From the
Folylpoly-
Folylpoly-
The existence of mitochondrial and cytosolic
folylpolyglutamate pools in mammalian cells and tissues has been known
for some time
(4, 5) . FPGS was previously thought to be
located only in the cytoplasm, but subsequently it has also been found
in mitochondria
(6) . It is not clear whether the FPGS found in
these two cellular compartments represents the same protein or how FPGS
is targeted to the mitochondria. Recently, a cDNA that encoded a human
FPGS was reported
(7) . Upon transfection into the AUXB1 cells,
this sequence complemented the auxotrophy for thymidine and purine, but
not the requirement for glycine which is known to be synthesized in the
mitochondria
(8, 9) . This was unexpected, in view of
the fact that AUXB1 cells transfected with high molecular weight human
DNA regain prototrophy for thymidine and purines coincident with that
for glycine and that such transfectants express FPGS in both cellular
compartments
(10) .
FPGS is abundant in tumors, and in normal
gut and bone marrow stem cells, liver, and kidney but is, otherwise,
not appreciably expressed in adult tissues
(11, 12, 13, 14) . From a recent study
in this laboratory
(13) , it was concluded that FPGS levels are
controlled by at least two mechanisms, one of which is linked to
proliferation and the other acts during differentiation and is
tissue-specific. Recent studies suggest that the sensitivity of human
tumors to anti-folate chemotherapy is related to the level of
expression of FPGS
(13, 14, 15, 16) . In
addition, initial evidence is supportive of the existence of isoforms
of FPGS which accept different spectra of folate analogs as substrates
(17) . Should appreciable differences exist in either the
isoform or the control of expression of FPGS in normal stem cells and
neoplastic cells, it would represent an opportunity for the design of
selective cancer chemotherapy.
In this manuscript, we report the
cDNA sequence corresponding to the 5` termini of mature FPGS
transcripts and the organization and structure of the corresponding
region of the human genomic locus. The sequences of the cDNA molecules
studied and the transcriptional start site usage in human cell lines
and in AUXB1 cells transfected with human genomic DNA indicated that
two forms of FPGS mRNA were made from a single gene. One form
corresponded to the published, apparently cytosolic form of the enzyme,
and the other contained an additional 5` sequence compatible with a
mitochondrial leader peptide which, when transfected into cells lacking
FPGS, complemented the entire mutant phenotype.
Nitrocellulose was from Schleicher and Schuell. T4 DNA ligase,
T4 polynucleotide kinase, restriction enzymes, and Taq polymerase were from Promega. MaeIII endonuclease was
from Boehringer Mannheim. Total RNA was prepared by the method of
Chomczynski and Sacchi
(18) . Poly(A)
The first 363 nt of the FPGS cDNA (Fig. 2)
were located within the 6.0-kb fragment, distributed onto three exons
that were 180, 129, and 54 bp in length. The sequences of these exons,
the positions of introns I and II which were 1039 and 85 nt long,
respectively, and 361 bp immediately upstream from exon I are shown in
Fig. 2
. Both upstream and downstream translational start sites
were located on exon I, an observation that rules out alternative exon
usage of this gene as the mechanism for the formation of cytosolic and
mitochondrial forms of FPGS. The 5`-flanking region of the FPGS gene did not contain a canonical TATA sequence nor a CCAAT motif.
However, two inverted CCAAT boxes, i.e. Y boxes, were present
at positions -57 and -20 nt (Fig. 2). The 5`-flanking
sequence and exon I were very GC-rich (74% from nt -221 to nt
+180) and contained eight forward (GGGCGG) and one reverse
(CCGCCC) SP1 binding sites.
The two
patterns expected for opposite single alleles were seen in the reaction
using RNA from the AUXB1 cells transfected with CEM DNA: RNA from the
FC2/2D cell line did not protect the probe at position 106 permitting
RNA cleavage at this site resulting in the presence of the 69-bp
fragment, whereas RNA from the FB1/2C transfectant showed a simple
pattern of multiple protected bands without the 69-bp fragment
(Fig. 3 a). The fragments protected by RNA from FC2/2D
cells ( lane 3, Fig. 3, hollow arrows) were
exactly 69 nt less than the three longest major products protected by
RNA from FB1/2C and appeared to be generated by cleavage at the tsp and at the polymorphic mismatch. The protection of RNA from six
cell lines differing in homozygosity at position 106 of the fpgs locus is shown in Fig. 3 b. RNA from homozygous cell
lines with sequence matching the probe (MCF7 and H209) did not result
in a 69-bp fragment, and, hence, the protected bands from lanes 11 and 12 in Fig. 3 b directly indicate
transcriptional start site usage. The reactions run with RNA isolated
from the homozygous cell lines bearing the opposite allelic variant at
position 106 resulted in the appearance of the 69-bp fragment
( lanes 7 and 8, Fig. 3 b). RNA from the
heterozygous cell lines CEM and LAN-5 ( lanes 9 and
10) showed protection of the longer bands and allowed the
cleavage of probe to the 69-bp fragment. The probe fragments protected
from ribonuclease by RNA from the MCF7 and H209 cells and from the
FB1/2C cells indicated the same set of multiple transcriptional start
sites in all of these cell lines; the major sites are mapped to the
upstream genomic structure of the FPGS gene in Fig. 2at
positions -2, +6, +46, and +78.
The 5`-RACE and
the ribonuclease protection experiments both indicate at least three
major and multiple minor transcriptional start sites. The positions of
the sites are consistent between the experiments within the constraints
of these techniques.
The ability of several
clonally isolated transfectant cell lines to form colonies with and
without glycine, adenosine, and thymidine is shown in . As
expected, the parental AUXB1 cells can form colonies only in the
presence of glycine, adenosine, and thymidine, whereas cells
transfected with pC-FPGS form colonies in the absence of nucleosides
but still require glycine. Cells transfected with pM-FPGS did not
require glycine or nucleosides, mimicking the wild type CHO and cells
transfected with CEM genomic DNA (FC2/2D cells).
We have shown that the human FPGS promoter drives
transcription from multiple tsps spread over approximately 80
bp. These mRNAs contain up to 99 bp of previously unreported 5`
sequence including an AUG codon which is in-frame with the previously
reported open reading frame and satisfies the requirements for
efficient translation defined by Kozak
(25) . Transfection of
the published FPGS cDNA complemented only the thymidine and purine
auxotrophy resultant from FPGS deficiency (7 and ).
However, transfection of a cDNA corresponding to the extended message
complemented the entire FPGS
In yeast there are several enzymes present in both the
cytoplasm and the mitochondria which originate from a single genetic
locus. These include histidine-tRNA synthetase
(29) ,
isopropylmalate synthetase
(30) , fumarase
(31) ,
valyl-tRNA synthetase
(32) , and
In
CHO cells, 50-65% of total cellular FPGS activity was reported to
be associated with the mitochondrial fraction
(6) . We
quantitated the intensity of RNase-protected bands in the G
Analysis of the putative FPGS promoter revealed a GC-rich region
with no canonical TATA or CCAAT sequences, a structure usually
associated with ``housekeeping'' genes and proto-oncogenes.
TATA-less promoters were previously believed to drive unregulated,
constitutive expression but have since been shown to respond to a
variety of stimuli with a range of transcriptional regulatory
responses. This group includes genes that are expressed differentially
during embryogenesis, are tissue-specific, and respond to viral and
pharmacological stimuli (reviewed in Ref. 37). Many of these genes are
growth-regulated with low levels in nongrowing cells which become
elevated as cells are stimulated to proliferate. This is consistent
with the distribution of FPGS activity in different cell types and
various growth and differentiation states
(12, 13) .
Numerous transcription factor binding sites were found in the promoter
region and in intron 1 (not shown), but their relative importance to
FPGS gene regulation remains to be determined. Of particular interest
will be the significance of the two Y boxes in the immediate promoter
area. In MHC II genes, the Y box consensus sequence is required for
constitutive and cytokine-mediated gene expression
(38) . Y box
consensus sequences in the proximal promoter region are also required
for the basal expression of the human MDR1 gene
(39) and the thymidine kinase gene
(40, 41) .
This study supports the hypothesis that the mitochondrial and
cytosolic forms of FPGS are derived from a single gene. The control of
expression of this gene is of practical significance: four recent
reports of pediatric acute lymphoblastic leukemia
(13, 14, 15, 16) have suggested a
correlation between levels of FPGS activity in leukemias and the
outcome of antifolate chemotherapy. Clearly, a basic understanding of
the regulation of this enzyme in normal stem cells and neoplastic cells
is now needed.
Colony-forming assays were
carried out in 100-mm tissue culture dishes using 10 ml of media per
plate. 150 cells were plated per dish in GAT media and left overnight.
The next day, cells were washed once in PBS, and the selective media
were applied. After 8 days, the colonies were stained and counted.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U14938 and U14939
(hamster and human sequences, respectively).
We thank Drs. Julie L.-C. Kan and Eric Westin for
their useful discussions throughout this work. We also appreciate the
guidance and encouragement of Dr. Norman Davidson during the early
phases of related work which led to this study.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-glutamate synthetase (FPGS) is essential for the
survival of proliferating mammalian cells and central to the action of
all ``classical'' folate antimetabolites. We report the
isolation of cDNAs corresponding to the 5` ends of FPGS mRNA from both
human and hamster cells which include a start codon upstream of and
in-frame with the AUG in the previously reported FPGS open reading
frame. The predicted hamster and human amino-terminal extension
peptides have features consistent with a mitochondrial targeting
sequence. Ribonuclease protection and 5`-rapid amplification of cDNA
ends assays indicated multiple transcriptional start sites consistent
with the sequence of the promoter region of this gene, which was highly
GC-rich and did not contain TATA or CCAAT elements. These start sites
would generate two classes of transcripts, one including the upstream
AUG and one in which only the downstream AUG would be available for
translation initiation. Transfection of the full length human cDNA into
cells lacking FPGS restored their ability to grow in the absence of
glycine, a product of mitochondrial folate metabolism, as well as of
thymidine and purines. Therefore, we propose that the mitochondrial and
cytosolic forms of FPGS are derived from the same gene, arising from
the use of the two different translation initiation codons, and that
the translation products differ by the presence of a 42-residue
amino-terminal mitochondrial leader peptide.
-glutamate synthetase (FPGS)
(
)
catalyzes the ATP-dependent formation of an amide bond
between the
-carboxyl group of the naturally occurring folates and
the amino group of glutamic acid. The addition of glutamic acid
moieties to folate compounds allows their intracellular retention and
concentration for both the naturally occurring folate compounds
(1, 2, 3) as well as for all of the
``classical'' folate antimetabolites studied to date. As a
result of its role in the retention of folate cofactors in the cell,
FPGS is essential for the survival of proliferating mammalian cells.
This concept is most directly supported by the phenotype of a mutant
CHO cell, AUXB1, which lacks measurable function of FPGS
(3) and, as a result, is auxotrophic for thymidine, glycine, and
purines
(2) , i.e. it cannot provide 1-carbon units via
folate metabolism.
-selected
RNA was isolated using the FastTrack
system (Invitrogen).
The human cell lines used were CEM (acute lymphoblastic leukemia, T
cell), MCF7 (breast adenocarcinoma), H209 (small cell lung cancer),
LAN-5 (neuroblastoma), COLO 205 (colon adenocarcinoma) and HT-29 (colon
adenocarcinoma). Growth conditions for these cells followed ATCC
recommendations.
5`-RACE and PCR
5`-RACE was carried out essentially as
described by Frohman et al. (23) . First strand cDNA
was synthesized using 1 µg of poly(A)-selected
CCRF-CEM RNA and 2 pmol of antisense primer F8
(5`-CTTGGTGAAGAGCTCAGGACTG-3`). PCR was carried out for 35 cycles (95
°C, 61 °C, and 72 °C for 1 min each) with 2.5 µl of the
poly(C)-tailed template in a 25-µl reaction with the anchored
primer and primer F21 (5`-ACTCCGTGCCAGGTACAGTTCCATG-3`). The PCR
products were ligated into the pCRII vector (Invitrogen) for
restriction analysis and double-stranded sequencing. The 5` region of
the hamster cDNA was PCR-amplified using the human FPGS primers Sta 1
(5`-ACTATGTCGCGGGCGCGGAGCCAC-3`) and FPE2
(5`-GGTACAGTTCCATGGCTTCCAACTGTGTCTGAGGG-3`) with cycling conditions as
before. From this sequence, hamster-specific primers were designed for
5`-RACE which was performed as described above. First strand synthesis
was from 1 µg of CHO poly(A)
-selected RNA with
primer HF1 (5`-TTTACCTGCTCCAGGTAGCTG-3`). PCR was then carried out with
the anchored primer and the nested primer HF2
(5`-AGCTGGCATTGGTCTGCAGGGTG-3`).
Cloning and Nucleotide Sequencing of the Human FPGS
Gene
A PCR-generated cDNA fragment representing the first 690 bp
of the published human FPGS open reading frame
(7) was used as
a probe to screen a human male placenta genomic library in the
Fix
II vector (Stratagene)
(24) . Restriction
analysis of positive clones demonstrated that the 5`-most sequence of
the FPGS cDNA lay on a 6.0-kb HindIII- NotI fragment
of
BL. This fragment was subcloned into pBluescriptII
SK
, and a 2-kb region was sequenced in both
directions.
Determination of the Transcriptional Start Sites Using
Ribonuclease Protection Analysis
Ribonuclease protection assays
were performed essentially as described
(24) . The probe was
synthesized from a genomic DNA template between two RsaI sites
and covered the first 175 nt of exon I and 203 nt immediately upstream
(see Fig. 2). 1-2.5 10
cpm of labeled
RNA was used per sample. Hybridization was performed overnight at 57
°C, and the mixture was digested in 350 µl of 100 µg/ml
RNase A for 30 min at 30 °C. Protected fragments were resolved on a
6% polyacrylamide/urea gel.
Figure 2:
Nucleotide sequence of the putative
promoter and first three exons of the human FPGS gene. The
thymidine which was the 5`-most transcription site defined by 5`-RACE
analysis is numbered +1. The major transcription start sites
defined by ribonuclease protection analysis are marked with
vertical arrows. Putative SP1 binding sites are
underlined, Y boxes are in bold type, and the two
proposed start methionines are boxed. The position of the
polymorphism is indicated at +106 as are the RsaI sites
used to synthesize the ribonuclease protection
probe.
Generation of AUXB1-Human Genomic DNA
Transformants
AUXB1 cells
(2) were transfected by
calcium phosphate precipitation
(19) , using high molecular
weight DNA from human CEM cells (20 µg/10cells) mixed
with the plasmid pY3 (5 µg/10
cells), which encodes
resistance to hygromycin B
(20) . Initial selection with 400
µg/ml hygromycin B was applied 48 h after transfection, and double
selection was applied 10-14 days later by the addition of media
lacking deoxyribo- and ribonucleosides, supplemented with 10% dialyzed
fetal bovine serum and with hygromycin B. The cloned cell lines used in
this study, FB1/2C and FC2/2D, were independently derived secondary
transfectants from DNA of primary transfectant cells generated in
separate experiments
(21) . FPGS expressed in both cell lines
was judged as CEM-derived on the basis of a ratio of activity with ATP
to that with dATP characteristic of human enzyme
(22) .
Generation of AUXB1-Human cDNA Transformants
The
published FPGS cDNA was generated by reverse transcriptase PCR
amplification of mRNA from CEM lymphoblasts and a fragment stretching
from the proposed
(7) downstream start methionine to 200 bp
into the 3`-untranslated region was inserted into the eukaryotic
expression vector pcDNA3 (Invitrogen) (pC-FPGS). To construct the
vector which would express the putative mitochondrial isoform
(pM-FPGS), the coding sequence identified in Fig. 1 a was
inserted into pC-FPGS so that the two vectors differed only by the
presence of the 42-codon putative leader sequence. Transfection was
carried out essentially as described above (5 µg of plasmid/5
10
cells), and selection was applied 48 h after
transfection using G418 (800 µg/ml) in either complete
-minimal essential medium or nucleoside-deficient medium. Three
independent clones were selected by two subcultures of individual
colonies from the pC-FPGS and pM-FPGS transfected AUXB1 cells surviving
double selection. In the colony-forming assays, combinations of glycine
(133 µ
M), adenosine (36 µ
M), and thymidine
(36 µ
M) were added to
-minimal essential medium
formulated without these components.
Figure 1:
a, transcriptional start sites for
human FPGS mRNA as determined by 5`-RACE analysis and comparison of
amino acid sequence with hamster FPGS. Arrows represent the
positions of the tsps; the numbers over the top represent site frequency (when >1) from 2 separate experiments.
The consensus sequences for translation initiation are underlined with the codons for the proposed start methionines in bold
type. Dots indicate identity between human and hamster
sequences. b, helical wheel analysis of the amino-terminal
extension peptide. Hydrophobic residues are boxed, and
positive charges are indicated.
The 5` Sequence of FPGS Transcripts
We used the
PCR-based 5`-RACE technique
(23) to determine whether there was
additional sequence upstream from the published GC-rich FPGS cDNA
(7) represented in poly(A)-selected mRNA from
CEM human leukemic cells. PCR with an anchor primer and the internally
nested primer, located 202 bp from the 5` end of the published
sequence, resulted in the amplification of a set of cDNAs ranging in
size from approximately 150-300 nt. These PCR products were
ligated into the pCR II plasmid; 25 clones were selected and sequenced
with some bias toward longer clones. All of the inserts examined
contained the published human FPGS sequence
(7) , but 20/25
cloned inserts extended further upstream, with as much as 99 additional
base pairs corresponding to the most 5` region of the CEM FPGS
transcripts. Three transcriptional initiation starts were repetitively
identified by this analysis (Fig. 1 a). A methionine codon was
found in this upstream sequence which fit the consensus for efficient
eukaryotic ribosome binding
(25) and was in-frame with the
previously defined
(7) downstream translational initiation
codon. The 42-amino acid sequence (Fig. 1 a) encoded by
the nucleotides between the two ATGs had a noticeable absence of acidic
residues, and a preponderance of arginine (6/42), serine (4/42), and
leucine (4/42) residues relative to what would be expected for the
amino terminus of cytosolic proteins. Helical wheel analysis of the
first 36 residues from the amino terminus revealed the potential to
form an amphipathic
-helix with positively charged and hydrophobic
faces (Fig. 1 b). These are all features thought to be
essential in a leader peptide needed for the passage of proteins
through the mitochondrial membranes
(26, 27, 28) . Reported vertebrate leader
peptides, which are proteolytically cleaved after entry into the
mitochondria, range in size from 20 to 60 amino acids
(26, 27) .
A Comparison of the 5` Termini of the FPGS Transcripts
Found in Human and Hamster Cells
We were able to amplify a
250-bp region of the hamster FPGS cDNA using primers chosen from the
upstream human cDNA sequence. The sequence of this region was
homologous to the human FPGS sequence (Fig. 1 a). When
the hamster sequence was extended using 5`-RACE, an upstream ATG was
found at an identical location as in the human sequence and, again,
this codon was in-frame with the downstream ATG and obeyed
Kozak's consensus sequence
(25) . The entire putative
mitochondrial leader peptide was predicted to be 78% identical in
hamster and human sequences. Downstream of the 3`-methionine, the amino
acid homology was 93% over the sequence studied.
Cloning of the Human FPGS Genomic Locus
Forty
positive plaques were isolated from a screen of 1.5
10
recombinants; three classes of overlapping inserts were
found which covered the entire human FPGS genomic locus. Mapping
studies indicated that one clone (
BL) contained the 5` end of the
FPGS open reading frame and at least 10 kb of sequence upstream. A
6.0-kb NotI- HindIII fragment of
BL which
hybridized with the most upstream probes from the FPGS cDNA was
subcloned into pBluescript II SK
for fine mapping and
partial sequencing.
Ribonuclease Protection Analysis of the Human FPGS
Gene
Initial experiments using total RNA from CEM cells (Fig.
3 b, lane 9) indicated multiple transcriptional start
points ( tsps) with major bands representing start sites at
positions -2, +6, +46, +78, and +107 (with
respect to the 5`-most transcription start site detected by RACE). The
abundance of the small protected fragment at 69 bp (indicated by the
hatched arrow on Fig. 3) had not been predicted by the
RACE experiments (Fig. 1 a). Analysis of the sequence of
the 5`-RACE-derived human cDNA clones demonstrated a single base pair
polymorphism 69 bp from the 3` end of the riboprobe in CEM cell RNA
(see Fig. 2): 63% of the clones analyzed had an A at this
position and 37% had a G residue ( n = 19) (the residue
present in the riboprobe). Because a single base mismatch is often
sufficient for ribonuclease cleavage, this polymorphism introduced an
uncertainty in the interpretation of these ribonuclease protection
experiments and suggested that the 69-bp band was artifactual.
Figure 3:
Definition of the transcriptional start
sites of the human FPGS gene by ribonuclease protection. The
RNA probe used was a 378-nt fragment positioned between the
RsaI sites indicated in Fig. 2. a, lane 1 contains 1000 cpm of undigested riboprobe. Protection analysis was
performed on 30 µg of yeast tRNA and on 2 µg of
poly(A)RNA from AUXB1/CEM DNA transfectants FC2/2D
RNA and FB1/2C RNA ( lanes 2-4, respectively).
b, analysis was also performed on 30 µg of yeast tRNA and
30 µg of L1210 murine total RNA as controls ( lanes 5 and
6) and on 30 µg of total RNA from COLO 205, HT-29, CEM,
LAN-5, MCF7, and H209 human cell lines ( lanes 7-12,
respectively). Filled arrows represent major transcription
start sites, the bracket represents a group of minor start
sites, the hatched arrow indicates the position of the major
band caused by the polymorphism, and the hollow arrows represent other cleavage products of the polymorphism, most
clearly visible in lane 3 but also evident in lanes 7 and 8. The marker set used was HinfI-digested
X174 DNA.
We
used two complementary approaches to determine which bands represented
bona fide tsps and which resulted from this polymorphism. In
the first, we used cell lines in which the fpgsphenotype of AUXB1 cells was complemented by calcium
phosphate-mediated transfection with high molecular weight DNA from CEM
cells
(21) . These transfectants would be expected to be haploid
for the human fpgs locus due to the low frequency of
complementation and the low amount of human DNA stably integrated per
transfectant
(19) . Two of these transfected cell lines were
screened for the polymorphism using the fact that one of the allelic
variants represented the recognition sequence (5`-GTNAC-3`) for the
restriction enzyme MaeIII. Cell line FB1/2C contained only the
human fpgs allele with a G at position 106, whereas FC2/2D
contained only the other allele. In the second approach, several human
cell lines were screened for the polymorphism using the MaeIII
site and a selection of cell lines found to be either homozygotic and
heterozygotic by this criterion were analyzed further.
Transfection of FPGS cDNAs into AUXB1 Cells
The
ability of FPGS cDNAs carrying the additional upstream ATG to
complement the adenosine, thymidine, and glycine auxotrophy in AUXB1
cells was evaluated using stable AUXB1 transfectants. The transfection
efficiency of AUXB1 cells with the pC-FPGS and pM-FPGS constructs
ranged between 1 in 400 to 1 in 800. Of the AUXB1 colonies which had
acquired G418 resistance from the FPGS constructs, approximately 80%
were viable on media lacking nucleosides.
phenotype including
auxotrophy for glycine, which is synthesized in the mitochondria. We
propose therefore that differential usage of the two translational
start codons determines the cytosolic or mitochondrial location of
FPGS.
-isopentenyl
pyrophosphate:tRNA isopentanyltransferase I
(33) . Each of these
genes contain two in-frame ATG codons, both of which can be used to
initiate translation resulting in the production of proteins differing
only by the presence or absence of an amino-terminal extension peptide.
Although less frequent, this phenomenon has also been documented in
mammals, examples of which include rat liver mitochondrial and
peroxisomal serine:pyruvate aminotransferase
(34) , and rat
cytosolic and mitochondrial fuma-rases
(35) . In both of these
cases, the two potential translation initiation codons are found within
exon I as in the FPGS gene. The mRNA which codes for rat
mitochondrial serine:pyruvate aminotransferase is transcribed from a
start site 70 bp upstream of that for the cytosolic form and encodes an
amino-terminal 22-residue peptide essential for the translocation of
the protein into the mitochondria. For the rat fumarases, it has been
proposed that both mitochondrial and cytosolic forms are translated
from one species of mRNA and that it is the secondary structure of the
5`-noncoding region which determines initiation codon usage
(35) . The ratio of cytosolic to mitochondrial FPGS levels may
therefore be determined simply by the relative levels of start site
usage or there may be a component of translational regulation.
homozygous cell lines using a phosphorimaging system. From four
evaluable cell lines, 66-81% of transcripts contain sufficient
upstream sequence for translation of the mitochondrial FPGS precursor.
In a recent study, Escherichia coli FPGS cDNA was transfected
into AUXB1 cells
(36) . E. coli FPGS was expressed only
in the cytosol, and this expression reversed the auxotrophy for purines
and thymidine of these cells, but not that for glycine, the synthesis
of which is thought to occur mainly in the mitochondria
(8, 9) . E. coli FPGS directed to the
mitochondria only, using a synthetic leader sequence, complemented all
three growth requirements. We now report the occurrence, sequence, and
function of the endogenous mitochondrial leader sequence for FPGS. It
would appear that folylpolyglutamates cannot enter the mitochondria,
but their function is required in that organelle for glycine synthesis.
Table:
Viability of CHO cell transfectants in media
with and without exogenous glycine
-glutamate synthetase; 5`-RACE, 5`-rapid amplification
of cDNA ends; tsp, transcription start point; PCR, polymerase
chain reaction; bp, base pair(s); kb, kilobase(s); nt, nucleotide(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.