From the Institute of Basic Medical Sciences,
¶ Laboratory Animal Research Center, University of Tsukuba,
Tsukuba, Ibaraki, 305-8575, Japan and the
Howard Hughes Medical
Institute and Department of Molecular Genetics, University of Texas
Southwestern Medical Center at Dallas, Dallas, Texas 75235-9050
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ABSTRACT |
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Orexin-A and -B are recently identified potent
orexigenic peptides that are derived from the same precursor peptide
and are highly specifically localized in neurons located in the lateral hypothalamic area, a region classically implicated in feeding behavior.
We cloned the whole length of the human prepro-orexin gene and
corresponding cDNA. The human prepro-orexin mRNA was predicted
to encode a 131-residue precursor peptide (prepro-orexin). The human
prepro-orexin gene consists of two exons and one intron distributed
over 1432 base pairs. The 143-base pair first exon includes the
5'-untranslated region and a small part of the coding region that
encodes the first seven residues of the secretory signal sequence. The
second exon contains the remaining portion of the open reading frame
and 3'-untranslated region. The 3.2 kilobase pairs of the 5'-upstream
region from a cloned human prepro-orexin gene promoter is sufficient to
direct the expression of the Escherichia coli
Orexins (orexin-A and -B) are neuropeptides that were identified
as endogenous ligands for an orphan G-protein-coupled receptor, which
was originally found as an expressed sequence tag from human brain (1).
Orexin-A and -B are derived from the same precursor peptide
(prepro-orexin) by proteolytic processing. They bind and activate two
closely related G-protein-coupled receptors, termed OX1 and
OX2 receptors. OX1 receptor is selective for
orexin-A, whereas OX2 receptor is a nonselective receptor
for both orexin-A and -B. Prepro-orexin mRNA and immunoreactive
orexin-A are highly specifically localized in neurons within and around
the lateral hypothalamic area
(LHA)1 in the adult rat
brain, a region implicated in feeding behavior (2-4).
Orexin-containing neurons diffusely innervate the entire brain,
including monosynaptic projections, to various regions of the cerebral
cortex, limbic system, and brain stem (5, 6). Orexins stimulate food
consumption when administered intracerebroventricularly (1). Orexin
gene expression is up-regulated upon fasting, suggesting the existence
of molecular mechanisms that control orexin gene expression according
to the nutritional status of the animal (1).
Expression of the orexin gene is highly restricted to neurons located
in the LHA, indicating the existence of molecular mechanisms by which
orexin gene transcription is highly specifically performed by the
distinct population of neurons in these areas (1, 5, 7). Radiation
hybrid mapping showed that the human prepro-orexin gene is located at
human chromosome 17q21 (1). We have already reported that human
prepro-orexin mRNA can also be exclusively detected in the
hypothalamus/subthalamic regions (1). The mechanisms by which orexin
gene expression is highly restricted to the distinct populations of
neurons in these regions is of interest.
As the first step toward unveiling these mechanisms, we cloned
fragments of the human prepro-orexin gene and its corresponding cDNA to determine their complete primary structures. One way to study the physiological roles of the orexin neuronal system would be to
examine the consequences of expression of exogenous genes in
orexin-producing neurons of transgenic mice, thereby manipulating the
cellular environment in vivo. However, such studies require the use of an appropriate gene promoter to direct gene expression to
orexin-producing neurons. Human prepro-orexin gene promoter is indeed a
good candidate for targeting gene expression to orexin-producing neurons.
From these points of view, we made a prepro-orexin-lacZ
fusion gene and tested it in transgenic mice to identify a DNA fragment containing all the necessary elements for appropriate orexin
expression. This approach would also be useful to examine the mechanism
by which orexin gene expression is highly restricted to the LHA and adjacent regions.
Cloning of Human Prepro-orexin Gene--
Because we found that
the full-length rat orexin cDNA, which contains CTG triplet repeats
(encoding the oligo-leucine stretch in the signal sequence), tends to
cross-hybridize with a number of unrelated genes, we used a 0.29-kb
segment of rat cDNA encoding Gln33-Ser128
of prepro-orexin as a probe (1). Approximately one million clones from
a human genomic library (CLONTECH) were screened by plaque hybridization with this cDNA probe. We isolated several clones, and one of the longest clones, Genomic Southern Blot Analysis--
A membrane that contains
human genomic DNA digested with designated restriction enzymes was
purchased from CLONTECH (Human GENO-BLOT; catalog
number 7700-1). The 544-bp PstI fragment of human
prepro-orexin gene was labeled by the random priming method with
[ Primer Extension Analysis--
An infrared dye (IRD41)-labeled
primer 5'-GTAGCCGGGAAAGGAGATGTCTGTGGTGG-3', which is complementary to
position 72 to 100 of human prepro-orexin gene, was hybridized to 1 µg of human whole brain poly(A+) RNA (purchased from
CLONTECH) in a solution containing 80% formamide, 400 mM NaCl, 10 mM EDTA, 40 mM
PIPES (pH 6.4) at 30 °C. The hybridized RNA/primer was precipitated
with ethanol and then subjected to reverse transcription with avian
myeloblastosis virus reverse transcriptase. The same primer was also
used for dideoxy sequencing reaction with Thermo Sequenase (Amersham
Pharmacia Biotech) using Cloning of Human Prepro-orexin cDNA--
Human cDNA
encoding prepro-orexin was cloned by the 3'-rapid amplification of
cDNA ends method using human hypothalamus Marathon-ready cDNA
(CLONTECH; catalog number 7429-1) as a template
following the manufacturer's instruction. We made an oligonucleotide,
5'-ACATCTCCTTTCCCGGCTACCCCAC-3', which corresponds to nucleotides
81-105 of human prepro-orexin gene (5'-untranslated region of human
prepro-orexin mRNA) and used it as a specific primer. We performed
the polymerase chain reaction using this primer and adaptor primer
(AP-1 primer; CLONTECH) under the following
conditions: 30 cycles at 94 °C for 1 min, 60 °C for 1 min, and
70 °C for 1 min. A discrete 0.6-kb product containing the correct
cDNA sequence was obtained and subcloned into pCR2.1 vector
(Invitrogen). The cDNA sequence was determined by sequencing the
products obtained from three independent 3'-rapid amplification of
cDNA ends reactions with a DNA sequencer (LI-COR IR4200) using T7
primer and M13 reverse primer.
Transgenic Mice with Human Prepro-orexin-nlacZ Fusion
Gene--
We performed polymerase chain reaction reaction using cloned
human prepro-orexin gene as a template with two oligonucleotide primers, 5'-GCAGCGGCCATTCCTTGG-3' and
5'-AAGTCGACGGTGTCTGGCGCTCAGGGTG-3'. The product corresponded to
position
The resulting gene fragment free of vector sequence was isolated and
injected into fertilized mouse eggs to generate transgenic founder
animals. The presence and copy numbers of the transgene was identified
by tail blot (9). Animals from these lines were examined by
LacZ Histochemical and Immunohistochemical Staining--
The
mice were perfused via the heart with phosphate-buffered saline
followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). Tissue fragments were further fixed for 60 min at 4 °C in
the same fixative buffer. They were then rinsed three times with a
solution containing 0.1 M phosphate buffer (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, and
0.02% Nonidet P-40. The staining reaction was then performed by
incubating the tissue fragments for 16-24 h at 37 °C in a solution
containing 0.1 M phosphate buffer (pH 7.5), 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02%
Nonidet P-40, 1 mg/ml X-gal, 5 mM
K3Fe(CN)6, and 5 mM
K4Fe(CN)6. Post fixation was performed for 24-48 h in 10%
formalin. Tissues were dehydrated and embedded in paraffin for sectioning.
For immunohistochemical staining, coronal sections of brain (40µm)
were incubated for 35 min in 0.6% hydrogen peroxide to eliminate
endogenous peroxide activity. Sections were rinsed in phosphate buffer
and incubated for 30 min in Tris-buffered saline containing 3% normal
goat serum and 0.25% Triton X-100. Thereafter, sections were incubated
with rabbit polyclonal anti-orexin antibody (1) or
anti-melanin-concentrating hormone (MCH) antibody diluted 1/2000 in
Tris-buffered saline containing 1% normal goat serum and 0.25% Triton
X-100 overnight at 4 °C. The primary antibody was localized with the
avidin-biotin system (Vector Laboratories). Bound peroxidase was
visualized by incubating sections with 0.01 M imidazole
acetate buffer containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.005% hydrogen peroxide.
Structure and Sequence of Human Prepro-orexin Gene and Its
Transcript--
By screening a human genomic library (constructed in
To locate the transcription initiation sites, we performed primer
extension analysis using poly(A+) RNA from whole human
brain or control yeast tRNA. Primer extension analysis with a 29-mer
oligonucleotide primer complementary to the 5'-untranslated region
(positions 23 to 51 upstream from the putative translation initiation
site) revealed that the main transcription initiation site is at
nucleotide 1 in Fig. 2. Although there is a TATAAA sequence in position 5-10, this result suggests that this
TATA box-like sequence is nonfunctional. However, there is a minor
signal at positions 33-35 (27-29 bases down stream of TATAAA
sequence), implying the possible existence of an additional transcription initiation site. Human prepro-orexin gene lacks a typical
CCAAT box. A potential site for the promoter region was predicted to
start at position
The restriction enzyme map and the structural organization of the human
prepro-orexin gene are schematically shown in Fig. 1. The complete
nucleotide sequence of the structural gene and the 5'- and 3'-flanking
regions determined from the results of primer extension analysis and
the cDNA sequence are shown in Fig. 2. The pghLig72Bam insert
contains a 3149-bp 5'-flanking region, a 1432-bp structural gene, and a
364-bp 3'-flanking region. The structural gene consists of two exons
(143 and 473 bp) and one intron (816 bp). Southern blots of human
genomic DNA were probed with the 544-bp PstI fragment of
The intron 1 starts and ends with the consensus sequence for the 5'-end
(KAG/GTRA) and 3'-end (YnNYAG/G, n = 10~12) of
U2-type GT-AG introns (10), respectively, and has a putative branch site (CTRAY) at nucleotides 549-553 (CTGAT) (Fig. 2).
A BLAST (blastn) search of the GenBankTM data bases with the sequence
presented in Fig. 3 failed to find a significantly similar sequence,
except for several highly repetitive elements of the primate
Alu family (nucleotides
Comparison of the gene and cDNA sequences, together with the
results form the primer extension analysis (Fig.
4), suggest that human prepro-orexin
mRNA, which is 616 nucleotides long excluding the
poly(A+) tail, is encoded by two exons distributed over
1432 base pairs of the human genome (Figs. 1 and 2). The 5'-most ATG
codon of the cDNA (nucleotides 123-125) was preceded by an
in-frame stop codon (TGA; nucleotides 108-110), and the sequence
around this initiation codon conformed well to Kozak's rules (11). The
open reading frame starting with this ATG encodes a 131-residue
polypeptide, human prepro-orexin (Fig. 2). The 5'-untranslated region
and the first 7 residues of the secretory signal sequence correspond to the 143-bp first exon. The 473-bp second exon contains the remaining portion of the open reading frame and the 102-bp 3'-untranslated region. Thus, the remaining portion of the signal sequence (residues 8-33) and pro-orexin are encoded in exon 2 (Fig. 2).
Structure and Sequence of Human Prepro-orexin--
The first 33 amino acids of human prepro-orexin exhibited characteristics of a
secretory signal sequence: a hydrophobic core followed by residues with
small polar side chains (12). The SignalP Server web
site4 predicted that
Ala33-Gln34 was the most likely site for
signal sequence cleavage. The orexin-A sequence starts with
Gln34, which is presumably cyclized enzymatically into the
N-terminal pyroglutamyl residue by transamidation (13, 14). Thus, the mature peptide directly follows the signal peptide cleavage site. The
last residue of the mature peptide is followed by Gly67,
which presumably serves as an NH2 donor for C-terminal
amidation by the sequential actions of peptidylglycine monooxygenase
and peptidylamidoglycolate lyase (15, 16). As expected,
Gly67 is followed by a pair of basic amino acid residues,
Lys68-Arg69, which constitutes a recognition
site for prohormone convertases (17). The last residue of the sequence
of orexin-B, Met96 residue, is again followed by
Gly-Arg-Arg. These observations suggest that human orexin-A and -B are
also C-terminally amidated like their counterparts in the rodent.
The predicted human orexin-A sequence was identical to rodent/bovine
orexin-A. Human orexin-B had two amino acid substitutions compared with
the rodent sequence. Overall, the human prepro-orexin sequences were
83% identical to the rat counterpart (1). The majority of amino acid
substitutions were found in the C-terminal part of the precursor, which
appears unlikely to encode for another bioactive peptide.
Expression of
The lacZ staining showed that the transgene was highly
specifically expressed in the LHA/subthalamic region in two lines
(Table I), suggesting that this fragment contains all the necessary elements for appropriate expression in these regions (Fig.
5). We observed that the neurons
expressing the transgene were only a subset of the neurons expected to
stain. Generally, only 30-50% of neurons containing immunoreactive
orexin were stained by
Line D5 showed ectopic expression of lacZ in several regions
that do not express orexins, including the arcuate nucleus,
periventricular nucleus, and preoptic nuclei (Table I). This ectopic
expression was only observed in line D5 and might be because of a
positional effect. We could not observe any ectopic expression of
transgene other than the eutropic expression in the LHA in line A3 and
line J2 (Table I).
We also examined whether MCH-containing neurons, which are also known
to be exclusively located in the LHA and adjacent regions, express the
transgene or not. As shown in Fig. 5, MCH-positive neurons did not show
lacZ activity, suggesting that this promoter does not direct
expression to the MCH neurons. We could not observe any positive
lacZ staining in tissues outside the brain.
Recent studies have identified several neuropeptide and receptor
systems in the hypothalamus that are critical in the regulation of body
weight (18). The LHA has long been considered essential in regulating
food intake and body weight, because cell specific lesions of this
region can result in decreased food intake and body weight (2), and
this region contains glucose-sensing neurons (4).
We recently identified a family of neuropeptides, orexins, which are
localized exclusively in neurons in the LHA. Orexins increase food
intake when administered intracerebroventricularly. The neurons
containing orexins (orexin neurons) diffusely innervate the entire
brain, including monosynaptic projections to the cerebral cortex,
limbic system, and brain stem (5-7). Therefore, orexin neurons may be
ideally positioned to regulate cognitive, motivational, emotional, and
autonomic aspects of food intake and body weight regulation.
Orexin neurons are shown to be highly specifically localized within and
around the LHA in rodents and humans (1, 5-7). These observations
suggest the existence of molecular mechanisms by which orexin
expression is highly restricted to distinct populations of neurons in
these regions. To examine these mechanisms, we first determined the
structure of the human prepro-orexin gene and its transcript.
The human prepro-orexin mRNA was predicted to encode a 131-residue
precursor peptide. The human prepro-orexin gene consists of two exons
and one intron distributed over 1432 bp (Fig. 1, 2). The 143-bp exon 1 includes the 5'-untranslated region and the coding region that encodes
the first 7 residues of the secretory signal sequence. Exon 2 contains
the remaining portion of the open reading frame and the 3'-untranslated
region. The predicted human orexin-A sequence was identical to
rodent/bovine/porcine orexin-A. Human orexin-B had two amino acid
substitutions compared with the rodent sequence, and one amino acid
substitution as compared with the porcine sequence (Fig. 2).
Considering the difference in species, the structures of both orexins
are strikingly conserved. This suggests the important physiological
roles of orexins.
To evaluate the function of the cloned human prepro-orexin gene
fragment, we generated transgenic mice using human orexin gene-nlacZ fusion gene. This construct utilizes the human
prepro-orexin gene fragment, which contains the 3,149-bp 5'-flanking
region and 122-bp 5'-noncoding region of exon 1 to drive the
nlacZ gene as a reporter protein (Fig. 1). We found that
this gene fragment is sufficient to direct the expression of human
prepro-orexin mRNA in the LHA and adjacent regions (Table I, Fig.
5). This finding suggests that this genomic fragment contains all the
necessary elements for appropriate expression of the gene. However, we
observed that the neurons expressing the transgene were only a subset
of the orexin-containing neurons (Fig. 5). Generally, only 30-50% of
neurons containing immunoreactive orexins were stained by
On the other hand, neurons containing MCH, which are also known to be
localized exclusively in the LHA, did not express lacZ in
the transgenic mice (Fig. 5). This observation suggests that this
promoter specifically directs expression to orexin neurons, and orexin
neurons and MCH neurons are distinct neuronal populations.
Transgenic line D5 showed ectopic expression in several regions, which
may be because of a positional effect (Table I). Alternatively, there
may be other necessary elements to ensure the proper gene expression in
other genomic regions. In this case, ectopic expression would be
eliminated by the inclusion of additional DNA for the endogenous gene,
leading to valuable insights into the regulatory mechanism of the
endogenous gene.
In any case, the 3.2-kb human prepro-orexin promoter we used in this
study was sufficient to direct expression of the exogenous gene in
orexin-producing neurons in transgenic mice. Therefore, this promoter
might be useful to examine the consequences of the expression of
exogenous molecules in orexin neurons of transgenic mice, thereby
manipulating the cellular environment in vivo. This genomic
fragment will also be useful for the targeted ablation of orexin
neurons by using it as a promoter that drives toxin expression
(20).
Prepro-orexin mRNA was shown to be up-regulated under fasting
conditions, indicating that these neurons somehow sense the nutritional
status of the animal. We have found that orexin gene expression is
influenced by plasma glucose and leptin
levels.5 Human prepro-orexin
nlacZ transgenic mice will also be useful to examine the
molecular mechanisms by which orexin gene expression is regulated.
-galactosidase (lacZ) gene in transgenic mice to neurons in the lateral hypothalamic area and adjacent regions. The
lacZ-positive neurons were positively stained with
anti-orexin antibody but not with anti-melanin-concentrating hormone
antibody. These findings suggest that this genomic fragment contains
all the necessary elements for appropriate expression of the gene and
will be useful for the targeted expression of the exogenous gene in
orexin-containing neurons. These mice might also be useful for
examining the molecular mechanisms by which orexin gene expression is regulated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ghLig72-7, was analyzed further. We subcloned the 4.9-kb BamHI fragment of
ghLig72-7 insert into the pBluescript SKII(+) vector
(Stratagene), which we termed pghLig72Bam, and subjected it to
further characterization by standard procedures. DNA sequencing was
performed with Thermo Sequenase (Amersham Pharmacia Biotech) and
analyzed with LI-COR 4000L or IR4200 DNA sequencer following the
manufacturer's instructions.
32P]dCTP to a specific activity of 5 × 108 cpm/mg and used as a probe. The membrane was incubated
at 65 °C in a solution containing 1 M NaCl, 1% SDS, 150 mg/ml salmon sperm DNA, and 5 ng/ml probe. The membrane was finally
washed in a solution containing 0.1× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate) and 0.1%
SDS at 50 °C and subjected to autoradiography at
80 °C for
48 h.
ghLig72-7 DNA as a template. The samples
were analyzed by 4000L DNA sequencer (LI-COR).
118 to 122 of human prepro-orexin gene shown in Fig. 2 and
had an artificial SalI site at the 3'-end. We digested this
fragment with PstI and SalI and ligated it to the
1.6-kb PstI fragment of the cloned prepro-orexin gene (from
1.7 kb to 72 bp shown in Fig. 2). This DNA fragment was then ligated
to BamHI-PstI (
3.15 -
1.7 kb) fragment of the
gene. The resulting DNA fragment, which has a 3.15-kb 5'-flanking
region and the whole length of the 5'-noncoding region, was used as the promoter that directs expression of the cloned Escherichia
coli lacZ gene with an inserted SV40 antigen nuclear
localization signal (nlacZ), derived from pnlacF (8). The
3'-end of nlacZ was ligated to murine protamine-1 (mPrm1)
gene fragment (from +95 relative to the transcription start site to
+625), which includes part of exon 1 and all of intron 1 and exon 2, including the poly(A+) adenylation signal and site.
-galactosidase histochemical technique to assess transgene
expression in the tissues.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EMBL3 SP6/T7 vector) with rat prepro-orexin cDNA probe, we
cloned a recombinant
phage clone
ghLig72-7, which has an
approximately 18-kb insert that contains the whole length of human
prepro-orexin gene. We subcloned the 4.9-kb BamHI fragment
of
ghLig72-7 insert into the pBluescript SKII(+) vector
(Stratagene), which we termed pghLig72Bam, and subjected it to further
analyses. Structural organization of the human prepro-orexin gene and
mRNA are shown in Fig. 1. We also
cloned a cDNA for human prepro-orexin by 3'-rapid amplification of
cDNA ends to determine the sequence of prepro-orexin mRNA.
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Fig. 1.
A, structural organization of human
prepro-orexin gene and mRNA. Exons are indicated by filled
boxes. The structural organization of the mRNA is presented
above the scheme of the gene structure. The translated region is shown
by a box. The coding sequences for mature orexin-A and
orexin-B are indicated by a filled box and a hatched
box, respectively. The putative cleavage site for the secretory
signal sequence is indicated by an arrow. B,
diagram of orexin-nlacZ transgene. mPrm1, murine protamine 1 fragment that contains part of exon 1, all of intron 1, and exon 2, including the polyadenylation signal and site.
291 by BCM Gene Finder web site.2 This program also
ignores the TATAAA sequence in position 5-10.
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Fig. 2.
Complete nucleotide sequence of human
prepro-orexin gene cloned in phLig72Bam. The exon sequence is
shown in capital letters, whereas introns and flanking
sequences are shown in lowercase letters. Nucleotide
residues are numbered positively, starting at the transcription
initiation site determined by the primer extension analysis shown in
Fig. 4 and negatively in the 5'-flanking sequence. Alu
repeat elements are indicated by dashed underlines. The
deduced amino acid sequence for prepro-orexin is shown under the
nucleotide sequence of the translated region. Amino acid residues are
numbered beginning with the initiative methionine residue. The
sequences of mature orexin-A and orexin-B are underlined.
The consensus sequence for the branch site of the intron is
double-underlined. The poly(A+) signal (AATAAA)
is indicated by thin underline. The nucleotide sequence has
been submitted to GenBankTM (accession number AF118885).
ghLig72-7, which contains a part of intron 1 and exon 2. Single
EcoRI (>23 kb), HindIII (7.0 kb),
BamHI (4.9 kb), and PstI (0.55 kb) fragments of
the genomic DNA hybridized with the probe, indicating that the cloned
DNA is an authentic copy of the genomic DNA (Fig.
3).
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Fig. 3.
Southern blot analysis of human DNA with
cloned human prepro-orexin gene fragment as a probe. Each
lane contains 5 µg of digested human genomic DNA. The
membrane was hybridized with a 544-bp PstI fragment of
ghLig72-7 insert. The positions of HindIII fragments of
l-DNA are shown as molecular weight markers.
2414 to
2143,
1916 to
1640,
1627 to
1343,
1292 to-1004, and
885 to
613) (searched with CENSOR Web Server3). These
regions show 64-85% nucleotide identity with the consensus sequence
of human Alu repeat.
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Fig. 4.
Mapping of sites of transcription initiation
by primer extension method. An infrared dye (IRD41)-labeled primer
(5'-GTAGCCGGGAAAGGAGATGTCTGTGGTGG-3') was hybridized with 1 µg of
human brain poly(A+) RNA or 30 µg of yeast tRNA. The
hybridized primer/RNA was extended with reverse transcriptase. The same
primer was used for DNA sequencing with Thermo Sequenase using
ghLig72-7 as a template. These samples were analyzed simultaneously
by a DNA sequencer (LI-COR). Asterisks show the
transcription start site.
-Galactosidase Gene in Transgenic Mice--
A
fragment of human prepro-orexin gene, which contains a 3.15-kb
5'-flanking region and the whole length of the 5'-noncoding region of
exon 1, was fused to the modified E. coli lacZ
gene, which has an SV40 T antigen nuclear localization signal
(nlacZ) (8) (Fig. 1). We generated transgenic mice using
this construct as a transgene. A total of six lines bearing the
transgene were examined, and lacZ expression could be
detected in three of these (Table I).
Characterization of six hOX-nlacZ-transgenic lines
-galactosidase histochemical technique. All
of the lacZ-positive neurons in the LHA contained
immunoreactive orexins (Fig. 5).
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Fig. 5.
Expression of E. coli
-galactosidase gene in the brain of the human
orexin-nlacZ fusion gene-transgenic mice line A3
(Table I), visualized by lacZ histochemistry. The
expression of transgene is observed in orexin-containing neurons
(A and B) but not in MCH-containing neurons
(C and D) in the LHA. A, visualization
of neurons containing E. coli
-galactosidase directed by
cloned human orexin gene promoter in the brain of transgenic mice. The
coronal section of brain stained with lacZ histochemistry
showing the bilateral and symmetrical distribution of labeled neurons
within and around the LHA (stained blue). No detectable
signal other than background was generated in transgene negative
controls (not shown). The section was then stained with anti-orexin
antibody (golden brown). B, higher magnification
of section shown in A. Note that all
lacZ-positive neurons (stained blue) contain
immunoreactive orexins (stained brown). C, the
lacZ-stained section was doubly stained with anti-MCH
antibody. D, higher magnification of section shown in
C. Note that MCH neurons do not overlap with
lacZ-positive neurons.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase histochemistry. This phenomenon has also been
described for hsp68-lacZ and dopamine
hydroxylase-lacZ transgene and is referred to as incomplete
penetrance (8, 19).
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. N. A. Tritos and T. Maratos-Flier for providing us anti-MCH antiserum. We thank Dr. W. Gray for reading our manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from the University of Tsukuba Project Research and by the Ministry of Education, Science, and Culture of Japan.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF118885.
§ To whom correspondence should be addressed: Tel.: 81-298-53-3276; Fax: 81-298-53-3039; E-mail: stakeshi{at}md.tsukuba.ac.jp.
2 http://defrag.bcm.tmc.edu:9503/genefinder/gf.html.
3 http://charon.lpi.org/~server/censor.html.
4 http://www.cbs.dtu.dk/services/SignalP/.
5 T. Sakurai, T. Moriguchi, and Yamanaka, A., unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
LHA, lateral
hypothalamic area;
MCH, melanin-concentrating hormone;
kb, kilobase pair(s);
bp, base pair(s);
PIPES, 1,4-piperazinediethanesulfonic acid;
nlacZ, E. coli -galactosidase gene with a
nuclear localization signal.
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REFERENCES |
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