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From the
GTP cyclohydrolase I is the first and rate-limiting enzyme for
the biosynthesis of tetrahydrobiopterin in mammals. Previously, we
reported three species of human GTP cyclohydrolase I cDNA in a human
liver cDNA library (Togari, A., Ichinose, H., Matsumoto, S., Fujita,
K., and Nagatsu, T. (1992) Biochem. Biophys. Res. Commun. 187,
359-365). Furthermore, very recently, we found that the GTP
cyclohydrolase I gene is causative for hereditary progressive dystonia
with marked diurnal fluctuation, also known as DOPA-responsive dystonia
(Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M.,
Nomura, Y., Endo, K., Tanaka, H., Tsuji, S., Fujita, K., and Nagatsu,
T. (1994) Nature Genetics 8, 236-242). To clarify the
mechanisms that regulate transcription of the GTP cyclohydrolase I gene
and to generate multiple species of mRNA, we isolated genomic DNA
clones for the human and mouse GTP cyclohydrolase I genes. Structural
analysis of the isolated clones revealed that the GTP cyclohydrolase I
gene is encoded by a single copy gene and is composed of six exons
spanning
GTP cyclohydrolase I (EC 3.5.4.16) catalyzes the formation of
D- erythro-7,8-dihydroneopterin triphosphate from GTP.
This is the first step for the de novo biosynthesis of
(6 R)-(L- erythro-1`,2`-dihydroxypropyl)-2-amino-4-oxo-5,6,7,8-tetrahydropteridine
(tetrahydrobiopterin)
(1) . Tetrahydrobiopterin has multiple
physiological functions. It acts as an essential cofactor for three
aromatic amino-acid monooxygenases, i.e. phenylalanine,
tyrosine, and tryptophan hydroxylases
(2, 3, 4) . These enzymes are essential for
synthesizing hormones and neurotransmitters such as dopamine,
noradrenaline, adrenaline, and serotonin. It has also been shown that
tetrahydrobiopterin acts as a cofactor for the generation of nitric
oxide from arginine
(5, 6) . Besides these cofactor
roles, tetrahydrobiopterin has been suggested to be involved in the
proliferation and growth of erythroid cells
(7) .
The de
novo biosynthesis of tetrahydrobiopterin is thought to be
regulated by the activity of GTP cyclohydrolase I
(1) , and the
GTP cyclohydrolase I activity increases in response to various stimuli.
For example, the addition of interferon-
There have
been several reports on tetrahydrobiopterin-dependent
hyperphenylalaninemia caused by a deficiency in GTP cyclohydrolase I
(11, 12, 13) . Hyperphenylalaninemia caused by
GTP cyclohydrolase I deficiency shows more severe symptoms than that
caused by a defect in phenylalanine hydroxylase because a deficiency in
GTP cyclohydrolase I impairs the biosynthesis of both catecholamines
and serotonin due to the lack of tetrahydrobiopterin. Patients show
severe retardation of development, severe muscular hypotonia of the
trunk and hypertonia of the extremities, convulsions, and frequent
episodes of hyperthermia without infections
(11, 12, 13) .
Very recently, we found that
the gene for GTP cyclohydrolase I is causative for hereditary
progressive dystonia with marked diurnal fluctuation (HPD),
GTP cyclohydrolase I cDNA was first isolated from a rat liver cDNA
library
(15) . Previously, we reported the isolation of three
GTP cyclohydrolase I cDNA clones from human liver
(16) . All
three cDNAs were identical at their central and 5`-regions, but
diverged at their 3`-ends. We designated these three cDNAs as types
1-3. Type 1 cDNA, which has the longest coding region, consisting
of 250 amino acids, corresponds to rat
(15) and mouse
(17) cDNAs.
For elucidation of the mechanisms regulating the
expression of the GTP cyclohydrolase I gene and the mechanism
generating the molecular heterogeneity of its mRNA, structural
information on the gene is indispensable. Furthermore, recent advances
in murine developmental biology have made it possible to knock out a
specific gene in mice. Mice having a defect in the GTP cyclohydrolase I
gene would be useful for analyzing physiological roles for
tetrahydrobiopterin and as an animal model of hyperphenylalaninemia
caused by GTP cyclohydrolase I deficiency or HPD/DRD.
In this study,
we isolated and characterized the mouse and human GTP cyclohydrolase I
genes. Furthermore, we assigned the chromosomal location of mouse GTP
cyclohydrolase I. This is the first report on the genomic structure of
the GTP cyclohydrolase I gene in mammals.
In the primer extension experiment, mouse brain poly(A)
For RT-PCR experiments, total RNA from mouse brain was
reverse-transcribed with random hexamer and amplified using primer
m-42R as an antisense primer and primer m-189F, m-131F, or m-122F as a
sense primer. Their sequences and positions on genomic DNA are shown in
Fig. 5
. To remove a trace amount of genomic DNA contaminating the
RNA preparation, the total RNA was subjected to digestion with
RNase-free DNase I (Boehringer Mannheim) prior to the reverse
transcription reaction. Amplification for 30 cycles was carried out as
follows: denaturation at 94 °C for 30 s and annealing and extension
at 60 °C for 1 min. The amplified DNA fragments were
electrophoresed on 4% NuSieve GTG agarose (FMC Corp. BioProducts).
The chromosomes
on slides were hardened at 65 °C for 2 h, denatured at 70 °C in
70% formamide in 2
Excitation was carried out at a
wavelength of 450-490 nm (Nikon filter set B-2A), and a near
365-nm filter (UV-2A) was used for observation. Kodak Ektachrome ASA
100 films were used for microphotography.
Patient M. K., a
female born near term weighing 2.7 kg, was identified by newborn
screening as having phenylketonuria with blood phenylalanine values
>2400 µmol/liter
(22) . In the first week of life, the
baby developed feeding problems, poor sucking, and poor muscle tone. At
the age of 6 months, in spite of good control of blood phenylalanine
levels, the baby was found to be delayed in development. By the age of
2 years, the child was unable to walk and developed seizures and
choreoathetosis. Urinary pterins showed a profound deficiency in
neopterin and biopterin (). The same deficiency was found
in the plasma (data not shown). Administration of tetrahydrobiopterin,
5-hydroxytryptophan, and L-DOPA/carbidopa reduced the blood
phenylalanine level and improved the choreoathetosis. Patient M. K.
died at the age of 10 years. Diagnosis was confirmed post-mortem by the
measurement of the enzyme activity in the liver.
Wild-type and mutant cDNAs were introduced into the EcoRI
site of a pGEX-2T vector (Pharmacia Biotech Inc.), which can express a
foreign gene as the fusion protein with glutathione
S-transferase. E. coli strain BL21 cells carrying
each plasmid were cultured at 37 °C to late-log phase, and the
expression of the desired protein was induced by the addition of
isopropyl-
We examined two patients with GTP cyclohydrolase I
deficiency (patients N. R. and M. K.) and normal individuals. The
patients had missense mutations in both alleles (data not shown).
Patient N. R. had an A nucleotide instead of the G nucleotide found in
normal subjects, and this mutation resulted in an amino acid
substitution of methionine with isoleucine at position 211 (M211I).
Patient M. K. showed a transition from G to A, and the arginine residue
was substituted with a histidine residue at position 184 (R184H). Both
patients were homozygous in terms of the mutations, and no other
mutations were found in the coding region of their GTP cyclohydrolase I
gene. These substitutions affect highly conserved amino acid residues
of GTP cyclohydrolase I.
We determined the entire structures of the mouse and human
GTP cyclohydrolase I genes and the chromosomal location of mouse GTP
cyclohydrolase I. This is the first demonstration of the genomic
structure of any vertebrate GTP cyclohydrolase I enzyme.
Togari
et al. (16) reported the isolation of three types of
cDNAs for human GTP cyclohydrolase I, designated as types 1-3,
from a human liver cDNA library. Recently, Gütlich et al. (29) confirmed the presence of both type 1 and 2 cDNAs in
their human liver cDNA library. They showed that only type 1 cDNA
encodes an active enzyme and that proteins corresponding to type 2 and
3 cDNAs have no GTP cyclohydrolase I activity. The present analysis of
genomic DNA revealed that these three types of cDNAs are produced from
a single gene. An alternative usage of the splicing acceptor site in
exon 6 generates type 2 cDNA, whereas type 3 cDNA contains the fifth
intron. Although we described the presence of all three mRNAs in human
liver in a previous paper
(16) , there is a possibility that
type 3 cDNA may be derived from an immature mRNA that has introns to be
spliced out.
On the other hand, Gütlich et al. (30) observed, by RNA blot hybridization analysis, that two
species (1.4 and 3.6 kb) of GTP cyclohydrolase I mRNA exist in rat and
mouse tissues, but they detected only one species (3.6 kb) in
human-derived cells. From our analysis of genomic DNA, a full-length
human type 1 mRNA should be
When we
compared the gene structure of the mouse and human GTP cyclohydrolase I
enzymes, we considered it noteworthy that the length of the sixth exon
is quite different. The human sixth exon is 2127 base pairs in length,
but the corresponding mouse exon has only 296 base pairs. Although no
sequence information on longer species of rodent cDNA has yet been
reported, alternative polyadenylation may produce longer species of
rodent mRNA as observed in RNA blot hybridization analysis by
Gütlich et al. (30) .
It is well known that GTP
cyclohydrolase I is induced by various stimuli, such as
interferon-
Genetic defects in patients with GTP cyclohydrolase I
deficiency were characterized for the first time in more detail.
Analysis of the genomic structure of GTP cyclohydrolase I enabled us to
determine mutations in patients with GTP cyclohydrolase I deficiency or
HPD/DRD. Here we demonstrated that patients with GTP cyclohydrolase I
deficiency are homozygous for a defect in the GTP cyclohydrolase I
gene, whereas HPD/DRD patients are known to be heterozygous. In HPD/DRD
patients, we had found three missense mutations (R88W, D134V, and
G201E) and a 2-base insertion that shifts the reading frame just after
the initiating methionine
(14) . We had expressed the mutated
enzymes with the R88W or G201E mutation in the same expression system
as employed in the present study. Transfection with cDNAs prepared from
patients with GTP cyclohydrolase I deficiency as well as from HPD/DRD
patients failed to give the increase in the GTP cyclohydrolase I
activity seen in the bacteria transfected with wild-type cDNA,
indicating that the mutated enzymes have practically no catalytic
activity. These results indicate that there is no qualitative
difference between the mutations in GTP cyclohydrolase I deficiency and
HPD/DRD. Since GTP cyclohydrolase I deficiency is a recessive disease
and both alleles of the gene are mutated as demonstrated in this paper,
patients with GTP cyclohydrolase I deficiency have no detectable amount
of the enzyme activity. On the other hand, autosomal dominant
inheritance with low penetrance is shown in HPD/DRD. Patients with
HPD/DRD carry a mutant gene only in one allele, and they have little
but some GTP cyclohydrolase I activity. Therefore, the difference in
the level of the enzyme activity would explain the differing clinical
presentations of these disorders, although mutations of both disorders
reduced the activity of mutant proteins to zero. GTP cyclohydrolase I
forms dihydroneopterin triphosphate from GTP through a very complex
mechanism
(1) , and the amino acid sequence of the enzyme is
highly conserved among different species. Thus, various amino acid
substitutions would be expected to result in a loss of the enzyme
activity.
Previously, we reported the chromosomal locus for human
GTP cyclohydrolase I to be at 14q22.1-q22.2
(14) . In the
present study, we assigned mouse GTP cyclohydrolase I to the C2-3
region of chromosome 14. This region of the mouse chromosome
corresponds to human chromosome 14. This result confirmed the
localization of the human gene.
There is a mutant mouse (hph-1) that
is thought to have a defect in GTP cyclohydrolase I
(34, 35) . This mouse was generated by treatment with
ethylnitrosourea, and the selected hph-1 mutant was screened for
hyperphenylalaninemia by the Guthrie assay. But the GTP cyclohydrolase
I activity in hph-1 can be detected later in life, i.e. adult
mice
(34) , and the phenotype of hph-1 mice is far from that of
GTP cyclohydrolase I deficiency seen in man. Human GTP cyclohydrolase I
deficiency results in severe symptoms, such as severe retardation of
development, severe muscular hypotonia of the trunk and hypertonia of
the extremities, and convulsions
(11, 12, 13) .
On the other hand, hph-1 mice show normal development and no sign of
illness except for hyperphenylalaninemia. The mutated locus for hph-1
had been assigned to the C2-3 region of chromosome 14
(36) .
This suggests that the genetic defect in the hph-1 mutant mouse would
be in the structural gene or cis-acting element for GTP
cyclohydrolase I itself, but not in its regulatory gene. The reason for
the species difference in the phenotypes of hph-1 mice and GTP
cyclohydrolase I deficiency in man is not clear. The metabolism of
tetrahydrobiopterin may be different between man and mouse. It is well
known that in mammals, except primates, no neopterin can be detected in
significant amounts. This would be due to the rate-limiting activity of
GTP cyclohydrolase I. In man, the rate-limiting step may be the
conversion of dihydroneopterin triphosphate to
6-pyruvoyltetrahydropterin by the enzyme 6-pyruvoyltetrahydropterin
synthase. Molecular characterization of the defect in the hph-1 mouse
will help to explain the species difference in the phenotype caused by
a defect in GTP cyclohydrolase I.
The
nucleotide sequences reported in this paper have been submitted to the
GSDB, DDBJ, GenBank
We are grateful to Takahide Nomura, Nakao Iwata,
Machiyo Shirakura, and Minae Takeno for help in this research.
Volume 270,
Number 17,
Issue of April 28, pp. 10062-10071, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
MUTATIONS IN PATIENTS WITH GTP CYCLOHYDROLASE I DEFICIENCY (*)
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
30 kilobases. We sequenced all exon/intron boundaries of
the human and mouse genes. Structural analysis also demonstrated that
the heterogeneity of GTP cyclohydrolase I mRNA is caused by an
alternative usage of the splicing acceptor site at the sixth exon. The
transcription start site of the mouse GTP cyclohydrolase I gene and the
5`-flanking sequences of the mouse and human genes were determined. We
performed regional mapping of the mouse gene by fluorescence in
situ hybridization, and the mouse GTP cyclohydrolase I gene was
assigned to region C2-3 of mouse chromosome 14. We identified missense
mutations in patients with GTP cyclohydrolase I deficiency and
expressed mutated enzymes in Escherichia coli to confirm
alterations in the enzyme activity.
(8) ,
phytohemagglutinin
(9) , or kit ligand
(10) greatly
induces the GTP cyclohydrolase I activity. It is thus of great
importance to clarify the regulatory mechanism(s) underlying the
inducible expression of the GTP cyclohydrolase I gene.
()
also known as DOPA-responsive dystonia (DRD)
(14) .
This disease is inherited as an autosomal dominant trait with a low
penetrance. We found that HPD/DRD patients have genetic defects in the
GTP cyclohydrolase I gene only in one allele and that their GTP
cyclohydrolase I activity in mononuclear blood cells is reduced to
<20% of that of normal individuals. If only one allele carries the
mutant gene, the enzyme activity would be expected to be decreased to
about half of the control value. We assumed that the expression of GTP
cyclohydrolase I in HPD/DRD patients would be lower than in normal
individuals. To elucidate the etiology of HPD/DRD in more detail, we
undertook molecular characterization of the GTP cyclohydrolase I gene.
Screening of a Genomic Library and Analysis of Phage
Clones
A mouse genomic library, constructed in Lambda FixII
phage vectors (Stratagene), was screened by use of a mouse GTP
cyclohydrolase I cDNA clone
(17) labeled with
[
-P]dCTP. Human genomic DNA libraries,
constructed in Charon 4A (donated by Dr. Tom Maniatis, Harvard
University) and in EMBL3 (LI 018; donated by the Japanese Cancer
Research Resources Bank), were screened with a human GTP cyclohydrolase
I cDNA clone
(16) used as a probe. DNAs were isolated from the
purified phage plaques following standard protocols
(18) , and
the fragments of interest were subcloned into pBluescript KS
(Stratagene) or pUC119 vectors for further structural analysis. The
nucleotide sequences of the clones were determined by the dideoxy chain
termination method
(19) using Sequenase (United States
Biochemical Corp.).
DNA Blot Hybridization Analysis
Mouse genomic DNA
(10 µg) was digested with EcoRI, HindIII, and
SacI; separated according to size on a 0.8% agarose gel; and
capillary-blotted onto a nylon membrane (Hybond-N,
Amersham Corp.). This membrane was hybridized with a
P-labeled full-length cDNA clone of mouse GTP
cyclohydrolase I
(17) in a solution consisting of 6
SSC
(1
SSC = 0.15 M NaCl and 0.015 M
sodium citrate), 5
Denhardt's solution, 0.5% SDS, and 0.1
mg/ml salmon sperm DNA. After hybridization at 65 °C overnight, the
membrane was washed twice with 2
SSC containing 0.05% SDS at
room temperature for 5 min and twice with the same solution at 42
°C for 15 min.
Transcription Initiation Site Mapping
Total RNA
was extracted from mouse brain with guanidinium thiocyanate followed by
centrifugation in cesium chloride solutions. The transcription
initiation site was determined by both primer extension analysis and
reverse transcription-polymerase chain reaction (RT-PCR) analysis.
RNA (1.5 µg) was annealed to
P-end-labeled
primer m-42R (5`-AACAAGCGCTGCGGCTCAGCT-3`) in 0.25 M KCl by
heating at 65 °C for 1 h, followed by incubation at room
temperature for 1.5 h. The annealed primer was extended at 45 °C
for 1 h in a reaction mixture containing 50 mM Tris-HCl (pH
8.3), 75 mM KCl, 3 mM MgCl
, 10
mM dithiothreitol, 0.25 mM dNTPs, 110 units of RNase
inhibitor, and 400 units of reverse transcriptase (SuperScript II, Life
Technologies, Inc.). Then the sample was precipitated by ethanol and
analyzed on a 7 M urea, 6% acrylamide sequencing gel. The DNA
fragment containing exon 1 was sequenced with primer m-42R at the same
time.
Figure 5:
Sequence of the 5`-flanking region of the
mouse GTP cyclohydrolase I gene. The nucleotide sequence is numbered
with the first base of the ATG initiation codon designated as position
+1. The location of the transcription initiation site determined
by RT-PCR and primer extension analyses is indicated by the
asterisk. Primers used for RT-PCR and primer extension are
designated by half-arrows. An AT-rich region and a CCAAT
sequence are boxed. An H1 box and consensus sequences for
IBP-1b- and GT-2B-binding proteins are
underlined.
Chromosomal Localization of the GTP Cyclohydrolase I Gene
by Fluorescence in Situ Hybridization
The direct R-banding
fluorescence in situ hybridization method
(20) was
used for chromosomal localization of the mouse GTP cyclohydrolase I
gene. Mouse lymphocyte cultures and preparation of R-banded chromosomes
were performed as described previously
(20) .
SSC, and then dehydrated in 70/85/100%
ethanol series at 4 °C. The HindIII-digested mouse genomic
DNA fragment (3.5 kb in length) containing exon 2 was labeled by nick
translation with biotinylated 16-dUTP (Boehringer Mannheim) following
the manufacturer's protocol. The labeled DNA fragments were
ethanol-precipitated and then denatured in 100% formamide at 75 °C
for 10 min. The denatured probe was mixed with an equal volume of
hybridization solution to make final concentrations of 50% formamide, 2
SSC, 10% dextran sulfate, and 1 mg/ml bovine serum albumin
(Sigma). Twenty microliters of the mixture containing 250 ng of probe
DNA was put on the denatured chromosomes, and the slides were then
covered with Parafilm and incubated overnight at 37 °C, after which
they were washed for 20 min in 50% formamide in 2
SSC at 37
°C and in 2
SSC and 1
SSC for 20 min each at room
temperature. After the slides had been rinsed in 4
SSC, the
chromosomes were heat-incubated under a coverslip with fluoresceinated
avidin (Vector Labs, Inc.) at a 1:500 dilution in 1% bovine serum
albumin, 4
SSC for 45 min at 37 °C. The slides were then
washed with 4
SSC, 0.1% Nonidet P-40 in 4
SSC and with
4
SSC for 10 min each on a shaker. After the excess liquid had
been drained from the slides, the chromosomes were finally stained with
0.75 µg/ml propidium iodide.
Case Reports
Patient N. R. is the second child of
healthy nonconsanguineous parents. Pregnancy and delivery at term were
uneventful; breast feeding was tolerated well, and the Guthrie
phenylketonuria screening test performed at the age of 4 days was
normal. The child was diagnosed as hyperphenylalaninemic at the age of
5 months, presenting progressive neurological deterioration, including
severe hypotonia and uncoordinated and generalized movements
(Parkinson-like symptoms). Metabolic studies including analysis of
pterins in urine and cerebrospinal fluid as well as measurement of GTP
cyclohydrolase I activity in a liver biopsy led to the diagnosis of GTP
cyclohydrolase I deficiency ()
(21) . Patient N. R.
is still living and is in good health
(21) .
Mutation Analysis
Genomic DNA was extracted from
primary skin fibroblasts. We amplified exons for GTP cyclohydrolase I
including splicing junctions using PCR on genomic DNA. Primer sequences
used for amplification of exons were as follows: exon 1,
5`-GTTTAGCCGCAGACCTCGAAGCG-3` and 5`-GAGGCAACTCCGGAAACTTCCTG-3`; exon
2, 5`-GTAACGCTCGCTTATGTTGACTGTC-3` and 5`-ACCTGAGATATCAGCAATTGGCAGC-3`;
exon 3, 5`-AGATGTTTTCAAGGTAATACATTGTCG-3` and
5`-TAGATTCTCAGCAGATGAGGGCAG-3`; exon 4, 5`-GTCCTTTTTGTTTTATGAGGAAGGC-3`
and 5`-GGTGATGCACTCTTATAATCTCAGC-3`; exon 5,
5`-GTGTCAGACTCTCAAACTGAGCTC-3` and 5`-TCACTTCTAGTGCACCATTATGACG-3`; and
exon 6, 5`-ACCAAACCAGCAGCTGTCTACTCC-3` and
5`-AATGCTACTGGCAGTACGATCGG-3`. PCR amplification was performed in a
reaction volume of 25 µl containing 100 ng of genomic DNA, 10 pmol
of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl,
1.5 mM MgCl, 5% dimethyl sulfoxide, 0.2 unit of
Perfectmatch (Stratagene), and 1.25 units of Taq polymerase
(Perkin-Elmer). After having been heated at 94 °C for 3 min, the
reaction mixture was cycled according to the following program: 30
cycles for 30 s at 94 °C and for 1 min at 60 °C and a final
extension at 60 °C for 6 min. The PCR products were sequenced
directly with the same primers used for amplification by use of an
automatic DNA sequencer (Perkin-Elmer Model 373A) and a DyeDeoxy
Terminator Cycle sequencing kit.
Site-directed Mutagenesis and Expression of GTP
Cyclohydrolase I in Escherichia coli
To perform
oligonucleotide-directed in vitro mutagenesis, we followed the
manufacturer's protocol for the Sculptor in vitro mutagenesis system (Amersham Corp.). We used plasmid Bluescript KS
containing human GTP cyclohydrolase I cDNA (hGCH1)
(16) as a
template for construction of mutant GTP cyclohydrolase I cDNAs by
site-directed mutagenesis. Although the hGCH1 cDNA clone lacked the
first 25 bases corresponding to nine amino acids from the starting
methionine, the first nine amino acids in the human sequence do not
seem to be important for the activity since mouse and rat enzymes lack
these nine residues. The mutagenesis primers were
5`-GCAACACACATATGTATGGTAATG-3` for the generation of the M211I mutation
and 5`-GTTCAGGAGCACCTTACAAAAC-3` for the generation of R184H (the
mutated nucleotides are underlined). The integrity of the newly
constructed mutant cDNAs was checked by sequencing the plasmids.
-D-thiogalactopyranoside. Expressed proteins
were analyzed by SDS-10% polyacrylamide gel electrophoresis and stained
with Coomassie Brilliant Blue R-250. GTP cyclohydrolase I activities
were measured as described previously
(14) with crude
homogenate used as an enzyme source.
Miscellaneous
Proteins were determined by the
method of Bradford
(23) with bovine -globulin used as a
standard. Student's t test was used for statistical
evaluation of the mutated enzyme activity expressed in E.
coli.
Isolation and Characterization of Mouse GTP
Cyclohydrolase I Genomic Clones
Using the cloned mouse GTP
cyclohydrolase I cDNA
(17) as a probe, we isolated five
overlapping genomic DNA clones by screening a mouse genomic library
constructed in FixII (mgGCH1-5) (Fig. 1). Structural
analysis of these clones revealed that the complete mouse GTP
cyclohydrolase I gene is
32 kb in length and consists of a total
of six exons. We sequenced the exons and their splice sites. As shown
in Fig. 2, each exon/intron boundary conformed perfectly to the
GT/AG rule
(24) .
Figure 1:
Structure of the mouse GTP
cyclohydrolase I gene. The structure of the mouse GTP cyclohydrolase I
gene is depicted at the top. The six exons are indicated by closed boxes and introns by open boxes. Five
isolated clones (mgGCH1-5) covering >30 kb of the mouse GTP
cyclohydrolase I gene are depicted below the structure of the gene.
EcoRI restriction sites ( E) are
indicated.
Figure 2:
Exon/intron boundary sequences of the
mouse GTP cyclohydrolase I gene. Exon sequences are written in
upper-case letters and intron sequences in lower-case
letters. Sequences are numbered with the first base of the ATG
initiation codon designated as position +1 (nucleotides within
introns are not numbered). Sizes of both exons and introns are
indicated. AATAAA as a polyadenylation signal (24) is
underlined. b, bases.
Genomic DNA Blot Hybridization Analysis
To
determine the copy number of the GTP cyclohydrolase I gene, we
performed genomic DNA blot hybridization analysis. High molecular
weight DNA samples were digested with the restriction enzymes
EcoRI, HindIII, and SacI; separated
electrophoretically on an agarose gel; and then transferred to a nylon
membrane. The membrane was then hybridized with the mouse cDNA clone
(Fig. 3). The sizes of bands showed good coincidence with those
predicted from the restriction enzyme site mapping of the genomic DNA
clones. This result suggests that GTP cyclohydrolase I is specified by
a single copy gene.
Figure 3:
Southern hybridization analysis of mouse
genomic DNA. Ten micrograms of mouse genomic DNA was digested with
EcoRI, HindIII, or SacI. After
electrophoresis on a 0.8% agarose gel, each digest was transferred to a
nylon membrane and hybridized with P-labeled mouse GTP
cyclohydrolase I cDNA as a probe.
Determination of the Transcription Start Site
The
transcription start site was determined by primer extension analysis
and was confirmed by RT-PCR analysis as described under
``Materials and Methods.'' Primer extension analysis was
performed with a P-end-labeled oligonucleotide (m-42R)
located in exon 1 and with poly(A)
RNA from mouse
liver. As shown in Fig. 4 A, this experiment yielded a
major primer-extended product of 125 nucleotides with minor products
ranging from 123 to 126 nucleotides. The length of the fragment mapped
the initiation site around the guanine nucleotide at position
164.
Figure 4:
Determination of the transcription start
site of the mouse GTP cyclohydrolase I gene. The transcription
initiation site was determined by primer extension ( A) and
RT-PCR ( B) analyses as described under ``Materials and
Methods.'' A, primer extension analysis was performed
with antisense primer m-42R and poly(A)RNA from mouse
liver. The reverse transcription reaction was performed with (+)
or without (
) reverse transcriptase. Asterisks indicate
the positions of extension products. The locations of the extension
stop points were determined by parallel lanes containing a
dideoxynucleotide-terminated sequence. B, total RNA from mouse
liver was reverse-transcribed by use of random hexamer
oligonucleotides. The resulting cDNA ( lanes 1-3) was
subjected to PCR amplification using primer m-42R as an antisense
primer and primer m-122F ( lanes 1 and 4),
m-131F ( lanes 2 and 5), or m-189F ( lanes 3 and 6) as a sense primer. A cloned genomic DNA
containing the 5`-flanking region was also subjected to PCR
amplification using the same primer sets ( lanes 4-6) to
monitor PCR. Lane M contains an
HaeIII-digested
X174 marker.
To confirm this result, we performed RT-PCR analysis
using m-42R as an antisense primer and m-122F, m-131F, or m-189F as a
sense primer (Fig. 4 B). Total RNA from mouse liver
generated amplified products of the expected size with primers m-122F
and m-131F, whereas primer m-189F showed no band
(Fig. 4 B, lanes 1-3). These three primers
correctly amplified the plasmid DNA containing exon 1
(Fig. 4 B, lanes 4-6). These results
indicate that the 5`-end of the mRNA is present between primers m-131F
and m-189F. This agreed with the result of the primer extension
analysis. On the basis of these two results, the transcription start
site of mouse GTP cyclohydrolase I mRNA was determined to be around
guanine at position 164, ranging from cytidine at position
162 to adenine at position
165.
DNA Sequence Analysis of the 5`-Flanking Region of the
Mouse GTP Cyclohydrolase I Gene
The nucleotide sequence of the
5`-flanking region was determined to 620 base pairs upstream from
the transcription start site and is shown in Fig. 5. This
sequence has been submitted to the DDBJ data base. No obvious TATA box
was observed in the promoter region. Instead, an AT-rich putative
promoter motif (ATAAAAA) or a CCAAT box was present just upstream or 50
base pairs upstream from the transcription start site, respectively. In
addition, H1 box
(25) -, IBP-1b
(26) -, and GT-2B
(27) -binding consensus sequences were found.
Chromosomal Mapping of the Mouse GTP Cyclohydrolase I
Gene
The direct R-banding fluorescence in situ hybridization method
(20) was performed for chromosomal
localization of the mouse GTP cyclohydrolase I gene, with a
HindIII-digested genomic DNA fragment (3.5 kb in length)
containing exon 2 used as a probe. The signals were localized to the
region of C2-3 on chromosome 14 (data not shown). The standard G- and
R-banded karyotypes have been reported by Matsuda et al. (20) .
Isolation and Characterization of Human GTP
Cyclohydrolase I Genomic Clones
We previously isolated three
species of cDNA encoding GTP cyclohydrolase I from a human liver cDNA
library
(16) . All three cDNAs were identical at their central
and 5`-regions, but diverged at their 3`-ends. To clarify the
mechanism(s) that generate this heterogeneity of GTP cyclohydrolase I
mRNA, we analyzed the human GTP cyclohydrolase I gene structure. Using
a cDNA clone as a probe, we isolated three genomic DNA clones from
genomic DNA libraries constructed in Charon 4A (hgGCH6) or EMBL3
(hgGCH5 and hgGCH10) (Fig. 6). Clone hgGCH5 contained exon 1;
hgGCH10 contained exon 2; and hgGCH6 contained exons 4-6. Exon 3
was missing in these clones, although the clones encompassed >30 kb
of human chromosomal DNA. To search for exon 3, we used PCR to amplify
genomic DNA from the end of hgGCH10 to exon 3. PCR amplified a DNA
fragment with a length of 1 kb. Sequence analysis of the fragment
revealed that the correct DNA region was amplified. The length between
exons 3 and 4 was determined by PCR. The recently developed long PCR
technology enabled us to amplify the entire DNA region between exons 3
and 4, which showed a length of 9 kb.
Figure 6:
Structure of the human GTP cyclohydrolase
I gene. Isolated clones (hgGCH1-3) of the human GTP
cyclohydrolase I gene are depicted. Coding regions are indicated by
closed boxes and untranslated regions by open boxes. Introns are represented by thin horizontal lines. EcoRI restriction
sites ( E) are indicated. The region amplified by PCR is shown
by a dashed line.
Structural analysis of these
clones revealed that the human GTP cyclohydrolase I gene consists of
six exons. We sequenced exon/intron boundaries, and these sequences
have been submitted to GenBank. Break points between exons
were completely identical to those of the mouse gene (Fig. 7).
Each exon/intron boundary conformed to the GT/AG rule
(24) .
Figure 7:
Exon/intron boundary sequences of the
human GTP cyclohydrolase I gene. Exon sequences are written in
upper-case letters and intron sequences in lower-case
letters. Sequences are numbered with the first base of the ATG
initiation codon designated as position +1 (nucleotides within
introns are not numbered). Sizes of both exons and introns are
indicated. ATTAAA as a polyadenylation signal (24) is
underlined. b, bases.
A marked difference between the human and mouse GTP cyclohydrolase I
genes was observed in the last exon (exon 6). We also isolated human
cDNAs from a human pheochromocytoma cDNA library and sequenced a cDNA
of 2.4 kb in length. The cDNA clone had an open reading frame
corresponding to type 1 (a common type in human, rat, and mouse) and an
extremely long 3`-noncoding region (2 kb) with a
poly(A)
stretch. The entire 3`-noncoding region was
encoded in exon 6. The nucleotide sequence of human exon 6 is shown in
Fig. 8
and has been submitted to the DDBJ data base.
Figure 8:
Entire sequence of exon 6. Nucleotides are
numbered above the DNA sequence with the first base of the ATG
initiation codon designated as position +1. ATTAAA as a
polyadenylation signal (24) is
underlined.
Analysis
of the genomic DNA structure revealed that three cDNAs diverged just
after the break point between exons 5 and 6. We found the type 2 cDNA
sequence at the middle of exon 6. The sequence at the 3`-splice site of
intron 5 was TATTTTGTAGAC, while the sequence around the splice
site for type 2 mRNA was TCATTTTCAG
GT. Thus, a splicing between
the 5`-splice site of intron 5 (AC
GTAAGTCTGC) and the 3`-splice
site of intron 5 produces type 1 mRNA, whereas a splicing between the
5`-splice site of intron 5 and the middle of exon 6 generates type 2
mRNA. On the other hand, the sequence of intron 5 completely matched
the divergent sequence of type 3 cDNA, indicating that intron 5 is not
spliced out in the transcript corresponding to type 3 cDNA. The
alternative splicing mechanism suspected to produce heterogeneity of
GTP cyclohydrolase I mRNAs is illustrated in Fig. 9.
Figure 9:
Mechanism generating human GTP
cyclohydrolase I mRNA heterogeneity. See ``Results'' for
details.
DNA Sequence Analysis of the 5`-Flanking Region of the
Human GTP Cyclohydrolase I Gene
The nucleotide sequence of the
5`-flanking region of exon 1 was determined to 600 base pairs
upstream from the first exon and was compared with that of the mouse
GTP cyclohydrolase I gene (Fig. 10). Both human and mouse
5`-flanking sequences have been submitted to DDBJ. Several highly
conserved regions are boxed in Fig. 10. The AT-rich
putative promoter motif and the CCAAT box found in the mouse gene were
conserved in the human GTP cyclohydrolase I gene. A consensus sequence
for the binding of GT-2B
(27) was found in the human gene as
well as in the mouse gene. The IBP-1b and H1 box consensus sequences
found in the mouse gene were not present in the human gene, whereas a
leader-binding protein-1
(28) -binding sequence motif
((A/T)CTGG, positions
479 to
475), a T-antigen-binding
motif (positions
277 to
272), a TGGCA box (positions
675 to
671), and an SP-1 consensus sequence (positions
245 to
236) were present in the human gene.
Figure 10:
Comparison of the 5`-flanking regions of
the human and mouse GTP cyclohydrolase I genes. Identical nucleotides
are shaded. Gaps (indicated by dashes) were
introduced to obtain maximum homology. Highly conserved regions are
boxed. The transcription start site in mouse GTP
cyclohydrolase I is indicated by the asterisk. AT-rich and
CAAT motifs and a binding consensus sequence for GT-2B (27) are
underlined.
Mutations in the GTP Cyclohydrolase I Gene in Patients
with GTP Cyclohydrolase I Deficiency
To search for mutations in
the coding region of the GTP cyclohydrolase I gene, we amplified exons
including splicing junctions from genomic DNA from the patients by PCR.
Amplified DNA fragments were directly sequenced with an automated DNA
sequencer.
Expression of Mutated Enzymes in E. coli
To prove
that the amino acid changes found in the patients result in functional
alterations, we expressed the wild-type and mutant proteins and
analyzed their GTP cyclohydrolase I activity. Expression in E. coli was carried out with the glutathione S-transferase gene
fusion system. This system expresses the introduced gene as a fusion
protein with glutathione S-transferase (molecular mass of
glutathione S-transferase is 26,000 Da). Since human GTP
cyclohydrolase I, composed of 250 amino acids, has a calculated
molecular mass of 27,903 Da, the fusion protein is expected to be
54,000 Da. Expression of foreign genes was examined by
SDS-polyacrylamide gel electrophoresis (Fig. 11 A).
Obvious extra bands at a molecular mass of
54,000 Da appeared in
E. coli extracts harboring the plasmid containing wild-type or
mutant GTP cyclohydrolase I genes (M211I and R184H). The amount of
expressed proteins was similar as judged by Coomassie Brilliant Blue
R-250 staining. Then we measured the GTP cyclohydrolase I activities in
the crude homogenates. E. coli extracts harboring wild-type
cDNA showed a high GTP cyclohydrolase I activity, whereas E. coli extracts harboring the expression vector without a cDNA insert
(pGEX-2T) had relatively low endogenous activity. Both the M211I and
R184H substitutions completely abolished the increase in the GTP
cyclohydrolase I activity shown with wild-type cDNA
(Fig. 11 B). These results demonstrate that the M211I and
R184H mutations are not simply polymorphisms.
Figure 11:
Overexpression of wild-type or mutant GTP
cyclohydrolase I in E. coli. A, SDS-polyacrylamide
gel electrophoresis. E. coli strain BL21 harboring the pGEX-2T
expression vector alone ( lane 1) or with wild-type
( lane 2), M211I mutant ( lane 3), or
R184H mutant ( lane 4) GTP cyclohydrolase I cDNA was
induced with isopropyl--D-thiogalactopyranoside. A crude
protein fraction was prepared, and 20 µg of total protein was
separated on each lane. The gel was stained with Coomassie Brilliant
Blue R-250. The arrowhead indicates the 54-kDa GTP
cyclohydrolase I protein fused with glutathione S-transferase
(26 kDa). B, GTP cyclohydrolase I activities in crude E.
coli BL21 extracts harboring the expression vector alone
( pGEX-2T) or with wild-type GTP cyclohydrolase I cDNA
( wild), M211I mutant cDNA, or R184H mutant cDNA. Values are
represented as means ± S.E. ***, p <
0.001.
3 kb in length. Probably the longer
species of mRNA (3.6 kb) observed by Gütlich et al. (30) in their blot hybridization analysis corresponds to type 1
mRNA because estimation of size by RNA blot hybridization using 18 S
and 28 S ribosomal RNAs is not accurate. Since type 2 mRNA is shorter
than type 1 mRNA by up to 1089 bases, type 2 mRNA should be
distinguishable from type 1 mRNA by RNA blot hybridization. Thus, the
result of RNA blot hybridization analysis suggests that the amount of
type 2 mRNA is very low compared with that of type 1 mRNA.
(8, 31) , bacterial lipopolysaccharide
(32) , and kit ligand
(10) . Here we found a sequence
similar to the IBP-1-binding site in the upstream region of the mouse
GTP cyclohydrolase I gene. IBP-1 is a DNA-binding factor induced by
treatment of HeLa cells with interferon-
and is thought to be
involved in the response to interferon-
(26) . Recently,
extensive studies were performed on the mechanism of transcriptional
regulation by interferon, and these defined the DNA sequence required
for activation of transcription by interferon-
, the
-activated site
(33) . So far, we have found no typical
-activated site-like sequence in the 5`-flanking region of the
mouse or human GTP cyclohydrolase I gene. The molecular mechanism for
transcriptional regulation of GTP cyclohydrolase I remains to be
clarified.
Table: Biochemical data on two GTP cyclohydrolase
I-deficient patients
/EMBL, and NCBI nucleotide sequence
data bases with accession numbers D38601-D38603 (sequences of the
mouse and human 5`-flanking regions and the sequence of the human sixth
exon) and U19256-U15259 (sequences of human exons including
introns).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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