(Received for publication, March 25, 1996, and in revised form, September 17, 1996)
From the Laboratory of Neuropsychopharmacology, Mayo Clinic, Jacksonville, Florida 32224
In the present study, we have cloned the human
neurotensin receptor (NTR) gene, determined its structure, demonstrated
that its promoter is functional in transfection experiments, and
identified the start site of transcription and a tetranucleotide repeat
polymorphism that locates at less than 3 kilobase pairs from the gene.
The gene contains three introns, all in the coding regions. Several differences in genomic clones and previously characterized cDNA sequences are reconciled. The 5 regulatory region, which is rich in
presumptive transcription factors, can drive luciferase expression in
transfected CHO-K1 cells. Stepwise 5
deletions identify a positive
modulator between
782 and
1309 and a negative modulator between
1309 and
1563. Southern blot analyses demonstrate a single copy
gene for the NTR. The tetranucleotide repeat polymorphism is highly
informative with at least 23 alleles and might serve as a very useful
marker for genetic study of the relationship between the NTR and
neuropsychiatric disorders.
Neurotensin (NT),1 an endogenous tridecapeptide, has a wide spectrum of biological activity in the central and peripheral nervous systems, including hypotension, hyperglycemia, hypothermia, antinociception, and regulation of intestinal motility and secretion (1). It is well established that the multiple functions of neurotensin are mediated through the neurotensin receptor (NTR), which is broadly distributed in central and peripheral tissues (2). Molecular biologists have recently cloned the rat (3) and human (4, 5) NTR. The human NTR cDNA encodes a 418-amino acid protein and rat a 424-amino acid protein. It has seven transmembrane spanning regions and a high degree of homology with other receptors that couple to G-proteins (guanine nucleotide-binding proteins). Therefore, it belongs to the large superfamily of receptors coupled to G-proteins. However, no information on the genomic structure of the NTR, the regulation of this gene, or genetic markers for this gene was available.
Growing evidence supports the hypothesis that NT and its receptor are pathophysiologically altered in some neuropsychiatric disorders such as Parkinson's disease and schizophrenia. For example, a significant decrease was observed in the concentration of immunoreactive NT in the hippocampus of Parkinson's disease patients but not other regions including substantia nigra (6). When 125I-NT was used as a radioligand to localize the NTR in postmortem brain tissues from Parkinson's patients, significant NTR decreases were found in the substantia nigra (7). Most recently, Wolf et al. (8), in an autoradiographic study, found a 40% reduction in neurotensin receptors in the entorhinal cortex of schizophrenics compared with controls. Yet the mechanisms underlying these alterations remain to be elucidated.
Our laboratory previously reported that mRNA for the rat NTR was elevated throughout the substantia nigra/ventral tegmental area after chronic treatment with haloperidol, a typical neuroleptic (9). Whether the increase in NTR mRNA is mediated via regulation by neuroleptics of the NTR gene is unknown because information about the genomic structure of the NTR gene, especially the promoter regulatory region, had not been available. Therefore, analysis of the molecular regulation of the NTR may advance our understanding of how these receptors are modified by disease and by pharmacological manipulation. To understand these regulatory mechanisms requires knowledge of the genomic structure, including the regulatory region of this gene.
Materials were obtained from the following
sources: human genomic libraries from Stratagene (La Jolla, CA);
Colony/Plaque Screen from DuPont NEN; Hybond N+ nylon
membranes and multiprime DNA labeling system from Amersham Corp.;
restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, and
luciferase assay kit from Promega (Madison, WI); pCR3lacZ plasmid from
Invitrogen (San Diego, CA); DNA sequencing kit from United States
Biochemical Corp.; GeneClean kit from Bio 101 (La Jolla, CA); G-25 spin
columns from 5 Prime 3 Prime Inc. (Boulder, CO); radionucleotides
[
-32P]dCTP, [
-32P]ATP (~3000
Ci/mmol; 1 Ci = 37 GBq) and
-35S-dATP (~1300
Ci/mmol) from DuPont NEN; Taq polymerase from Fisher; Total
RNA isolation kit from Ambion (Austin, TX); SuperscriptTM
reverse transcriptase and RNase H from Life Technologies, Inc.; HT-29
(human adenocarcinoma cell line) and CHO-K1 cells from American Type
Culture Collection (Rockville, MD); Dulbecco's modified Eagle's medium from Life Technologies, Inc.; Fetal Clone II from Hyclone Laboratories, Inc. (Logan, UT); human cerebral cortex from Brain Bank
of Mayo Clinic, Jacksonville, FL; the blood samples from subjects in a
study conducted by Dr. Neill R. Graff-Radford (Mayo Clinic,
Jacksonville, FL); and two CEPH families from Bios Laboratories (New
Haven, CT). All reagents were of molecular biology grade.
Approximately 1 × 106 colony forming
units of a human placenta genomic library in the cosmid SuperCos 1 vector (Stratagene) were screened at high stringency with the
32P-labeled full-length human NTR cDNA probe using the
previously reported methods (10). Filters were hybridized in 50%
formamide, 5 × SSC, 5 × Denhardt's, 0.1 mg/ml sheared and
denatured salmon sperm DNA, 0.5% SDS, and 50 mM sodium
phosphate, pH 7.0, and washed in 2 × SSC (1 × SSC = 0.15 M NaCl and 0.015 M sodium citrate), 0.1%
SDS at room temperature for 10 min each, then in 0.1% SSC, 0.1% SDS
at 65 °C for 20 min. Ten positive colonies were selected and
purified to homogeneity. Restriction enzyme digestion products of each
were hybridized with the NTR cDNA probe and with oligonucleotide probes from the extreme end of 5- and 3
-noncoding regions and coding
regions of the NTR cDNA. The hybridizing genomic fragments were
subcloned into pBluescript for further restriction enzyme analysis and
sequencing. DNA sequencing was carried out by the standard dideoxy
chain termination method using an automated DNA sequencer.
Sequence-specific oligonucleotides were synthesized by Ransom Hill
Bioscience, Inc. (Ramona, CA).
PCR was used to determine the
intron size with the following three sets of oligonucleotides as
primers: 1) 5-TGGACGTGAACACCGACATCTAC-3
(nucleotide 161-183 in Fig.
1A) and 5
-GGGAATATGAAGGACATGAAGGTG-3
(743 to 720); 2)
5
-CAAGCTGACCGTCATGGTACG-3
(783-803) and 5
-GAGTCCACTGCTCATCCGAG-3
(1006 to 987); 3) 5
-GTGGTCATCGCCTTTGTGGTC-3
(922-942) and
5
-TGGCAGAGACGAGGTTGTAC-3
(1108 to 1089). For intron 1, PCR was
performed according to the previous methods (11) with a slight
modification. Briefly, genomic DNA (100 ng) was amplified in a 25-µl
reaction buffer that contained 50 mM KCl, 10 mM
Tris-HCl, pH 8.3, 2 mM MgCl2, 5 pmol of
synthetic oligonucleotides (~100 ng), 200 µM of each
dNTP, 0.5 mM dithiothreitol, and 1.0 unit of AmpliTaq
polymerase. Samples were overlaid with approximately 50 µl of mineral
oil and then heated to 95 °C for 5 min. The PCR program consisted of
35 cycles as follows: 94 °C for 1 min, 55 °C for 1 min, and
72 °C for 1 min. For introns 2 and 3, PCRs were carried out using
eLONGaseTM Amplification System provided by Life
Technologies, Inc. Cosmid DNA (100 ng), which contained the genomic NTR
clone, was amplified in a 50-µl reaction buffer that contained 30 mM Tris SO4, pH 9.1, 16 mM
(NH4)2SO4, 2 mM
MgSO4, 100 ng of synthetic oligonucleotides, 200 µM dNTP mix, and 2 µl of eLONGase Enzyme Mix, which
contained a Taq/Pyrococcus species GB-D DNA
polymerase mixture. The PCR program included pre-amplification
denaturation for 1 min at 94 °C followed by 35 thermal cycles;
94 °C for 40 s, 55 °C for 40 s, and 70 °C for 8 min.
The PCR products were fractionated on 1% agarose gel and stained with
ethidium bromide. To confirm whether the PCR products were specific
ones, PCR products were transferred to Hybond-N+ membrane
with 0.4 M NaOH and then hybridized with the internal oligonucleotide probes. The intron sizes were determined by comparison of the products to the DNA markers.
A, the nucleotide and deduced amino acid sequence of the human NTR gene. The deduced amino acid sequence is shown below the nucleotide sequence. The exon sequences are shown in uppercase letters. The intron and the flanking sequences are in lowercase letters. The size of each intron is shown in parentheses. Nucleotides of the transcript are numbered sequentially from the first nucleotide of the methionine initiation codon. The different nucleotides and amino acids (at AA200), compared with the sequence of the NTR cDNA reported by Vita et al. (5), are bold. The polyadenylation signal is bold and underlined. Four GT clusters are underlined. B, the intron positions in deduced amino acid sequence of the human NT receptor. The interruption by three introns of the deduced amino acid sequence is shown by arrows.
Cell Culture
HT29 or CHO-K1 cells were cultured at 37 °C under 10% CO2 in minimum essential medium supplemented with 5% Fetal Clone II and 1% minimum Eagle's medium nonessential amino acids. HT29 cells were cultured for RNA preparation and transient expression assays and CHO-K1 cells only for transient expression assays.
RNA PreparationTotal RNA preparation from human cerebral cortex was carried out using phenol/chloroform methods as described in a previous publication (12). For preparation of total RNA from HT29 cells, an RNA isolation kit was used. Approximately 5 × 107 to 5 × 108 cells were used for RNA isolation. The purity and integrity of RNA were analyzed by measuring the absorbance ratio at A260/A280 as well as by electrophoresis on a 1% agarose gel.
Primer ExtensionPrimer extensions were carried out using
an oligonucleotide that corresponded to 283 to
309 base pairs (bp)
upstream of the first methionine codon of the NTR
(5
-TCCAGGTGCCTCCGATCTCCAAACCG-3
), under conditions described in the
instruction manual along with a kit for first strand cDNA synthesis
(Life Technologies, Inc.). Briefly, the oligonucleotides were
end-labeled with [
-32P]ATP, hybridized to 30 µg of
total RNA from the human cerebral cortex or from HT29 human
adenocarcinoma cell line, and extended using 200 units of SuperScript
II reverse transcriptase in 20 µl of reaction solution. Thirty µg
of yeast tRNA was used as a negative control. The extension reaction
was incubated at 42 °C for 60 min and then terminated at 70 °C
for 15 min followed by addition of 1 µl of RNase H to digest mRNA
at 37 °C for 30 min. The primer-extended products were separated on
an 8 M urea, 6% polyacrylamide gel and then analyzed by
autoradiography. Sequence of a single-stranded DNA from bacteriophage
M13mp18 was used as a DNA marker.
To construct nested deletions in the
promoter of the NTR gene, four DNA fragments, whose sizes were 303, 770, and 1297, and 1873 bp, were generated by PCR using sense primers
located at 315 to
296,
782 to
762,
1309 to
1289, and
1885
to
1866, and an antisense primer at
10 to
28 nucleotide of the
promoter region of the NTR gene (Fig. 2). The first three fragments
were subcloned into the pGL3 luciferase reporter vector (pLUC-Basic), yielding clone pLUC(
315), pLUC(
782), and pLUC(
1309). The last fragment (1873 bp) was cut with HindIII. A large piece of
DNA (
1563 to
10) was purified and subcloned into the pLUC-Basic, yielding a clone pLUC(
1563). The clones were then sequenced to determine correct orientation. pLUC-Basic, which lacked a promoter and
an enhancer, was used for base-line luciferase activity.
Transient Cell Transfections and Luciferase Assays
Transient transfections of CHO-K1 and HT29 cells were
performed by the calcium phosphate procedure as described by Chen and Okayama (13). Briefly, 2 µg of promoter/luciferase plasmid DNA, along
with 0.1 µg of pCR3lacZ plasmid DNA to normalize for transfection efficiency, were co-precipitated using CaPO4. Cells were
harvested 72 h after transfection in lysis buffer and assayed for
luciferase expression according to the manufacturer's instructions
(Promega, Madison, WI). -Galactosidase was assayed using a
chemiluminescent detection procedure according to the manufacturer's
instructions (Tropix, Bedford, MA), except that all reagents were
scaled down to one-half.
10 µg of genomic DNA bought
from Promega (Madison, WI) was digested overnight with the restriction
endonucleases described in the legend to Fig. 8. The digested samples
were subjected to electrophoresis on a 1% agarose gel and then
transferred to Hybond-N+ membrane using the alkaline (0.4 M NaOH) transfer protocol. The DNA samples were
prehybridized and probed using the 32P-labeled NTR
cDNA. The hybridization and wash conditions were the same as those
described in the section on "Isolation and Characterization of
Genomic Clones."
Identification of Microsatellite Repeats
To determine whether any microsatellite repeat sequences were present in the sequence of or flanking the NTR gene, the cosmids containing the NTR gene were digested with Sau3AI and screened for the presence of di-, tri-, or tetranucleotide repeats by hybridization with 32P-labeled dinucleotide repeat (AC)15 or trinucleotide repeat (GGC)10, (GGT)10, (GTT)10, (TAA)10, (GAA)10, (GAT)10, (GTA)10, (GCA)10, (GAC)10, and (GGA)10 oligonucleotide probes or tetranucleotide repeat (AGAT)5, (AAAT)5, (CTTT)5, (CCTT)5, (ATCC)5, and (ACAG)5 oligonucleotide probes. The Sau3AI restriction fragments hybridized to (CCTT)5 and (CTTT)5 oligonucleotide probes were subcloned into pBluescript vector and sequenced.
Radioactive PCR Assays for Polymorphism AnalysisThe
genomic DNAs, which were prepared from the blood samples of 105 unrelated individuals according to a previously published method (11),
were amplified by PCR using two primers flanking the microsatellite
(5-CCTCATCAGCTCAGAAGCAGATG-3
and 5
-CCTGGGTGACAAGAGCAAGAAC-3
). PCR
amplification was carried out in a 25-µl reaction buffer that contained 100 ng of genomic DNA, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 4% dimethyl
sulfoxide, 100 ng each unlabeled primer, 100 µM of each
dNTP, 1 µCi of [32P]dCTP, and 1.0 unit of AmpliTaq
polymerase. Samples were heated to 95 °C for 5 min to denature the
DNA. The PCR program consisted of 35 cycles as follows: 94 °C for 1 min, 66 °C for 40 s, and 72 °C for 40 s. The amplified
PCR products were migrated on a 6% denaturing polyacrylamide gel for
5 h at 70 watts. The gel was dried and autoradiographed overnight.
Sizes of the alleles were determined by comparison to M13mp18 DNA
sequencing ladders.
We
screened approximately 1 × 106 recombinants from a
human placenta genomic library using the 32P-labeled human
NTR cDNA probe and isolated ten positive colonies. These colonies
were digested with endonucleotide restriction enzyme EcoRI
and then the products were fractionated on 1% agarose gel, followed by
hybridization with the NTR cDNA probe and with oligonucleotide probes from the extreme end of 5 and 3
noncoding regions of the NTR
cDNA. All ten positive clones contained the NTR sequence, among
which we found that two clones, clones 1 and 8, were identical and
contained exon 1, including the 5
-flanking region and part of the
coding region. The other eight clones all contained exons 2, 3, and 4 including the remainder of the coding region, 3
-untranslated region,
and the 3
-flanking region. The hybridizing genomic fragments from
clones 1 and 5 were subcloned into pBluescript for further analysis and
sequencing.
The NTR gene spanned
more than 10 kb. Except for the intron sequence, the entire sequence of
the NTR gene, including 5- and 3
-flanking regions, coding region, and
exon-intron junctions, was determined (Fig.
1A). Comparison of the sequence of the NTR gene from the genomic clones with that of the cDNA showed that the
NTR gene consisted of four exons and three introns. All three introns
were located within the coding region (Fig. 1B). The
sequences of exon-intron boundaries and the placement of each intron
interrupting the coding sequence are shown in Fig. 1. All of the
exon-intron junction site sequences followed the consensus "AG-GT"
rule. The sizes of the introns determined by PCR were approximately
0.3, 4.1 and 1.8 kb, respectively.
Comparison of the NTR genomic sequence with the sequence of the
published NTR cDNA (5) revealed some minor differences (Fig.
1A). Three nucleotide differences were observed in the
coding region, two conservative substitutions of G for A and A for G at
the third base of codon 63 (nucleotide 189) and at the third base of
codon 92 (nucleotide 276). A non-conservative substitution of G for A
was found at the first base of codon 200 (nucleotide 598), which gave
rise to a change from Thr to Ala at residue 200. Additionally, two
nucleotide differences were found in the 5-untranslated region with
the substitution of A for G at nucleotide
309 and A for T at
472.
We
sequenced the 5-flanking region 1887 bp 5
of the first translation
codon, ATG (Fig. 2). A comparison of the 5
-flanking sequence with established consensus sequences in a transcription factors data base revealed that the 5
-flanking region lacked a typical
TATA box or CAAT box but contained several potential Sp1 binding sites.
It also revealed the presence of the putative binding sites for several
other transcription factors, which included AP2 (14), GATA motifs (15),
E47 (16), myeloid zinc finger gene 1 (17), P300 (18), histone
promoter-associated nuclear factor-A (19), histone H4 transcription
factor-2 (20), late SV40 transcription factor (21), and cAMP response
element binding protein (22). In addition, at
1765 there was an
octanucleotide sequence of CCATGTGC at
1765, which is an inverted
sequence of the MyoD-E2A binding site (23), and at
1770, 1715,
1672, and
1039 four hexanucleotide sequences of TCCCAG, which is an
inverted sequence of the "acute-phase reactant regulatory element"
(24) (Fig. 2).
To determine the transcription initiation site, we performed primer
extension experiments using a primer (282 to
309), which was
located 281 bp 5
of the first codon, ATG. The results showed that an
extended product was found at
438 with total RNA from both human
cerebral cortex and HT29 cells used as templates. No signal was
observed in the control groups, where the same amount of yeast tRNA was
used as a negative control (Fig. 3).
Analysis of the 3
There was a potential polyadenylation signal 30 bp 5 of the
polyadenylation site of the cDNA, which was followed by four GT
clusters (Fig. 1A). These two features have been reported to represent conserved areas for transcription termination and
3
-processing (25).
To determine the
promoter region that controls the human NTR gene expression, several 5
deletion mutants of the 1.887-kb promoter were generated. The deleted
constructs, fused 5
to the luciferase gene of a reporter plasmid, were
transfected into CHO-K1 cells for the transient expression analysis.
Results of the luciferase assays in CHO-K1 cells (Fig.
4) indicated that the highest promoter activity was
produced with the clone pLUC(
1309), containing a 1300-bp region (
10
to
1309) of the NTR promoter. Deletion of the promoter region 5
to
782 bp significantly decreased the promoter activity. When the
deletion reached
315 bp, the promoter activity was completely
abolished. Interestingly, the longest luciferase construct
pLUC(
1563), which contained a 1554-bp region (
10 to
1563) of the
NTR promoter, had less promoter activity than did pLUC(
1309),
indicating that silencer elements existed in the region between
1309
and
1563 (Fig. 4).
Identification of Microsatellite Repeat Sequences in the NTR Gene
To determine whether any microsatellite repeat sequences
were present in the NTR gene or in the sequences flanking the NTR gene,
we digested the cosmids containing the NTR gene with Sau3AI and screened for the presence of microsatellite repeats by
hybridization with 32P-labeled di-, tri- or tetranucleotide
repeat oligonucleotide probes. Hybridization to the (CCTT)5
and (CTTT)5 oligonucleotide probes was observed in a
fragment from clones 3 and 4. This microsatellite repeat, which was
located at less than 3 kb from the poly(A) site (data not shown), was
subcloned into pBluescript vector and then sequenced. Sequence analysis
revealed an almost exclusive CT region (~360 bp), in which there
existed two tetranucleotide microsatellite repeats. The first
microsatellite had 9 perfect "CCTT" repeats and the second had 17 perfect "CTTT" repeats (Fig. 5).
Analysis of the Tetranucleotide Repeat Polymorphism in the NTR Gene
Using PCR with two primers flanking the tetranucleotide
repeat to amplify the genomic DNA from 105 unrelated individuals, we
found that this repeat was very polymorphic (Fig. 6).
Further study demonstrated that 23 alleles were found in 210 chromosomes from 105 unrelated individuals who were 97 Caucasian, 2 Black, 4 Asian, and 2 others. Many alleles had very low frequencies, among which 11 were below 2%, 8 were between 2 and 8%, and only 4 were over 10% (range 11.90-12.86%) (Table I). The
estimated heterozygosity was 0.914, and polymorphism information
content value was 0.906. This tetranucleotide repeat polymorphism was also found to be stable and inherited in a Mendelian fashion in two
three-generation pedigrees (Fig. 7).
|
Southern Blot Hybridization Analyses of the Human NTR Gene
To determine the number of genes encoding the human NTR, we digested human genomic DNA with EcoRI, HindIII, KpnI, or XbaI restriction enzymes, whose restriction sites were not present in the sequence of the NTR cDNA, and then probed with the NTR cDNA probe. A single band was detected in KpnI and XbaI digests, and two bands were detected in EcoRI and HindIII digests (Fig. 8). Since the NTR genomic clones, digested with EcoRI and HindIII, also showed two bands (data not shown), this indicated that the restriction sites for EcoRI and HindIII were in introns. Therefore, the Southern analysis revealed that only a single gene encoded the receptor.
In the present study we have clarified the organization, genomic
structure, and promoter function of the human NTR gene. We have also
identified a microsatellite polymorphism that locates at less than 3 kb
from the poly(A) site. The present study demonstrates that the human
NTR gene contained three introns, all of which were in the coding
region (Fig. 1). The placement of these introns (Fig. 1B) is
interesting, since we have shown that the intracellular loop 3 is
involved with coupling to release of inositol phosphates (26) and that
the extracellular loop 3 is likely involved with binding of agonists
(27, 28). Among the molecularly cloned G-protein-coupled receptors,
some, such as the 2- and
-adrenergic receptors (29,
30, 31), the D1-dopamine receptor (32), muscarinic
receptors (33), the platelet-activating factor receptor (34), and IL-8
receptor A (35) have no introns in their coding regions. Others have an
intron or multiple introns in the coding region, such as
A2a adenosine receptor (10), dopamine D2,
D3, and D4 receptors (36, 37, 38, 39), the
5-HT2 receptor (40), the substance K receptor, the
substance P receptor (41), the endothelin-A receptor (42), the
neuromedin K receptor (43), opsins (44), and the luteinizing hormone
receptor (45). Therefore, the human NTR gene elucidated in the present
study belongs to the family of G-protein-coupled receptor genes that
contain intronic sequences within their coding regions.
Comparison of the NTR genomic sequence with the sequence of the NTR
cDNA from HT29 cells (5) revealed several nucleotide differences
between the two sequences. Two were found in the 5-noncoding region
and three in the coding region (Fig. 1A). Compared with the
coding region of the NTR cDNA published by our group (4), the
genomic sequence showed two nucleotide differences. Table II summarizes the differences occurring in the coding
region. Both the present results and the results from the previous work of our group (4) indicated that the third base of codon 63 (nucleotide
189) and the first base of codon 200 (nucleotide 598) were G rather
than A, compared with the sequence reported by Vita et al.
(5). By carefully resequencing the NTR cDNA clones from both the
HT29 cell line and the substantia nigra (in the case of
AA200 two more human DNA samples were sequenced), Watson
et al. (4) suggested that the base changes at
AA63 and AA200 were probably sequencing errors
in the NTR sequence reported by Vita et al. (5). This
suggestion was confirmed by the present study. Furthermore, we note
that when the first base at AA200 is G and not A, not only
is there an amino acid change but also there is a BglI
restriction site introduced.
|
In order to be certain that the first base at AA200 was G rather than A in our genomic DNA, we further examined this DNA by a combination of PCR technique and restriction enzyme analysis. A 606-bp fragment including nucleotide 598 was amplified by PCR from the genomic DNA of 15 unrelated individuals. The resulting PCR products were digested with BglI. The results demonstrated that all samples tested had a BglI cutting site, which indicates all 15 samples had the base G at nucleotide 598 (Table II).
With the nucleotide difference at the first base of codon 194 (nucleotide 580), where C or T was observed in two published cDNA
sequences, it was suggested that this is a polymorphic site (4).
Further study in our group found that this polymorphic site has no
effect on ligand binding, although it causes an amino acid change, Leu
or Phe (1). The genomic clone sequenced in this study had a C base in
this position or Leu for the amino acid. Another nucleotide difference
was found at AA92 (nucleotide 276), where the third base
was A in the genomic DNA, whereas it was G in the cDNA. However,
this was only a conservative change. Additionally, two nucleotide
differences were found in the 5-untranslated region with the
substitution of A for G at nucleotide
309 and A for T at
472.
Whether these nucleotide difference are polymorphic sites remains to be
determined.
In order to study the promoter function for the NTR gene, we sequenced
the promoter region 1515 bp 5 of the cDNA sequence thus far
available (Fig. 2). We found that the 5
-flanking region was GC-rich.
The overall GC content was greater than 68%. Eighty percent GC content
was found in the region encompassing the transcription initiation site
(±130 bp), which was located at 438 bp 5
of the methionine initiation
codon, as determined by the primer extension experiments (Fig. 3). This
phenomenon seems to fit with the observation that the promoter of a
gene is usually surrounded by GC-rich sequences.
The 5 regulatory region of the NTR gene is high in GC content and
lacks a typical TATA or CAAT box but contains multiple potential Sp1
binding sites, among which two were found to be located within 40 bp 5
of the transcription start site (Fig. 2). Functional study of the
promoter region revealed that the two Sp1 transcription factors were
very important for the activation of transcription. In addition, a
large number of binding sites for other putative transcription factors
are present in the promoter region of the NTR gene, suggesting that the
regulation of this gene involves a complex array of regulatory
factors.
We further investigated the promoter function of the human NTR gene
using a transient expression system in CHO-K1 cells to define the areas
of the 5-flanking region that play a vital role in transcriptional
regulation. Maximal luciferase activity was found in the plasmid
construct pLUC(
1309) containing the 1300-bp promoter region.
Construct pLUC(
315) bearing 306 bp of the promoter, not including the
transcription start site, had no promoter activity. The other two
constructs, pLUC(
782) and pLUC(
1563) having 773 and 1554 bp of the
promoter, respectively, were found to have less luciferase activity,
compared with the construct pLUC(
1309). This study, on the one hand,
supported the location for the transcription start site determined by
primer extension experiments. On the other hand, it suggested that the
region between
315 and
782 plays an essential role in
transcriptional initiation and activation, that the region from
782
to
1309 possesses positive regulatory elements, which enhance gene
expression, and that the region between
1390 and
1563 contains
negative regulatory elements.
The NTR gene is expressed in a tissue-specific manner. For example, results from our group demonstrated that NTR mRNA exists at high levels in substantia nigra pars compacta and the nucleus paranigralis but at background levels in the nucleus ruber, the colliculus inferior, the nucleus caudatus, the putamen, and the nucleus accumbens of both human and rat brain (46). To determine whether the portion of the promoter we cloned contains the sequence information for tissue-specific/cell type-specific function, we tried to analyze the expression of the luciferase reporter in HT29 cells that are known to express the NTR. This would enable us to compare the promoter activity in HT29 cells with that in CHO-K1 cells that do not express the NTR naturally. However, repeated attempts to detect promoter activity in HT29 cells were not successful. It may be that the HT29 cell line is not suitable for the transient transfection assays in this case.
Recently, microsatellite polymorphisms have received more and more attention in mapping defective genes for genetic diseases, since they are highly informative, abundant, and uniformly distributed genetic markers. With the microsatellite polymorphisms used as genetic markers, the locations of genes for cystic fibrosis, Duchenne muscular dystrophy, and Huntington's disease have been found.
In the present study, we demonstrated a tetranucleotide repeat
polymorphism present in the 3-flanking region of the NTR gene (less
than 3 kb from the poly(A) site). This microsatellite polymorphism has
at least 23 alleles as determined in 210 chromosomes from 105 unrelated
individuals, which we examined. Many alleles were of very low
frequencies (Table I). This polymorphism also had a high heterozygosity
(0.914) and polymorphism information content value (0.906) with a
Mendelian inheritance. Therefore, this is a highly informative
polymorphism and will be very useful as a genetic marker.
The accumulated evidence has shown that there are striking interactions between NT or the NTR and central dopaminergic systems (47), suggesting that NT and the NTR play an important role in the etiology of some neurological and psychiatric disorders. These include Parkinson's disease (46) and schizophrenia (48, 49). Especially for schizophrenia, studies have shown that genetic factors play an important role in the etiology of this disease (50), at least for some subgroups of schizophrenia. Therefore, the search for markers of vulnerability has been a recent focus in schizophrenia research.
In light of the studies suggesting an association between NT and the NTR with schizophrenia (8, 48, 49, 50), it is reasonable to hypothesize that the gene for NT or the NTR receptor might be a candidate gene for schizophrenia. Recently, the NTR gene was mapped to chromosome 20q13 (51). It is noteworthy that some deletions involving the long arm of chromosome 20 have also been reported. These deletions seem to be associated with myeloid disorders, mental retardation, and severe malformation of the limbs (52). Therefore, it would be very interesting to look at the association genetically between the NTR and schizophrenia. However, until the present study, no genetic markers were available for NT or the NTR gene. With the availability of a highly informative microsatellite polymorphism in the NTR gene as demonstrated here, the genetic study of the relationship between the NTR gene and schizophrenia or other neuropsychiatric disorders has become feasible.
AcknowledgmentWe thank Terrance Souder for his assistance in some techniques.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U69634[GenBank] and U59381[GenBank].