Note: Descriptions are shown in the official language in which they were submitted.
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W O 97131015 PCTAUS97102397
TITLE OF THE INV~NTION
RAT OB RECEPTORS AND NUCLEOTIDES ENCODING THEM
CROSS-REFERENCE TO RELATED APPLICATIONS
S ~his application is a continuation-in-part of Provisional
patent application Serial No. , (Attorney Docket No. l 9642PV)
filed February 22, l 996, which is hereby incorporated by reference.
STATEMENT REGARDING FEDERALLY-SPONS()RED R&D
Not Applicable
REFERENCE TO MICROFICHE APPENDIX
Not Applicable
15 FIELD OF THE INVENTION
This invention relates to rat ~b receptor proteins, to DNA
and RNA sequences encoding them, and to assays using rat receptor
proteins.
20 BACKGROUND OF THE INVENTION
Recently the identification of mutations in several gene~s
involved in the onset of obesity in rodent~s have been identified. Of
particular interest are mutation.s discovered in the peptide hormone,
leptin, which is a component of a novel signal transduction pathway that
25 regulates body weight (Zhang et al. 1994, Nature 372:425-432; Chen et
al. 1996, Cell ~4:491-495). Leptin was initially discovered by the
positional cloning of the obesity gene, vb, in mice. Two different ob
alleles have been identified: one mutation cau~es the premature
termination of the leptin peptide resulting in a truncated protein, and the
30 other mutation changes the transcriptional activity of the obesity (oh)
gene, resulting in a reduced amount of circulating leptin.
There ïs a correlation between a decrease in the levels of
biologically active leptin and the overt obese phenotype observed in
l)htob mice. Recombinant leptin has been shown to induce weight loss in
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the ~blo~ mouse but not in the diabetic phenotype d~ldh mouse
~Campfield et al. 1995, Science 269: 546-549; Halaas et al. 1995,
Science 269: 543-546; Pellymounter et al. 1995, Science 269:540-543;
Rentsch et al . 1995 , Biochem. Biophys. Res. Comm. 214: 131 - 136; and
5 Weigle et al. 1995, J. Clin. Invest. 96:2065-2070).
Although the synthesis of leptin occurs in the adipocyte, its
ability to decrease food intake and increase metabolic rate appears to be
mediated centrally by the hypothalamuls. Injection of recombinant leptin
into the third ventricle of the brain elicit,s a similar re.sponse as
10 peripheral admini,stration of leptin. Furthermore, the recent cloning of
the human receptor for the leptin, the ob-receptor (OB-R), reveals that
it is transcribed in the hypothalamuls (Tartaglia et al. 1995, Cell
~3:1263-1271; Stephen,s et al. 1995, Nature 377: 530-532). In addition,
a mutation that results in premature termination of the long-fo~n of the
15 mouse OB-R, which is preferentially expressed in the hypothalamus,
appears to be responsible for the obese phenotype of the dbldl~ mouse
(Lee e~ al. 1996, Nature 379:632-635; Chua et al. 1996, Science
271 :994-996; and Chen et al. 1996, Cell ~4:491 -495).
The f~ mutation is a recessive allele that arose
20 spontaneously in the 13M rat strain and was first repolted in 1961
(Zucker et al. 1961, J. Heredity 52: 275-27~. The onset of obesity in
thefalfa Zucker rat is at 5-7 weeks of age and progres~ses with age. The
mature fatty rat is approximately twice the weight of lean litter mates
and over 40% of its body weight is adipose tissue (Zucker et al. 1962,
25 Proc. Soc. Exp. Biol. Med. 110: 16~-171, Zucker et al. 1963, J.
N~trition ~0:6-19). Thefa/fa Zucker rat exhibits hypercholesterolemia,
hyperlipemia? and hyperglycemia and has been used extensively as an
~nim~l model for human cardiovascular disease and diabetes. Most of
the fatty Zucker rat colonies have been maintained by outbreeding in
30 order to retain heterozygousity at as many loci as possible. However,
certain stock.s have been inbred to produce ~nim~ls such as the Zucker
diabetic fatty (ZDF) rat which exhibits a more profound diabetic
phenotype than the outbred falfa Zucker rat (Clark, et al. 19~3~ Pr~( .
Soc E~p. Biol. Me~l 173: 6~-75).
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Thefà mutation maps to rat chromosome S in a region that
is syntenic with the dh allele on mouse chromosome 4 (Truett, et al.
1991, Proc. Natl. Acc~d. Sci. 8~: 7X06-7~09). This observation, in
conjunction with the ~imilar phenotypes of thefcllfa rat and the d~lclh
5 mou~e, led to the proposal that thef~ gene was the rat homologue of the
~lb gene. Higher resolution genetic mapping supports the contention
that thef~ mutation is located in the gene encoding the rat OB-R (Chua
et al. S~ ien~ ~ 271: 994).
It would be de,~iirable to be able to further experiment with
10 the rodent model system for obesity, and to be able to clone and produce
purified rat oh receptot to u~ie in assays for the identification of ligand~
which may be useful in understanding obesity and for its prevention and
treatment.
15 SUMMARY OF THE ~VENTION
Not Applicable
BRIEF DECRIPTION OF THE DRAWrNG~
FIGURE I i~i the amino acid ~equence of the rat OB-
20 receptor.
FIGURE 2 is the cDNA seL~uence of the rat OB-receptor.
FIGURE 3 is a table of primers u,sed for the PCR reaction~
detailed in the Exan~ples.
FIGURE 4 show.s the gels demonstrating the analy~is of the
25 A8~0 to C mutation identified in the OB-receptor from hypothalamic
cDNA and genomic DNA obtained from lean and f~/fà rats.
FIGURE 5 compares the amino acid se4uence between
human cytokine receptor gpl 30 (Humgp 130), the mou~ie OB-R
(MousOBR)~ hum~n OB-R (HumOBR) and lean rat OB-R (RatOBR).
30 The numbering refers to the location in the protein, and the cytokine
motif GXWSXWS can be .seen.
A.~; used througout the .specification and claims, the
fvllowing definitioll~ apply:
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"Substantially free from associated rat membrane proteins"
means that the rat receptor protein is not in physical contact with any rat
membrane proteins.
"Substantially purified rat OB-receptor" means that the rat
5 receptor protein is at least 90% and preferably at least 95% pure.
"Wild type" means that the gene or protein is .substantially
the ,~ame as that found in a rat which is not considered to have a
mutation for that gene or protein. It is also referred to as "lean"
throughout the .specification and claims.
'fa" means that the gene or protein is substantially the same
as that found in a rat homologous for thefatty mutation.
"Substantially the sarne" when referreing to a nucleic acid
or amino acid se~uenGe means either it is the same a~s the reference
seyuence, or if not exactly the same, contains change,s which do not
15 affect its biologic~l activity or function. Although thefa and wild type
rat OB-R genes dilfer by only one nucleotide, they are not considered
"substantially the same" as the biological activity and functions of their
encoded proteins are very different.
The rat OB~R is a member of the cytokine receptor family.
20 Motifs that are characteristic of the cytokine receptors such as the motif
WSXWS (where W is the amino acid residue tryptophan, S is the amino
acid residue ~erine and X is any amino acid.) were found to be
con.served in the rat OB-R.
One a~spect of this invention is the molecular cloning of a
rat OB-R. The nucleotide ~equence for the rat OB-R from both lean
andfa/fa rat hypoth~lamic cDNA was deterrnined and compared. In the
talfa rat, there was a single nucleotide change, an A to C at nucleotide
8~0 resulting in an amino acid change at glutamine 269 to proline. The
mutation introduces an Msp I site (CC~G) that was utilized to genotype
a number of lean control and fatty :~nim:~ls. The re~sult~ indicate that the
mutation is tightly linked to thefa allele. Thus, it is likely that thefa
mutation lies in the 013-R receptor cDNA and that the A to C
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W O 97131015 PCTrUS97/023g7
transver.sion at ba~e pair ~0 is responsible for the obese phenotype.
Both rat OB-R allele,~, i.e. the OB-R containing a glutamine 269 and the
allele containing proline 269 are part of thi~s invention, a,s are all nucleic
acid,s which can encode them.
The nucleotide sequence of the wild type rat OB-R cDNA
obtained in accordance with this invention has 3650 nucleotides. as
shown in FIGURE 2. This DNA sequence contain.s an open reading
frame from nucleotide 75 to 3653 that encodes a protein of 1162 amino
acid~i. The open reading frame extending from nucleotide 75 to 3653
makes up one aspect of thi.s invention.
The wild type andfa receptor proteins contain an
extracellular, a transmembrane domain. The extracellular domain
extends from amino acids 1-~s30; the transmembrane domain is from
l;S amino acids ~39-~60; and the cytoplasmic domàin is from amino acids
~60-1162. This invention also includes proteins which lack one or more
of these domains. Such deleted proteins are use~ul in assay.s for
identifying ligands and their binding activity.
It ha,s also been found that alternate splicing can occur in
the receptor gene processing. Thi~ can occur at base pair 2742
(lysine~89). The alternative se(luence ~for both the wild type and fa)
gene,s and receptors, is shown below and forms another aspect of this
invention:
AGA GCG GAC ACT CTT TGA ATA TCT
R A D T L STOP
Amino acids 1-2~ form a signal sequence; thu,s the mature
protein~i extend from amino acids 2~-1162. The mature proteins form
yet another aspect of thi,s invention. Thi~ differs from the signal
30 se4uence of 1-22 reported for mouse and human OB-r; this may be
explained by the u,~e of ~ different analysis program.
Comparison of wild type rat OB-R to known OB-R
receptor.s of different ,~pecies has revealed some ,similaritie~s. For
example, the rat OB-R nucleotide se~uence is 93~O identical to the
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W O 9713101~ PCT~US97/02397
mouse OB-R and Xl % identical to the human OB-R sequences. The
deduced amino acid sequence of the rat OB receptor is 93% identical to
the mouse and 76% identical to the hllm~n OB-R.
The slze of the open reading frame of the rat OB-receptor
5 of this invention, (1162 amino acid.s) is similar to that of the human OB-
R (1165 amino acids) reported by Toriaglla et al. 1995, Cell 83:1-20.
Both the rat OB-R of this invention and the human OB-R contain a large
cytoplasmic domain. In contrast, the mouse OB-receptor of f~94 amino
acids has a relatively ,short cytoplasmic domain.
One of the mo.st notable and susprising aspects of this
invention is that there i.s only a single nucleotide difference between the
wild type rat cDNA and the fà/fa rat cDNA for the OB-R. PCR
fragments obtained from fa/f~ cDNA were sequenced. A single
1~ nucleotide change relative to the lean cDNA sequence wa.s observed in
the hypothalamus. An A to C transversion at bp ~80 results in an amino
acid change of glutamine to proline at amino acid residue 268. Every
tissue examined in thef~l~ rat was found to be homozygous for this A
to C mutation at nucleotide 880. The A to C change in the sequence
20 introduce.~ a MspI re.striction endonuclease site (CCGG) into the
sequence, and this is the ba.~ of an assay for presence of the mutation.
Thus another aspect of this invention is an assay to
determine the genotype of a OB-R DNA, suspected of having an A to C
mutation at bp ~80, comprising digesting the OB-R DNA with MspI.
25 and comparing the restriction products so producted. In a preferred
embodiment, the assay comprises generating PCR products of the OB-R
DNA, digesting the PCK products with MspI, and comparing the
restriction product~ so produced with those obtained f'rom a rat
containing a wild-type OB-R gene. The gene from a rat which ha~ a
30 wild-type OB-R will yield two re.striction products, 1774 and 289 bp
long. The gene fro1n thefa rat will have three restriction products:
747, 1027 and 289 bp long. The,se are easily observed using standard
gel techniques.
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W O 97131015 PCTrUS97tO23g7
The ()B-R gene can be introduced into virtually any host
cell using known vectors. Preferred host cells include ~ coli as well as
m~nm~lian and yeast cell lines.
One of ordinary skill in the art is able to choo,~e a known
:~ vector which is appropriate for a given host cell; generally pla~mids or
viral vector,s are preferred. The OB-R gene may be present in the
vector in its native form, or it may be under the control of a
heterologous promoter, and if desired, one or more enhancers, or other
~equences known tO regulate tran,scription or translation. The host cell
10 containing the OB-R gene is cultured, and the OB-R gene i,~; expressed.
After a suitable period of time the OB-R protein may be harvested from
the cell using conventional separation techniques.
A further aspect of this invention iS the u~;e of rat OB-R in
assay,s to identify OB-K ~igands. A ligand binds to the OB-R, and in
1~ vivo may or may not result in an activation of the receptor. Ligands
may be agonists of the receptor (i.e. stimulate its activity), antagonists
(inhibit its activity) or they may bind with little or no effect upon the
receptor activity.
In an assay for ligands, the rat OB-R of this invention is
20 expo.sed to a putative ligand, and the amount of binding is measured.
The amount of binding may be measured in many ways; for example,
ligand or the OB-R being investigated may he labeled with a
conventional label (such as a radioactive or fluore~cent label) and then
put in contact with the OB-R under binding conditions. After a suitable
25 time, the unbound ligand is saparated from the OB-R and the amount of
ligand which has bound can be mea.sured. Thi,s can be performed with
either the wild-type OB-R or thefà OB-R of thi~? invention; alternatively
the amount of bindin~ to the two allele.~ can be compared. ln a
competitive a.~isay, both the putative ligand and a known ligand are
~0 present, and the a]nount of binding of the putative ligand iS compared to
the amount of binding to a known ligand. Alternatively? the putative
ligand's ability to displace previously bound known ligand (or vice-
versa) may be mea~?ure(i. In yet other embodiments, the assay may be a
heterogeneou~ one where the OB-R may be bound to a .surface, and
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W O 97/31015 PCTAUS97102397
contacted with putative ligand.s. Dectection of binding may be by a
variety of method.s, including labelling, reaction with antibodies, and
chomophores.
~S DETAILED DESCRIPTION OF THE INVENTION
This invention relates to a rat o~ receptor which is
substantially free from associated rat membrane proteins. It also relate~
to substantially purified rat ob receptor ("rat OB-R" or "rat OB-
receptor") protein. One of the rat OB-R,s of this invention is obt~ined
10 from a rat which ha.~i a wild-type OB-R. Another rat OB-R of this
invention is obtained from a rat which has the fcl mutation.
Another aspect of this invention is to nucleic acids which
encode a rat OB receptor. The nucleic acid m~y be any nucleic acid
which can encode a protein, such as genomic DNA, cDNA, or any of the
15 various forms of RNA. Preferably, the nucleic acid is cDNA.
This invention also includes vectors containing a rat OB-R
gene, host cells containing the vectors, and methods of making
susbstantially pure rat OB-R protein compri,~iing the steps of introducing
a vector comprising a rat OB-R gene into a host cell, and cultivating the
20 host cell under appropriate conditions such that rat OB-R is produced.
The rat OB-R so produced may be harvested from the host cells in
conventional ways~
Yet anothel aspect of this invention are a.ssays which
employ a rat OB-R. In these assays, various molecules, suspected of
2~ being rat OB-R ligand~ are contacted with a rat OB-R, and their binding
is detected. In thi.s way agonists, antagonists, and ligand mimetics may
be identified. A further aspect of this invention are the ligands so
indentified.
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The following non-limiting Example~ are presented to
better illustrate the invention.
EXAMPLE I
s
Preparation of mRNA and cDNA from rat tissues
Tissue~ were collected from lean andfa/fu Zucker rats and
snap frozen in 1i4uid nitrogen. The tissue,s collected included:
hypothalarnus, pituitar~,, lung, liver, kidney, heart, adrenal glands,
10 .smooth mu,scle, skeletal muscle, and adipo,se ti.ssue. The tissues were
homogenized with a Brinkmann Polytron homogenizer in the pre~ence
of guanadinium i~othiocyanate. mRNA was prepared from
hypoth~l~mus, lun~g, and kidney according to the instruction.s provided
with the me,ssenger RNA i~olation kit (Stratagene, L~ Jolla, CA). cl)NA
15 wa~ prepared from approximately 2 ~g of mRNA with the
SuperScriptTM choice ,system (Gibco/BRL Gaithersburg, MD). The first
strand cDNA ,synthesi,s was primed using 1 ug of oligo(dT)12-lx primer
and 25 ng of random hexamer~; per reaction. Second ~trand cDNA
sythesi~ was performed according to the manufacturer's instructions.
20 The ~uality of the cDNA wa~s asses~ed by labeling an ~liqout (l/lOth) of
the ,~iecond strand reaction with approximately I ~Ci of [a-32PJdCTP
(3000 Ci/rnmol). The labeled products were separated on an agaro~ie gel
and detected by autoradiography.
EXAMPLE 2
Amplification of Lean Rat OB-receptor cDNA u~in~,~ PCR
The initial portion of the rat OB receptor wa~i obtained by
PCR using degenerate primer~ ba.sed on the mou~ie and hl~m~n OB-
30 receptor amino acid sequence,s. A .set of 9 oligonucleotide primer~,ROBR 1-9, shown in FIGURE 3, were de~signed to region.s with low
codon degeneracy. The pairing of the forward primer~; ROBR 2 (5'-
CAY TGG GAR TTY C'TI TAY GT-3') and ROBR 3 (S'-GAR TGY
TGG ATG AAY GG-3') corre,sponding to mou,~e amino acid se~luence,~
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W O 97/31015 PCTAUS97102397
- 10-
HWEFLYV and E~CWMKG, with reverse primers ROBR 6 (5 '-ATC
CAC ATI GTR TAI CC-3'), 7(~'-CTC CAR TTR CTC CAR TAI CC-
3'), and ~ (5'-ACY TTR CTC AT~ GGC CA-3') repre.senting mouse
amino acids, GYTMWI, VYWSNWS, and WPMSKV provided good
5 yields of the appropriately sized product~. The fragment~ of interest
were amplified as long polymerase chain reaction (PCR) products by a
modifying the method of Barnes (1994, Pro~. Natl. Acad. S~i. 91:2216-
2220, which i~; hereby incorporated by reference. Ln order to obtain the
required long PCR fragment.s, Taq Extender (Stratagene, La Jolla CA.)
10 and the Expand Long Template PCR System (Boehringer Mannheim,
lndianapolis, IN) were used in combination. The standard PCR reaction
mix, in a final volume of 20 ,ul, contained 5 ng of template (lean rat
cDNA), 100 ng of primers, 500 ,uM dNTPs, I X Buffer 3 from the
Expand kit, 0.1 ,ul each of Taq Polymerase and Ta~ Expander.
1~ Reactant.s were as~embled in thin walled reaction tubes.
The amplification protocol wasl cycle of 92~C for ~0 ~iec., followed by
32 cycles at 92~C for 30 sec., 45~C for 1 min. and 6~"C for 3 min. using
a Perkin-Elmer (Norwalk, CT) 9600 Thermal Cycler.
This .strategy produced a series of PCR products with the
20 largest being approximately 2.2 Kbp amplified from primer~ ROBR 2
and ROBR ~. These product~ were .subcloned for DNA ~equence
analysis as described below.
EXAMPLE 3
Subcloning of PCR products
PCR products of the appropriate size were prepared for
~subcloning by .separation on an agarose gel, excising the band, and
extracting the DNA using Prep-A-Gene (BioRad, Richmond, CA). PCR
30 product~ were ligated into pCRTMII (Invitrogen, San Diego, CA)
according to the instructions provided by the manufacturer. The
ligation was transformed into INVaF' cell~ and plated on Luria-Bertani
plate.~i containing 100 ~g/ml ampicillin and X-Gal (32 ~l of ~0 mg/ml X-
Gal (Promega, Madison, WI). White colonie~ were picked and grown
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W O 97/31015 PCTrUS97102397
overnight in Luri~ -Bertani broth plus 100 ~g/ml ampicillin. Plasmid
DNAs were prepared using the Wizard miniprep kit (Promega,
Madison, WI). Lnserts were analyzed by digesting the plasmid DNA
with EcoRI and ~ieparating the re.striction endonulease dige~stion products
on an agaro,se gel.
Plasmid DNA was prepared for DNA ~e~luencing by
ethanol precipitation and resuspending in water to achieve a final DNA
concentration of 100 ~g/ml. DNA sequence analysis was perfonned
using the ABI PRISMTI'I dye terminator cycle sequencing ready reaction
10 kit with AmpliTa~l DNA polymerase, FS. The initial DNA ~equence
analysis was perfonned with M13 forward and reverse primers,
.subsequently primers based on the rat OB-R ~e~uence were utilized.
Following amplification in a Perkin-Elmer 9600, the extension product~
were purified and analyzed on an ABI PRISM 377 automated se~uencer
15 (Perkin Elrner, Norwalk, CT). DNA sequence data was analyzed with
the Sequencher program. Due to the unknown genotype of the lean
Zucker rat for the t~/ allele, either (+/+ or +/fa) the DNA sequence of
multiple .subclone~ of each fragment wa.s analyzed to determine the
cDNA sequence of the lean rat OB-R.
EXAMPLE 4
Arnplification and DNA sequence analysis of lean and fàlfa with
prirners ROBR 10 and 17
Once specific lean rat sequence had been obtained from the
ROBR 2-~ PCR fragment, rat specific primers ROBR 10(5'-CTG CAC
TTA ACC TGG CCT ATC-3') and ROBR 17 (5'-GGC CA~ AAC TGT
AAC AGT GTG-3') were synthesized. Using primer~ ROBR 10 and 17,
PCR products were amplified from rat lean hypothalamus, lean lung,
30 fàlfa hypothalamu.i andJalJ'à kidney cDNAs. The PCR condition.~ u.sed
for thi~s reaction were a l'CR reaction mix with a total volume of 50 ~1
containing 5 ng of template (various rat cDNA.s mentioned above), 200
ng of primers 500 ,uM dNTPs, 1 X Buffer 3 from the Expand kit, 0.2
~1 each of Taq Polvmerase and Taq Expander. Reactant~ were
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W O 97/31015 PCTAUS97/02397
assembled in thin walled reaction tube,s. The amplification protocol was
1 cycle of 92~C for 30 sec., followed by 32 cycles at 92~C for 30 ~sec.,
60~C for I min. and 6~~C for 4 min. using a Perkin Elmer 9600
Thermal Cycler.
EXAMPLE 5
Amplification of the 3' portion of the rat OB-R cDNA using
Semi-nested PCR
The 3' end of both the lean and fàlfà rat OB-receptors was
obtained by the PCR with an initial amplification of the rat cDNA u.sing
a rat specific 5' primer paired with either a degenerate primer that
corresponds to the cytoplasmic domain of the hum~n OB-receptor or the
3' UTR of the human or mouse sequences. Thi~s was followed by a
.second short round of amplification with either one of the original
primers paired with a nested primer positioned within the original1y
amplified fragment, or with two nested primers.
Rat specific primers ROBR 15 (~ TCA CCT TGC TTT
GGA AGC C-3'), ROBR ~6 (5'-GAC ATG GTC ACA AGA TGT
GGG-3') and ROBR 23 (5'-CCT GGA CAC TGT CAC CTG ATG-3')
were paired in different combinations with human de~enerate primers
located in the cytoplasmic domain of the human OB receptor; HOBR S
(~'-CAT CAT YTC RTC YTT RTT YTT CCA-3'), HOBR 6 (~S'-GTY
TGR AAY TGI GGC AT-3') and HOBR 7 (~'-TCR CAC ATY TTR
TTY TCC AT-3') which correspond to aminn acids WKNKDEMM,
MPQFQT, and MENKMCD, respectively. Primers from the 3' ends of
the human, HOBR I R (5'-TCT CTC CCA CCC ACA ACT AT-3'), and
mouse, MOBR IR (s~-TGG GTT CAT CTG TAG TGG TC-3'), OE~
receptor~ were also paired with rat specific primers.
PCR reactions were perfolmed with various combinations
of the above primer sets in a total volume of 20 ,ul containin~ 5 ng of
template (lean and fillf~ hypoth~l~mus cDNA~s), 100 ng of primers, 500
!lM dNTPs, 1 X But'fer 3 from the Expand kit, 0.1 !11 each of Ta4
Polymerase and Taq Expander. Reactant,s were assembled in thin walled
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W O 97/31015 PCT~US97102397
reaction tubes for the Perkin Elmer 9600 The~nal cycler. The
amplification protocol was 1 cycle of 92~C for 30 sec.7 followed by 32
cycle.~ at 92~C for 30 ~ec., 45~C for I min. and 6~~C for 4 min. u,~ing a
Perkin Elmer 960() Thermal Cycler.
S Product,s were then purified, removing all nucleotide~i and
primers, using the QlAquick PCR purihcation kit according to the
m~nufacturer's .specified protocols and resuspended in 30 ~1 of water.
The second PCR step was then perfo~ned using the first PCR reaction
a,~ the template and a nested rat specific primer paired with the original
10 3' primer as outlined above. The reaction condition~i were ~ 50 ~1
reaction containing 5 ~1 of template (from the purified PCR product),
20() ng of primeri? .S00 ~M dNTPs, I X Buffer ~ from the Expand kit,
0.25 ~1 each of Ta~l Polymerase and Taq Expander. Reactants were
as~embled in thin walled reaction tubes for the Perkin Elmer 9600
15 Thermal cycler. The amplification protocol wa~s I cycle of 92~C for 30
sec., followed by 25 cycles at 92~C for 30 sec., 45~C for I min. and
6Ps~C for 4 min. using a Perkin Elmer 9600 Thermal Cycler.
The large~it fragment that wa,~i generated u.~iing the .~trategy
wa.~i a fragment produced from ROBR 16 and HOBR I R that was
20 approximately 1500 bp in length. The mouse 3' UTR which pre.sumably
encode~ a smaller isofo~n generated by ~Itemative splicing, produced a
fragment that was about 650 bp long.
EXAMPLE 6
Amplification of 5' end of the rat OB receptor
The 5' end of the rat OB receptor wa.~ obt~ined by u.~ing
semi-ne~sted PCR in a m~mner analogous to that described above for the
3' end. ln this case the rat specific primer.s are the 3' primer~ that were
30 combined with primer~ from the 5' UTRs of the hum~n OB-receptor.
The primers utilized were HOBR IF (5'-CTT ATG CTG GGA TGT
GCC-3') and HOBK lF-2 (5'-TCG TGG CAT TAT CC~T TCA G-3')
paired with either ROBR 11 (5'-GAT A(IG CCA GCiT TAA GTG
CAG-3') or ROBR 12 (5'-GAG TGC GGA GCA GTT TTG AC-3).
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W O 97/31015 PCTAUS97/02397
- 14 -
The largest product, HOBR lF-2 and ROBR 11, yielded a 500 bp
fr~gment that covers the region and incluces an initiator methionine
codon.
EXA MPLE 7
Identification of a nucleotide chan~e in thefa/fà cDNA
PCR fragments obtained fromfa/fa cDNA were prepared
for DNA .sequence analysis by separating the PCR products on an
10 agaro,se gel, excising the band of interest, and extracting the DNA using
Prep-A-Gene (BioRad). Se4uencing results of the PCR product
generated fromfalfa hypoth~l~mic cDNA identified ~ single nucleotide
change relative to the lean cDNA sequence. An A to C tran.liversion at
bp ~X0 results in an amino acid change of glutamine to proline at amino
1~ acid residue 26~. The A to C change in the sequence introduces a MspI
restriction endonuclease site (CCGG) into the sequence.
Several independent PCR products were amplified from
hypothalamus, lung and kidney cDNA from lean and falf~l tissues using
the primer pair ROBR ] 0 and 17. This product contains only one
20 endogenous Msp I site at nucleotide 1907. Restriction digestion of the
PCR products in a reaction that consisted of ~ 1 of the PCR reaction, 4
~1 of water and I ~l of the restriction endonuclease M5'p I. These were
mixed, incubated for I hr at 37~C and analyzed on a l % agarose gel.
The PCR products ~rom the lean rat cDNAs contained only the
25 endogenous Msp I site and generated product,s of 1774 and 2~9 bp. In
contrast the PCR products from thefa/fà cDNAs contained an additional
Msp I site identified during the sequencing of ROBR 10/17 and
generated products of 747, 1027, and 2P~9. Thus~ every tissue examined
in thefa/fa rat wa~ homozygous for the A to C mutation at nucleotide
30 8~0.
CA 02246226 1998-08-14
W O 97131015 PCT~S97/02397
- 15 -
EXAMPLE
Genotype analysis of lean andfalfa rats
Genomic DNA was prepared from a 2 cm portion of the
tail from ten lean and tenfalfa Zucker rats and 2 lean and ~ fà/fà ZDF
rats. The tissue was digested overnight at ~5~C using 0.3 ~g of
Proteinase K in 0.7 ml buffer containing 50 mM Tri,s, pH 8.0, 100 rnM
EDTA, and 0.5% SDS. The DNA was extracted two times with
phenol/chloroform and one time with chloroform. The DNA wa.s
10 precipitated by adding NaCI to achieve a concentration of 0.3M and then
adding an equal volume of 100% ethanol. The DNA was transferred to
a 70% wa,sh and th~n resuspended in 10 mM Tri.s, 1 mM EDTA.
Genornic ONA, obtained as outlined above from variou,s
sources, was diluted in water to a final concentration of approximately
100 ng/ul. In thi.s experiment, the reaction condition,s were a 20 ~l
reaction containing 1 !11 of genomic DNA template. 1()0 ng of primers,
~00 ,uM dNTP.s, I X Buffer 3 from the Expand kit, 0.25 ,ul each of Taq
Polymera,se and Ta(l Expander. Reactant,s were as,sembled in Perkin
Elmer 0.5 ml thin walled reaction tube~. The amplification protocol for
20 a Perkin Elmer 4~() Therrnal Cycler wa.~ 32 cycles of 92~C for 30 ,sec.7
54~C for 1 min. and 6~''C for 5 min. Primers ROBR 27 (5'-GTT TGC
GTA TGG AAG TCA C'AG-3') and ROBR 2~S (5~-ACC AGC AGA
GAT GTA TCC GAG-3') were used to amplify a 1.~ Kbp fragment that
must contain approximately 1.65 Kbp of intronic se~uence since these
25 primer,s only produce a 156 bp PCR fragment when amplifying cDNA.
After PCR amplification, an M5p I restriction endonuclease
digestion of the products wa~ undertaken. The reaction contained 5 !11
of the PCR reaction. 4 ~1 of water and 1 ~1 of the restriction
endonuclea~se Msp 1. These were mixed and incubated for I hr at 37"C.
30 The products were then analyzed on a 1% agarose gel. The PCR
product~s contained an endogenou,s M5p 1 site that cleave.~i the fragment
somewhere in the intron and produces a 700 bp fragment. Thus, the
Msp I restriction endonuclea.se digestion of the 1~00 bp ROBR 27/2g
PCR product from ~ homo~ygous lean r~t yields two fr~gment.~ of l l00
CA 02246226 1998-08-14
W O 97131015 PCTAUS97/02397
- 16 -
bp and the endogenous 700 bp fragment. In contrast~ Msp I digestion of
PCR products from a fa/fa ROBR 27/2X PCR amplification, which
contains the A to C mutation, introduces an additional Msp I site that
cleaves the 1100 bp band to produce a 950 bp and a small fragment of
5 130 bp. The genomic analysis of the lean Zucker and ZDF rats also
demon,strated that Fa/fa heterozygotes where present as illustrated by
Msp I restriction endonuclease digestion patterns that ,showed that the.se
rats had the 1100 bp fragments as well as the 950 mutant fragment.
