Note: Descriptions are shown in the official language in which they were submitted.
: 2157807
MUTANT DNA ENCODING PROTEIN PHOSPHATASE 1 G-SUBUNIT
FIELD OF THE INVENTION
The present invention relates to a mutant DNA sequence encoding
protein phosphatase 1 G-subunit, a method of detecting a
5 mutation in the gene encoding protein phosphatase 1 G-subunit,
as well as a diagnostic composition and a test kit for use in
the method.
BACKGROUND OF THE INVENTION
The human protein phosphatase 1 (PPl) enzyme complex has been
10 shown to comprise at least three isoforms of the catalytic
subunit: PPlC~, PPlC~ and PPlCy encoded by different genes (13-
15). All three subtypes of PPlC exhibit a wide tissue
distribution and they all bind to the glycogen associated
targeting subunit of PPl (16). The human G-subunit is probably
15 encoded by a single gene and as determined for rabbit PPlG-
subunit it is expressed in skeletal, heart and diaphragm muscle
tissues (17). A different subtype of PP1-G is expressed in
liver (1). The rabbit skeletal muscle PPl-G has been shown to
undergo in vivo and in vitro phosphorylation at several serine
20 residues most of which are located near the NH2-terminus ( for
review, see 1). Cyclic AMP-dependent protein kinase
phosphorylates PPlG-subunit at Ser 46 (site 1) and Ser 65 (site
2). Phosphorylation of site 2 promotes dissociation of the C-
subunit and its translocation from the glycogen-protein
25 particles to the cytosol, where it is likely to be inactivated
by a cytosolic protein termed inhibitor-1. Thus,
phosphorylation of PPlG-subunit by cyclic AMP-dependent protein
kinase results in an immediate inhibition of glycogen synthesis
and a stimulation of glycogenolysis. Insulin stimulates
30 glycogen synthesis and inhibits glycogenolysis in skeletal
muscle and this is thought to be mediated by the activation of
PPlG as a result of the phosphorylation of site 1 on the G-
subunit catalysed by an insulin stimulated protein kinase (18).
215~807
The latter was subsequently identified as the Rsk-2 isoform of
ribosomal S6 kinase (19) and was also shown to inactivate
glycogen synthase kinase-3 (GSK-3) in vitro (20). Since GSK-3
phosphorylates the sites in glycogen synthase which are
S dephosphorylated in response to insulin, inhibition of GSK-3 by
this hormone, which has been demonstrated in vivo (21), may
also contribute to the activation of glycogen synthesis. A
further complication is that GSK-3 phosphorylates the PPlG-
subunit at Ser38 and Ser42in vitro (22), but the relevance of
10 this to insulin action has still to be evaluated.
The glycogen-associated form of protein phosphatase 1 (PPl G-
subunit) derived from skeletal muscle is a heterodimer composed
of a 37 kDa catalytic subunit (C) and a 124 kDa targeting and
regulatory subunit (G) (1). PPl-G not only binds to muscle
15 glycogen with high affinity and thus enhances dephosphorylation
of glycogen bound PPl substrates such as glycogen synthase and
glycogen phosphorylase kinase but also plays an essential role
in the control of glycogen metabolism by different hormones
(1). Phosphorylation at Ser46 (site 1) of the G-subunit in
20 response to insulin enhances the activity of PPl-G towards
glycogen bound substrates (stimulation of glycogen synthesis
and inhibition of glycogenolysis) while phosphorylation at Ser65
(site 2) of PPl-G in response to adrenaline causes
dissociation of PPlC from the targeting G-subunit thereby
25 inhibiting glycogen synthesis and stimulating glycogenolysis
(1) .
In subsets of patients with widespread disorders like obesity,
non-insulin dependent diabetes mellitus (NIDDM), essential
hypertension, dyslipidaemia, and premature atherosclerosis,
30 impaired insulin stimulated non-oxidative glucose disposal,
which primarily reflects insulin resistance of skeletal muscle
glycogen synthesis, has repeatedly been reported (2,3).
Resistance to the action of insulin on muscle glycogen
synthesis has therefore been proposed as the inherited basis
35 for subsets of disorders in the insulin resistance syndrome. In
2157~0~
support of this hypothesis, defective insulin mediated
activation of muscle glycogen synthase has been found in
glucose tolerant but insulin resistant first degree relatives
of Caucasian NIDDM patients (4,5). Also in severely insulin
5 resistant Pima Indians a reduced basal and insulin stimulated
activity of protein phosphatase 1 in muscle tissue has been
demonstrated, providing a mechanism by which glycogen synthase
activation by insulin is reduced in these subjects (6,7).
Obviously, numerous genes encoding proteins participating in
10 insulin signaling and glucose processing in muscle may be
involved in the pathogenesis of insulin resistance and given
the heterogeneity of disorders the inherited component of the
impaired insulin stimulated glycogen synthesis may well
comprise a series of different genes. Previously, several
15 candidate genes have been examined for mutations including
insulin receptor substrate-1 (IRS-1) (8), insulin sensitive
glucose transporter Glut 4 (9), insulin stimulated protein
kinase-1 (10), three catalytic subunits of PP1-C (10) and
glycogen synthase (9). Except for two non-conservative amino
20 acid polymorphisms in IRS-1 (8) no frequent mutations have been
found. The gene which encodes the G-subunit of PP1 was
therefore considered a possible candidate for inherited insulin
resistance of muscle glycogen synthesis. The human PP1 G-
subunit cDNA has recently been cloned (11). Applying Single
25 Strand Conformation Polymorphism (SSCP) scanning of the PP1 G-
subunit cDNA a heterozygous missense mutation (Ala931~Glu) in one
out of 30 insulin resistant NIDDM patients (11). The carrier of
this mutation had an insulin stimulated non-oxidative glucose
metabolism in the lower part of the range for NIDDM patients
30 compatible with a defect in the insulin activation of the PP1
G-subunit (11).
SUMMARY OF THE INVENTION
`- - 2157807
The gene encoding the PPl G-subunit was investigated for
further mutations by heteroduplex formation analysis (12)
resulting in the identification of a mutation in codon 905.
Accordingly, the present invention relates to a DNA isolate
5 comprising a DNA sequence encoding the protein phosphatase type
1 (PPl) G-subunit, the DNA sequence containing a mutation of G
to T in the first position of codon 905 such that aspartic
acid905 in the expressed PPl G-subunit is substituted by
tyrosine, or comprising a fragment of the DNA sequence spanning
10 said mutation.
The aspartic acid to tyrosine polymorphism at codon 905 of PPl
G-subunit has been found to occur with a genotype prevalence of
13 % in the examined group of 313 NIDDM patients and 18 % in
the examined group of 150 healthy Danish Caucasians. Studies in
15 a subgroup of healthy subjects applying the euglycaemic
hyperinsulinaemic clamp technique in combination with indirect
calorimetry demonstrate that this allelic variation of PPl G-
subunit has functional importance in that carriers of this
mutation have been found to exhibit insulin resistance of non-
20 oxidative glucose metabolism as well as an increased basalglucose oxidation rate. The individuals who were heterozygous
for the Asp905/Tyr905 polymorphism had, on average, a 54% increase
in whole body basal glucose oxidation rate and a 26% reduction
in insulin stimulated nonoxidative glucose disposal rate
25 compared to homozygous Asp905 individuals. A case-control study
of 313 NIDDM patients and 150 controls showed that the
prevalence of the PPlG-subunit Tyr905 variant allele was similar
in NIDDM and control subjects.
The results obtained in apparently healthy individuals suggest,
30 that the PPl G-subunit variant is associated with an increased
whole body glucose oxidation in the fasting state as well as an
impaired nonoxidative glucose metabolism after 4 h of
euglycaemia and hyperinsulinaemia. In this context it is of
interest that studies in rat skeletal muscle have provided
- - ~15780~
experimental evidence that under basal insulin conditions a
large portion of the intracellular glucose-6-phosphate (G6P)
is derived from glycogen rather than from plasma glucose (23).
In addition, experiments in the same animal model demonstrated
5 that skeletal muscle glycogenolysis is more sensitive to
insulin than is glucose uptake and glycogen synthesis (23).
Therefore, the present finding of an increased glucose
oxidation rate in Tyr905 subunit carriers may in part be
secondary to a diminished ability of the mutant PPlG-subunit to
10 mediate the inhibitory effect of fasting insulin on glycogen
breakdown in peripheral tissues thereby causing elevated
intracellular levels of G6P for glucose oxidation.
Alternatively and/or additionally an increased glycogenolysis
might be caused by an enhanced sensitivity to ~-adrenergic
15 stimulation of mutant PPl G-subunit (1).
The surprising finding that the Tyr905 PPl G-subunit associates
with a whole body insulin resistance which is specific for the
glycogen synthesis pathway of peripheral tissues is compatible
with a diminished effect of insulin on the mutant PPl G-subunit
20 and consequently a reduced activation of glycogen synthesis. It
has been recognized for a lond time that whole body insulin
sensitivity is affected by both nongenetic components (i.e.
physical activity and diet) and genetic factors, the latter
elucidated in studies of genetic admixture and familial
25 transmission in various ethnic groups (24,25). The genetics
behind the physiologic variation in the population is at
present unknown. In this connection, the Asp905/Tyr9os
polymorphism of the PPl G-subunit offers the first important
contribution to explain the large variation in insulin
30 stimulated glycogen synthesis of peripheral tissues in the
general Caucasian population.
In another aspect, the present invention relates to a living
system containing a DNA isolate of the invention and capable of
expressing the PPl G-subunit wherein Asp905 is substituted by
35 Tyr. The living system, which may comprise a cell or a
` - 2 1 ~
multicellular organism containing the appropriate signal
transduction pathway, may be used to screen for substances
which have an effect on insulin resistance of non-oxidative
glucose metabolism.
5 In a further aspect, the present invention relates to a method
of detecting the presence of a mutation of G to T in the first
position of codon 905 of the gene encoding the PPl G-subunit,
the method comprising obtaining a biological sample from a
subject and analysing the sample for said mutation. Based on
10 current knowledge, it is assumed that this method may be used
to diagnose predisposition to insulin resistance of non-
oxidative glucose metabolism in a subject as well as other
disorders resulting from insulin resistance, such as obesity,
essential hypertension, dyslipidemia or premature
15 atherosclerosis. Furthermore, it cannot be excluded that the
Tyr905 PPlG-subunit variant may act in combination with mutations
in other insulin resistance genes to present a significant risk
for the onset of some subtypes of NIDDM.
Biological samples may, for instance, be obtained from blood,
20 serum, plasma or tissue.
The invention further relates to a diagnostic composition and
a test kit for use in the method.
DETAILED DESCRIPTION OF THE INVENTION
In a preferred embodiment, the DNA isolate of the invention
25 comprises the DNA sequence shown in the Sequence Listing as SEQ
ID NO:l, in which the G to T mutation occurs in nucleotide
2711.
The length of the DNA isolate may vary widely depending on the
intended use. For use as an oligonucleotide probe for
30 hybridization purposes, the DNA fragment may be as short as 17
nucleotides. For expression in a living system as defined
21~7807
above, the DNA isolate will typically comprise the full-length
DNA sequence encoding the PPl G-subunit mutant.
The DNA isolate of the invention comprising the mutation of G
to T in the first position of codon 905 in the DNA sequence
5 encoding the PP1 G-subunit may suitably be of genomic or cDNA
origin, for instance obtained by preparing a genomic or cDNA
library and screening for DNA sequences coding for all or part
of the the PPl G-subunit by hybridization using synthetic
oligonucleotide probes in accordance with standard techniques
10 (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual,
2nd Ed., Cold Spring Harbor, 1989). The probes used should be
specific for the mutation. Alternatively, the DNA sequence
encoding wild-type PPl G-subunit may be modified by site-
directed mutagenesis using synthetic oligonucleotides
15 containing the mutation for homologous recombination in
accordance with well-known procedures.
The DNA sequence may also be prepared by polymerase chain
reaction (PCR) using specific primers, for instance as
described in US 4,683,202, or Saiki et al., Science 239, 1988,
20 pp. 487-491, or PCR Protocols, 1990, Academic Press, San Diego,
California, USA.
The DNA isolate of the invention may also be prepared
synthetically by established standard methods, e.g. the
phosphoamidite method described by Beaucage and Caruthers,
25 Tetrahedron Letters 22 (1981), 1859 - 1869, or the method
described by Matthes et al., EMBO Journal 3 (1984), 801 - 805.
According to the phosphoamidite method, oligonucleotides are
synthesized, e.g. in an automatic DNA synthesizer, purified,
annealed and ligated. This procedure may preferably be used to
30 prepare fragments of the PPl G-subunit encoding DNA sequence.
The recombinant vector into which the DNA isolate is inserted
may be any vector which may conveniently be subjected to
recombinant DNA procedures. The choice of vector will often
-- 2157807
depends on the host cell into which it is to be introduced.
Thus, the vector may be an autonomously replicating vector,
i.e. a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
5 e.g. a plasmid. Alternatively, the vector may be one which,
when introduced into a host cell, is integrated into the host
cell genome and replicated together with the chromosome(s) into
which it has been integrated (e.g. a viral vector).
The recombinant vector is preferably an expression vector in
10 which the DNA sequence encoding the PP1 G-subunit mutant is
operably connected to additional segmants required for
transcription of the DNA. In general, the vector is derived
from plasmid or viral DNA or may contain elements of both. The
term "operably linked" indicates that the segments are arranged
15 so that they function in concert for their intended purposes,
e.g. transcription initiates in a promoter and proceeds through
the DNA sequence coding for the PPl G-subunit mutant.
The promoter may be any DNA sequence which shows
transcriptional activity in the host cell of choice and may be
20 derived from genes encoding proteins either homologous or
heterologous to the host cell. Examples of suitable promoters
for directing the transcription of the mutant DNA encoding IRS-
1 in mammalian cells are the SV40 promoter (Subramani et al.,
Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein
25 gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814)
or the adenovirus 2 major late promoter.
The mutant DNA sequence encoding the PPl G-subunit may also be
operably connected to a suitable terminator, such as the human
growth hormone terminator (Palmiter et al., op. cit.). The vec-
30 tor may further comprise elements such as polyadenylationsignals (e.g. from SV40 or the adenovirus 5 Elb region),
transcriptional enhancer sequences (e.g. the SV40 enhancer) and
translational enhancer sequences (e.g. the ones encoding
adenovirus VA RNAs).
2 1 ~ 7 8 0 7
The recombinant vector may further comprise a DNA sequence
enabling the vector to replicate in the host cell in question.
An example of such a sequence is the SV40 origin of
replication. The vector may also comprise a selectable marker,
5 e.g. a gene the product of which complements a defect in the
host cell, such as the gene coding for dihydrofolate reductase
(DHFR) or one which confers resistance to a drug, e.g.
neomycin, hygromycin or methotrexate.
The procedures used to ligate the DNA sequences coding for the
10 PPl G-subunit, the promoter and the terminator, respectively,
and to insert them into suitable vectors containing the
information necessary for replication, are well known to
persons skilled in the art (cf., for instance, Sambrook et al.,
op.cit.).
15 In a further aspect, the present invention relates to a variant
of the PPl G-subunit in which aspartic acid905 is substituted by
tyrosine, or a fragment thereof containing said substitution.
The living system into which the DNA isolate of the invention
is introduced may be a cell which is capable of producing the
20 PPl G-subunit and which has the appropriate signal transduction
pathways. The cell is preferably a eukaryotic cell, such as a
vertebrate cell, e.g. a Xenopus laevis oocyte or mammalian
cell, in particular a mammalian cell. Examples of suitable
mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL
25 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell
lines. Methods of transfecting mammalian cells and expressing
DNA sequences introduced in the cells are described in e.g.
Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621;
Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327 - 341;
30 Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422 - 426;
Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson,
Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb,
Virology 52 (1973), 456; and Neumann et al., EMB0 J. 1 (1982),
841 - 845.
-
2157807
The mutant DNA sequence encoding the PPl G-subunit may then be
expressed by culturing a cell as described above in a suitable
nutrient medium under conditions which are conducive to the
expression of the PPl G-subunit-coding DNA sequence. The medium
5 used to culture the cells may be any conventional medium
suitable for growing mammalian cells, such as a serum-
containing or serum-free medium containing appropriate
supplements. Suitable media are available from commercial
suppliers or may be prepared according to published recipes
10 (e.g. in catalogues of the American Type Culture Collection).
The living system according to the invention may also comprise
a transgenic animal. A transgenic animal is one in whose genome
a DNA sequence encoding the PPl G-subunit mutant has been
introduced. In particular, the transgenic animal is a
15 transgenic non-human mammal, mammals being generally provided
with appropriate signal transduction pathways. For the present
purpose, it is generally preferred to employ smaller mammals,
e.g. rodents such as mice, rabbits or rats.
For expression of the PPl G-subunit mutant in transgenic
20 animals, a mutant DNA sequence encoding the PPl G-subunit
mutant is operably linked to additional DNA sequences required
for its expression to produce expression units. Such additional
sequences include a promoter as indicated above, as well as
sequences providing for termination of transcription and
25 polyadenylation of mRNA. Construction of the expression unit
for use in transgenic animals may conveniently be done by
inserting a DNA sequence encoding the mutant PPl G-subunit into
a vector containing the additional DNA sequences, although the
expression unit may be constructed by essentially any sequence
30 of ligations.
The expression unit is then introduced into fertilized ova or
early-stage embryos of the selected host species. Introduction
of heterologous DNA may be carried out in a number of ways,
including microinjection (cf. US 4,873,191), retroviral
- 2157807
infection (cf. Jaenisch, Science 240, 1988, pp. 1468-1474) or
site-directed integration using embryonic stem cells (reviewed
by Bradley et al., Bio/Technoloqy 10, 1992, pp. 534-539). The
ova are then implanted into the oviducts or uteri of
5 pseudopregnant females and allowed to develop to term.
Offspring carrying the introduced DNA in their germ line can
pass the DNA on to their progeny, allowing the development of
transgenic populations.
General procedures for producing transgenic animals are known
10 in the art, cf. for instance, Hogan et al., Manipulating the
Mouse Embryo: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1986; Simons et al., Bio/TechnoloqY 6, 1988, pp.
179-183; Wall et al., Biol. Reprod. 32, 1985, pp. 645-651;
Buhler et al., Bio/TechnoloqY 8, 1990, pp. 140-143; Ebert et
15 al., Bio/Technology 6: 179-183, 1988; Krimpenfort et al.,
Bio/Tecnoloqy 9: 844-847, 1991, Wall et al., J. Cell. Biochem.
49: 113-120, 1992; US 4,873,191, US 4,873,316; WO 88/00239, WO
90/05188; WO 92/11757 and GB 87/00458. Techniques for
introducing heterologous DNA sequences into mammals and their
20 germ cells were originally developed in the mouse. See, e.g.
Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980,
Gordon and Ruddle, Science 214: 1244-1246, 1981; Palmiter and
Brinster, Cell 41: 343-345, 1985; Brinster et al., Proc. Natl.
Acad. Sci. USA 82: 4438-4442, 1985; and Hogan et al. (ibid.).
25 In brief, in the most efficient route used to date in the
generation of transgenic mice, several hundred linear molecules
of the DNA of interest are injected into one of the pro-nuclei
of a fertilized egg according to techniques which have become
standard in the art. Injection of DNA into the cytoplasm of a
30 zygote can also be employed. Similar procedures may be employed
for the production of trangenic individuals of other species.
In one embodiment of the present method of detecting the
presence of the mutation of G to T in the first position of
codon 905 of the PPl G-subunit gene, a biological sample is
35 obtained from a subject, DNA (in particular genomic DNA) is
21S7807
12
isolated from the sample and digested with a restriction
endonuclease which cleaves wild-type PP1 G-subunit DNA at a
recognition site eliminated by the mutation of G to T in the
first position of codon 905 of the PP1 G-subunit gene so that
5 the mutant PPl G-subunit DNA is not cleaved at this site, and
the PP1 G-subunit DNA is analysed for cleavage at this site.
After digestion, the resulting DNA fragments may be subjected
to electrophoresis on an agarose gel. The restriction pattern
obtained may be analysed, e.g. by staining with ethidium
10 bromide and visualising bands in the gel by means of UV light.
The restriction pattern of the DNA after digestion with the
restriction endonuclease may then be compared to the
restriction pattern obtained with a negative control comprising
at least a portion of wild-type DNA encoding the PP1 G-subunit
15 spanning codon 905, and/or with a positive control comprising
at least a portion of DNA encoding the PP1 G-subunit and
containing the mutation. For instance, the number and size of
restriction fragments may be compared on the agarose gel, from
which carriers of the mutation may be identified, and from
20 which heterozygous carriers may be distinguished from
homozygous carriers.
An example of a suitable restriction endonuclease is Dde 1
which has the recognition sequence CTNAG, cutting after C (at
position 2709 of the PP1 G-subunit gene) and leaving a 3 base
25 5' overhang. This recognition sequence is abolished by the
mutation of G to T in position 2713.
In a variant of this embodiment, the DNA isolated from the
sample may be amplified prior to digestion with the restriction
endonuclease. Amplification may suitably be performed by
30 polymerase chain reaction (PCR) using oligonucleotide primers
based on the appropriate sequence of PP1 G-subunit spanning the
site(s) of mutation, essentially as described by Saiki et al.,
Science 230, 1985, pp. 1350-1354. After amplification, the
amplified DNA may be digested with the appropriate restriction
- 2157807
endonuclease and subjected to agarose gel electrophoresis. As
a control, wild-type DNA encoding the PPl G-subunit (i.e. not
containing the mutation) may be subjected to the same
procedure, and the restriction patterns may be compared.
5 A further embodiment of the method of the invention is an
adaptation of the method described by U. Landegren et al.,
Science 241, 1988, pp. 1077-1080, which involves the ligation
of adjacent oligonucleotides on a complementary target DNA
molecule. Ligation will occur at the junction of the two
10 oligonucleotides if the nucleotides are correctly base paired.
In a still further embodiment of the present method, the DNA
isolated from the sample may be amplified using oligonucleotide
primers corresponding to segments of the gene coding for the
PPl G-subunit. The amplified DNA may then be analysed by
15 hybridisation with a labelled oligonucleotide probe comprising
a DNA sequence corresponding to at least part of the gene
encoding the PPl G-subunit and containing the mutation of G to
T in the first position of codon 905. As a control, the
amplified DNA may furthermore be hybridised with a labelled
20 oligonucleotide probe comprising a DNA sequence corresponding
to at least part of the wild-type gene encoding the PP1 G-
subunit spanning codon 905. This procedure is, for instance,
described by DiLella et al., Lancet 1, 1988, pp. 497-499.
Another PCR-based method which may be used in the present
25 invention is the allele-specific PCR method described by R.
Saiki et al., Nature 324, 1986, pp. 163-166, or D.Y. Wu et al.,
Proc. Natl. Acad. Sci. USA 86, 1989, pp. 2757-2760, which uses
primers specific for the mutation in the PPl G-subunit gene.
Other methods of detecting mutations in DNA are reviewed in U.
30 Landegren, GATA 9, 1992, pp. 3-8. A currently preferred method
of detecting mutations is by single stranded conformation
polymorphism (SSCP) analysis substantially as described by
Orita et al., Proc. Natl. Acad. Sci. USA 86, 1989, pp. 2766-
2770, or Orita et al., Genomics 5, 1989, pp. 874-879.
2157807
14
The mutation may also be detected by heteroduplex formation
analysis. Heteroduplex formation scanning is a technique that
utilizes the formation of DNA heteroduplexes from alleles
differing at one or a few nucleotides during the multiple
5 denaturation-annealing steps in normal PCR-amplifications (12).
During non-denaturing gel electrophoresis the normal double
stranded homoduplex DNA segments migrate at a different speed
than their heteroduplex counterparts. Thus, heterozygous
carriers of mutations in a specifically amplified gene may be
10 identified as having two bands of double stranded DNA instead
of one. Homozygous mutation carriers, however, cannot be
detected by this technique because no heteroduplexes are
formed.
The label substance with which the oligonucleotide probe is
15 labelled is preferably selected from the group consisting of
enzymes, coloured or fluorescent substances, or radioactive
isotopes.
Examples of enzymes useful as label substances are peroxidases
(such as horseradish peroxidase), phosphatases (such as acid or
20 alkaline phosphatase), B-galactosidase, urease, glucose
oxidase, carbonic anhydrase, acetylcholinesterase, glu-
coamylase, lysozyme, malate dehydrogenase, glucose-6-phosphate
dehydrogenase, ~-glucosidase, proteases, pyruvate de-
carboxylase, esterases, luciferase, etc.
25 Enzymes are not in themselves detectable but must be combined
with a substrate to catalyse a reaction the end product of
which is detectable. Examples of substrates which may be em-
ployed in the method according to the invention include
hydrogen peroxide/tetramethylbenzidine or chloronaphthole or o-
30 phenylenediamine or 3-(p-hydroxyphenyl) propionic acid or
luminol, indoxyl phosphate, p-nitrophenylphosphate, nitrophenyl
galactose, 4-methyl umbelliferyl-D-galactopyranoside, or
luciferin.
- - 2157~07
Alternatively, the label substance may comprise coloured or
fluorescent substances, including gold particles, coloured or
fluorescent latex particles, dye particles, fluorescein,
phycoerythrin or phycocyanin.
5 In a particularly favoured embodiment, the probe is labelled
with a radioactive isotope. Radioactive isotopes which may be
used for the present purpose may be selected from I-125, I-131,
In-lll, H-3, P-33, C-14 or S-35. The radioactivity emitted by
these isotopes may be measured in a beta- or gamma-counter or
10 by a scintillation camera in a manner known per se.
For use in the present method, the invention further relates to
a test kit for detecting the presence of a mutation of G to T
in the first position of codon 905 of the gene encoding the PPl
G-subunit, the kit comprising
15 (a) a restriction endonuclease which which cleaves wild-type
PPl G-subunit DNA at a recognition site eliminated by the
mutation of G to T in the first position of codon 905 of the
PPl G-subunit gene so that the mutant PPl G-subunit DNA is not
cleaved at this site,
20 (b) a first DNA sequence corresponding to at least part of the
wild-type gene encoding the PPl G-subunit spanning codon 905
and/or
(c) a second DNA sequence corresponding to at least part of the
gene encoding the PPl G-subunit and containing a mutation of G
25 to T in the first position of codon 905.
The first DNA sequence may, for instance, be obtained from
genomic DNA or cDNA encoding the PPl G-subunit obtained from a
healthy subject (a non-mutation carrier). The second DNA
sequence may conveniently be a DNA isolate according to the
30 invention.
- 21~7807
16
For use in the present method, the invention further relates to
a test kit for detecting the presence of a mutation of G to T
in the first position of codon 905 of the gene encoding the PP1
G-subunit, the kit comprising
S (a) means for amplifying DNA, and
(b) a labelled oligonucleotide probe comprising a DNA sequence
corresponding to at least part of the gene encoding the PPl G-
subunit and containing a mutation of G to T in the first
position of codon 905.
10 Appropriate means for amplifying DNA (typically genomic DNA
isolated from the biological sample) include, for instance,
oligonucleotide primers, appropriate buffers and a thermostable
DNA polymerase.
The test kit may further comprise a labelled oligonucleotide
15 probe comprising a DNA sequence corresponding to at least part
of the wild-type gene encoding the PPl G-subunit spanning codon
905.
The invention is further illustrated in the following example
which is not intended in any way to limit the scope of the
20 invention as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is described in detail with reference to
the appended drawings, wherein
Fig.la is an autoradiogram showing heteroduplex formation in
25 the double stranded DNA bands (ds) corresponding to the
nucleotide 2584-2844 cDNA segment of the human PPlG-subunit
from two heterozygous subjects (lanes 2,3) compared to wild
types (lanes 1,4).
2157807
17
Fig. lb is an autoradiogram with sequence ladder of a part of
the PPlG-subunit cDNA segment 2378-2844 of the coding strand
showing the G2713~ T mutation in a heterozygous carrier (lane 1)
compared to the wild type sequence (lane 2). This predicts a
5 codon change : Asp90s~ Tyr.
Fig. 2 is a print from an automated laser fluorescence DNA
sequencer using the "DNA fragment manager" software. The RFLP
examples of the three different genotypes are visible as peaks
of fluorescence. Upper lane (8) shows a heterozygous (G2713~T)
10 RFLP consisting of a 127 and a 147 bp. fragment, middle lane
(10) shows the wild type (G2713) RFLP with one 127 bp. fragment,
and bottom lane (18) shows a homozygous (G27l3~T) RFLP with one
147 bp. fragment.
EXAMPLE
15 MATERIALS AND METHODS
Subjects. Heteroduplex formation scanning was carried out in 30
NIDDM patients on whom we previously performed SSCP scanning
of the coding region of the PP1 G-subunit gene (11). These
Danish Caucasian diabetic patients were insulin resistant (11)
20 as estimated by the euglycaemic hyperinsulinaemic clamp
technique and all had a fasting serum C- peptide level above
0.3 nM. The NIDDM patients were treated with diet alone or with
a combination of diet and oral hypoglycaemic drugs.
An association study was performed in 313 NIDDM patients (mean
25 age 55 + 8 yr) and 150 healthy control subjects without known
predisposition of NIDDM (mean age 53 + 10 yr). The NIDDM
patients (109 females, 204 males) had an average diabetes
duration of 6.3 years (+ 6.1 yr), fasting plasma glucose of
10.7 mM (+ 3.6 mM) and HbAlC of 8.3% (+ 1.7%). The control
30 subjects (62 females, 88 males) had an average fasting plasma
glucose of 4.9 mM (+ 0.6 mM) and a HbA~C of 5.4 % (+ 0.5%).
21~7807
18
Twenty-seven of the 150 healthy controls volunteered for a
euglycaemic hyperinsulinaemic clamp investigation in
combination with indirect calorimetry to relate the values of
whole body glucose metabolism to the PPl G-subunit genotype.
5 Prior to the participation in the investigation the purpose and
risks of the study were carefully explained both vocally and in
writing to all study paticipants and their informed consent was
obtained. The protocol was approved by the committee of ethics
in Copenhagen and was in accordance with the Helsinki
10 Declaration.
Euglycaemic hyperinsulinaemic clamp. The 27 healthy subjects
who were examined did not differ in phenotype characteristics
from those who did not participate in the clamp protocol. The
studies were initiated in the morning at 8.00 a.m. after a 10
15 h overnight fast. Each clamp comprised of a 2 h basal period
followed by a 4 h hyperinsulinaemic, euglycaemic clamp. Details
of the clamp technique have been decribed previously (27). To
assess total peripheral glucose uptake, (3-3H) glucose was
infused throughout the study period. Tritiated glucose was
20 administered as a primed (25 ~Ci) continuous (0.25 ~Ci/min)
infusion. The clamp was performed by continuous infusion of 2
mU insulin-kg ~1 min -1 (Actrapid, Novo Nordisk A/S, Bagsværd,
Denmark), and euglycemia was maintained by a variable infusion
of 20% glucose at a rate determined by measurement of the
25 plasma glucose concentration at 5- to 10- min intervals. Total
glucose disposal rate was calculated from the plasma
concentrations of [3-3H]glucose and plasma glucose with Steele s
non-steady state equations (28). In these calculations, the
distribution volume of glucose was taken as 200 ml/kg body
30 weight and the pool fraction as 0.65. At the highest steady-
state level, where the hepatic glucose production is nil,
glucose infusion rates were used to calculate the glucose
disposal rate.
Glucose and lipid oxidation. Indirect calorimetry was performed
35 using flow-through canopy gas analyzer system (Deltatrac
- 2157807
19
Metabolic Monitor, Datex, Helsinki, Finland). After an
equilibration period of 10 min, the average gas exchange rates
recorded over the two 30 min steady state periods were used to
calculate rates of glucose oxidation and lipid oxidation.
5 Protein oxidation rate was estimated from urea nitrogen
excretion (lg nitrogen = 6.25 g protein). Rates of oxidation
were calculated from Frayn s equation (29) The nonoxidative
glucose metabolism was calculated as the difference between the
total glucose disposal rate and the glucose oxidation rate as
10 determined by indirect calorimetry.
Heteroduplex formation analysis. Heteroduplex formation
scanning is a technique that takes advantage of-the formation
of DNA heteroduplexes from alleles differing at one or a few
nucleotides during the multiple denaturation-annealing steps in
15 normal PCR-amplifications (12). The formation of heteroduplexes
can be facilitated if the PCR-amplification is terminated with
inclusion of a final denaturation step followed by slowly
cooling to ambient temperature. Alternatively, the PCR products
in loading buffer can be heat-denatured followed by cooling on
20 ice for 10 min immediately prior to loading on the gel. During
non-denaturing gel electrophoresis the normal double stranded
homoduplex DNA segments migrate at a different speed than their
heteroduplex counterparts. Thus, heterozygous carriers of
mutations in a specifically amplified gene can be identified as
25 having two bands of double stranded DNA instead of one.
Homozygous mutation carriers, however, cannot be detected by
this technique because no heteroduplexes are formed. Since we
have used a denaturing loading buffer we could also detect
single stranded DNA during electrophoresis. In our hands this
30 method has also proved successful in previous studies (8).
Heteroduplex formation scanning was carried out after segmental
PCR amplification of PPl G-subunit cDNA in 15 different
overlapping segments with the overlaps varying from 16 to 78 bp
as described previously (11) but with the addition of a final
35 denaturation step in the PCR reaction at 99C for 10 min
2157807
followed by slowly cooling to ambient temperature. One ~1 of
the reaction mixture was mixed with 3 ~1 of sequencing stop
solution (0.3 M NaOH, 0.1 M EDTA, 10% glycerol, and 0.03%
xylene cyanol/bromphenol blue). This mixture was loaded onto 38
5 (height) x 31 (width) x 0.03 cm 5% polyacrylamide gel (49:1,
acrylamide:bisacryl-amide) in 90 mM Tris-borate, 2.5mM EDTA,
with 5% glycerol. Electrophoresis was carried out at room
temperature (25-29C) powered by a temperature controlling
device (Stratagene, La Jolla, CA). The gels were transferred to
10 a 3 MM filter paper, covered with plastic wrap, and
autoradiographed at -80C with intensifying screens for 8-12 h
(Fig. la).
Isolation of genomic DNA from blood. Genomic DNA was isolated
from human leucocyte nuclei isolated from whole blood by
15 proteinase K digestion followed by phenol-chloroform extraction
on an Applied Biosystems 341 Nucleic Acid Purification System
(Foster City, CA).
Isolation of total RNA from human skeletal muscle. In the
fasting state at 0800 after an overnight fast a percutaneous
20 biopsy (about 500 mg) of vastus lateralis muscle was taken
under local anesthesia (1% lidocaine without epinephrine) about
20 cm above the knee using a modified Bergstrom needle. Muscle
biopsies were homogenized in a 4 M guanidinium thiocyanate
solution, and subsequently total RNA was isolated on an Applied
25 Biosystems 341 Nucleic Acid Purification System (Applied
Biosystems Inc., Foster City, CA.).
Oligonucleotides used for amplification. Oligonucleotides, 20-
22 mers in length, were synthesized on an Applied Biosystems
394 DNA/RNA Synthesizer and purified on a NABTM-10 column
30 (Pharmacia P-L Biochemicals Inc., Milwaukee, WI). Fluorescein
labeling of primers were done during synthesis using the
FluorePrimeTM Amidite (Pharmacia).
21a7~07
21
cDNA synthesis. cDNA was synthesized in volumes of 25 ~1 con-
taining (in final concentrations) 50 mM Tris-HCl (pH 8.3), 75
mM KCl, 3 mM MgCl2, 10 mM DDT, 0.2 mM dNTP's, 40 units of RNasin
(Promega, Madison, WI), 0.625 micrograms of Oligo (dT) 18~ 400
5 units of M-MLV RT (Life Technologies Inc., Grand Island, NY)
and 1.0 ~g of total muscle RNA. The reactions were performed at
37C for 1 h, followed by enzyme inactivation during 10 min
incubation at 95C.
Direct DNA sequencing. Single stranded DNA for sequencing was
10 generated by using biotinylated oligonucleotide primers and
streptavidine-coated magnetic beads according to the
manufacturer's recommendations (Dynal A/S Oslo,Norway). PCR
reactions were performed with 5 pmol of biotinylated primers.
Dideoxynucleotide sequencing using sequenase 2.0 (United States
15 Biochemical Corporation, Cleveland, OH) and (~_35S) dATP
(Amersham International plc, Buckinghampshire, Great Britain)
was performed according to standard procedures. Heteroduplex
forming PCR segments were sequenced on both strands by
amplifying a biotinylated 2378-2844 segment and use of nested
20 sequencing primers (b = biotin):
PCR primers:
2378TAGGTGAATCTATGACAATG2398
2844bCGTAGAAATAGGTTGGCTAGC2824
Sequencing primers:
2s22ATGGTAGTAACTGCATTc2805
2584CTGGATTTACAGTTGGGAATG2604
Amplification of genomic DNA. Each PCR-reaction was carried out
with 100 ng of genomic DNA as template. The assay conditions
were: 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.1 ~
30 Triton X-100, 0.2 mM dNTP's, 0.2 ~M of each oligonucleotide
primer and 0.6 unit of Taq DNA polymerase (Promega), all in
final concentrations and 25 ~1 reaction volumes. The mixture
was overlaid with 18 ~1 of mineral oil and after initial
denaturation at 95C for 3 min, the samples were subjected to
35 40 cycles of amplification: annealing at 55C for 1 min,
- 21S7807
extension at 72C for 1 min and denaturation at 94C for 1 min.
Oligonucleotide primers used to amplify nucleotide segment
2584-2844 were (the A-primer was fluorescein labeled)
PPlG-A: 2584CTGGATTTACAGTTGGGAATG2604
PPlG-B: 2844CGTAGAAATAGGTTGGCTAGC2824
Restriction fragment length polymorphism (RFLP). Dde
restriction enzyme (New England Biolabs, Inc., MA) digestion of
PCR amplified nucleotide fragment 2584-2844 was carried
according to manufacturer's recommendations: five ,ul PCR
10 reaction mixture was digested by 2-4 U Dde 1 in a total volume
of 15 ,ul at 37C for 4-8 h. This reaction mixture was mixed with
an equal volume of stop buffer (formamide with 5 mg/l dextran
blue), denatured at 90C for 3 min and subsequently loaded onto
a 6~6 denaturing acrylamide PreMix Long Ranger (HydroLink, AT
15 Biochem, PA) on an automated laser fluorescence DNA sequencer
(Pharmacia). This type of gel can be repetitively loaded 6
times.
The 2584-2844 nt DNA segment of PPl G-subunit contains two
cutting sites for Dde 1 restriction enzyme at base positions
20 2709 and 2729, respectively. The recognition sequence is CITNAG
cutting after the C and leaving a 3 base 5 primed overhang. The
change of a G2713 ~ T deletes this Dde 1 recognition site in the
PPlG-subunit segment 2584-2844. Dde 1 digestion of the wild-
type 2584-2844 DNA segment results in three DNA fragments of
25 127 bp, 115 bp, and 20 bp, respectively, whereas a heterozygous
carrier will generate four different DNA fragments of 147 bp,
127 bp, 115 bp, and 20 bp. A homozygous carrier of the G2713~ T
mutation will generate two DNA fragments of 147 bp and 115 bp.
When the DNA segment is fluorescein labeled in the 5'-end,
30 electrophoresis on an automated laser fluorecsence DNA
sequencer will reveal a wild type as one single peak
corresponding to the 127 bp fragment, whereas a heterozygote
will appear as two peaks corresponding to the 127 bp and 147 bp
fragments. A homozygote for the T2713 will appear as one peak
35 corresponding to the 147 bp fragment (Fig. 2).
- 2157807
Other analyses. Glucose in plasma and urine was measured by a
hexokinase method. Serum insulin and C-peptide concentrations
were analyzed by RIA (30,31). HbAlc was measured by a HPLC
method, normal range 4.1-6.1%. Body fat mass was measured with
5 an impedance technique (32) and fat free mass was calculated by
substrating fat mass from body mass.
Statistics. Chi-square analysis was applied to test for
significance of differences in allelic frequencies among NIDDM
patients and matched controls. The Mann-Whitney U test was used
10 for comparison of unpaired data. Data are means + SE. P< 0.05
was considered significant.
RESULTS
Heteroduplex formation analysis and nucleotide sequencing. When
15 overlapping segments of the PPl G-subunit cDNA were examined
15 in 30 NIDDM patients two cases of heteroduplex formation were
identified in the same 2584-2844 nt. PCR segment (Fig la).
Nucleotide sequencing revealed that both patients were
heterozygous carriers of the same G2713~ T mutation encoding an
Asp905~ Tyr amino acid substitution (Fig lb).
20 Studies of whole body glucose metabolism. To study the
potential influence of the Tyr 905 substitution on whole body
glucose metabolism, 27 healthy control subjects, who
volunteered for a 4 h euglycaemic hyperinsulinaemic clamp in
combination with indirect calorimetry, were examined. PPl-G
25 genotyping showed that 6 individuals were heterozygous for the
Asp905 /Tyr905 polymorphism and 21 were wild type (Asp905)
carriers. The two groups did not differ statistically regarding
age, body mass index, fat free mass, fasting level of plasma
glucose or fasting serum levels of insulin and C-peptide (Table
30 1). Furthermore, the two groups of volunteers did not differ
significantly in basal or insulin stimulated whole body glucose
disposal rates (Table 1). Importantly, however, the routing of
glucose in whole body metabolism differed in the Tyr905 carriers
21a7807
24
when compared to the wild type carriers. Thus, basal glucose
oxidation was significally increased by 54 % (p < 0.04)
whereas insulin stimulated nonoxidative glucose metabolism was
decreased significantly by 25 % (p< 0.04) in mutation carriers.
5 Association study. By means of RFLP analysis as described in
Methods and depicted in Fig. 2 an association study was
performed in 313 NIDDM patients and 150 control subjects. Four
of the NIDDM patients were homozygous (ho=1%) for the Tyr905
substitution and 37 were heterozygous (he=12%) for the same
10 mutation whereas 272 were wild types (wt=87%). Among the
controls, one subject was homozygous (ho=1%) and 26 were
heterozygous (he=17%) whereas 123 were wild types (wt=82%). The
genotype (ho+he) frequency in NIDDM patients (13%) did not
differ significantly from the genotypic frequency in control
15 subjects (18%). (X 2 = 1.94, p = 0.16).
2157807
References
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21S7807
29
SEQUEN OE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Novo Nordisk A/S
(B) S'l'K~'l': Novo Alle
(C) CITY: Bagsvaerd
(E) CCUNTRY: Denmark
(F) POSTAL CODE (ZIP): 2880
(G) ~ T~pHoNE +45 4444 8888
(H) '1'~1 ~FAX: +45 4449 3256
(ii) TITLE OF INVENTION: Mutant DNA encoding Protein ph~h~tase 1
G-Subunit
(iii) NUMBER OF SEOUENCES: 1
(iv) C~MP~TER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) OCMP~T~R: I~M PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFrW~RE: PatentIn Release #1.0, Version #1.25 (EPO)
(2) INFORMATION FOR SEO ID NO: 1:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 4322 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOL0GY: linear
(ii) MOLECUIE TYPE: cDNA
(iii) HYPOq~ETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOUROE :
(A) ORGANISM: Homo sapiens
(xi) SEOUENCE DESCRIPTION: SEO ID NO: 1:
GGAGCCITCT GAAGTACCTA GTCAGATTAG CAAAGATAAT T m TAGAAG TICCTAATTT 60
ATCTGACTCT ~l'l'l~l~AAG ATGAAGAAGT TACTTICCAA CCqX~FFrCT CCCCTCAACC 120
AAGTAGACGA G~ ATT CTICIGAAGA CATATACCTG GATACCCCAT CTTCAGGTAC 180
TAGAAGAGTT TCATTTGCTG ATTCcTTTaG ATrCAATCIT ~l~ l~ AAGAATITGA 240
TIX~qK~GAA TTACCGAGTG CTTCAACCAC TTTTGAC~ A G5GACGGACA ~ ~ACAC 300
- 2157807
AGAAGAATAT GTIITAGCCC CA~ l~A CqqX~X~rrCT TCAAAAGAAG ATCITATGCA 360
ACAACTCCAA ATACAGAAAG CAATACrGGA GTCAACrGAG 'l~'l~'l'l~TrG GZTCTACAAG 420
TATCAAGGGT ATTATrCGAG TTrrGAAT&T 'l'l~'l'l'l'l~AG AAGITAGTAT ATGTAAGAAT 480
~'l~'l'l'l'AGAT GACIGGCAGA CACATTATGA CAITITAGCA GA~ aTrc CTAATrCATG 540
TGATGZTGAA ACTGACCAGT TCTCCTTTAA GATTGTATrG Gaq~X~DOCTT ATCAAAAAGA 600
TGGCAGTAAA GTrGA~l'l'l'l' GTATACGITA T&AAACTTCT GTTGGTACAT 'l'l'l~ AA 660
TAATAATGGC ACAAATTATA CATrCATITG TCAAAAGAAA GAACAAGAGC CGGAGCCTGT 720
AAAACCATGG AAAGAAGTTC CTAACAGACA AATAAAAGZC TGCITAAAGG TAAAATCAAG 780
TAAAGAAGAA TCATCAGTAA CATCAGAAGA AAATAA~'l'l'l' GA&AATCCAA AGAATACAGA 840
TACCTATATC CCAACAATCA 'l'l'l~'l'l~'l~'A TGAGGACAAG GAAGATr~GG AAGCCAGTAA 900
TCGAAATGTA AAAGATGTAA ACAGGGAACA TGAT&AACAT AATGAAAAAG AATTAGAGIT 960
GATGATAAAT CAACACITAA TAAGAACCAG AAGTACIGCT TCCAGAGATG AAAGGAATAC 1020
ATTITCAACA GATCCAGTCA A'l'l'l'l~AAA TAAAGCAGAG GZGITAGAGA AGAAGCAAAT 1080
CCATGG~GAA ATATGTACTG ACTr~TTCC~ AAGGTirCTG TCrCCAAGlT CATCAGCAGA 1140
AAGCrCCGTA AAGZGAGATT TITACIGCAA TGAAAAATAT TCCTCAGGAG ATGACrGTAC 1200
ACATCAACCT TCAGAGGAAA CTACITCAAA TATGGGAGAA ATCAAGCCAT CATIGGGAGA 1260
TACTAGTAGT GATGAACTAG TGCAATTACA TACrGGCAGC AAAGAAGTCC TGGATGATAA 1320
TGCTAATCCA GCCCATGGCA ATGZCACAAT GCAAATACCT IG~XXX~rCTT CAGATCAACT 1380
AATGGCAGGA AACCITAATA AAAAACATGA AGZAGGAGCT AAAAAAATrG AAGTAAAAGA 1440
TTIGGGATGT TTACGAAGAG ATTTCCATrC AGATACGTCG GCATGTCTCA AAGAATCAAC 1500
AGAAGAAGGA TCTTCTAAGG AAGATrATTA TGGCAATGGT AAGGATGATG AAGAACAAAG 1560
AATATATTTA GZIGITAATG A~AAACAAAG AAA~AATrTC CAAACAATCT TACATGACCA 1620
AGAAAGGAAG ATGZGTAACC CTAAAATAAG TGTGGCAGZG ATrGGAGCTA GTAACAGAGA 1680
CCrGGCTACT CrGCTGAGCG AACATACCGC AATCCCCACC CGZGCAATCA CAGCAGATGT 1740
GTCTCATrCA CCAAGGACAA A'l'l'l'AAGTTG GZAAGAAGCT GTGITAACCC CAGAGCATCA 1800
TCATTrGACT AGTGAAGZCA GCGCTITAGG AGZGATAACT G&TCAA~l'l'l' GTrCATCAAG 1860
AACrGGAAAT ~'l'l'l'l~AGGA ATGATTATCT TTTCCAA~l~l~ GAA~U~AAT CAGGTGGGAT 1920
TAATTCTGAA GATCAG&ATA ATAGCCCACA GCATAAACAA AGTIGGAATG 'l'l~'l~AAAG 1980
-
- 21S7807
31
TCAG&&AAAA TCAAGAGAGA ATAAGACAAA CATAACAGAG CATATCAAAG GACAAACAGA 2040
TTGTGAAGAC GTY~X~GAA AAAGAGATAA TACGAG&AGT TrGAAAGCTA CTACAGAAGA 2100
A'l'l~'l'l'l~CC T&CCAAGAAA CAGTGIGCqG T&AACTGTCT TCrCTAGCTG AI~P~ CAT 2160
TACTGAGAAA GCAGAAGCTG GTACAGCCTA TATAATTAAG ACAACATCAG AAAGTACTCC 2220
AGAAAGCATG TCIGCTAGAG AAA~AGC~AT AATIGCTAAG CTACCTCAAG AGACAGCACG 2280
AAGTGACAGG CCCATCGAGG TAAAG&AAAC AG~ AT CCACAT&AAG G&AGAAAT&A 2340
T&ATTCACAT TATACCCIIT GTCAACGAGA TACAGTAG&T GTAATCTATG ACAAT&ATTT 2400
T&AAAAG&AA TCACGIITAG GTATIT&TAA T&TAC&TGTA GAT&AAATGG AGAAG&AA.&A 2460
AACCAT&TCT ATGTACAATC CTAG&AAGAC ACAT&ACAG& GAGAAAT&TG GCACTGGAAA 2520
TATAACATCT GT&&AAGAAT CCTCAT&G&T CATTACAGAA TATCAAAAAG CAACTTCAAA 2580
ACTG&ATITA CAGqlXX~iAA T&TTACCAAC AGACAAAACT GTATrTTCAG AAAACAGAGA 2640
TCATAGGCAG GTTCAAGAAT TATCAAAGAA AACAGACTC& GATGCCATT& TGCATTCTGC 2700
rl'l'l'l'AACTCA TACACTAATA GAGCTOCTCA GAATAGCTCT C~'l'l'l'l'l~A AACATCATAC 2760
T&AAATITCA GT&TCAACTA AT&AGCAGGC AATT&CT&TA GAGAATGCAG TTACTACCAT 2820
G&CTAGCCAA CCTATrTCTA C&AAATCAGA AAATATIT&T AATICAACAA GAGAAATCCA 2880
GGGTATT&AG AAGCACCCTT ATCCTGAGTC TAAACCT&AA GAAGTITCCA GAAGTTCAGG 2940
AATAGT&ACA TCAG&TAGTA GA~AAGAAAG AT&CATAGGC CAGA'l'l'l'l~C AAACAGAAGA 3000
GTATAGT&TG GAAAAATCTC TAGGGCCAAT GA'l'l'l'lAATC AACAAACCTC TT&AGAATAT 3060
G&AAGAAGCA AG&CATGAAA ATGAAGGATT AGTAAGCTCT GGGCAATCAC TATACACTTC 3120
AGG~ LAAG GAATCT&ACA GCTCT~CTTC TACTAGTCTT C~'l~'l'l~AGG AAAGTCAAGC 3180
TCAAG&CAAC GAA'l~'l'l'l~'l' TTTCPAAATA TACCAACTCT AAAATACCTT Al'l'l~'l'l'l'l' 3240
GD~FX~iGATA 'l'l'l~ll~TAA ~'l~'l~'l'ACCA TTAT&ACITA ATGATrG&CT T&ACATTCTA 3300
C~'l'l'l'l~'l~A TI~XX~nGGC TATCCq~GGA AGAG&&TAGA CAAAAAGAGT CqGaY~AAA 3360
GAAGTAACCT CAGCACTACT A'l'l~'l~l~'l'l' AAAAGATAAG CTATTTAACC CCAAACAlll 3420
G&ATTG&T&A AT&G&ACTAT TCATrGTTCA AAGATCCAGT GCAGTTITTC TCTT&AAG&A 3480
TCATITAAAA AGG&AC&C&A ATAAGTITGC TCCTTCATAT AAGTAATTAT TCTATATAGG 3540
ACCATTATGT TIG&ATCATT AAATACCTAT ATGAATAT&A GATCTi AAEC ACGTCAAGTT 3600
GAAATTAG&T ACAGCT&IT& CTCCITAGCA G&CTATGAAG TTGCAAT&CT TCACATCTCT 3660
2157807
32
TCACTACTTA AAGT&CTATT TCITGCATTC A m CTCTIG CAATAAAGCT TCAll~lllll~ 3720
'l'l~C~ A GAGTATATAT m CCCCTAC GGTTITAAAA AACAGATAAA ACATGGACAA 3780
TGGCAGAGGA ~'l'l'l'l'l'l~'l' CTIITAGTAT TGAACATGAA ATrTGTATIT AACACTGTAT 3840
CAl'l'l'ATCAG GATTCATTGA TC~AATA m CAAC~llll~ ATATTTITAA GAAAACACCC 3900
ATATATATTG AAATGCAAAC TTAAACATAT 'lW ~l'l'CACT TGAGTGTAAT ACITGATGCA 3960
TGCACACACA CACACACATA C~'l'l~'l'l'l'AT AGCTACAAAG TCAGGGCGTT CATAGGCAAA 4020
ATCTGACAAG ACTGAAACAA TIGGGAGITA I~qCF~G~ATT CTGAAAAATT CqYD~ 3G 4080
AA'l~'l~l'l'l'A AGCAAT~CIT 'l'l~'l~'l'l~'lG AGGGAGACTG TAACACAGCT GAACTATTTC 4140
AGTITCAACT ATTCAGAATT GAAAAT~lAA ATTAAAATIT CCACAAGCAT TGCITCAGAA 4200
TGAATTTGTA C~TATAAGCA TAAGGCATTA AATGACAATA AAAATICCAA ATGGACTATT 4260
~ ACAT T~'l'AT m TG TCTTCAAATA TrTTCTAAGA GAATGAATTA TCXXX~3GG 4320
AA 4322
