From the
In Aspergillus nidulans, loss-of-function mutations in
the uapA and azgA genes, encoding the major uric
acid-xanthine and hypoxanthine-adenine-guanine permeases, respectively,
result in impaired utilization of these purines as sole nitrogen
sources. The residual growth of the mutant strains is due to the
activity of a broad specificity purine permease. We have identified
uapC, the gene coding for this third permease through the
isolation of both gain-of-function and loss-of-function mutations.
Uptake studies with wild-type and mutant strains confirmed the genetic
analysis and showed that the UapC protein contributes 30% and
8-10% to uric acid and hypoxanthine transport rates,
respectively. The uapC gene was cloned, its expression
studied, its sequence and transcript map established, and the sequence
of its putative product analyzed. uapC message accumulation
is: (i) weakly induced by 2-thiouric acid; (ii) repressed by ammonium;
(iii) dependent on functional uaY and areA regulatory
gene products (mediating uric acid induction and nitrogen metabolite
repression, respectively); (iv) increased by uapC gain-of-function mutations which specifically, but partially,
suppress a leucine to valine mutation in the zinc finger of the protein
coded by the areA gene. The putative uapC gene
product is a highly hydrophobic protein of 580 amino acids
( M
The ascomycete Aspergillus nidulans is able to utilize
purines as nitrogen sources. The pathway of purine catabolism has been
studied genetically and physiologically (Scazzocchio and Darlington,
1968; Scazzocchio et al., 1982). The expression of the genes
involved is subject to pathway-specific induction by uric acid
(Scazzocchio, 1973; Sealy-Lewis et al., 1978), mediated by the
uaY-positive regulatory gene product (Scazzocchio et
al., 1982; Suárez et al., 1991a, 1991b) and to
nitrogen metabolite repression mediated by the GATA-like,
areA-positive regulatory gene product (Arst and Cove, 1973;
Kudla et al., 1990).
Early genetic evidence suggested the
existence of two specific, active transport systems responsible for the
uptake of various purines and their analogues: (i) a system responsible
for the uptake of uric acid and xanthine, their thioanalogues
2-thiouric acid and 2-thioxanthine, allopurinol, and oxypurinol
(Darlington and Scazzocchio, 1967; Arst and Cove, 1973; Arst and
Scazzocchio, 1975; Scazzocchio and Arst, 1978). The uapA gene
has been genetically identified as the gene coding for the major
permease of this system. Similar to previously isolated
uapA
The mutation
areA102 is a leucine to valine change in the zinc finger of
the AreA regulatory protein (Kudla et al., 1990) altering
specifically the expression of several genes under areA control. It results in enhanced expression of a number of genes,
abolishes the expression of others, and does not affect the expression
of some others (Hynes and Pateman, 1970; Arst and Cove, 1973; Arst and
Scazzocchio, 1975). Although areA102 mutants show normal
expression of all genes encoding enzymes involved in purine catabolism,
they cannot grow on uric acid or xanthine as nitrogen sources due to
reduced uptake of these purines (Arst and Scazzocchio, 1975; Gorton,
1983). Strains carrying this mutation show unimpaired growth on the
uric acid precursor, hypoxanthine. We have shown that the areA102 mutation leads to drastically reduced uapA gene
expression (Arst and Scazzocchio, 1975; Diallinas and Scazzocchio,
1989; Gorfinkiel et al., 1993). The inability of the strains
carrying areA102 mutation to grow on uric acid or xanthine is
absolute, in contrast to strains carrying uapA
We have previously referred to the gene uapC (Scazzocchio and Gorton, 1977; Diallinas and Scazzocchio, 1989;
Gorfinkiel et al., 1993, Scazzocchio, 1994) as a gene which
might code for a secondary uric acid-xanthine permease. Here, we
present rigorous genetic and biochemical evidence that the uapC gene product is a wide specificity purine permease responsible for
the leaky phenotypes of uapA
Extragenic revertants of a
uapA24 (loss-of-function) areA102 double mutant
selected on uric acid or xanthine should include gain-of-function
mutations, analogous to uap-100, in the gene(s) coding for the
secondary permease(s) which are not expressed in an areA102 background. Indeed, we isolated a number of suppressors which
defined uapC, a new gene involved in uric acid-xanthine
uptake. The mutation uapC201 exemplifies this class of
suppressors (see Fig. 1); it was mapped by mitotic haploidization
(McCully and Forbes, 1965) to chromosome I but recombines freely with
uapA mutations (results not shown). Four phenotypically
identical revertant mutations were closely linked to uapC201 (results not shown). By analogy to the uap-100 mutation,
the specific suppression of the areA102 allele might result
from regulatory mutations in the promoter of the uapC gene. In
agreement with this assumption, we showed (results not shown) that: (i)
in an areA
We
also crossed the putative uapC
The uapC
While we have measured the uptake rates only for
hypoxanthine and uric acid, results cited above show that UapA is
specific for oxydized purines and their analogues, AzgA for
hypoxanthine, adenine, guanine, and their analogues while UapC can
accept all these purines. The uptake rates and the growth tests
indicate, however, that UapC plays a more important role in the uptake
of the oxydized than the reduced purines.
We demonstrated that this region contains the
uapC gene as follows. First, we have shown that a vector
carrying the 3.3-kb SalI- SalI genomic fragment of
A. nidulans DNA present in clone LAN903 is sufficient to
complement a uapC
We further established the identity of the
pAN903 insert by inactivation of the resident A. nidulans gene. A KSII+ vector carrying a 0.7-kb insert isolated from
the PstI- PstI 1.6-kb restriction fragment (by
removing the internal 0.9-kb XhoI- XhoI restriction
fragment; see Fig. 3) was used, together with a plasmid carrying
the argB gene, to cotransform a uapC201areA102argB2 strain (complete genotype under ``Experimental
Procedures''). This strain utilizes uric acid as nitrogen source
only because the uapC201 gain-of-function mutation suppresses
the phenotype of areA102 on uric acid (see above). Thus the
utilization of uric acid by this strain is strictly dependent on the
integrity of the UapC permease. Among 1500 argB
The 5`-non-coding region of the
uapC gene contains consensus sequences of putative regulatory
importance (see Fig. 5). A 5`-TATA-3`box (Proudfoot, 1979) is
found at position -113/-110 from the putative start of
translation. The consensus sequence 5`-WGATAR-3` (both in the coding
and non-coding strand) is found five times (positions
-189/-184, -212/217, -250/-245, -524/529,
and -562/567). These are candidate target sequences for AreA
binding (Fu and Marzluf, 1990a; 1990b).
The 3`
end of the uapC transcript was mapped by determining the size
of an appropriate cDNA clone. This was constructed by reverse
transcribing induced uapC RNA with a 15-(dT) oligonucleotide
and amplifying the first DNA strand with a uapC specific
primer (O11), in the presence of radiolabeled
[
We have also
obtained a uapC cDNA clone including a nearly complete
(lacking only the last four, C-terminal, codons) uapC open
reading frame (see ``Experimental Procedures''). This clone
was completely sequenced (as described for genomic DNA sequencing) and
allowed us to confirm the presence and the position of a 46-bp short
intron predicted from genomic DNA sequencing. The position of this
uapC cDNA clone is shown in Figs. 3 and 5. Thus, our results
lead to an estimation of 1837/1840 nucleotides for the uapC major transcript, a size in agreement with that estimated from
Northern blot analyses.
We compared the UapC deduced amino acid
sequence to that of all known proteins in data bases using the programs
FASTA (Pearson and Lipman, 1988) and BLAST (Altschul et al.,
1990) and further calculated similarity scores using the programs
GCG/BESTFIT and GCG/GAP (Devereux et al., 1984). The most
striking observation was the similarity of UapC to the UapA amino acid
sequence (Fig. 7). These two purine permeases share 58.4% identical and
78.3% similar amino acid residues throughout their lengths. Identity
reaches nearly 70% within hydrophobic segments and is lowest in the N-
and C-terminal regions of the proteins. An extremely conserved
hydrophilic region (amino acid residues 355-386 in UapC,
377-409 in UapA) is also present, and its putative functional
significance is considered below. It is also of interest that the UapC
protein lacks three acidic amino acid residues which comprise a
significant part of the acidic amphipathic
Fig. 8 shows other
sequences providing significantly high scoring segment pairs with the
UapC amino acid sequence. These are the YicE 48.9 kDa hypothetical
protein of unknown function, encoded by an open reading frame 3` to the
gene coding for the glutamine transporter gene of E. coli (34%
identity overall; Burland et al., 1993), a putative
polypeptide coded by an open reading frame in the 5` region of the
cpe gene encoding an enterotoxin in C. perfrigens (31% identity; Brynestad et al., 1994), the Bacillus
subtilis pyrimidine (uracil) permease (23% identity; Quinn et
al. 1991; Turner et al., 1994), the Bacillus
caldolyticus uracil permease (26% identity; Ghim and Neuhard,
1994), and the E. coli uracil permease (26% identity; X73586
in data bases). Interestingly, the segment showing the highest score
between UapC (or UapA) and any of these bacterial transporters is a
hydrophilic region in YicE (48% identity, 65% similarity over 52 amino
acid residues) overlapping the best conserved hydrophilic region common
to the UapC and UapA amino acid sequences described above (see Figs. 7
and 8).
A number of other transporters and membrane proteins show
scattered regions with less significant amino acid sequence identity
scores (19-21%; results not shown) when compared to the UapC or
UapA sequences. Such similarities probably reflect common structural
determinants in hydrophobic regions of membrane proteins.
The genetic identification, uptake studies, molecular
cloning, regulation of expression, genomic and cDNA sequence and
transcript map of the uapC gene, and an analysis of its
putative translation product have been presented. Our results provide
compelling evidence that the uapC gene codes for a wide
specificity, low efficiency, permease transporting all natural purines
as well as purine analogues. The specificity of the UapC transporter
completely overlaps the transport specificities of UapA and UapC, the
two specific, high efficiency purine permeases of A. nidulans.
It was described previously (Diallinas and
Scazzocchio, 1989; Gorfinkiel et al., 1993) that extragenic
suppression of the inability of an areA102 mutant to grow on
uric acid or xanthine as sole nitrogen source resulted from positive
regulatory mutations in the promoter region of the uapA gene
coding for the major uric acid xanthine permease. Here we describe
extragenic suppressors of an areA102 mutant carrying a
uapA
An analysis of uric
acid and hypoxanthine uptake directly confirmed the role of the
uapC gene product as established by genetic studies. Both
uptake studies and growth phenotypes showed that the UapC protein
contributes significantly to uric acid uptake and weakly to
hypoxanthine uptake. Transport assays showed that the UapC protein
accounts for 30 and 10% of uric acid and hypoxanthine uptake rates,
respectively. These studies also confirmed the roles of the AzgA and
UapA permeases as established previously as specific for uric
acid-xanthine and for hypoxanthine-adenine-guanine, respectively
(Darlington and Scazzocchio, 1967; Arst and Scazzocchio, 1975;
Scazzocchio and Arst, 1978).
The uapC gene was cloned by
low stringency hybridization of an A. nidulans genomic library
to a radiolabeled uapA-specific probe. The vector constructed
(pAN903) from the clone isolated was shown to include a complete
uapC gene by complementation of a uapC
Although uapC expression
is regulated very similarly to uapA gene expression in
response to ammonium repression and the presence of the areA102 allele (dramatically reduced transcript levels), it shows a
different response to pathway-specific induction than the uapA gene. Non-induced uapC transcript levels are higher than
uapA non-induced transcript levels (Diallinas and Scazzocchio,
1989; Gorfinkiel et al., 1993). On the other hand, 2-thiouric
acid induction affects more strongly uapA than uapC message accumulation (6-8-fold versus 2-3-fold; Diallinas and Scazzocchio, 1989; Gorfinkiel et
al., 1993).
The sequence
of genomic and cDNA uapC clones has been established and,
together with experiments which mapped the 5` and 3` ends of the
uapC transcript, it allowed us to determine the uapC transcript sequence, identify the uapC open reading
frame, and to conduct a detailed, computer-aided analysis of its
derived translation product. The UapA and UapC putative permeases share
approximately 58% identical amino acids throughout their amino acid
sequences. Similarity scores reach 78%. Both proteins are predicted,
based on hydropathy profiles, to have extremely similar higher
structures (see Fig. 7). Of greatest interest, is the presence of
a long hydrophilic segment located between putative transmembrane
domains 9 and 10 showing
Conservation of transporter amino acid sequences has been previously
described for the sugar facilitators of higher eukaryotes and the sugar
active transporters of lower eukaryotes and prokaryotes (Marger and
Saier, 1993). In contrast, amino acid transporters or facilitators of
higher eukaryotes form many distinct groups which do not show
significant sequence similarities with the family of fungal amino acid
permeases, the plant, or the bacterial amino acid transporters
(Sophianopoulou and Scazzocchio, 1989; Christensen, 1990; Reizer et
al., 1993; Grenson, 1992).
The fungal purine permeases and the bacterial pyrimidine
transporters shown in Fig. 8exhibit nearly superimposable
hydropathy profiles predicting 12-14 transmembrane segments
(results not shown). Most prokaryotic and eukaryotic transporters are
predicted to consist of a structural motif of 12 tightly packed
transmembrane domains (Griffith et al., 1992; Saier, 1994).
The sequence, showing neither significant similarities to any
previously described family of transporters nor internal repeats, and
the predicted structure, with possibly 14 putative transmembrane
segments, of UapC and similar permeases described in this article,
reveal a new family of prokaryotic and eukaryotic transporters.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) X79796.
We are grateful to Claire Rousseau for her excellent
secretarial assistance, to Annie Glatigny and Teresa Suarez for their
help in Northern blot analysis, and Fathia Mejjad for her technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 61,251) including 12-14 putative
transmembrane segments. The UapC protein is highly similar (58%
identity) to the UapA permease and significantly similar (23-34%
identity) to a number of bacterial transporters. Comparisons of the
sequences and hydropathy profiles of members of this novel family of
transporters yield insights into their structure, functionally
important residues, and possible evolutionary relationships.
loss-of-function mutations (Darlington
and Scazzocchio, 1967), uapA insertional inactivation
mutations (Diallinas and Scazzocchio, 1989) result in leaky growth on
uric acid or xanthine as nitrogen sources and in partial resistance to
their thioanalogues, allopurinol and oxypurinol. These observations
suggested the existence of, at least, one other permease with similar
specificity with the uapA gene product. The uapA gene
has been cloned (Diallinas and Scazzocchio, 1989), its sequence
determined, and its expression studied at the level of message
accumulation (Gorfinkiel et al., 1993). uapA codes
for a highly hydrophobic protein with 12-14 putative
transmembrane domains. Its expression is inducible by 2-thiouric acid
(and by implication by uric acid), repressible by ammonium, and almost
absolutely dependent on functional uaY and areA gene
products. (ii) A system specific for the uptake of adenine, guanine,
hypoxanthine, and the analogues 8-azaguanine and purine;
loss-of-function mutations in the corresponding gene, azgA,
led to azaguanine resistance and to reduced growth on adenine, guanine,
and hypoxanthine as nitrogen sources. In contrast to the highly
regulated expression of the uapA gene, azgA expression is probably constitutive (Scazzocchio and Darlington,
1967). Similar to uapA
mutants,
azgA
loss-of-function mutants have leaky
phenotypes, suggesting the existence of a ``secondary''
permease transporting the same purines and analogues as the azgA gene product (Darlington and Scazzocchio, 1967).
mutations. Interestingly, the areA102 mutation also
eliminates the residual growth of azgA
mutants on hypoxanthine (or adenine or guanine) (see below).
These observations suggested that this mutation affects similarly the
expression of uapA and of the gene(s) coding for the uptake
proteins responsible for the residual growth of strains carrying the
uapA
and azgA
mutations. As areA102 single mutants are not affected in
their utilization of hypoxanthine it can be concluded that the azgA gene is normally expressed in the presence of the areA102 mutation.
and
azgA
mutants on various purines as nitrogen
sources. We report the sequence of the uapC gene and analyze
its putative product which shows approximately 58% identity to the UapA
permease and significant similarities to a number of bacterial
pyrimidine trans-porters. Our analyses also suggested that two
previously reported open reading frames of unknown function in
Escherichia coli and Clostridium perfrigens might
encode purine transporters. Finally, we study the regulation of
uapC message accumulation under different physiological
conditions of growth and in different regulatory mutants.
Media and Growth Conditions
Standard media and
growth conditions of A. nidulans were previously described
(Cove, 1966; Scazzocchio et al., 1982). Supplements were added
when necessary. Non-inducing, inducing, and inducing-repressing
conditions have been previously established (Suárez et
al., 1991a).
Strains
The following A. nidulans strains
were used: biA1; pabaA1; yA2 pantoB100; fwA1
pyroA4 areA102; uapA24 pabaA1; uapA24 azgA4 pabaA1;
uap-100 biA1; uap-100 areA102 pabaA1; uapC201biA1; fwA1 uap-100 uapC201
biA1; uapC201 areA102 pabaA1; azgA4 pabaA1; azgA4 areA102 pyroA4
pabaA1; fwA1 uapC201 areA102 azgA4 pyroA4 pabaA1; uaY207 pabaA1;
uapC201 uapC401 pabaA1; uapC201 uapC401 uapA24 pabaA1; uapC201 uapC401
azgA4 pabaA1; uapC201 uapC401 uapA24 azgA4 pabaA1; uapC201 areA102
argB2 pabaA1; biA1, pabaA1, pyroA4, pantoB100 and argB2 indicate auxotrophies for biotin, p-aminobenzoic acid,
pyridoxine, pantothenic acid, and arginine, respectively. yA2 and fwA1 result in yellow and fawn conidia, respectively.
All these markers do not affect the regulation of gene products
involved in purine catabolism. areA102 is described in the
Introduction and has also been described previously (Kudla et
al., 1990). uapC201 and uapC401 are described in
this article. uapA24, azgA4, and uaY207 (Scazzocchio et al., 1982; Suarez et al., 1991a,
1991b) are non-revertible loss-of-function mutations in the
corresponding genes. uap-100 is an up-promoter regulatory
mutation described previously (Arst and Scazzocchio, 1975; Diallinas
and Scazzocchio, 1989; Gorfinkiel et al., 1993). Double and
triple mutants were constructed by standard A. nidulans genetic techniques (Pontecorvo et al., 1953; McCully and
Forbes, 1965).
Purine Transport Assays
Purine uptake was assayed
by a novel method.(
)
This method measures uptake
in germlings. These are conidiospores incubated for a short time in
nutrient media and thus committed to germination and nuclear division
but where the germinal tube is not yet visible. Preliminary work in a
variety of systems, including purine permeation, demonstrated that
germlings showed the same response to induction as mycelia.
This method avoids all the complications of uptake measurements
in a mycelial mat (Scazzocchio and Arst, 1975). The appropriate strains
of A. nidulans were grown for 3 days on complete media at 37
°C. Conidiospores were collected and used to inoculate liquid
cultures of 200 ml of minimal medium with 5 m
M urea as sole
nitrogen source, all necessary supplements, and 27 µ
M of
2-thiouric acid as a gratuitous inducer. Before inoculation the spores
are counted and the concentration adjusted so as to give a final
concentration of
5
10
conidiospores/ml in the
culture media. Inoculated cultures were incubated at 120
revolutions/min for 210 min at 37 °C. At the end of this incubation
step, conidiospores were filtered through millipore filters (0.45
µm) and washed with minimal medium plus supplements. Washed conidia
were resuspended in 10 ml of minimal medium plus supplements. This
gives a concentration of
10
conidia/ml. 0.5 ml-aliquots
of this suspension were distributed in 2-ml Eppendorf tubes and
equilibrated for 10 min at 37 °C. Uptake was initiated by the
addition of radiolabeled uric acid or hypoxanthine at a final
concentration of 20 µ
M. Uptake assays were terminated by
the addition of cold uric acid or hypoxanthine, at a final
concentration of 0.8 m
M, after 15, 30, 60, and 120 s.
Conidiospores were then washed three times with 2 ml of ice-cold
minimal medium plus 0.8 m
M uric acid or hypoxanthine, boiled
in 5% trichloroacetic acid, and soluble radioactivity was measured by
liquid scintillation counting. The uptake rates of uric acid and
hypoxanthine were linear throughout the sampling period. We have also
determined that, under the above conditions, uptake is directly
proportional with the number of conidiospores in the suspension. The
actual viable conidiospore number was determined, for each assay, by
removing and plating on complete medium, serial dilutions of an aliquot
obtained before the final washing step. Initial uptake rates are
expressed in p
M substrate incorporated/min 10
viable conidiospores. Uniformly labeled
[8-
C]uric acid (specific activity 1.94 GBq/mmol)
and [G-
H]hypoxanthine (specific activity 244
GBq/mmol) were obtained from Isotopchim, Ganagobie-Peyruis, France and
Amersham International, United Kingdom.
Transformation Techniques
A. nidulans and
E. coli transformations were performed as described by Tilburn
et al. (1983) and Hanahan (1983), respectively.
Nucleic Acid Extraction and Manipulations
Total
RNA and genomic DNA isolation from A. nidulans strains has
been previously described by Lockington et al. (1985). Plasmid
isolation from E. coli strains and DNA manipulations were
performed as described by Sambrook et al. (1989).
Nucleic Acid Hybridizations
Southern and Northern
blot analyses were performed, basically, as described by Sambrook
et al. (1989), in 0.5
M sodium orthophosphate, pH 7,
1 m
M EDTA, 7% sodium dodecyl sulfate, and 1% bovine serum
albumin, at 65 °C for 18 h. [P]ATP-labeled
DNA molecules used as gene-specific probes were prepared by using
random hexanucleotide primers following the supplier's
instructions (Amersham). Cloning of the uapC gene involved
screening of a
EMBL4 genomic library of A. nidulans (a
gift from C. M. Lazarus) in the same buffer as that described for
Northern or Southern blots, at 58 °C, and filters were then washed
twice in 2
SSC, at 58 °C.
DNA Sequencing
DNA was sequenced using the
dideoxynucleotide chain termination method (Sanger et al.,
1977) on pAN903 and bluescript KSII+ double-stranded subclones of
the uapC locus (see Fig. 3) and a series of
oligonucleotides specific for both strands of the uapC locus.
The oligonucleotides used were the following:
5`-CTGCAGCCTCTCGAAGC
-3` (O1),
5`-
ACCGTCAATCCCCGCCC
3` (O2),
5`-
CGATCAATCTCGACGTA
3` (O3),
5`-
GCGCATGCTGCAATCCC
3` (O4),
5`-
CGAAGTTCCTACCACCG
3` (O5),
5`-
CGGTGGTAGGAACTTCG
-3` (O6),
5`-
CCCAGATGAAGGATGCG
-3` (O7),
5`-
CATTCAGGGTGGCGTCC
-3` (O8), 5`-
GATAACGCCGTTGTTCTG
-3` (O9), 5`-
CGTTCCTCTTCTCCTCCG
-3` (O10),
5`-
GGAGGAGGATGTTGTGG
-3` (O11),
5`-
GACCAGAGGAACCAGGC
-3` (O12),
5`-
CTTTCATTGCGTTGCCG
-3` (O13),
5`-
GGAGTTAGCAAAGCG
-3` (O14),
5`-
GC-CGTTCGTGTGCGAGC
-3` (O15),
5`-
GATCCCACTGGTCCTCG
-3` (O16), and the T3
and T7 primers (Pharmacia).
Figure 3:
Physical
map of the uapC gene region cloned in pAN903. Coding sequences
are shown as white bars. Non-coding sequences, including a
short intron, are shown as black bars. The direction of
transcription is indicated by an arrow. uapC subclones used in determining the uapC nucleotide
sequence and a nearly complete uapC cDNA clone used in
determining the presence of introns (see ``Experimental
Procedures'') are shown by gray and hatched
bars, respectively. The sequencing strategy for both strands is
shown with arrows (internal primers: filled
triangles, universal; T7 and T3 primers, open triangles).
S, SalI; P, PstI; Xb,
XbaI; Xh, XhoI; H,
HindIII;
cDNA Clones
5 µg of total RNA extracted from
induced wild-type mycelia were denatured by heating for 5 min at 70
°C and reverse transcribed, in the presence of 1 unit of RNasin
(Boehringer Mannheim), with 20 units of avian myeloblastosis virus
reverse transcriptase (Boehringer Mannheim), using the oligonucleotide
O3, for 1 h at 45 °C. The first strand thus synthesized was
directly annealed with the oligonucleotide O12, 1 unit of Taq DNA polymerase was added, and reiterative polymerase chain
reaction cycles were performed as described in the protocol of the
GeneAmp DNA Amplification Reagent kit 5 (Perkin-Elmer-Cetus
Instruments). The program used included 30 cycles of denaturation at 94
°C for 1 min, annealing at 52 °C for 2 min, and elongation at
72 °C for 3 min. The resulting DNA product consisted of 1793
bp(
)
and included the complete 5`-non-coding
region of the uapC transcript and a nearly complete uapC open reading frame (the last four, C-terminal, codons are
missing). This cDNA fragment was cloned by standard procedures in
bluescript KSII+ digested with the SmaI restriction
enzyme and sequenced as described previously for the genomic uapC clones. To map the 3` end of the uapC transcript, a
reverse transcription reaction was performed, as described above, using
an oligo(dT) primer, and then the DNA single strand products were
amplified with oligonucleotide O11 in the presence of radiolabeled
[
S]dATP (Amersham). The size of the radiolabeled
uapC-specific DNA product was estimated (139 and 142 bp) by 6%
polyacrylamide-urea gel electrophoresis, using as size markers a
uapC DNA sequence reaction with the same oligonucleotide
(O11).
5` Primer Extension
Primer extension was performed
using 5 µg of either non-induced or induced total RNA and the
oligonucleotide O4 in the presence of 20 units of reverse
transcriptase, 1 unit of RNasin, a ``cold'' nucleoside
mixture (20 µ
M) of dATP, dCTP, dGTP, and dTTP, and 5
µCi of radiolabeled [S]dATP, in a standard
extension buffer, for 1 h at 45 °C. The size of the synthesized
products was estimated by standard polyacrylamide-urea gel
electrophoresis, using a sequence reaction as DNA size ladder.
Sequence Analyses
DNA and protein sequence
analyses were performed with the DNA Strider1.2 program
(Marck, 1988). DNA and protein searches, comparisons, and alignments
were performed using the FASTA (Pearson and Lipman, 1988), BLAST
(Altschul et al., 1990), GAP, BESTFIT, PILEUP, and PRETTYBOX
(Smith and Waterman, 1981; Devereux et al., 1984) programs and
the data bases SwissProt, PIR, and the translation of the EMBL/GenBank
nucleotide sequences.
Genetic Identification of the uapC Gene: Isolation of
uapC Gain-of-function Mutants
Arst and Scazzocchio (1975)
previously described the isolation of uap-100, defined as an
initiator constitutive, up-promoter mutation in the uapA gene.
This mutation was later shown to be a 164-bp tandem duplication located
103 bp upstream from the uapA translation start resulting in a
pronounced increase in the steady state uapA mRNA levels under
both non-induced and induced conditions (Diallinas and Scazzocchio,
1989; Gorfinkiel et al., 1993). The uap-100 mutation
was selected as a mutation suppressing the inability of an areA102 mutant to grow on uric acid.
background, the uapC201 mutation leads to increased sensitivity to 2-thioxanthine and
2-thiouric acid even in the presence of strongly repressing
concentrations (10 m
M) of ammonium as a nitrogen source; (ii)
a uapC201 uapA24 double mutant grows clearly better than a
uapA24 single mutant on uric acid or xanthine as nitrogen
sources; and (iii) a uapC201 uap-100 double mutant shows an
additive phenotype in respect to 2-thioxanthine (or 2-thiouric acid)
sensitivity suggesting that two different uptake systems are involved.
These results strongly suggested that the uapC gene codes for
a permease able to take up, at least, uric acid, xanthine,
2-thioxanthine, and 2-thiouric acid. As areA102 eliminates the
residual growth of azgA
mutants on
hypoxanthine, adenine, or guanine as nitrogen source, it could be asked
whether the UapC permease is also responsible for the residual uptake
of these purines. A triple mutant, areA102 azgA4 (loss-of-function) uapC201, was constructed and tested on
various purines as nitrogen sources. Fig. 1shows that
uapC201 suppresses partially the inability of an azgA4
areA102 double mutant to utilize hypoxanthine (and also adenine
and guanine, results not shown) as sole nitrogen source and leads to
reduced resistance to 8-azaguanine (not shown).
Figure 1:
Growth tests of a uapC gain-of-function mutant. The strains tested (see diagram) are
described under ``Results'' and ``Discussion.''
Complete genotypes are described under ``Experimental
Procedures.'' Petri dishes contain 1% glucose-minimal medium plus
necessary supplements (see ``Experimental Procedures'') and
either 3.3 m
M ammonium tartrate ( N), or 10 m
M uric acid ( UA), or 10 m
M hypoxanthine
( H), as sole nitrogen source. Growth was scored after 48 h at
37 °C.
Thus, our results
show that: (i) uapC codes for a permease able to transport all
naturally occurring purines, thus overlapping the specificities of the
uapA and azgA gene products; (ii) uapC gene
expression is under areA control and is absent (as judged by
growth tests) in strains carrying the areA102 allele; and
(iii) uapC gain-of-function mutations, such as
uapC201, specifically but partially suppress the effect of the
areA102 allele.
Isolation and Genetic Characterization of uapC
Loss-of-function Mutants
In order to substantiate the role of
the uapC gene in purine uptake, we isolated loss-of-function
mutants. A uapC201 areA102 mutant strain, which is sensitive
to 0.1 mg/ml 2-thioxanthine in the presence of 3 m
M ammonium
as nitrogen source, was mutagenized by UV light or
N-methyl- N-nitro- N-nitrosoguanidine and
2-thioxanthine-resistant colonies were selected as described previously
(Alderson and Scazzocchio, 1967; Diallinas and Scazzocchio, 1989). Such
mutants could, in principle, be the result of loss-of-function
mutations in a number of genes involved in purine catabolism
( uaY, hxA, hxB, cnx; Diallinas and
Scazzocchio, 1989), as well as, loss-of-function mutations in the
uapC or areA genes. However, very simple growth tests
previously described (Alderson and Scazzocchio, 1968; Diallinas and
Scazzocchio, 1989) allowed us to distinguish uptake mutants (and thus
potential uapCmutants) from all other
classes. These mutants should not grow on uric acid or xanthine, grow
less well than wild-type strains on hypoxanthine, adenine, or guanine,
and show resistance to 2-thioxanthine, 2-thiouric acid, and
allopurinol. Five strains with the expected phenotype were isolated. In
order to investigate whether the putative uapC
mutations ( uapC401, uapC402, uapC403,
uapC404, and uapC405) were genetically linked to the
uapC gene (identified by the uapC gain-of-function
mutations), we crossed all putative uapC
uapC201 areA102 mutants to a uapC
areA102 strain. Only uapC
uapC201areA102 recombinants should be able to grow on uric
acid as a nitrogen source. All putative uapC
mutations showed very tight linkage to the uapC201 mutation; mutations uapC403, uapC404,
uapC405 did not recombine with mutation uapC201 while
mutations uapC401 and uapC402 recombined at low
frequencies (
10
to 10
).
mutants with
a wild-type strain ( uapC
uapC201areA102
uapC
areA
, see
``Experimental Procedures'' for full genotype of strains) and
in no case isolated strains showing a uapC201 phenotype among
the progeny tested (100 progeny/cross). These crosses further confirmed
the tight genetic linkage of the uapC loss-of-function and
gain-of-function mutations.
mutations were placed in an areA
background and all possible combinations of double and triple
mutants carrying the uapA
, azgA
, and uapC
loss-of-function mutations were constructed. Fig. 2 shows some
crucial growth tests. Our results confirm that the uapC gene
codes for a wide specificity purine permease overlapping the uptake
activities of the uapA and azgA permeases. As
uapC
single mutations impair growth on
hypoxanthine (and adenine and guanine) considerably less than
azgA
mutations and on uric acid (and
xanthine) less than uapA
mutations, but
eliminate the leaky growth of strains carrying either
uapA
or azgA
single mutations, it can be concluded that the uapC gene
codes for a permease of wide specificity but having lower capacity for
transport.
Purine Transport in Wild-type and Mutant
Strains
We examined the rates of purine uptake in wild-type and
all single, double, and triple purine permease loss-of-function mutants
described above. Radiolabeled uric acid and hypoxanthine initial uptake
rates were determined by a novel method using germlings (germinated
conidiospores) incubated under induced conditions (see
``Experimental Procedures''). Table I shows a typical
experiment of a series performed with higly consistent results. The
rate of uptake of hypoxanthine is about five times higher than the rate
of uptake of uric acid. Transport rates of uric acid and hypoxanthine
uptake in mutant strains were in perfect agreement with the growth
tests presented above. Uric acid incorporation is not altered in
azgAstrains. It is nearly completely
eliminated in strains carrying both uapA and uapC loss-of-function mutations. uapA
and
uapC
single mutants show approximately 30
and 50-55% uric acid uptake rates, respectively, as compared to
the wild-type. Hypoxanthine incorporation is dramatically reduced in
azgA
mutants (8-10% of wild-type
rates). It is not altered in uapA
single
mutants. The rate of uptake of uapC
and
uapA
uapC
mutants is not
distinguishable from the wild-type rate. However, the contribution to
hypoxanthine uptake of the uapC permease is revealed by the
azgA
uapC
double mutant (and
the azgA
uapC-uapA
triple
mutant). While the contribution to hypoxanthine uptake of the UapC
permease is masked by the much more efficient AzgA permease, it becomes
obvious in its absence. This was predicted from the growth tests. Fig.
2 shows that in a uapC
single mutant strain
growth on hypoxanthine as nitrogen source is not impaired. However, the
residual growth of an azgA
strain is
abolished by the introduction of an uapC
mutation.
Molecular Cloning of the uapC Gene
Diallinas
(1989) observed that in Southern blot analyses of A. nidulans DNAs, a uapA-specific probe detects weakly hybridizing
non- uapA sequences. Given the close functional relationship of
the uapA and uapC gene products, we considered the
possibility that a uapA clone might cross-hybridize with
uapC DNA sequences thus allowing the cloning of uapC by screening an A. nidulans genomic EMBL4 bank with a
uapA-radiolabeled probe at low stringency hybridization
conditions. We screened 5000 clones and isolated six,
non- uapA, positively hybridizing plaques (see
``Experimental Procedures''). Restriction and Southern blot
analyses identified two overlapping clones corresponding to the same
genomic region of A. nidulans (results not shown). The
restriction profiles of these clones was different from that expected
if the phages contained the uapA genomic region of A.
nidulans.
loss-of-function mutation.
To this end, we constructed plasmid pAN903, carrying this fragment
cloned into Bluescript KSII+ (Fig. 3). This plasmid was used to
transform both a strain which cannot take up and utilize uric acid,
uapC401 uapC201 uapA24, and a strain that cannot take up or
utilize either uric acid or hypoxanthine ( uapC401 uapC201 uapA24
azgA4, see ``Experimental Procedures'' for full
genotypes). Transformants were selected, respectively, on uric acid and
on either uric acid or hypoxanthine as nitrogen sources. In each
experiment, plasmid pAN503 (Diallinas and Scazzocchio, 1989) carrying
an intact uapA gene was used as a positive control. The
plasmid containing the putative uapC insert, pAN903,
transformed both recipient strains for growth on uric acid at low
frequencies comparable to those obtained with the plasmid pAN503 (about
10 transformants/µg of DNA). As expected, pAN903 also transformed
the uapC401 uapC201 uapA24 azgA4 recipient strain for growth
on hypoxanthine. Southern blot analysis of transformants obtained from
the latter strain showed that heterologous integration of a single copy
of pAN903 in the genome of A. nidulans is sufficient to
complement the mutations carried by this strain for growth on uric acid
or hypoxanthine (results not shown). This, together with sequencing
data (see below), strongly suggests that the entire uapC gene
is included in the 3.3-kb SalI- SalI restriction
fragment of pAN903.
transformants analyzed, two showed an apparent
uapC
phenotype (scored by lack of growth on
uric acid as a nitrogen source and resistance to the analogue
2-thioxanthine). Southern blot analysis (not shown) confirmed that the
recipient genomic 1.6-kb PstI- PstI restriction
fragment was disrupted. Sequence analysis (see below) has confirmed
that the disrupted sequences map within the uapC open reading
frame. Both inactivation and complementation studies show that the
sequences cloned contain the uapC gene. Further evidence as to
the identity of the pAN903 insert came from studies on the regulation
of expression and the sequence of the uapC gene described
below.
Regulation of uapC Message Accumulation
We studied
uapC message accumulation in wild-type and mutant strains
grown under different physiological conditions by Northern blot
analyses, as described under ``Experimental Procedures.'' Our
results are summarized in Fig. 4. Fig. 4 a shows that (i)
uapC maximum message accumulation is dependent on a functional
uaY product and (ii) ammonium drastically represses uapC message accumulation. Fig. 4 b shows that uapC message accumulation is (i) weakly (2-3-fold) inducible by
the gratuitous inducer 2-thiouric acid, (ii) increased (5-fold) in
a uapC201 mutant strain, (iii) drastically diminished in an
areA102 mutant strain, and (iv) significantly higher in a
uapC201 areA102 double mutant than in an areA102 single mutant (but still lower than in a wild-type strain). These
results further confirm that we have cloned the uapC gene and
reveal how uapC message accumulation is regulated in response
to pathway-specific induction and nitrogen metabolite repression.
Figure 4:
Northern blot Analyses of uapC message accumulation. Approximately 5 µg of total RNA were
loaded in each lane as described under ``Results'' and
``Experimental Procedures.'' Panel a, wild-type
( lanes 1-3) and uaY207 (loss-of-function)
mutant ( lanes 4-6) strains grown under non-inducing
( lanes 1 and 4), inducing-repressing ( lanes 2 and 5), and inducing ( lanes 3 and 6)
conditions, as described previously (Suárez et al.,
1991b). Panel b, wild-type ( lanes 1 and 2),
uapC201 mutant ( lanes 3 and 4), uapC201
areA102 mutant ( lanes 5 and 6), and areA102 mutant ( lanes 7 and 8) strains grown under
non-inducing ( lanes 1, 3, 5, and 7)
and inducing ( lanes 2, 4, 6, and 8)
conditions. Below both panels a and b, are the same
blots, washed and rehybridized using the A. nidulans actin
gene (Fidel et al., 1988) as a
probe.
Sequence of the uapC Gene
We have shown that the
entire uapC gene is included within the 3.3-kb
SalI- SalI restriction fragment of pAN903. The
complete sequence of this DNA fragment was established for both strands
(see Fig. 5). The sequencing strategy is described under
``Experimental Procedures.'' Our results revealed a single
long open reading frame of 1737 bp interrupted by a single putative
short intron of 46 bp recognized by the 5` and 3` consensus sequences
of fungal introns (Gurr et al., 1987) whose existence is
confirmed below. The direction of this open reading frame is consistent
with the direction of uapC transcription established by
Northern blot analysis using single-stranded radiolabeled probes
(results not shown). The high GC content of the uapC sequence
is in agreement with all previously characterized A. nidulans genes (Lloyd and Sharp, 1991).
(
)
The
sequence 5`-
CGGTGCCGCCCG
-3` at positions
-280/-269 is a candidate sequence for the target site of
the UaY regulatory protein as deduced by in vitro protection
and interference studies conducted with fusion proteins containing the
UaY zinc finger.
(
)
Finally, a sequence of unknown
significance, 5`-TTTGCATTA-3` at positions -180/-172 is
also found, similarly positioned (-125/-117), in the
uapA 5`-non-coding region (Gorfinkiel et al., 1993).
Figure 5:
Nucleotide Sequence of the uapC gene and amino acid sequence of its putative translation product.
The nucleotide sequence shown was determined by sequencing both strands
of a genomic clone and a nearly complete cDNA clone (see
``Experimental Procedures''). Restriction sites PstI
and XbaI are indicated to allow the orientation of this
sequence with respect to the physical map shown in Fig. 1. Putative
AreA binding 5`-GATA-3` sites are shown by horizontal arrows.
A putative UaY binding 5`-CGGTXCCCG-3` site is indicated by
an open box. A 5`-TATA-3` box and a sequence similar to a
uapA 5` non-coding sequence are shown overlined. The
translation start codon and the intron 5` and 3` consensus sequences
are shown underlined, and the translation stop codon is
indicated with an asterisk. The major start and stop points of
transcription are shown by black triangles while a possible
minor start of transcription is shown by a white triangle. A
putative 3` end polyadenylation signal is also
underlined.
The 3`-non-coding region of the uapC gene contains the
sequence 5`-TATGTA-3` which has been described previously as a
3`-polyadenylation signal in Saccharomyces cerevisiae (Russo
et al., 1991; Irniger and Braus, 1994). However, its location
downstream from the end of the uapC transcript (see below) and
its absence from other genes makes it rather improbable to be involved
in polyadenylation in A. nidulans.
Transcript Mapping of the uapC Gene
The 5` and 3`
ends of the uapC transcript as well as the presence and
position of a single short intron were established. The 5` start of
uapC transcription was mapped by primer extension experiments
using total RNAs extracted from both non-induced and induced wild-type
strains and a uapC-specific oligonucleotide (O4) close to the
start of uapC translation (see ``Experimental
Procedures''). Fig. 6 shows a major start site corresponding to
nucleotide -64 numbering from the ATG (see Fig. 5) for both
non-induced and induced mycelia. Induction by 2-thiouric acid led to
approximately 3-fold increased accumulation of uapC-specific
primer extension products, a result in agreement with our Northern blot
analyses (see Fig. 4, a and b). At position
-80, another minor transcription startpoint is detected under
non-inducing conditions. Within the transcribed sequence, an ATG codon
is present 34 nucleotides upstream from the proposed start codon, but
it leads to an open reading frame of only five amino acids.
S]dATP (results not shown). Fig. 5shows
the 3` end points of uapC transcription located 33 and 36
nucleotides downstream from the translation stop codon.
Analysis of the Predicted uapC Protein
The
uapC open reading frame encodes a protein of 580 amino acids
with a predicted molecular mass of 61251 Da (Fig. 5). Codon usage
is not biased. As expected for a transporter, the UapC protein is
extremely hydrophobic. The UapC hydropathy profile is practically
superimposable on that described for the UapA protein (Gorfinkiel
et al., 1993), independently of the algorithm and the
``window'' used to calculate the hydropathic character (Kyte
and Doolittle, 1982; Eisenberg et al., 1982); it shows
hydrophilic N and C termini and 12-14 (depending on the window
used) putative, -helical, transmembrane segments. No putative
glycosylation sites (Struck et al., 1978) were found in the
UapC amino acid sequence.
-helical region of UapA
(residues 530-547; Gorfinkiel et al., 1993) overlapping
with the last hydrophobic segment of the protein.
loss-of-function mutation
( uapA24), for growth on uric acid, which define uapC,
a gene mapping on chromosome I but recombining freely with the uapA gene. We constructed a series of double mutants carrying the
uapC201 gain-of-function mutation and loss-of-function
( uapA24, azgA4) or other gain-of function
( uap100) mutations in the genes coding for the two specific
purine permeases in A. nidulans and showed that uapC gain-of function mutations affect the transport of all purines and
analogues transported by the uapA and azgA gene
products. We further established the contribution of the uapC gene product to purine uptake by isolating and genetically
characterizing a series of apparent uapC
loss-of-function mutations, which were shown to be tightly linked
to the uapC gain-of-function mutations.
mutation and by insertional inactivation of the
uapC
resident gene. In Northern blots, the
cloned sequence detects a
2.0-kb RNA transcript whose level of
accumulation is differentially affected by uric acid induction and
ammonium repression or by the presence of an uapC gain-of-function mutation, the areA102 allele or a
uaY loss-of-function mutation. Finally, uapC sequencing has shown that this gene encodes a protein with a
plausible structure for a transporter and a very similar amino acid
sequence with the UapA permease.
(
)
Interestingly, we observed
that the putative UaY-binding site in the 5` regulatory region of the
uapC gene shows differences with the UaY target sites
established for the uapA, hxA (encoding purine
hydroxylase I) or uaZ (encoding urate oxidase; Oestreicher and
Scazzocchio, 1993) genes.
The putative uapC UaY-binding site is 5`-
CGGTGCCGCCCG
-3`
while the target sites in the regulatory regions of the other three
genes conform to the sequence
5`-
CGGAX
GCCG
-3`.
70% identical (90% similar) amino acids
residues in the UapA and UapC permeases. Another hydrophilic segment,
located between putative transmembrane domains 5 and 6, also shows
significant similarity (
56% identity) while the remaining, shorter,
hydrophilic segments are less similar in the two transporters. While
sequence similarities of hydrophobic segments might provide clues to
the structure or the topology of transport proteins, hydrophilic
segments with important similarities should, in principle, be relevant
to substrate recognition or to the mechanism of transport. If the UapC
protein includes 14 transmembrane segments (using an 11-amino-acid
``window'' to calculate its hydropathy), the two long
hydrophilic segments conserved in UapA and UapC would be exposed to the
exterior of the cell and are thus good candidates to be involved in
purine recognition and binding. The UapA and UapC permeases show
overlapping specificities and, probably, different capacities for
purine uptake. A few amino acid changes in these hydrophilic segments
might be responsible for these differences. We have noted that the UapC
permease lacks three acidic amino acid residues of the C-terminal,
amphipathic domain present in the UapA permease. Other, more subtle,
amino acid changes can be identified (for example, residue 372 in UapC
is a glutamate, while the corresponding residue in UapA is an arginine)
and might be of great help in the design of site-directed mutagenesis
and domain-swapping experiments for testing structure-function
relationships. The importance of the described hydrophilic region of
greatest similarity in the UapA and UapC permeases is further suggested
by the observation that this same region shows the highest similarity
score with YicE, a non-identified open reading frame of E. coli which may well be a purine transporter (see below).
Figure 7:
Amino acid sequence similarity alignment
of the UapC and UapA permeases by the GCG/BESTFIT program. Identical
amino acids are indicated by vertical lines, and similar amino
acids are indicated by two dots or a single dot. Gaps
are introduced by the program to maximize similarity scores. For both
permeases, hydrophobic segments which are considered putative
transmembrane domains are shown boxed. The UapC permease is
shown on top.
We have
also shown that the A. nidulans purine transporters are
similar to pyrimidine transporters of E. coli and Bacillus species (see Fig. 8). 22 amino acid residues are identical
and 42 additional amino acid residues are similar in all seven
transporters analyzed. Pair-wise comparisons show amino acid identity
scores from 22 to 60% (similarities from 51 to 78%) and suggest that
this group of proteins might be made of two subgroups, the purine and
the pyrimidine transporters. Interestingly, although the E. coli YicE protein is equally similar to the A. nidulans purine
permeases (59% similarity) and to the bacterial pyrimidine transporters
(53-58% similarity), it shares significantly more identical amino
acid residues with the first (33-34% identity) than with the
second (23-27% identity). This observation suggests that this
protein of unknown function might be the first prokaryotic purine
permease identified. If this is true, the alignment of the YicE and
UapA or UapC sequences should be revealing for the identification of
amino acid ``signature'' residues determining the ability of
a transporter to take up purines in both prokaryotes and eukaryotes.
Moreover, the putative product of an open reading frame of unknown
function in C. perfrigens was also found to belong to
the group of transporters described here. This open reading frame
consists of a previously described 567-bp open reading frame plus 366
bp of what has been proposed to be its immediate 5` upstream region
(Brynestad et al., 1994). We obtained a continuous 933-bp open
reading frame (whose putative 311 amino acid translation product is
shown in Fig. 8) by assuming a frameshift sequencing mistake, and
adding an ``X'' to ``re-establish'' the reading
frame, immediately upstream of the previously proposed ATG start codon.
This makes an incomplete amino acid sequence of a protein with a
significant identity similarity score (30-60%, respectively) with
all the purine-pyrimidine transporters described here. Based on
specific amino acid similarities, we believe that the C.
perfrigens DNA sequence might correspond to an incomplete
sequence of a purine transporter gene.
Figure 8:
Alignment of the UapC permease sequence
with all significantly similar (according to FASTA and BLAST programs)
proteins from data bases (SwissProt, Pir, GenEmbl) using the program
PILEUP. Identical amino acids are shown in black boxes and
similar amino acids in gray boxes ( light or deep
gray according degree of similarity). Gaps are introduced to
maximize similarity. The protein sequences shown are the following:
pyrp-Bs, pyrimidine transporter of B. subtilis (accession number in data bases P25982); pyrp-Bc, uracil
transporter of B. caldolyticus (X76083), uraa; uracil
transporter of E. coli (X73586); Uapa (X71807) and
Uapc permeases of A. nidulans described in the text;
Yice (P27432) a hydrophobic E. coli protein of
unknown function; Cpx, a putative protein of unknown function
from C. perfrigens (X71844).
We found no significant
sequence similarities to other prokaryotic or eukaryotic proteins,
including the purine-cytosine, allantoin, or allantoic acid
transporters of S. cerevisiae (Rai et al.,
1988; Yoo et al., 1992), or the nucleoside transporters of
bacteria (Craig et al., 1994) or mammalian cells (Pajor and
Wright, 1993; Huang et al., 1994). A purine-pyrimidine
transporter has been recently identified in mammalian cells (Griffith
and Jarvis, 1993) but the corresponding gene has not been cloned.
(
)
It will be of
great interest to know whether, in cases of transporter sequences
conserved between prokaryotes and eykaryotes, functional
complementation of prokaryote mutants can be achieved with eukaryotic
genes and vice versa. This would imply conservation in
structure and function of the proteins involved in transporter
translocation (Ljungdahl et al., 1992; Green et al.,
1992).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.