From the Rheumatology Section, Department of Medicine, The University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Phosphoribosylpyrophosphate (PRPP) synthetase
(PRS) superactivity is an X-linked disorder characterized by gout with
overproduction of purine nucleotides and uric acid. Study of the two
X-linked PRS isoforms (PRS1 and PRS2) in cells from certain affected
individuals has shown selectively increased concentrations of
structurally normal PRS1 transcript and isoform, suggesting that this
form of the disorder involves pretranslational dysregulation of
PRPS1 expression and might be more appropriately termed
overactivity of normal PRS. We applied Southern and Northern blot
analyses and slot blotting of nuclear runoffs to delineate the process underlying aberrant PRPS1 expression in fibroblasts and
lymphoblasts from patients with overactivity of normal PRS. Neither
PRPS1 amplification nor altered stability or processing of
PRS1 mRNA was identified, but PRPS1 transcription was
increased relative to GAPDH (3- to 4-fold normal in
fibroblasts; 1.9- to 2.4-fold in lymphoblasts) and PRPS2.
Nearly coordinate relative increases in each process mediating transfer
of genetic information from PRPS1 transcription to maximal
PRS1 isoform expression in patient fibroblasts further supported the
idea that accelerated PRPS1 transcription is the major
aberration leading to PRS1 overexpression. In addition, modulated
relative increases in PRS activities at suboptimal Pi concentration and in rates of PRPP and purine nucleotide synthesis in
intact patient fibroblasts indicate that despite an intact allosteric
mechanism of regulation of PRS activity, PRPS1
transcription is a major determinant of PRPP and purine synthesis. The
genetic basis of disordered PRPS1 transcription remains
unresolved; normal- and patient-derived PRPS1s share
nucleotide sequence identity at least 850 base pairs 5' to the
consensus transcription initiation site.
Phosphoribosylpyrophosphate
(PRPP)1 is a substrate in the
synthesis of virtually all nucleotides (1) as well as an important regulator of rates of the de novo pathways of purine and
pyrimidine nucleotide synthesis (2-4). PRPP synthesis from MgATP and
ribose-5-phosphate is catalyzed in mammalian cells by a family of PRPP
synthetase (PRS; EC2.7.6.1) isoforms in
reactions requiring Mg2+ and Pi as activators
and subject to inhibition by purine, pyrimidine, and pyridine
nucleotides (5-8). Of the three highly homologous human PRS isoforms
identified to date, PRS1 and PRS2 are expressed in all tissues (9, 10)
and are encoded by genes (PRPS1 and PRPS2) that
map, respectively, to the long and the short arms of the X chromosome
(11, 12). PRS3 expression is detectable only in the testes and is
encoded autosomally (9, 13).
Superactivity of PRS is an X chromosome-linked human disorder (14)
characterized by PRPP, purine nucleotide, and uric acid overproduction
(15-17), gout (15, 18), and, in some affected families,
neurodevelopmental impairment (18-21). The kinetic mechanisms underlying inherited PRS superactivity are diverse and include defective allosteric regulation of PRS1 activity (regulatory defects) (15-19, 21, 22), increased apparent affinity of PRS for the substrate
ribose-5-phosphate (23), and increased activity of the normal PRS1
isoform (formerly called catalytic superactivity) (24-27). Study of
the genetic and mechanistic bases of the heterogeneous kinetic
alterations associated with PRS superactivity has shed light on the
manner in which the synthesis of PRPP is regulated (3, 16, 21). In the
case of regulatory defects, for example, patient-derived PRS1 cDNAs
bear point mutations encoding recombinant mutant PRS1s with altered
allosteric properties (resistance to noncompetitive purine nucleotide
inhibition and increased sensitivity to Pi activation)
characteristic of those of PRS in cells from the respective affected
individual (21). This finding provides evidence that allosteric control
of PRS1 activity is important in regulating PRPP synthesis in human cells.
In contrast, overexpression of normal PRS1 transcript as well as PRS1
isoform has been demonstrated in cells from patients with overactivity
of normal PRS (24). The association of increased PRS1 transcript level
with increased PRS1 isoform content and enzyme activity suggests a
pretranslational defect in the expression of PRPS1 in this
type of inherited PRS superactivity (24). In studies aimed at further
defining the process responsible for overexpression of normal PRS1
transcript and isoform, we have found selective acceleration of
PRPS1 transcription in this disorder as well as evidence
that in fibroblasts from affected individuals the rate of transcription
of PRPS1 serves as a major determinant of PRPP and purine
nucleotide production rates despite intact allosteric regulation of PRS activity.
Cell Lines--
Fibroblast strains initiated from skin biopsies
obtained from five normal individuals and three unrelated males with
overexpression of normal PRS (25, 26) were propagated in monolayer in
Eagle's minimal essential medium containing 10% fetal bovine serum, 2 mM L-glutamine, nonessential amino acids,
penicillin (100 units/ml) and streptomycin (100 µg/ml). B lymphoblast
lines, derived as described (28) from two normal individuals and two of
the affected males, were propagated in RPMI 1640 medium containing 10%
fetal bovine serum, 2 mM L-glutamine, and
penicillin and steptomycin. Conditions for growth and study of cell
cultures and procedures for preparing cell extracts for PRS activity
determinations and PRS isoform analyses were as described (16, 28). The
extraction buffer was 8 mM sodium phosphate, 1 mM dithiothreitol, 1 mM EDTA (pH 7.5).
PRS Activity and Isoform Analyses--
PRS activities were
determined in crude cell extracts by a two-step procedure previously
described in detail (24). In the first step, PRPP was generated at pH
7.5 in the presence of either 1.0 or 32.0 mM
Pi, saturating substrate concentrations (500 µM MgATP; 350 µM ribose-5-phosphate), and
5.0 mM MgCl2. Activities of PRS are expressed
in units/mg of protein, where 1 unit is defined as 1 µmol of PRPP
formed/min at 37 °C. Separation and quantitation of PRS1 and PRS2
isoforms in cell extracts were accomplished by a polyacrylamide-urea
isoelectric focusing-immunoblotting procedure recently described in
detail (24). The intensities of bands of identical mobility in samples
of cell extracts and of purified recombinant PRS1 and PRS2 isoforms (8)
were quantitated on a Molecular Dynamics (Sunnyvale, CA) computing
densitometer. Cell extract band densities were related to those of the
respective purified recombinant PRS isoform and to the amount of
protein in the sample applied to the isoelectric focusing gel,
permitting determination of the concentration of each isoform in the
corresponding sample. Protein concentrations were determined by the
method of Lowry et al. (29).
PRPP Generation and Purine Synthesis de Novo--
Rates of PRPP
generation were calculated as described (16, 23) from simultaneously
measured values for intracellular PRPP concentrations and rates of
incorporation of [14C]Ade into intracellular purine
compounds. Incorporation of the purine precursor
[14C]formate into intracellular purines and purines
excreted into the culture medium was measured to estimate rates of
purine synthesis de novo in intact cells (16, 28).
PRPS1 Genomic DNA Sequencing--
Human genomic DNA clones of
8.5 and 13.6 kilobases were isolated and purified by repeated plaque
hybridization (with a 450-bp oligo-[32P]-labeled 5' PRS1
cDNA HindIII fragment) from a fetal fibroblast genomic
DNA library in the vector
Genomic DNAs isolated from three normal fibroblast strains and three
strains derived from affected individuals (1-2 × 107
cells each) (30) served as templates for sequential polymerase chain
reaction amplifications utilizing a nested primer approach to prepare
PRPS1 genomic DNA segments inclusive of 850 bp of proximal 5'-untranscribed DNA and the 5'-transcribed but untranslated region of
the gene (24). The amplification primers utilized are listed in Table
I, with their respective locations relative to the PRS1 cDNA
translation initiation site. Sequencing of the proximal untranscribed 5' genomic DNA and of the 144-bp 5' DNA segment encompassing the proposed human PRPS1 transcription initiation sites (31, 32) to the translation initiation codon was carried out on both DNA strands
from all strains, utilizing appropriately oriented primer sequences
(Table I) from the PRPS1
promoter region and exon 1 of PRPS1. Sequence identity has
previously been established for the 997-bp 3'-untranslated segment of
the transcribed region of normal and patient cell-derived
PRPS1 DNAs (24).
PRS1 and PRS2 Transcript Levels--
Steady state levels of PRS1
and PRS2 transcripts were estimated by Northern blot analysis (30)
after electrophoresis and transfer of cellular total RNA samples to
nitrocellulose filters, prehybridization, and hybridization, all as
described previously (24). Probes for hybridization were
oligo[32P]-labeled human PRS1 cDNA (2.3 kilobases),
PRS2 cDNA (2.7 kilobases), and glyceraldehyde-3-phosphate (GAPDH)
cDNA (1.8 kilobases). After washing at suitable stringency,
radioactivities in the regions of the membrane corresponding to PRS,
and control transcripts were quantitated on a PhosphorImager (Molecular
Dynamics) for 12 h before exposure of the membrane to x-ray
film for 24-72 h at
To estimate PRS transcript stability, relative PRS mRNA levels were
determined in lymphoblasts incubated with the RNA polymerase II
inhibitor, actinomycin D. Identical cultures of each lymphoblast line
(20 × 106 cells in 20 ml of growth medium) were
incubated at 37 °C for 0 to 24 h after the addition of
actinomycin D (final concentration, 5 µg/ml). At appropriate times,
cultures were harvested by centrifugation, and cells were washed twice
in ice-cold serum-free medium before extraction of RNA (30). Northern
blot analysis was carried out as described above, except that the
nitrocellulose filters were probed with a labeled cDNA probe (1.4 kilobases) for 18 S ribosomal RNA as well as labeled PRS cDNA
probes. Values for PRS transcript levels are expressed relative to that
of the 18 S ribosomal RNA level measured in the respective sample.
DNA Preparation and Filter Hybridization--
Normal male and
patient fibroblast genomic DNAs were digested with BamHI and
HindIII. After electrophoresis of equal amounts of DNA in
0.8% agarose gels, DNAs were transferred to GeneScreen nylon membrane
filters (NEN Research Products) under alkaline conditions (33). Blots
were hybridized with oligo[32P]-labeled PRS1 cDNA
probes (12), including a full-length (2.3 kilobase) PRS1 cDNA and a
HindIII fragment containing the 5' 450 bp of PRS1 cDNA.
Conditions of hybridization, washing, and exposure of filters to x-ray
film were as described (12).
Rates of PRPS Gene Transcription--
Transcription rates were
estimated in fibroblasts and lymphoblasts by nuclear runoff analysis
(34). Nascent RNA transcripts were [32P]UTP-labeled in
and isolated from fibroblast (7 to 9 × 107
cells/assay) and lymphoblast (1.8 to 2.2 × 108
cells/assay) nuclei prepared as described (30, 34). RNAs in each
nuclear preparation were hybridized to linearized plasmid pGEM3Z or to
pGEM3Z-borne PRS1, PRS2, and GAPDH cDNAs immobilized by slot
blotting onto a nitrocellulose filter (30). Binding of label to pGEM3Z
without a cDNA insert served as background control, and a 28 S
ribosomal RNA cDNA probe (35) was applied to the filter to bind
labeled ribosomal RNA and thus reduce nonspecific binding elsewhere on
the filter. After hybridization and washing at suitable stringencies,
label corresponding to each slot was quantitated on a PhosphorImager,
and the filter was exposed to x-ray film for 48 to 72 h at
PRPS1 Genomic DNA Sequencing--
The 1161-bp sequence of normal
human PRPS1 preceding the translation initiation ATG triplet
is shown in Fig.
1.2
Sequence identity between normal- and patient-derived PRPS1
genomic DNA was confirmed for the 5'-transcribed but -untranslated
region of the gene from the 3 affected and 3 normal individuals,
supporting the contention (24) that PRS1 transcript structure is normal in hemizygous males with overactivity of normal PRS1. In addition, the
sequence of polymerase chain reaction-amplified PRPS1 DNA corresponding to the 850 bp immediately 5' to the consensus
transcription initiation site (nucleotides Genomic DNA Hybridization--
Southern blots of restriction
enzyme-digested patient and normal male genomic DNAs showed identical
patterns and intensities of hybridizing bands when filters were probed
with either full-length normal PRS1 cDNA (Fig.
2) or a HindIII fragment
containing only the 5' 450 bp of normal PRS1 cDNA (not shown).
These findings exclude gene amplification as a likely basis for
PRPS1 overexpression in this form of PRS superactivity.
PRS Transcript Levels and Stability--
Northern blot analysis
confirmed (24) increased concentrations of PRS1 but not PRS2 transcript
in extracts of patient fibroblasts and, to a lesser
degree, lymphoblasts when expressed relative to levels of either GAPDH transcript (Table
II)3 or 18 S ribosomal RNA
(Fig. 3A) in the same extract.
Nevertheless, rates of decrement of PRS1 mRNA (Fig. 3, A
and B) and of PRS2 mRNA (Fig. 3, A and
C) (relative to those of 18 S ribosomal RNA) from normal and
patient lymphoblasts during incubation with actinomycin D were
virtually indistinguishable. PRS1 transcript half-lives were 10.8 ± 1.4 h and 11.1 ± 0.9 h (mean ±S.D. of 3 determinations in each cell line), respectively, in normal and patient
cells. Corresponding mean half-lives for PRS2 transcripts were 13.1 and 12.2 h. In all instances, PRS1 and PRS2 transcripts were
detectable only as single hybridizing bands at 2.3 and 2.7 kilobases
respectively, and no additional hybridizing bands suggestive of
immature or alternatively processed PRS transcripts were observed.
Rates of PRPS Gene Transcription--
Slot-blot analyses of
specific RNAs labeled in nuclei isolated from cultured normal and
patient cells showed consistent differences with respect to relative
rates of labeling of PRS1 mRNA (Fig. 4; Table
III). In fibroblasts from 3 affected
males, rates of PRS1 transcript labeling relative to those of GAPDH
transcript labeling were 3- to 4-fold greater than in cells from 5 normal individuals (Table II; Fig. 4A). In lymphoblasts, the
corresponding increases in relative labeling of PRS1 transcript were
1.9- to 2.4-fold (Table II; Fig. 4B). Relative rates of
labeling of PRS2 mRNA were indistinguishable in normal and patient
cells, a point more readily apparent in lymphoblasts, which have
substantially higher relative rates of PRS2 transcription than
fibroblasts (Table III). In conjunction with the preceding studies
excluding PRPS1 gene amplification or altered PRS1
transcript structure or stability, these findings suggest that
increased expression of PRPS1 in overactivity of normal PRS1
results at least in major part from selective acceleration of
PRPS1 gene transcription.
PRPS1 Gene Transcription, PRS Expression, and Purine Nucleotide
Synthesis--
If accelerated transcription of PRPS1 is the
major determinant of PRS overactivity in fibroblasts and lymphoblasts
from affected patients, correspondence between rates of
PRPS1 transcription and PRS1 mRNA levels, PRS1 isoform
concentrations, PRS enzyme activities, and rates of PRPP and purine
nucleotide synthesis should be demonstrable. Such correspondence would
support the view that PRPS1 transcription rate can determine
expression of PRS1 activity even when allosteric regulation of enzyme
activity is intact. To assess these relationships, we compared relative rates of PRPS1 transcription, relative PRS1 mRNA
concentrations, PRS isoform concentrations, and PRS activities in
fibroblast strains and lymphoblast lines derived from normal
individuals and affected patients. In addition, PRPP generation and
rates of purine synthesis de novo were determined in these cells.
Differences between normal and patient fibroblasts with respect to
PRPS transcription rates, PRS mRNA and isoform levels, and maximal PRS enzyme activities are presented in Table II, where values for individual patient-derived strains are expressed relative to
the respective mean values for the group of 5 normal fibroblast strains. For each patient-derived cell strain, nearly coordinate increases are apparent in all processes relating PRPS1
transcription to PRS activity measured at 32 mM
Pi, a concentration of Pi at which allosteric
inhibition of PRS activity by endogenous purine nucleotides is minimal
(16). As is also shown in Table II, intact patient-derived fibroblasts
synthesize PRPP and purine nucleotides at increased relative rates,
which are, however, more modest than the increased relative rates of
PRPS1 gene transcription or the increased relative levels of
PRS1 transcript or isoform, or maximal PRS activities. Relative
increases in PRPP and purine nucleotide synthesis, in fact, more
closely parallel relative increases in PRS activities measured at 1.0 mM Pi, a concentration closer to that in intact
fibroblasts (36) and at which allosteric inhibition by endogenous
nucleotides is potent (16). The modulated relative increases of PRPP
and purine nucleotide synthesis in patient cells thus appears to
reflect both the operation of allosteric inhibition of PRS activity and
the higher intracellular concentrations of purine nucleotide inhibitors
in intact patient fibroblasts (16).
Similar relationships are detectable but less immediately apparent in
lymphoblast lines (Table II) than in fibroblast strains, first, because
of smaller differences in all measurements comparing patient and normal
lymphoblasts, and, second, because the contribution of PRS2 to total
PRS isoform content and PRS activity is substantially greater in
lymphoblasts than fibroblasts (24). In fact, as previously noted (24,
28), the small increases in maximal PRS activities measured in
lymphoblast extracts from affected patients appear insufficient to
drive excessive production of PRPP or purine nucleotides in intact cells.
The current studies provide evidence for a selectively increased
rate of transcription of PRPS1 as the pretranslational
aberration underlying increased expression of the normal PRS1 isoform
in inherited PRS overactivity. In each of the fibroblast strains and
lymphoblast lines cultured from affected individuals, transcription of
PRPS1 was consistently greater relative to that of
GAPDH than was the case in corresponding normal cells.
Although the relative increases in PRPS1 transcription rates
were greater in patient fibroblasts than lymphoblasts, study of the
latter cell type permitted a more accurate demonstration of the
selectivity of accelerated PRPS1 transcription. That is,
relative rates of PRPS2 transcription in normal cells were
substantially greater in lymphoblasts than in fibroblasts, but for both
lymphoblasts and fibroblasts, relative rates of PRPS2
transcription in normal and patient cells were indistinguishable.
Consistency was also found in the relative differences between normal
and patient cells in each of the processes mediating flow of genetic
information from PRPS1 transcription to maximal PRS1 isoform
expression (enzyme activity at 32 mM Pi). This
finding, in conjunction with the demonstration that patient and normal
cells did not differ in PRS1 mRNA or isoform structure or in
alternative pretranslational mechanisms that might otherwise explain
PRS1 isoform overexpression, supports the contentions that PRS1 isoform
concentrations are determined, at least in major part, at the level of
transcription and that an inherited increase in PRPS1
transcription rate provides the basis for the increase in the
concentration of the normal PRS1 isoform in cells of affected individuals.
The genetic basis of inherited acceleration of PRPS1
transcription remains to be determined. Sequence identity of patient and normal PRPS1 DNAs in the 850-bp region 5' to the
consensus transcription initiation site (32) excludes mutation in the gene promoter and immediate 5' PRPS1 flanking sequence, for
which examples of transcriptional dysregulation have been established, as in the thalassemias (37-39) and hereditary persistence of fetal hemoglobin (40, 41). Among alternative possibilities to explain accelerated PRPS1 transcription are mutations in a more
remote promoter element, either in contiguity with the immediate
5'-flanking sequence (42) or even substantially distant (43); in a
cis-acting element within or adjacent to the PRPS1 gene,
such as an intronic enhancer or suppressor (44) or a 3'-flanking DNA
sequence (45); or in a trans-acting gene influencing the regulation of
PRPS1 transcription. In any case, X chromosome-linked transmission of PRS catalytic superactivity (14) favors the view that the primary defect altering PRPS1 transcription is itself X-linked. In
addition to extended PRPS1 5'-flanking region sequencing,
functional analysis of the PRPS1 promoter and adjacent
5'-flanking DNA, comparing PRPS1 promoter-plasmid construct
expression in normal and patient cells, should prove helpful in
distinguishing among these possibilities.
Prior studies (3, 16) comparing mechanisms of purine nucleotide
overproduction in fibroblasts from individuals with PRS superactivity
(either catalytic or regulatory defects in PRS1) and severe deficiency
of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) have
confirmed that the rate of the pathway of purine synthesis de
novo is controlled at the sequential PRS and amidophosphoribosyltransferase (EC 2.4.2.14) reactions, the latter the
first committed step in the pathway. Within this regulatory domain,
purine nucleotides inhibit both reactions (3, 16, 36), exerting more
potent inhibition of amidophosphoribosyltransferase by antagonizing
PRPP activation of this reaction (3, 46). Although fibroblasts with
either regulatory defects in PRS or overactivity of normal PRS share
the biochemical hallmarks of PRS superactivity, increased rates of PRPP
and purine synthesis and increased intracellular purine nucleotide
concentrations, defect-specific differences in the intracellular
control of PRPP and purine nucleotide synthesis are apparent (16).
In the case of PRS regulatory superactivity, where PRS activity in cell
extracts or purified enzyme preparations are resistant to purine
nucleotide inhibition, accelerated rates of intracellular PRPP and
purine nucleotide synthesis are refractory to inhibition by exogenous
purine base precursors of purine nucleotides or by endogenous increases
in purine nucleotide concentrations (16). Thus, in cells bearing PRS1s
with any of an array of point mutations (21), in vitro
defects in allosteric regulation of PRS1 activities are paralleled by
dysregulation of PRPP and purine synthesis. In contrast, allosteric
regulation of PRS activity is normal in enzyme preparations from
fibroblasts with overactivity of normal PRS, and suppression of PRPP
and purine nucleotide synthesis in response to purine base addition is
intact in the corresponding cells (16). Nevertheless, these cells
express increased rates of PRPP and purine nucleotide synthesis.
The apparent paradox of increased rates of PRPP and purine nucleotide
synthesis in fibroblasts with overactivity of normal PRS catalytic
superactivity despite increased purine nucleotide inhibitor pools and
normal allosteric regulation of PRS activity (16) is best resolved by
the view that the increased concentration of the normal PRS1 isoform
(24) results in a rate of PRPP synthesis sufficient to activate
amidophosphoribosyltransferase despite coexisting increased levels of
inhibitory purine nucleotides (16). Consistent with this formulation
are the substantially more modest increases in rates of PRPP and purine
nucleotide synthesis in fibroblasts with intact allosteric regulation
than in cells in which mutations in PRPS1 impair this
control mechanism (16). Thus, although intact allosteric inhibition
apparently modulates expression of PRPP and purine overproduction in
fibroblasts with excessive PRS1 isoform, this regulatory mechanism is
insufficient to overcome the excessive expression of enzyme activity
resulting from acceleration of PRPS1 transcription. The
current studies provide, then, an example of a circumstance in which
transcription of PRPS1 is a major determinant of PRPP and
purine nucleotide synthetic rates.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
FixI (Stratagene, Menasha, WI) (30). A
2.2-kilobase SacI fragment common to human DNA in both
clones was subcloned into pGEM3Z, and the identity of the cloned DNA
was confirmed by establishing sequence identity with that of exon 1 of
PRPS1 and the adjacent 5' proximal genomic and 3' intron 1 DNA sequences published to date (31, 32). The 2.2-kilobase
PRPS1 genomic DNA was amplified in Escherichia
coli and served as template in sequencing reactions utilizing SP6
and PRPS1-specific oligonucleotide primers (see Table
I).
Oligonucleotides used for polymerase chain reaction amplification of 5'
promoter and 5'-untranslated regions of PRPS1 and for sequencing
amplified and cloned PRPS1 DNAs
70 °C. Values for PRS1 and PRS2
transcript levels in a cultured cell total RNA sample are
expressed relative to the GAPDH transcript level measured in that sample.
70 °C for densitometric quantitation. Rates of PRPS1
and PRPS2 transcription are expressed relative to
transcription of GAPDH, determined on the same filter.
RESULTS
117 to
115, Fig. 1; Ref.
32) was identical regardless of whether normal- or patient-derived genomic DNA served as template.
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Fig. 1.
Sequence of normal human PRPS1
extending 1161 bp 5' to the translation initiation ATG
codon. Numbering is relative to the A of the ATG codon
(+1). Transcription initiation sites identified by primer extension and
S1 nuclease protection assays (32) are indicated by
arrowheads. The 5' end of the human PRS1 cDNA ( 122)
(cloned from normal human B lymphoblasts) is denoted by the
horizontal arrow. With exception of a single base (C)
addition at
410, the sequence from
437 to +3 is as previously
published (32). An identical sequence was found in the interval from
967 (vertical arrow) to +3 when 3 normal and 3 patient
fibroblast-derived PRPS1 DNAs were amplified and
sequenced.
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Fig. 2.
Southern blot analysis of restriction
enzyme-digested human genomic DNA extracted from a normal male B
lymphoblast line (lanes 1 and 2) and
a lymphoblast line derived from patient TB with overactivity of normal
PRS (lanes 3 and 4). DNAs were
digested with BamHI (lanes 1 and 3)and
HindIII (lanes 2 and 4). Each
lane contained 10 µg of enzyme-restricted DNA, and after
overnight electrophoresis in 0.8% agarose and transfer to a
nitrocellulose filter, hybridization was carried out with a full-length
oligo-labeled (2.3 kilobase) PRS1 cDNA. The filter was washed (33)
and exposed to a PhosphorImager for 4 h. Shown on the right are
the locations of bands corresponding to DNA standards of known size
(kilobases).
Relationships between accelerated PRPS1 transcription and PRS activity,
PRPP generation, and rates of purine synthesis in human fibroblasts
with overactivity of normal PRS
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Fig. 3.
PRS1 and PRS2 mRNA half-life
determinations in normal and patient lymphoblasts. A,
identical cultures of normal (LIV) and of patient (TB) lymphoblasts
were incubated with 5 µg/ml actinomycin D for the indicated time
intervals. RNA was isolated, and 10 µg was run in each
lane of a denaturing gel, blotted onto a nitrocellulose
membrane, and sequentially probed, first with full-length PRS1 and PRS2
cDNAs and then with an 18 S rRNA cDNA. B and
C, PRS1 and PRS2 mRNAs and 18 S rRNA levels were
quantitated using a PhosphorImager, and PRS1 and PRS2 mRNA
expression was normalized for 18 S rRNA expression. , normal cells;
, patient cells.
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Fig. 4.
Slot blot analysis of rates of transcription
of PRPS1, PRPS2, and GAPDH were
determined in normal and patient fibroblasts (A) and
lymphoblasts (B) by nuclear runoff (30, 34).
Arrows indicate sites of binding of linearized pGEM3Z
containing the respective human cDNAs to nitrocellulose filters.
Slots containing linearized pGEM3Z only were removed after 18 h of
exposure of the filters to a PhosphorImager screen, and values in these
control slots were subtracted from those measured in slots with the
respective human cDNAs. In this experiment, rates of
PRPS1 and PRPS2 transcription, expressed as
percent of GAPDH transcription are, respectively, 4.8 and
1.1 in normal (LEO) fibroblasts, 17.5 and 1.0 in patient (SS)
fibroblasts, 9.5 and 10.3 in normal (LIS) lymphoblasts, and 18.3 and
8.4 in patient (TB) lymphoblasts.
Relative rates of PRPS1 and PRPS2 transcription in cultured human
fibroblasts and lymphoblasts
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Danette Shine for excellent manuscript preparation. We appreciate the helpful comments of Drs. Craig B. Thompson, Tullia Lindsten, and Harinder Singh (University of Chicago) during the course of this work.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant DK28554 and a grant from the Arthritis Foundation, Greater Chicago Chapter.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) AF104626.
To whom correspondence should be addressed: University of Chicago
Medical Center, MC 0930, 5841 South Maryland Ave., Chicago, IL 60637. Tel.: 773-702-6899; Fax: 773-702-3467; E-mail:
mbecker{at}medicine.bsd.uchicago.edu.
2
The only sequence in the data base with
significant relatedness to the sequence in Fig. 1 is the sequence
gb/M31078 for rat PRPS1, exon 1, that shows 94% identity in
the region corresponding to 149 to
94 in human
PRPS1.
3 Normal and patient cell strains are designated by initials of donors in Figs. 3 and 4 and Tables II and III.
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ABBREVIATIONS |
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The abbreviations used are: PRPP, 5-phosphoribosyl 1-pyrophosphate; PRS, phosphoribosylpyrophosphate synthetase; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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REFERENCES |
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