primer 42ctgttgcatt
ggataaaagg c 214319DNAArtificial SequenceDescription of Artificial
Sequence Primer 43gatgaatgga gagggctgg 194421DNAArtificial
SequenceDescription of Artificial Sequence Primer 44ctgcggtaag
gtaggatagg g 214521DNAArtificial SequenceDescription of Artificial
Sequence Primer 45cacaccgttt acatagaggg c 214619DNAArtificial
SequenceDescription of Artificial Sequence Primer 46cctctcccct
cctttcctg 194719DNAArtificial SequenceDescription of Artificial
Sequence Primer 47gtcagtttcc gtaggcttc 194819DNAArtificial
SequenceDescription of Artificial Sequence Primer 48tggggccatg
taattaggc 194920DNAArtificial SequenceDescription of Artificial
Sequence Primer 49tgggaaagag tagacagccg 205018DNAArtificial
SequenceDescription of Artificial Sequence Primer 50actgaacctg
gtgtgggg 185119DNAArtificial SequenceDescription of Artificial
Sequence Primer 51tatctctgcc tgtgcccag 195220DNAArtificial
SequenceDescription of Artificial Sequence Primer 52gtaagatcag
ctgctggaag 205320DNAArtificial SequenceDescription of Artificial
Sequence Primer 53gaagctctcc tgcaccaagc 205418DNAArtificial
SequenceDescription of Artificial Sequence Primer 54aggtaccccc
acaatgcc 185522DNAArtificial SequenceDescription of Artificial
Sequence Primer 55tcattgtggt tccagtactc ag 225622DNAArtificial
SequenceDescription of Artificial Sequence Primer 56tttttgcaac
tatatagcca gg 225720DNAArtificial SequenceDescription of Artificial
Sequence Primer 57agcctgtgtg agtagccatg 205819DNAArtificial
SequenceDescription of Artificial Sequence Primer 58gcatcagggc
gaggctgtc 195920DNAArtificial SequenceDescription of Artificial
Sequence Primer 59cccagcaata ctgggagatg 206019DNAArtificial
SequenceDescription of Artificial Sequence Primer 60ggtaacctca
cagtcttcc 196119DNAArtificial SequenceDescription of Artificial
Sequence Primer 61gggaacgatg gctttttgc 196220DNAArtificial
SequenceDescription of Artificial Sequence Primer 62tcccattatg
aagcaatacc 206319DNAArtificial SequenceDescription of Artificial
Sequence Primer 63ccttagactt tcgagatgg 196423DNAArtificial
SequenceDescription of Artificial Sequence Primer 64gctaccagcc
tggtatttca ttg 236520DNAArtificial SequenceDescription of
Artificial Sequence Primer 65gttataaccc atgcctgaag
206618DNAArtificial SequenceDescription of Artificial Sequence
Primer 66tgcacgcgca cgtgtgac 186721DNAArtificial
SequenceDescription of Artificial Sequence Primer 67tgaaggtccc
agtgaagtgg g 216820DNAArtificial SequenceDescription of Artificial
Sequence Primer 68cagcagctat ccagtaaagg 206919DNAArtificial
SequenceDescription of Artificial Sequence Primer 69aacgcctgcc
atcttgaac 197020DNAArtificial SequenceDescription of Artificial
Sequence Primer 70gttgggcaca attcttatgc 207120DNAArtificial
SequenceDescription of Artificial Sequence Primer 71gttgtttgga
ggtcaggtac 207221DNAArtificial SequenceDescription of Artificial
Sequence Primer 72aacatcaccc agctgttcca g 217320DNAArtificial
SequenceDescription of Artificial Sequence Primer 73actcaggaga
taccagggac 207422DNAArtificial SequenceDescription of Artificial
Sequence Primer 74ggaagacaac aagcagtttc ac 227520DNAArtificial
SequenceDescription of Artificial Sequence Primer 75atctactgcc
ctgatcatac 207621DNAArtificial SequenceDescription of Artificial
Sequence Primer 76aagactgaga cttcagtctt c 217722DNAArtificial
SequenceDescription of Artificial Sequence Primer 77ggtgtgcctt
ttaaaagtgt gc 227822DNAArtificial SequenceDescription of Artificial
Sequence Primer 78ttcatgtttc cctacaaaac cc 227922DNAArtificial
SequenceDescription of Artificial Sequence Primer 79catgagagtt
tctcattcat gg 228022DNAArtificial SequenceDescription of Artificial
Sequence Primer 80tgtttacatg gtttttaggg cc 228119DNAArtificial
SequenceDescription of Artificial Sequence Primer 81ttcagcagga
ggagggatg 198222DNAArtificial SequenceDescription of Artificial
Sequence Primer 82cctttccttc actgatttct gc 228318DNAArtificial
SequenceDescription of Artificial Sequence Primer 83aatcagcact
tcgcggtg 188419DNAArtificial SequenceDescription of Artificial
Sequence Primer 84tgtaaggcct tcccaaagc 198520DNAArtificial
SequenceDescription of Artificial Sequence Primer 85tggtccttca
gcgcacacac 208620DNAArtificial SequenceDescription of Artificial
Sequence Primer 86cattttgcag agctggcagc 208720DNAArtificial
SequenceDescription of Artificial Sequence Primer 87cttctgtcag
gagatgatcc 208821DNAArtificial SequenceDescription of Artificial
Sequence Primer 88ggagtgcatt atatccagac g 218920DNAArtificial
SequenceDescription of Artificial Sequence Primer 89cctggctctg
cttgaccaac 209020DNAArtificial SequenceDescription of Artificial
Sequence Primer 90tgctgtcctg tgagagcatc 209119DNAArtificial
SequenceDescription of Artificial Sequence Primer 91gtaaccctcc
cagctttgg 199220DNAArtificial SequenceDescription of Artificial
Sequence Primer 92cagttcccac ataaggcctg 209319DNAArtificial
SequenceDescription of Artificial Sequence Primer 93cagttctgga
tgccctgag 199422DNAArtificial SequenceDescription of Artificial
Sequence Primer 94gaagagaggt cccatggaaa gg 229522DNAArtificial
SequenceDescription of Artificial Sequence Primer 95gcttgcataa
gcatatcaat tg 229621DNAArtificial SequenceDescription of Artificial
Sequence Primer 96ctcctaaacc atcctttgct c 219718DNAArtificial
SequenceDescription of
Artificial Sequence Primer 97aggcaggcac aagagctg
189818DNAArtificial SequenceDescription of Artificial Sequence
Primer 98cttaccctgg ggcctgac 189921DNAArtificial
SequenceDescription of Artificial Sequence Primer 99ctcagagcca
ccctactata g 2110020DNAArtificial SequenceDescription of Artificial
Sequence Primer 100gaagcttctc cagccctagc 2010120DNAArtificial
SequenceDescription of Artificial Sequence Primer 101tgcactctca
tgaaacaggc 2010220DNAArtificial SequenceDescription of Artificial
Sequence Primer 102gtttggggtg tttgcttgtc 2010321DNAArtificial
SequenceDescription of Artificial Sequence Primer 103acctctttcc
ccaacccaga g 2110420DNAArtificial SequenceDescription of Artificial
Sequence Primer 104gaagcagtaa tcagaagggc 2010521DNAArtificial
SequenceDescription of Artificial Sequence Primer 105gcctcacatt
cttccatgct g 2110620DNAArtificial SequenceDescription of Artificial
Sequence Primer 106tcacatccca caggcaagag 2010721DNAArtificial
SequenceDescription of Artificial Sequence Primer 107ttccaagtgt
caatggagaa c 2110820DNAArtificial SequenceDescription of Artificial
Sequence Primer 108attaccttag gcccaaccac 2010919DNAArtificial
SequenceDescription of Artificial Sequence Primer 109acactgggtg
ttctggacc 1911019DNAArtificial SequenceDescription of Artificial
Sequence Primer 110gtgtagggtg gtgttttcc 1911120DNAArtificial
SequenceDescription of Artificial Sequence Primer 111aagcccagtg
aaccagctgg 2011219DNAArtificial SequenceDescription of Artificial
Sequence Primer 112tcagctgagt gcccttcag 1911321DNAArtificial
SequenceDescription of Artificial Sequence Primer 113aggtgagcaa
gtcagtttcg g 2111420DNAArtificial SequenceDescription of Artificial
Sequence Primer 114ggtcttcgtg tgtggtcatt 2011518DNAArtificial
SequenceDescription of Artificial Sequence Primer 115ggtccagttc
ttccagag 1811622DNAArtificial SequenceDescription of Artificial
Sequence Primer 116atcctctgac tcagcaatca ca 2211721DNAArtificial
SequenceDescription of Artificial Sequence Primer 117ttgcaattac
aaatgcaatg g 211182261PRTMurine sp.MOD_RES(4)..(4)Variable amino
acid 118Met Ala Cys Xaa Pro Gln Leu Arg Leu Leu Leu Trp Lys Asn Leu
Thr1 5 10 15Phe Arg Arg Arg Gln Thr Cys Gln Leu Leu Leu Glu Val Ala
Trp Pro 20 25 30Leu Phe Ile Phe Leu Ile Leu Ile Ser Val Arg Leu Ser
Tyr Pro Pro 35 40 45Tyr Glu Gln His Glu Cys His Phe Pro Asn Lys Ala
Met Pro Ser Ala 50 55 60Gly Thr Leu Pro Trp Val Gln Gly Ile Ile Cys
Asn Ala Asn Asn Pro65 70 75 80Cys Phe Arg Tyr Pro Thr Pro Gly Glu
Ala Pro Gly Val Val Gly Asn 85 90 95Phe Asn Lys Ser Ile Val Ser Arg
Leu Phe Ser Asp Ala Gln Arg Leu 100 105 110Leu Leu Tyr Ser Gln Arg
Asp Thr Ser Ile Lys Asp Met His Lys Val 115 120 125Leu Arg Met Leu
Arg Gln Ile Lys His Pro Asn Ser Asn Leu Lys Leu 130 135 140Gln Asp
Phe Leu Val Asp Asn Glu Thr Phe Ser Gly Phe Leu Gln His145 150 155
160Asn Leu Ser Leu Pro Arg Ser Thr Val Asp Ser Leu Leu Gln Xaa Asn
165 170 175Val Gly Leu Gln Lys Val Phe Leu Gln Gly Tyr Gln Leu His
Leu Ala 180 185 190Ser Leu Cys Asn Gly Ser Lys Leu Glu Glu Ile Ile
Gln Leu Gly Asp 195 200 205Ala Glu Val Ser Ala Leu Cys Gly Leu Pro
Arg Lys Lys Leu Asp Ala 210 215 220Ala Glu Arg Val Leu Arg Tyr Asn
Met Asp Ile Leu Lys Pro Val Val225 230 235 240Thr Lys Leu Asn Ser
Thr Ser His Leu Pro Thr Gln His Leu Ala Glu 245 250 255Ala Thr Thr
Val Leu Leu Asp Ser Leu Gly Gly Leu Ala Gln Glu Leu 260 265 270Phe
Ser Thr Lys Ser Trp Ser Asp Met Arg Gln Glu Val Met Phe Leu 275 280
285Thr Asn Val Asn Ser Ser Ser Ser Ser Thr Gln Ile Tyr Gln Ala Val
290 295 300Ser Arg Ile Val Cys Gly His Pro Glu Gly Gly Gly Leu Lys
Ile Lys305 310 315 320Ser Leu Asn Trp Tyr Glu Asp Asn Asn Tyr Lys
Ala Leu Phe Gly Gly 325 330 335Asn Asn Thr Glu Glu Asp Val Asp Thr
Phe Tyr Asp Asn Ser Thr Thr 340 345 350Pro Tyr Cys Asn Asp Leu Met
Lys Asn Leu Glu Ser Ser Pro Leu Ser 355 360 365Arg Ile Ile Trp Lys
Ala Leu Lys Pro Leu Leu Val Gly Lys Ile Leu 370 375 380Tyr Thr Pro
Asp Thr Pro Ala Thr Arg Gln Val Met Ala Glu Val Asn385 390 395
400Lys Thr Phe Gln Glu Leu Ala Val Phe His Asp Leu Glu Gly Met Trp
405 410 415Glu Glu Leu Ser Pro Gln Ile Trp Thr Phe Met Glu Asn Ser
Gln Glu 420 425 430Met Asp Leu Val Arg Thr Leu Leu Asp Ser Arg Gly
Asn Asp Gln Phe 435 440 445Trp Glu Gln Lys Leu Asp Gly Leu Asp Trp
Thr Ala Gln Asp Ile Met 450 455 460Ala Phe Leu Ala Lys Asn Pro Glu
Asp Val Gln Ser Pro Asn Gly Ser465 470 475 480Val Tyr Thr Trp Arg
Glu Ala Phe Asn Glu Thr Asn Gln Ala Ile Gln 485 490 495Thr Ile Ser
Arg Phe Met Glu Cys Val Asn Leu Asn Lys Leu Glu Pro 500 505 510Ile
Pro Thr Glu Val Arg Leu Ile Asn Lys Ser Met Glu Leu Leu Asp 515 520
525Glu Arg Lys Phe Trp Ala Gly Ile Val Phe Thr Gly Ile Thr Pro Asp
530 535 540Ser Val Glu Leu Pro His His Val Lys Tyr Lys Ile Arg Met
Asp Ile545 550 555 560Asp Asn Val Glu Arg Thr Asn Lys Ile Lys Asp
Gly Tyr Trp Asp Pro 565 570 575Gly Pro Arg Ala Asp Pro Phe Glu Asp
Met Arg Tyr Val Trp Gly Gly 580 585 590Phe Ala Tyr Leu Gln Asp Val
Val Glu Gln Ala Ile Ile Arg Val Leu 595 600 605Thr Gly Ser Glu Lys
Lys Thr Gly Val Tyr Val Gln Gln Met Pro Tyr 610 615 620Pro Cys Tyr
Val Asp Asp Ile Phe Leu Arg Val Met Ser Arg Ser Met625 630 635
640Pro Leu Phe Met Thr Leu Ala Trp Ile Tyr Ser Val Ala Val Ile Ile
645 650 655Lys Ser Ile Val Tyr Glu Lys Glu Ala Arg Leu Lys Glu Thr
Met Arg 660 665 670Ile Met Gly Leu Asp Asn Gly Ile Leu Trp Phe Ser
Trp Phe Val Ser 675 680 685Ser Leu Ile Pro Leu Leu Val Ser Ala Gly
Leu Leu Val Val Ile Leu 690 695 700Lys Leu Gly Asn Leu Leu Pro Tyr
Ser Asp Pro Ser Val Val Phe Val705 710 715 720Phe Leu Ser Val Phe
Ala Met Val Thr Ile Leu Gln Cys Phe Leu Ile 725 730 735Ser Thr Leu
Phe Ser Arg Ala Asn Leu Ala Ala Ala Cys Gly Gly Ile 740 745 750Ile
Tyr Phe Thr Leu Tyr Leu Pro Tyr Val Leu Cys Val Ala Trp Gln 755 760
765Asp Tyr Val Gly Phe Ser Ile Lys Ile Phe Ala Ser Leu Leu Ser Pro
770 775 780Val Ala Phe Gly Phe Gly Cys Glu Tyr Phe Ala Leu Phe Glu
Glu Gln785 790 795 800Gly Ile Gly Val Gln Trp Asp Asn Leu Phe Glu
Ser Pro Val Glu Glu 805 810 815Asp Gly Phe Asn Leu Thr Thr Ala Val
Ser Met Met Leu Phe Asp Thr 820 825 830Phe Leu Tyr Gly Val Met Thr
Trp Tyr Ile Glu Ala Val Phe Pro Gly 835 840 845Gln Tyr Gly Ile Pro
Arg Pro Trp Tyr Phe Pro Cys Thr Lys Ser Tyr 850 855 860Trp Phe Gly
Glu Glu Ile Asp Glu Lys Ser His Pro Gly Ser Ser Gln865 870 875
880Lys Gly Val Ser Glu Ile Cys Met Glu Glu Glu Pro Thr His Leu Arg
885 890 895Leu Gly Val Ser Ile Gln Asn Leu Val Lys Val Tyr Arg Asp
Gly Met 900 905 910Lys Val Ala Val Asp Gly Leu Ala Leu Asn Phe Tyr
Glu Gly Gln Ile 915 920 925Thr Ser Phe Leu Gly His Asn Gly Ala Gly
Lys Thr Thr Thr Met Ser 930 935 940Ile Leu Thr Gly Leu Phe Pro Pro
Thr Ser Gly Thr Ala Tyr Ile Leu945 950 955 960Gly Lys Asp Ile Arg
Ser Glu Met Ser Ser Ile Arg Gln Asn Leu Gly 965 970 975Val Cys Pro
Gln His Asn Val Leu Phe Asp Met Leu Thr Val Glu Glu 980 985 990His
Ile Trp Phe Tyr Ala Arg Leu Lys Gly Leu Ser Glu Lys His Val 995
1000 1005Lys Ala Glu Met Glu Gln Met Ala Leu Asp Val Gly Leu Pro
Pro 1010 1015 1020Ser Lys Leu Lys Ser Lys Thr Ser Gln Leu Ser Gly
Gly Met Gln 1025 1030 1035Arg Lys Leu Ser Val Ala Leu Ala Phe Val
Gly Gly Ser Lys Val 1040 1045 1050Val Ile Leu Asp Glu Pro Thr Ala
Gly Val Asp Pro Tyr Ser Arg 1055 1060 1065Arg Gly Ile Trp Glu Leu
Leu Leu Lys Tyr Arg Gln Gly Arg Thr 1070 1075 1080Ile Ile Leu Ser
Thr His His Met Asp Glu Ala Asp Ile Leu Gly 1085 1090 1095Asp Arg
Ile Ala Ile Ile Ser His Gly Lys Leu Cys Cys Val Gly 1100 1105
1110Ser Ser Leu Phe Leu Lys Asn Gln Leu Gly Thr Gly Tyr Tyr Leu
1115 1120 1125Thr Leu Val Lys Lys Asp Val Glu Ser Ser Leu Ser Ser
Cys Arg 1130 1135 1140Asn Ser Ser Ser Thr Val Ser Cys Leu Lys Lys
Glu Asp Ser Val 1145 1150 1155Ser Gln Ser Ser Ser Asp Ala Gly Leu
Gly Ser Asp His Glu Ser 1160 1165 1170Asp Thr Leu Thr Ile Asp Val
Ser Ala Ile Ser Asn Leu Ile Arg 1175 1180 1185Lys His Val Ser Glu
Ala Arg Leu Val Glu Asp Ile Gly His Glu 1190 1195 1200Leu Thr Tyr
Val Leu Pro Tyr Glu Ala Ala Lys Glu Gly Ala Phe 1205 1210 1215Val
Glu Leu Phe His Glu Ile Asp Asp Arg Leu Ser Asp Leu Gly 1220 1225
1230Ile Ser Ser Tyr Gly Ile Ser Glu Thr Thr Leu Glu Glu Ile Phe
1235 1240 1245Leu Lys Val Ala Glu Glu Ser Gly Val Asp Ala Glu Thr
Ser Asp 1250 1255 1260Gly Thr Leu Pro Ala Arg Arg Asn Arg Arg Ala
Phe Gly Asp Lys 1265 1270 1275Gln Ser Cys Leu His Pro Phe Thr Glu
Asp Asp Ala Val Asp Pro 1280 1285 1290Asn Asp Ser Asp Ile Asp Pro
Glu Ser Arg Glu Thr Asp Leu Leu 1295 1300 1305Ser Gly Met Asp Gly
Lys Gly Ser Tyr Gln Leu Lys Gly Trp Lys 1310 1315 1320Leu Thr Gln
Gln Gln Phe Val Ala Leu Leu Trp Lys Arg Leu Leu 1325 1330 1335Ile
Ala Arg Arg Ser Arg Lys Gly Phe Phe Ala Gln Ile Val Leu 1340 1345
1350Pro Ala Val Phe Val Cys Ile Ala Leu Val Phe Ser Leu Ile Val
1355 1360 1365Pro Pro Phe Gly Lys Tyr Pro Ser Leu Glu Leu Gln Pro
Trp Met 1370 1375 1380Tyr Asn Glu Gln Tyr Thr Phe Val Ser Asn Asp
Ala Pro Glu Asp 1385 1390 1395Met Gly Thr Gln Glu Leu Leu Asn Ala
Leu Thr Lys Asp Pro Gly 1400 1405 1410Phe Gly Thr Arg Cys Met Glu
Gly Asn Pro Ile Pro Asp Thr Pro 1415 1420 1425Cys Leu Ala Gly Glu
Glu Asp Trp Thr Ile Ser Pro Val Pro Gln 1430 1435 1440Ser Ile Val
Asp Leu Phe Gln Asn Gly Asn Trp Thr Met Lys Asn 1445 1450 1455Pro
Ser Pro Ala Cys Gln Cys Ser Ser Asp Lys Ile Lys Lys Met 1460 1465
1470Leu Pro Val Cys Pro Pro Gly Ala Gly Gly Leu Pro Pro Pro Gln
1475 1480 1485Arg Lys Gln Lys Thr Ala Asp Ile Leu Gln Asn Leu Thr
Gly Arg 1490 1495 1500Asn Ile Ser Asp Tyr Leu Val Lys Thr Tyr Val
Gln Ile Ile Ala 1505 1510 1515Lys Ser Leu Lys Asn Lys Ile Trp Val
Asn Glu Phe Arg Tyr Gly 1520 1525 1530Gly Phe Ser Leu Gly Val Ser
Asn Ser Gln Ala Leu Pro Pro Ser 1535 1540 1545His Glu Val Asn Asp
Ala Ile Lys Gln Met Lys Lys Leu Leu Lys 1550 1555 1560Leu Thr Lys
Asp Thr Ser Ala Asp Arg Phe Leu Ser Ser Leu Gly 1565 1570 1575Arg
Phe Met Ala Gly Leu Asp Thr Lys Asn Asn Val Lys Val Trp 1580 1585
1590Phe Asn Asn Lys Gly Trp His Ala Ile Ser Ser Phe Leu Asn Val
1595 1600 1605Ile Asn Asn Ala Ile Leu Arg Ala Asn Leu Gln Lys Gly
Glu Asn 1610 1615 1620Pro Ser Gln Tyr Gly Ile Thr Ala Phe Asn His
Pro Leu Asn Leu 1625 1630 1635Thr Lys Gln Gln Leu Ser Glu Val Ala
Leu Met Thr Thr Ser Val 1640 1645 1650Asp Val Leu Val Ser Ile Cys
Val Ile Phe Ala Met Ser Phe Val 1655 1660 1665Pro Ala Ser Phe Val
Val Phe Leu Ile Gln Glu Arg Val Ser Lys 1670 1675 1680Ala Lys His
Leu Gln Phe Ile Ser Gly Val Lys Pro Val Ile Tyr 1685 1690 1695Trp
Leu Ser Asn Phe Val Trp Asp Met Cys Asn Tyr Val Val Pro 1700 1705
1710Ala Thr Leu Val Ile Ile Ile Phe Ile Cys Phe Gln Gln Lys Ser
1715 1720 1725Tyr Val Ser Ser Thr Asn Leu Pro Val Leu Ala Leu Leu
Leu Leu 1730 1735 1740Leu Tyr Gly Trp Ser Ile Thr Pro Leu Met Tyr
Pro Ala Ser Phe 1745 1750 1755Val Phe Lys Ile Pro Ser Thr Ala Tyr
Val Val Leu Thr Ser Val 1760 1765 1770Asn Leu Phe Ile Gly Ile Asn
Gly Ser Val Ala Thr Phe Val Leu 1775 1780 1785Glu Leu Phe Thr Asn
Asn Lys Leu Asn Asp Ile Asn Asp Ile Leu 1790 1795 1800Lys Ser Val
Phe Leu Ile Phe Pro His Phe Cys Leu Gly Arg Gly 1805 1810 1815Leu
Ile Asp Met Val Lys Asn Gln Ala Met Ala Asp Ala Leu Glu 1820 1825
1830Arg Phe Gly Glu Asn Arg Phe Val Ser Pro Leu Ser Trp Asp Leu
1835 1840 1845Val Gly Arg Asn Leu Phe Ala Met Ala Val Glu Gly Val
Val Phe 1850 1855 1860Phe Leu Ile Thr Val Leu Ile Gln Tyr Arg Phe
Phe Ile Arg Pro 1865 1870 1875Arg Pro Val Lys Ala Lys Leu Pro Pro
Leu Asn Asp Glu Asp Glu 1880 1885 1890Asp Val Arg Arg Glu Arg Gln
Arg Ile Leu Asp Gly Gly Gly Gln 1895 1900 1905Asn Asp Ile Leu Glu
Ile Lys Glu Leu Thr Lys Ile Tyr Arg Arg 1910 1915 1920Lys Arg Lys
Pro Ala Val Asp Arg Ile Cys Ile Gly Ile Pro Pro 1925 1930 1935Gly
Glu Cys Phe Gly Leu Leu Gly Val Asn Gly Ala Gly Lys Ser 1940 1945
1950Thr Thr Phe Lys Met Leu Thr Gly Asp Thr Pro Val Thr Arg Gly
1955 1960 1965Asp Ala Phe Leu Asn Lys Asn Ser Ile Leu Ser Asn Ile
His Glu 1970 1975 1980Val His Gln Asn Met Gly Tyr Cys Pro Gln Phe
Asp Ala Ile Thr 1985 1990 1995Glu Leu Leu Thr Gly Arg Glu His Val
Glu Phe Phe Ala Leu Leu 2000 2005 2010Arg Gly Val Pro Glu Lys Glu
Val Gly Lys Phe Gly Glu Trp Ala 2015 2020 2025Ile Arg Lys Leu Gly
Leu Val Lys Tyr Gly Glu Lys Tyr Ala Ser 2030 2035
2040Asn Tyr Ser Gly Gly Asn Lys Arg Lys Leu Ser Thr Ala Met Ala
2045 2050 2055Leu Ile Gly Gly Pro Pro Val Val Phe Leu Asp Glu Pro
Thr Thr 2060 2065 2070Gly Met Asp Pro Lys Ala Arg Arg Phe Leu Trp
Asn Cys Ala Leu 2075 2080 2085Ser Ile Val Lys Glu Gly Arg Ser Val
Val Leu Thr Ser His Ser 2090 2095 2100Met Glu Glu Cys Glu Ala Leu
Cys Thr Arg Met Ala Ile Met Val 2105 2110 2115Asn Gly Arg Phe Arg
Cys Leu Gly Ser Val Gln His Leu Lys Asn 2120 2125 2130Arg Phe Gly
Asp Gly Tyr Thr Ile Val Val Arg Ile Ala Gly Ser 2135 2140 2145Asn
Pro Asp Leu Lys Pro Val Gln Glu Phe Phe Gly Leu Ala Phe 2150 2155
2160Pro Gly Ser Val Leu Lys Glu Lys His Arg Asn Met Leu Gln Tyr
2165 2170 2175Gln Leu Pro Ser Ser Leu Ser Ser Leu Ala Arg Ile Phe
Ser Ile 2180 2185 2190Leu Ser Gln Ser Lys Lys Arg Leu His Ile Glu
Asp Tyr Ser Val 2195 2200 2205Ser Gln Thr Thr Leu Asp Gln Val Phe
Val Asn Phe Ala Lys Asp 2210 2215 2220Gln Ser Asp Asp Asp His Leu
Lys Asp Leu Ser Leu His Lys Asn 2225 2230 2235Gln Thr Val Val Asp
Val Ala Val Leu Thr Ser Phe Leu Gln Asp 2240 2245 2250Glu Lys Val
Lys Glu Ser Tyr Val 2255 22601191472PRTMurine
sp.MOD_RES(250)..(250)Variable amino acid 119Gln Ala Cys Ala Met
Glu Ser Arg His Phe Glu Glu Thr Arg Gly Met1 5 10 15Glu Glu Glu Pro
Thr His Leu Pro Leu Val Val Cys Val Asp Lys Leu 20 25 30Thr Lys Val
Tyr Lys Asn Asp Lys Lys Leu Ala Leu Asn Lys Leu Ser 35 40 45Leu Asn
Leu Tyr Glu Asn Gln Val Val Ser Phe Leu Gly His Asn Gly 50 55 60Ala
Gly Lys Thr Thr Thr Met Ser Ile Leu Thr Gly Leu Phe Pro Pro65 70 75
80Thr Ser Gly Ser Ala Thr Ile Tyr Gly His Asp Ile Arg Thr Glu Met
85 90 95Asp Glu Ile Arg Lys Asn Leu Gly Met Cys Pro Gln His Asn Val
Leu 100 105 110Phe Asp Arg Leu Thr Val Glu Glu His Leu Trp Phe Tyr
Ser Arg Leu 115 120 125Lys Ser Met Ala Gln Glu Glu Ile Arg Lys Glu
Thr Asp Lys Met Ile 130 135 140Glu Asp Leu Glu Leu Ser Asn Lys Arg
His Ser Leu Val Gln Thr Leu145 150 155 160Ser Gly Gly Met Lys Arg
Lys Leu Ser Val Ala Ile Ala Phe Val Gly 165 170 175Gly Ser Arg Ala
Ile Ile Leu Asp Glu Pro Thr Ala Gly Val Asp Pro 180 185 190Tyr Ala
Arg Arg Ala Ile Trp Asp Leu Ile Leu Lys Tyr Lys Pro Gly 195 200
205Arg Thr Ile Leu Leu Ser Thr His His Met Asp Glu Ala Asp Leu Leu
210 215 220Gly Asp Arg Ile Ala Ile Ile Ser His Gly Lys Leu Lys Cys
Cys Gly225 230 235 240Ser Pro Leu Phe Leu Lys Gly Ala Tyr Xaa Asp
Gly Tyr Arg Leu Thr 245 250 255Leu Val Lys Gln Pro Ala Glu Pro Gly
Thr Ser Gln Glu Pro Gly Leu 260 265 270Ala Ser Ser Pro Ser Gly Cys
Pro Arg Leu Ser Ser Cys Ser Glu Pro 275 280 285Gln Val Ser Gln Phe
Ile Arg Lys His Val Ala Ser Ser Leu Leu Val 290 295 300Ser Asp Thr
Ser Thr Glu Leu Ser Tyr Ile Leu Pro Ser Glu Ala Val305 310 315
320Lys Lys Gly Ala Phe Glu Arg Leu Phe Gln Gln Leu Glu His Ser Leu
325 330 335Asp Ala Leu His Leu Ser Ser Phe Gly Leu Met Asp Thr Thr
Leu Glu 340 345 350Glu Val Phe Leu Lys Val Ser Glu Glu Asp Gln Ser
Leu Glu Asn Ser 355 360 365Glu Ala Asp Val Lys Glu Ser Arg Lys Asp
Val Leu Pro Gly Ala Glu 370 375 380Gly Leu Thr Ala Val Gly Gly Gln
Ala Gly Asn Leu Ala Arg Cys Ser385 390 395 400Glu Leu Ala Gln Ser
Gln Ala Ser Leu Gln Ser Ala Ser Ser Val Gly 405 410 415Ser Ala Arg
Gly Glu Glu Gly Thr Gly Tyr Ser Asp Gly Tyr Gly Asp 420 425 430Tyr
Arg Pro Leu Phe Asp Asn Leu Gln Asp Pro Asp Asn Val Ser Leu 435 440
445Gln Glu Ala Glu Met Glu Ala Leu Ala Gln Val Gly Gln Gly Ser Arg
450 455 460Lys Leu Glu Gly Trp Trp Leu Lys Met Arg Gln Phe His Gly
Leu Leu465 470 475 480Val Lys Arg Phe His Cys Ala Arg Arg Asn Ser
Lys Ala Leu Cys Ser 485 490 495Gln Ile Leu Leu Pro Ala Phe Phe Val
Cys Val Ala Met Thr Val Ala 500 505 510Leu Ser Val Pro Glu Ile Gly
Asp Leu Pro Pro Leu Val Leu Ser Pro 515 520 525Ser Gln Tyr His Asn
Tyr Thr Gln Pro Arg Gly Asn Phe Ile Pro Tyr 530 535 540Ala Asn Glu
Glu Arg Gln Glu Tyr Arg Leu Arg Leu Ser Pro Asp Ala545 550 555
560Ser Pro Gln Gln Leu Val Ser Thr Phe Arg Leu Pro Ser Gly Val Gly
565 570 575Ala Thr Cys Val Leu Lys Ser Pro Ala Asn Gly Ser Leu Gly
Pro Met 580 585 590Leu Asn Leu Ser Ser Gly Glu Ser Arg Leu Leu Ala
Ala Arg Phe Phe 595 600 605Asp Ser Met Cys Leu Glu Ser Phe Thr Gln
Gly Leu Pro Leu Ser Asn 610 615 620Phe Val Pro Pro Pro Pro Ser Pro
Ala Pro Ser Asp Ser Pro Val Xaa625 630 635 640Pro Asp Glu Asp Ser
Leu Gln Ala Trp Asn Met Ser Leu Pro Pro Thr 645 650 655Ala Gly Pro
Glu Thr Trp Thr Ser Ala Pro Ser Leu Pro Arg Leu Val 660 665 670His
Glu Pro Val Arg Cys Thr Cys Ser Ala Gln Gly Thr Gly Phe Ser 675 680
685Cys Pro Ser Ser Val Gly Gly His Pro Pro Gln Met Arg Val Val Thr
690 695 700Gly Asp Ile Leu Thr Asp Ile Thr Gly His Asn Val Ser Glu
Tyr Leu705 710 715 720Leu Phe Thr Ser Asp Arg Phe Arg Leu His Arg
Tyr Gly Ala Ile Thr 725 730 735Phe Gly Asn Val Gln Lys Ser Ile Pro
Ala Ser Phe Gly Ala Arg Val 740 745 750Pro Pro Met Val Arg Lys Ile
Ala Val Arg Arg Val Ala Gln Val Leu 755 760 765Tyr Asn Asn Lys Gly
Tyr His Ser Met Pro Thr Tyr Leu Asn Ser Leu 770 775 780Asn Asn Ala
Ile Leu Arg Ala Asn Leu Pro Lys Ser Lys Gly Asn Pro785 790 795
800Ala Ala Tyr Xaa Ile Thr Val Thr Asn His Pro Met Asn Lys Thr Ser
805 810 815Ala Ser Leu Ser Leu Asp Tyr Leu Leu Gln Gly Thr Asp Val
Val Ile 820 825 830Ala Ile Phe Ile Ile Val Ala Met Ser Phe Val Pro
Ala Ser Phe Val 835 840 845Val Phe Leu Val Ala Glu Lys Ser Thr Lys
Ala Lys His Leu Gln Phe 850 855 860Val Ser Gly Cys Asn Pro Val Ile
Tyr Trp Leu Ala Asn Tyr Val Trp865 870 875 880Asp Met Leu Asn Tyr
Leu Val Pro Ala Thr Cys Cys Val Ile Ile Leu 885 890 895Phe Val Phe
Asp Leu Pro Ala Tyr Thr Ser Pro Thr Asn Phe Pro Ala 900 905 910Val
Leu Ser Leu Phe Leu Leu Tyr Gly Trp Ser Ile Thr Pro Ile Met 915 920
925Tyr Pro Ala Ser Phe Trp Phe Glu Val Pro Ser Ser Ala Tyr Val Phe
930 935 940Leu Ile Val Ile Asn Leu Phe Ile Gly Ile Thr Ala Thr Val
Ala Thr945 950 955 960Phe Leu Leu Gln Leu Phe Glu His Asp Lys Asp
Leu Lys Val Val Asn 965 970 975Ser Tyr Leu Lys Ser Cys Phe Leu Ile
Phe Pro Asn Tyr Asn Leu Gly 980 985 990His Gly Leu Met Glu Met Ala
Tyr Asn Glu Tyr Ile Asn Glu Tyr Tyr 995 1000 1005Ala Lys Ile Gly
Gln Phe Asp Lys Met Lys Ser Pro Phe Glu Trp 1010 1015 1020Asp Ile
Val Thr Arg Gly Leu Val Ala Met Thr Val Glu Gly Phe 1025 1030
1035Val Gly Phe Phe Leu Thr Ile Met Cys Gln Tyr Asn Phe Leu Arg
1040 1045 1050Gln Pro Gln Arg Leu Pro Val Ser Thr Lys Pro Val Glu
Asp Asp 1055 1060 1065Val Asp Val Ala Ser Glu Arg Gln Arg Val Leu
Arg Gly Asp Ala 1070 1075 1080Asp Asn Asp Met Val Lys Ile Glu Asn
Leu Thr Lys Val Tyr Lys 1085 1090 1095Ser Arg Lys Ile Gly Arg Ile
Leu Ala Val Asp Arg Leu Cys Leu 1100 1105 1110Gly Val Cys Val Pro
Gly Glu Cys Phe Gly Leu Leu Gly Val Asn 1115 1120 1125Gly Ala Gly
Lys Thr Ser Thr Phe Lys Met Leu Thr Gly Asp Glu 1130 1135 1140Ser
Thr Thr Gly Gly Glu Ala Phe Val Asn Gly His Ser Val Leu 1145 1150
1155Lys Asp Leu Leu Gln Val Gln Gln Ser Leu Gly Tyr Cys Pro Gln
1160 1165 1170Phe Asp Val Pro Val Asp Glu Leu Thr Ala Arg Glu His
Leu Gln 1175 1180 1185Leu Tyr Thr Arg Leu Arg Cys Ile Pro Trp Lys
Asp Glu Ala Gln 1190 1195 1200Val Val Lys Trp Ala Leu Glu Lys Leu
Glu Leu Thr Lys Tyr Ala 1205 1210 1215Asp Lys Pro Ala Gly Thr Tyr
Ser Gly Gly Asn Lys Arg Lys Leu 1220 1225 1230Ser Thr Ala Ile Ala
Leu Ile Gly Tyr Pro Ala Phe Ile Phe Leu 1235 1240 1245Asp Glu Pro
Thr Thr Gly Met Asp Pro Lys Ala Arg Arg Phe Leu 1250 1255 1260Trp
Asn Leu Ile Leu Asp Leu Ile Lys Thr Gly Arg Ser Val Val 1265 1270
1275Leu Thr Ser His Ser Met Glu Glu Cys Glu Ala Leu Cys Thr Arg
1280 1285 1290Leu Ala Ile Met Val Asn Gly Arg Leu His Cys Leu Gly
Ser Ile 1295 1300 1305Gln His Leu Lys Asn Arg Phe Gly Asp Gly Tyr
Met Ile Thr Val 1310 1315 1320Arg Thr Lys Ser Ser Gln Asn Val Lys
Asp Val Val Arg Phe Phe 1325 1330 1335Asn Arg Asn Phe Pro Glu Ala
His Ala Gln Gly Lys Thr Pro Tyr 1340 1345 1350Lys Val Gln Tyr Gln
Leu Lys Ser Glu His Ile Ser Leu Ala Gln 1355 1360 1365Val Phe Ser
Lys Met Glu Gln Val Val Gly Val Leu Gly Ile Glu 1370 1375 1380Asp
Tyr Ser Val Ser Gln Thr Thr Leu Asp Asn Val Phe Val Asn 1385 1390
1395Phe Ala Lys Lys Gln Ser Asp Asn Val Glu Gln Gln Glu Ala Glu
1400 1405 1410Pro Ser Ser Leu Pro Ser Pro Leu Gly Leu Leu Ser Leu
Leu Arg 1415 1420 1425Pro Arg Pro Ala Pro Thr Glu Leu Arg Ala Leu
Val Ala Asp Glu 1430 1435 1440Pro Glu Asp Leu Asp Thr Glu Asp Glu
Gly Leu Ile Ser Phe Glu 1445 1450 1455Glu Glu Arg Ala Gln Leu Ser
Phe Asn Thr Asp Thr Leu Cys 1460 1465 14701201704PRTHomo sapiens
120Met Ala Val Leu Arg Gln Leu Ala Leu Leu Leu Trp Lys Asn Tyr Thr1
5 10 15Leu Gln Lys Arg Lys Val Leu Val Thr Val Leu Glu Leu Phe Leu
Pro 20 25 30Leu Leu Phe Ser Gly Ile Leu Ile Trp Leu Arg Leu Lys Ile
Gln Ser 35 40 45Glu Asn Val Pro Asn Ala Thr Ile Tyr Pro Gly Gln Ser
Ile Gln Glu 50 55 60Leu Pro Leu Phe Phe Thr Phe Pro Pro Pro Gly Asp
Thr Trp Glu Leu65 70 75 80Ala Tyr Ile Pro Ser His Ser Asp Ala Ala
Lys Thr Val Thr Glu Thr 85 90 95Val Arg Arg Ala Leu Val Ile Asn Met
Arg Val Arg Gly Phe Pro Ser 100 105 110Glu Lys Asp Phe Glu Asp Tyr
Ile Arg Tyr Asp Asn Cys Ser Ser Ser 115 120 125Val Leu Ala Ala Val
Val Phe Glu His Pro Phe Asn His Ser Lys Glu 130 135 140Pro Leu Pro
Leu Ala Val Lys Tyr His Leu Arg Phe Ser Tyr Thr Arg145 150 155
160Arg Asn Tyr Met Trp Thr Gln Thr Gly Ser Phe Phe Leu Lys Glu Thr
165 170 175Glu Gly Trp His Thr Thr Ser Leu Phe Pro Leu Phe Pro Asn
Pro Gly 180 185 190Pro Arg Glu Pro Thr Ser Pro Asp Gly Gly Glu Pro
Gly Tyr Ile Arg 195 200 205Glu Gly Phe Leu Ala Val Gln His Ala Val
Asp Arg Ala Ile Met Glu 210 215 220Tyr His Ala Asp Ala Ala Thr Arg
Gln Leu Phe Gln Arg Leu Thr Val225 230 235 240Thr Ile Lys Arg Phe
Pro Tyr Pro Pro Phe Ile Ala Asp Pro Phe Leu 245 250 255Val Ala Ile
Gln Tyr Gln Leu Pro Leu Leu Leu Leu Leu Ser Phe Thr 260 265 270Tyr
Thr Ala Leu Thr Ile Ala Arg Ala Val Val Gln Glu Lys Glu Arg 275 280
285Arg Leu Lys Glu Tyr Met Arg Met Met Gly Leu Ser Ser Trp Leu His
290 295 300Trp Ser Ala Trp Phe Leu Leu Phe Phe Leu Phe Leu Leu Ile
Ala Ala305 310 315 320Ser Phe Met Thr Leu Leu Phe Cys Val Lys Val
Lys Pro Asn Val Ala 325 330 335Val Leu Ser Arg Ser Asp Pro Ser Leu
Val Leu Ala Phe Leu Leu Cys 340 345 350Phe Ala Ile Ser Thr Ile Ser
Phe Ser Phe Met Val Ser Thr Phe Phe 355 360 365Ser Lys Ala Asn Met
Ala Ala Ala Phe Gly Gly Phe Leu Tyr Phe Phe 370 375 380Thr Tyr Ile
Pro Tyr Phe Phe Val Ala Pro Arg Tyr Asn Trp Met Thr385 390 395
400Leu Ser Gln Lys Leu Cys Ser Cys Leu Leu Ser Asn Val Ala Met Ala
405 410 415Met Gly Ala Gln Leu Ile Gly Lys Phe Glu Ala Lys Gly Met
Gly Ile 420 425 430Gln Trp Arg Asp Leu Leu Ser Pro Val Asn Val Asp
Asp Asp Phe Cys 435 440 445Phe Gly Gln Val Leu Gly Met Leu Leu Leu
Asp Ser Val Leu Tyr Gly 450 455 460Leu Val Thr Trp Tyr Met Glu Ala
Val Phe Pro Gly Gln Phe Gly Val465 470 475 480Pro Gln Pro Trp Tyr
Phe Phe Ile Met Pro Ser Tyr Trp Cys Gly Lys 485 490 495Pro Arg Ala
Val Ala Gly Lys Glu Glu Glu Asp Ser Asp Pro Glu Lys 500 505 510Ala
Leu Arg Asn Glu Tyr Phe Glu Ala Glu Pro Glu Asp Leu Val Ala 515 520
525Gly Ile Lys Ile Lys His Leu Ser Lys Val Phe Arg Val Gly Asn Lys
530 535 540Asp Arg Ala Ala Val Arg Asp Leu Asn Leu Asn Leu Tyr Glu
Gly Gln545 550 555 560Ile Thr Val Leu Leu Gly His Asn Gly Ala Gly
Lys Thr Thr Thr Leu 565 570 575Ser Met Leu Thr Gly Leu Phe Pro Pro
Thr Ser Gly Arg Ala Tyr Ile 580 585 590Ser Gly Tyr Glu Ile Ser Gln
Asp Met Val Gln Ile Arg Lys Ser Leu 595 600 605Gly Leu Cys Pro Gln
His Asp Ile Leu Phe Asp Asn Leu Thr Val Ala 610 615 620Glu His Leu
Tyr Phe Tyr Ala Gln Leu Lys Gly Leu Ser Arg Gln Lys625 630 635
640Cys Pro Glu Glu Val Lys Gln Met Leu His Ile Ile Gly Leu Glu Asp
645 650 655Lys Trp Asn Ser Arg Ser Arg Phe Leu Ser Gly Gly Met Arg
Arg Lys 660 665 670Leu Ser Ile Gly Ile Ala Leu Ile Ala Gly Ser Lys
Val Leu Ile Leu 675 680 685Asp Glu Pro Thr Ser Gly Met Asp Ala Ile
Ser Arg Arg Ala Ile Trp 690 695 700Asp Leu Leu Gln Arg Gln Lys Ser
Asp Arg Thr Ile Val Leu Thr Thr705 710 715 720His Phe Met Asp Glu
Ala Asp Leu Leu Gly Asp Arg Ile Ala Ile Met 725 730 735Ala Lys Gly
Glu Leu Gln Cys Cys Gly Ser Ser Leu Phe Leu Lys Gln 740 745 750Lys
Tyr Gly Ala Gly Tyr
His Met Thr Leu Val Lys Glu Pro His Cys 755 760 765Asn Pro Glu Asp
Ile Ser Gln Leu Val His His His Val Pro Asn Ala 770 775 780Thr Leu
Glu Ser Ser Ala Gly Ala Glu Leu Ser Phe Ile Leu Pro Arg785 790 795
800Glu Ser Thr His Arg Phe Glu Gly Leu Phe Ala Lys Leu Glu Lys Lys
805 810 815Gln Lys Glu Leu Gly Ile Ala Ser Phe Gly Ala Ser Ile Thr
Thr Met 820 825 830Glu Glu Val Phe Leu Arg Val Gly Lys Leu Val Asp
Ser Ser Met Asp 835 840 845Ile Gln Ala Ile Gln Leu Pro Ala Leu Gln
Tyr Gln His Glu Arg Arg 850 855 860Ala Ser Asp Trp Ala Val Asp Ser
Asn Leu Cys Gly Ala Met Asp Pro865 870 875 880Ser Asp Gly Ile Gly
Ala Leu Ile Glu Glu Glu Arg Thr Ala Val Lys 885 890 895Leu Asn Thr
Gly Leu Ala Leu His Cys Gln Gln Phe Trp Ala Met Phe 900 905 910Leu
Lys Lys Ala Ala Tyr Ser Trp Arg Glu Trp Lys Met Val Ala Ala 915 920
925Gln Val Leu Val Pro Leu Thr Cys Val Thr Leu Ala Leu Leu Ala Ile
930 935 940Asn Tyr Ser Ser Glu Leu Phe Asp Asp Pro Met Leu Arg Leu
Thr Leu945 950 955 960Gly Glu Tyr Gly Arg Thr Val Val Pro Phe Ser
Val Pro Gly Thr Ser 965 970 975Gln Leu Gly Gln Gln Leu Ser Glu His
Leu Lys Asp Ala Leu Gln Ala 980 985 990Glu Gly Gln Glu Pro Arg Glu
Val Leu Gly Asp Leu Glu Glu Phe Leu 995 1000 1005Ile Phe Arg Ala
Ser Val Glu Gly Gly Gly Phe Asn Glu Arg Cys 1010 1015 1020Leu Val
Ala Ala Ser Phe Arg Asp Val Gly Glu Arg Thr Val Val 1025 1030
1035Asn Ala Leu Phe Asn Asn Gln Ala Tyr His Ser Pro Ala Thr Ala
1040 1045 1050Leu Ala Val Val Asp Asn Leu Leu Phe Lys Leu Leu Cys
Gly Pro 1055 1060 1065His Ala Ser Ile Val Val Ser Asn Phe Pro Gln
Pro Arg Ser Ala 1070 1075 1080Leu Gln Ala Ala Lys Asp Gln Phe Asn
Glu Gly Arg Lys Gly Phe 1085 1090 1095Asp Ile Ala Leu Asn Leu Leu
Phe Ala Met Ala Phe Leu Ala Ser 1100 1105 1110Thr Phe Ser Ile Leu
Ala Val Ser Glu Arg Ala Val Gln Ala Lys 1115 1120 1125His Val Gln
Phe Val Ser Gly Val His Val Ala Ser Phe Trp Leu 1130 1135 1140Ser
Ala Leu Leu Trp Asp Leu Ile Ser Phe Leu Ile Pro Ser Leu 1145 1150
1155Leu Leu Leu Val Val Phe Lys Ala Phe Asp Val Arg Ala Phe Thr
1160 1165 1170Arg Asp Gly His Met Ala Asp Thr Leu Leu Leu Leu Leu
Leu Tyr 1175 1180 1185Gly Trp Ala Ile Ile Pro Leu Met Tyr Leu Met
Asn Phe Phe Phe 1190 1195 1200Leu Gly Ala Ala Thr Ala Tyr Thr Arg
Leu Thr Ile Phe Asn Ile 1205 1210 1215Leu Ser Gly Ile Ala Thr Phe
Leu Met Val Thr Ile Met Arg Ile 1220 1225 1230Pro Ala Val Lys Leu
Glu Glu Leu Ser Lys Thr Leu Asp His Val 1235 1240 1245Phe Leu Val
Leu Pro Asn His Cys Leu Gly Met Ala Val Ser Ser 1250 1255 1260Phe
Tyr Glu Asn Tyr Glu Thr Arg Arg Tyr Cys Thr Ser Ser Glu 1265 1270
1275Val Ala Ala His Tyr Cys Lys Lys Tyr Asn Ile Gln Tyr Gln Glu
1280 1285 1290Asn Phe Tyr Ala Trp Ser Ala Pro Gly Val Gly Arg Phe
Val Ala 1295 1300 1305Ser Met Ala Ala Ser Gly Cys Ala Tyr Leu Ile
Leu Leu Phe Leu 1310 1315 1320Ile Glu Thr Asn Leu Leu Gln Arg Leu
Arg Gly Ile Leu Cys Ala 1325 1330 1335Leu Arg Arg Arg Arg Thr Leu
Thr Glu Leu Tyr Thr Arg Met Pro 1340 1345 1350Val Leu Pro Glu Asp
Gln Asp Val Ala Asp Glu Arg Thr Arg Ile 1355 1360 1365Leu Ala Pro
Ser Pro Asp Ser Leu Leu His Thr Pro Leu Ile Ile 1370 1375 1380Lys
Glu Leu Ser Lys Val Tyr Glu Gln Arg Val Pro Leu Leu Ala 1385 1390
1395Val Asp Arg Leu Ser Leu Ala Val Gln Lys Gly Glu Cys Phe Gly
1400 1405 1410Leu Leu Gly Phe Asn Gly Ala Gly Lys Thr Thr Thr Phe
Lys Met 1415 1420 1425Leu Thr Gly Glu Glu Ser Leu Thr Ser Gly Asp
Ala Phe Val Gly 1430 1435 1440Gly His Arg Ile Ser Ser Asp Val Gly
Lys Val Arg Gln Arg Ile 1445 1450 1455Gly Tyr Cys Pro Gln Phe Asp
Ala Leu Leu Asp His Met Thr Gly 1460 1465 1470Arg Glu Met Leu Val
Met Tyr Ala Arg Leu Arg Gly Ile Pro Glu 1475 1480 1485Arg His Ile
Gly Ala Cys Val Glu Asn Thr Leu Arg Gly Leu Leu 1490 1495 1500Leu
Glu Pro His Ala Asn Lys Leu Val Arg Thr Tyr Ser Gly Gly 1505 1510
1515Asn Lys Arg Lys Leu Ser Thr Gly Ile Ala Leu Ile Gly Glu Pro
1520 1525 1530Ala Val Ile Phe Leu Asp Glu Pro Ser Thr Gly Met Asp
Pro Val 1535 1540 1545Ala Arg Arg Leu Leu Trp Asp Thr Val Ala Arg
Ala Arg Glu Ser 1550 1555 1560Gly Lys Ala Ile Ile Ile Thr Ser His
Ser Met Glu Glu Cys Glu 1565 1570 1575Ala Leu Cys Thr Arg Leu Ala
Ile Met Val Gln Gly Gln Phe Lys 1580 1585 1590Cys Leu Gly Ser Pro
Gln His Leu Lys Ser Lys Phe Gly Ser Gly 1595 1600 1605Tyr Ser Leu
Arg Ala Lys Val Gln Ser Glu Gly Gln Gln Glu Ala 1610 1615 1620Leu
Glu Glu Phe Lys Ala Phe Val Asp Leu Thr Phe Pro Gly Ser 1625 1630
1635Val Leu Glu Asp Glu His Gln Gly Met Val His Tyr His Leu Pro
1640 1645 1650Gly Arg Asp Leu Ser Trp Ala Lys Val Phe Gly Ile Leu
Glu Lys 1655 1660 1665Ala Lys Glu Lys Tyr Gly Val Asp Asp Tyr Ser
Val Ser Gln Ile 1670 1675 1680Ser Leu Glu Gln Val Phe Leu Ser Phe
Ala His Leu Gln Pro Pro 1685 1690 1695Thr Ala Glu Glu Gly Arg
1700
* * * * *
References