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J Biol Chem, Vol. 273, Issue 44, 29118-29126, October 30, 1998
From the Department of Molecular Microbiology and Immunology,
Oregon Health Sciences University, Portland, Oregon 97201
We have studied the structure and expression of
glucose transporter genes in the parasitic protozoan Leishmania
mexicana. Three distinct glucose transporter isoforms, LmGT1,
LmGT2, and LmGT3, are encoded by single copy genes that are clustered
together at a single locus. Quantitation of Northern blots reveals that LmGT2 mRNA is present at ~15-fold higher level in
promastigotes, the insect stage of the parasite life cycle, compared
with amastigotes, the intracellular stage of the life cycle that lives
within the mammalian host. In contrast, LmGT1 and
LmGT3 mRNAs are expressed at similar levels in both
life cycle stages. Transcription of the LmGT genes in
promastigotes and axenically cultured amastigotes occurs at similar
levels, as measured by nuclear run-on transcription. Consequently, the
~15-fold up-regulation of LmGT2 mRNA levels in
promastigotes compared with amastigotes must be controlled at the
post-transcriptional level. Measurement of LmGT2 RNA decay in promastigotes and axenic amastigotes treated with actinomycin D
suggests that differential mRNA stability may play a role in regulating glucose transporter mRNA levels in the two life cycle stages.
Leishmania are important human pathogens (1) whose life
cycle involves transmission of an extracellular flagellate
(promastigote) from the alimentary tract of a sandfly vector to a
mammalian host, where the parasite multiplies intracellularly in
macrophages as an aflagellate (amastigote) (2). The pronounced
biological changes that occur during the life cycle underscore the
importance of developmental regulation of gene expression to parasite
survival in these contrasting and hostile environments. Yet, the
mechanisms responsible for regulation of gene expression in
Leishmania and related kinetoplastid protozoa have not been
extensively studied. To probe the mechanisms controlling gene
expression during the parasite life cycle, we are studying a family of
glucose transporters that contains both developmentally regulated and
constitutively expressed members.
Metabolism of glucose is of considerable interest in these parasites
since it provides a major source of energy to the promastigote (3), and
it involves some novel biochemical features such as specialized
membrane-bound organelles called glycosomes (4) that contain many of
the glycolytic enzymes. In Leishmania species, the permeases
for glucose are structurally related to the 12 transmembrane-spanning mammalian facilitative glucose transporters (5, 6). Previous studies
with Leishmania enriettii (5) have shown that the mRNAs encoding these glucose transporters are present at relatively abundant
levels in promastigotes but are dramatically down-regulated in
amastigotes. Down-regulation of glucose transporter expression in the
amastigote presumably reflects the lower glucose concentration that the
parasite encounters in the macrophage. Indeed, Leishmania amastigotes transport much less glucose than promastigotes (7) and
derive metabolic energy primarily from fatty acid oxidation (8). Thus,
the glucose transporter genes encode proteins that are likely to play
an important role in parasite life cycle adaptation, and they present
an attractive model in which to address the molecular mechanisms of
gene regulation during the parasite life cycle.
However, the study of developmentally regulated gene expression in
Leishmania has been hampered by the inherent difficulty in
obtaining substantial numbers of viable, pure amastigotes. Furthermore,
experiments involving expression of wild-type or altered genes from
extrachromosomal expression vectors (9) are impractical in
intracellular amastigotes, since transfected parasites would have to be
cultured in the presence of selective drugs, such as neomycin or
hygromycin, which are toxic to the host macrophages. One way to
overcome the technical difficulties of studying gene expression in
amastigotes is to use axenically cultured amastigotes as a model system
(10-12). Leishmania mexicana is one species that can be
grown in axenic culture and exhibits many of the morphological and
biochemical properties of intracellular amastigotes (13). To study the
developmentally regulated expression of glucose transporter genes in
this axenic model system, we have cloned glucose transporter genes from
L. mexicana. Mapping and sequencing of genomic clones
encoding these transporters (referred to hereafter as
LmGT1 genes,
indicating their identity as L. mexicana glucose
transporters) demonstrate that there are three genes that encode three
distinct isoforms of this permease, LmGT1, LmGT2, and LmGT3.
LmGT2 mRNA is strongly regulated during the parasite
life cycle, since it is present at a ~15-fold higher level in
promastigotes compared with amastigotes. In contrast, the less abundant
LmGT1 and LmGT3 mRNAs are present at similar
levels in both life cycle stages. Transcription of the LmGT
genes is constitutive in promastigotes and axenic amastigotes;
consequently, expression of LmGT2 is
post-transcriptionally regulated. Measurement of LmGT2
mRNA half-life suggests a role for differential mRNA turnover
in the control of glucose transporter gene expression during the
parasite life cycle. These results underscore the importance of
post-transcriptional regulatory mechanisms in Leishmania
parasites and also reveal for the first time the existence of both
developmentally regulated and constitutively expressed glucose
transporters in the genus.
Growth of Parasites and Isolation of Nucleic Acids--
L.
mexicana promastigotes (WHO reference strain M379) were grown at
26 °C in Dulbecco's modified Eagle's medium-L (14) containing 10%
heat-inactivated fetal calf serum. Axenic amastigotes were grown in a
simplified version of JH-30 medium (15), developed by Dr. David Russell
(Washington University, St. Louis) and designated JH-30A, consisting of
M199 (11 g·liter
In vivo amastigotes were grown in a macrophage-like cell
line, J774.G8 (16). J774.G8 cells were maintained in RPMI 1640 growth
medium supplemented with 20 mM HEPES buffer, 2 mM L-glutamine, and 20% heat-inactivated fetal
calf serum, at 35 °C in a humidified atmosphere containing 5%
CO2. When monolayers of J774.G8 were confluent, they were
infected with early stationary phase promastigotes at a ratio of 50 parasites per host cell. Infected monolayers were incubated for 12-24
h at 32.5 °C before unincorporated parasites were removed by washing
and replacing the growth medium. After incubation for an additional
24 h, monolayers were washed twice with ice-cold
phosphate-buffered saline, pH 7.3 and detached by scraping. J774.G8
cells were lysed by repeated passage through a 30-gauge needle, and the
amastigotes were purified by Percoll density gradient centrifugation
(16).
Isolation of genomic DNA and preparation of Southern and Northern blots
were performed as described (17). Total RNA was prepared as reported
(18). The phage library of EcoRI-digested L. mexicana genomic DNA in the Lambda DASH II vector (Stratagene) was
prepared according to protocols supplied by the manufacturer, and
screening, isolation, and subcloning were performed by standard protocols (19).
DNA Sequencing and Analysis--
DNA sequences were obtained
from the two KpnI fragments and the two
KpnI/EcoRI fragments, which together span the
LmGT locus (Fig. 1B) using an ABI 373 automated
sequencer (Perkin Elmer). Protein-coding regions and 5'-UTRs were
sequenced completely on both strands, whereas only partial sequence was
obtained for 3'-UTRs and intergenic regions. DNA sequences of cloned
PCR products and from other genomic clones derived from the
LmGT locus were obtained using the Sequitherm EXCEL DNA
sequencing kit (Epicentre Technologies Inc.).
Comparisons of the LmGT-predicted protein sequences to protein
sequences in the GenBank data base were performed using the FASTA
algorithm (20). Predicted membrane-spanning domains were determined
using the Eisenberg algorithm (21).
Use of the Polymerase Chain Reaction to Map the 5' Ends of LmGT
Transcripts--
Reverse transcription of total promastigote RNA and
PCR amplification of resulting cDNA was performed as described
previously (6). The forward primer was the 39-nucleotide sequence of
the spliced leader from Leishmania donovani (22).
Gene-specific reverse primers were the reverse complement of the
nucleotide sequences encoding divergent regions within the
NH2 terminus of the putative protein-coding regions for
LmGT1, LmGT2, and LmGT3. Thus, the
reverse primers were 5'-ATAGCTCCTTATTTGGGGATATAGACTCC-3' (LmGT1),
5'-GCTGCAGGATCCCGTGAATGGGCTCCTTTTCGGACCTAGAGC-3'
(LmGT2), and
5'-GCTGCAGGATCCCGTGCCGCTCCTCGGACAACCAGTTCGAGC-3'
(LmGT3), where the underlined nucleotides represent a
BamHI site introduced to facilitate subcloning of the PCR
products. Amplification products were either digested with
BamHI and subcloned into the BamHI site of the
plasmid vector pBluescript (Stratagene) or they were subcloned directly
into the pGEMT vector (Stratagene), and the nucleotide sequence
was determined to reveal the site for spliced leader addition.
Use of a Nested Polymerase Chain Reaction to Clone the 3' End of
the LmGT3 Transcript--
Since the sequence encoding the
COOH-terminal region of the LmGT3 isoform was not contained within the
~14-kb genomic EcoRI fragment obtained from the genomic
library (Fig. 1B), this region of the LmGT3 gene
was obtained by PCR amplification of cDNA. Reverse transcription of
total promastigote RNA was performed as described previously (6), using
the following oligonucleotide designed to prime from the poly(A)
tail: 5'-GGCCGGATCCGAATTCTTTTTTTTTTTTTTTTT-3'. Two consecutive
PCRs were performed, both using the above oligonucleotide as a reverse
primer. The first 13 nucleotides in this primer provided an
"anchor" that facilitated performing the PCR at a higher annealing temperature than is possible using oligo(dT) alone. The forward primer
for the first reaction, primed with cDNA, was 21 nucleotides from
the sequence that encodes the predicted hydrophilic loop between
transmembrane segments 10 and 11, identical in all three LmGT genes (primer 1, Fig. 1B:
5'-CTGGACTGACCGCGAGAGGCGC-3'). The forward primer for the second
reaction, templated with a small aliquot of the product from the first
reaction, was the 21 nucleotides upstream of the 3' end of the
EcoRI site near the predicted COOH terminus of LmGT3 (primer
2, Fig. 1B: 5'-AGGGAGCTGAGCGGGGAATTC-3'). This primer is
specific for LmGT3. The product of this PCR, called PCR1,
was sequenced directly to obtain the sequence of the COOH terminus (the
last six amino acids) and part of the 3'-UTR of the LmGT3
gene. The observation that PCR1 is only 1.8 kb, whereas LmGT3 mRNA is 7.5 kb long and hence contains a 3'-UTR of
almost 6 kb, indicates that the oligo(dT)-containing primer described above that was used to template cDNA synthesis did not actually prime from the poly(A) tail but initiated synthesis at an internal site
within the 3'-UTR.
To confirm that this PCR product represented a sequence that was
contiguous with the rest of the LmGT3 gene, PCR was
exploited to amplify from cDNA the sequences flanking the
EcoRI site within the predicted COOH terminus of LmGT3. The
forward primer was the oligonucleotide used in the first reaction above
(primer 1) and contained within the body of the LmGT3 coding
region, whereas the reverse primer was the reverse complement of the
nucleotide sequence ~200-220 nucleotides downstream of the putative
translation stop for LmGT3 (primer 3, Fig. 1B:
5'-AAAGGCGAAAGCCAACGGAGTG-3'), obtained from the sequence of PCR1.
Sequence analysis of this second PCR product, PCR2, confirmed that the
body of the LmGT3 protein-coding region was linked to the
COOH-terminal coding region originally obtained by sequencing PCR1.
Probes Used for Detection of LmGT1, LmGT2, and LmGT3
RNAs--
The LmGT coding region probe used for detection
of RNAs on Northern blots (Fig. 4A) was an antisense RNA of
~1.2 kb, derived from cloned DNA between the BglII site
upstream of the LmGT2 open reading frame and the
PstI site within the LmGT2 open reading frame
(Fig. 1B). The LmGT1 probe (Fig. 4B)
was a DNA probe of ~1.2 kb, encompassing sequence from a
SmaI site ~1 kb downstream of the LmGT1 open
reading frame to the KpnI site ~2.2 kb downstream of the
LmGT1 open reading frame (Fig. 1B). The
LmGT2 probe (Fig. 4C) was an antisense RNA of
~1.4 kb, derived from cloned DNA between the ScaI site at
the downstream end of the LmGT2 open reading frame and a
SmaI site ~200 base pairs downstream of the
KpnI site in the intergenic region between LmGT2
and LmGT3 (Fig. 1B). The LmGT3 probe
(Fig. 4D) was an antisense RNA of ~1.8 kb, derived from
the cloned product of PCR1 (see above).
Nuclear Run-on Transcription--
Crude nuclei were prepared
from promastigotes and axenically cultured amastigotes by detergent
lysis (23). Cells were harvested by centrifugation and washed twice in
ice-cold phosphate-buffered saline. Approximately 5 × 108 cells were resuspended in 4 ml of lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Nonidet P-40) and
vortexed gently for 10 s before incubation for 5 min on ice.
Lysates were centrifuged for 5 min at 500 × g at
4 °C, and the pellet was resuspended in 4 ml of lysis buffer and
centrifuged again as above. The pellet was resuspended in 1 ml of
storage buffer (50 mM Tris-Cl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA) and frozen
in 0.1-ml aliquots in liquid nitrogen.
Run-on transcription was performed using freshly thawed nuclei in a
300-µl reaction volume containing 50 mM Tris-Cl, pH 8.0, 25 mM NaCl, 2 mM dithiothreitol, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 2 mM each of rATP, rCTP, and rGTP, 10 µM rUTP, 250 µCi [
Labeled transcripts were hybridized to slot blots of linearized,
alkaline-denatured plasmid DNA (~5 µg per slot) (23). Slot blots
were prehybridized for at least 2 h at 42 °C in a sealed plastic bag containing 5 ml of 50% formamide, 5 × SSC, 50 mM NaPO4 (pH 7.0), 1% SDS, 200 µg·ml Analysis of mRNA Decay--
We first determined that 9 µg·ml Transport Assays in Xenopus Oocytes--
A
BglII-ClaI restriction fragment containing the
protein-coding region of the LmGT2 gene (labeled BC in Fig.
1B) was subcloned into the Bluescript SK+ plasmid
(Stratagene), linearized with ClaI, and transcribed
in vitro with T3 RNA polymerase (Life Technologies, Inc.) as
described (24). Stage V-VI Xenopus oocytes were injected with 40 nl containing ~3 ng of RNA, incubated for 3 days at 18 °C, and assayed for uptake of 50 µM
2-deoxy-D-[3H]glucose as described previously
(24).
Immunoblot Analysis--
Lysates of promastigotes and axenic
amastigotes were prepared by dissolving the cell pellet in Laemmli
sample buffer (19) at a concentration of 2.5-5 × 105
cells·µl Cloning and Structure of LmGT Glucose Transporter Genes from L. mexicana--
To study expression of glucose transporter genes using
the L. mexicana axenic amastigote system, we first cloned
these genes from L. mexicana. Southern blots of
restriction-digested L. mexicana genomic DNA, probed with a
protein-coding region of the L. enriettii Pro-1 glucose
transporter gene, revealed the presence of single hybridizing bands in
EcoRI, HindIII, EcoRV, and
BamHI digests, suggesting that most or all of the
LmGT locus was contained within the ~14-kb
EcoRI fragment (Fig.
1A). Other Southern blots
revealed that there were no other smaller bands that hybridized to the Pro-1 probe in genomic DNA digested with these four enzymes
(data not shown). Therefore, a phage library of
EcoRI-digested L. mexicana genomic DNA was
screened with the L. enriettii Pro-1 probe, and 11 positive
clones were isolated, all with an insert of ~14 kb. Restriction
mapping of one of these inserts revealed three restriction fragments of
5.2 kb (EcoRI/KpnI), 3.8 kb (KpnI),
and 2.7 kb (KpnI/EcoRI), which hybridized with
the Pro-1 probe (Fig. 1B). Each of these restriction fragments was subcloned, partially sequenced, and found to
contain a single copy of an L. mexicana Pro-1 homologue. The
results indicate that this locus contains three closely related glucose
transporter genes, with significant identity to L. enriettii Pro-1. The deduced amino acid sequences (Fig.
2) for these three genes indicates that
they differ from each other most markedly within their predicted
NH2-terminal hydrophilic domains (thought to be located on
the cytoplasmic side of the membrane (27)). LmGT1 has an
NH2 terminus that is 45 amino acids longer than that of
either LmGT2 or LmGT3, which have NH2 termini of similar
length but divergent sequence. Downstream of the predicted
NH2-terminal domains, all three sequences are more than
90% identical, and all have the same predicted membrane-spanning
topology. However, there are 24 additional amino acid positions that
are divergent among the three isoforms. Thus, LmGT1 and LmGT2 have
identical COOH termini, whereas LmGT3 differs in 8 of the last 9 amino
acids, and LmGT1 has a divergent cluster of 7 amino acids toward the cytoplasmic end of the predicted transmembrane domain IV. In addition, there are nine other non-identical amino acids distributed throughout the primary amino acid sequence. Among the three isoforms, LmGT2 is the
most closely related to L. enriettii Pro-1 (LmGT1, LmGT2, and LmGT3 have 78.0, 85.2, and 83.1% identity, respectively, to L. enriettii Pro-1 isoform 2).
5'- and 3'-UTRs of LmGT Genes--
To define the 5' end of the
mRNA from LmGT1, LmGT2, and LmGT3,
we amplified, subcloned, and sequenced cDNAs representing the 5'
ends of each mRNA (6). cDNA synthesized using a random
oligonucleotide primer was amplified in the PCR using a reverse primer
complementary to the unique NH2-terminal coding region of
each mRNA and a forward primer representing the 39-nucleotide
spliced leader (22) present on all L. donovani mRNAs
(see "Experimental Procedures"). The sequences of the cDNA
subclones revealed that the spliced leaders were
trans-spliced onto the 5' ends of the LmGT2 and
LmGT3 mRNAs at identical positions, 170 base pairs
upstream of the first ATG codon. Comparison of the 5'-UTRs of the
LmGT2 and LmGT3 mRNAs revealed that they were
identical. In contrast, the 5'-UTR of LmGT1 showed no
sequence homology with that of LmGT2 and LmGT3, and the site of spliced leader addition was 386 base pairs upstream of
the first ATG codon. For each predicted transporter protein, the
indicated initiation codon is the first in frame methionine codon in
the RNA. The limited sequence we have obtained for the 3'-UTRs of each
mRNA indicates that all three are divergent.
Functional Expression of LmGT2 Gene--
To confirm that the
LmGT genes encode bona fide glucose transporters,
we expressed the LmGT2 gene in Xenopus oocytes
and assayed for uptake of the glucose analog
2-deoxy-D-[3H]glucose (24). Oocytes injected
with LmGT2 RNA exhibited robust linear uptake of the
radiolabeled analog for at least 3 h, whereas oocytes injected
with water did not take up this compound (Fig. 3).
Regulation of LmGT Gene Expression in Promastigotes and Axenic
Amastigotes of L. mexicana--
Biological criteria suggest that
axenically cultured amastigotes of L. mexicana present a
reasonable model for in vivo amastigotes (12, 13). However,
if these axenic amastigotes are to be used as a model for investigating
the regulation of LmGT gene expression, it is essential to
establish that LmGT gene expression is correctly regulated
in these cells. Consequently, we have used Northern blots to compare
the expression of RNAs from several genes in promastigotes, axenically
cultured amastigotes, and amastigotes derived from cultures of
macrophage-like cells (Fig. 4). In these experiments, equal amounts of total RNA from promastigotes,
macrophage-derived amastigotes, and axenic amastigotes were separated
by gel electrophoresis. Following blotting and hybridization, the
signals for each mRNA were quantitated by phosphorimaging and
normalized to those for rRNA (28), allowing quantitative comparison of
mRNA levels in each cell type. For all of the genes investigated,
expression by axenically cultured amastigotes was very similar to
expression by intracellular amastigotes, supporting the validity of
these axenic amastigotes as a model for intracellular amastigotes.
Northern blots reveal that LmGT1, LmGT2, and LmGT3 are encoded by three
different mRNAs of ~8.0, ~3.5, and ~7.5 kb, respectively (Fig. 4). Hybridization with probes from the 3'-UTR of each gene detect
an ~8.0-kb transcript for LmGT1 (Fig. 4B), a
~3.5-kb transcript for LmGT2 (Fig. 4C), and a
~7.5-kb transcript for LmGT3 (Fig. 4D).
Hybridization with the LmGT2 protein-coding region alone detects each of these (Fig. 4A) and an additional ~6.0-kb
transcript (labeled "X") of unknown origin. Expression of
LmGT2 RNA is down-regulated ~15-fold in both axenically
cultured amastigotes and in intracellular amastigotes, compared with
promastigotes, as determined by phosphorimaging of the 3.5-kb signal
(Fig. 4A), indicating that the LmGT2 transcript is strongly developmentally regulated. In contrast, the larger LmGT1 and LmGT3 transcripts are present at similar levels in
promastigotes, axenic amastigotes, and intracellular amastigotes,
indicating that these mRNAs are constitutively expressed.
Expression of Other mRNAs in Promastigotes and Axenic
Amastigotes--
To further investigate axenic amastigotes as a model
for studying gene regulation, we have examined the relative levels of expression of mRNAs from several other genes (Fig. 4,
E-H). The mRNA encoding another developmentally
regulated protein, the paraflagellar rod protein PFR-1 (29), was also
down-regulated in both types of amastigote (Fig. 4D). In
contrast, the mRNA for the constitutively expressed Transcription of LmGT Genes in Promastigotes and Axenic
Amastigotes--
To determine whether the LmGT genes are
regulated at the transcriptional or post-transcriptional levels, we
have quantitated the relative rates of transcription of several genes
in promastigotes and axenically cultured amastigotes by nuclear run-on
transcription. Radiolabeled run-on transcripts from promastigote or
amastigote nuclei were hybridized with plasmid DNA immobilized on nylon
filters, and the amount of labeled transcript in each hybrid was
quantitated by phosphorimaging. The rate of transcription of each gene
was determined relative to the rate of rRNA transcription by
normalizing each hybridization signal to that obtained for the rRNA
hybrid in each run-on transcription. Hence, the run-on transcriptions have been normalized to the same standard used to normalize the relative levels of mRNA abundance, as determined by quantitation of
hybridization signals on Northern blots (Fig. 4). Probes for genes
whose relative rates of transcription were measured in these experiments include the LmGT genes (3.6-kb PstI
fragment labeled P in Fig. 1B), L. enriettii
In the preceding experiments, we hybridized the nascent transcripts to
a restriction fragment that contains both glucose transporter protein-coding regions and the intergenic region between the
LmGT2 and LmGT3 protein-coding regions. This
probe will hybridize most strongly to the LmGT2 transcript,
since the probe contains the entire unique 3'-UTR of this mRNA, but
it will also cross-hybridize to the LmGT1 and
LmGT3 transcripts due to the high sequence identity of all
three transcripts in the protein-coding regions. This cloned fragment
was chosen, rather than a shorter target representing the unique 3'-UTR
of LmGT2, to ensure that a detectable level of signal for
LmGT2 transcription could be obtained in the run-on experiments. Partial cross-hybridization of the probe to the
unregulated LmGT1 and LmGT3 transcripts would not
alter the interpretation of the results, since these RNA levels are not
regulated during the life cycle. It is thus clear that the 15-fold
regulation of LmGT2 mRNA steady-state levels (Fig.
4A) cannot be attributed to regulation of LmGT2
transcription.
Decay of LmGT2 mRNA in Promastigotes and Axenic
Amastigotes--
To determine whether decreased RNA stability could
contribute to the down-regulation of LmGT2 mRNA in
amastigotes, we measured LmGT2 mRNA decay in
promastigotes and axenic amastigotes that had been treated with 10 µg·ml Expression of LmGT Proteins in Promastigotes and Axenic
Amastigotes--
To determine whether LmGT proteins are down-regulated
upon the transformation of promastigotes to amastigotes, we performed immunoblots on lysates containing similar amounts of protein from promastigotes and axenic amastigotes. These blots were probed (Fig.
7) with the P1L antibody (26) directed
against the peptide T76-Y143 (5) from the large extracellular loop of
the L. enriettii Pro-1 glucose transporters, which is highly
related in sequence (48 of 68 amino acids are identical) to the
corresponding regions of the LmGT proteins. As in the case of L. enriettii, the L. mexicana lysates revealed a major
band (Fig. 7, solid arrow) of 50-55 kDa that was not
competed by GST (Fig. 7, lanes 1 and 2) but was
competed by the GST-fusion protein used to generate the antiserum (Fig. 7, lanes 3 and 4), confirming that this band
represents LmGT glucose transporters. This band presumably corresponds
to both LmGT2 and LmGT3, since both proteins are of the same predicted
molecular weight and contain the same sequence within the region
recognized by the P1L antiserum (Fig. 2A). The intensity of
this band was considerably greater in lysates from promastigotes (Fig.
7, lane 1) compared with axenic amastigotes (Fig. 7,
lane 2). Furthermore, a background band that was not
competed by either GST or the GST-fusion protein (Fig. 7, open
arrow) was of similar intensity in the promastigote and axenic
amastigote lanes, confirming that similar amounts of cellular protein
had been loaded onto each lane. This result establishes that expression
of the LmGT2 protein and the LmGT2 mRNA is similarly regulated during the parasite life cycle.
Coordinate regulation of gene expression in kinetoplastids is
central to the complex life cycle switches that are characteristic of
these organisms. Although higher eukaryotes control much of their gene
expression at the level of transcription, current data suggest that in
the Kinetoplastida, transcriptional regulation may not play a major
role in expression of many genes (33). Though current examples are
limited, expression of several genes in the related organism
Trypanosoma brucei seems to be regulated primarily at a
post-transcriptional level (34-38). In L. amazonensis, temperature-dependent induction of the developmentally
regulated hsp83 is post-transcriptionally controlled and involves a
change in the rate of mRNA turnover (39, 40).
Developmentally regulated changes in mRNA stability have also been
implicated in regulation of expression of the surface glycoprotein gp63
in Leishmania chagasi (41). Since the LmGT2
glucose transporter gene is among a limited number of strongly
developmentally regulated genes that have been identified in
Leishmania parasites, it provides a valuable model for
probing the molecular mechanisms that control gene expression during
the parasite life cycle.
An important reason for the current paucity of information regarding
leishmanial gene regulation is the technical difficulty of working with
amastigotes grown in host cells. Although it is possible to obtain
sufficient numbers of amastigotes from infected macrophages to isolate
nucleic acids and measure the differences in levels of specific
mRNAs in amastigotes compared with promastigotes, more
sophisticated experiments that probe the mechanism for regulating gene
expression are very difficult to perform with amastigotes isolated from
macrophages. Nuclear run-on transcriptions would be cumbersome to
perform with amastigotes isolated from macrophages, as it would be hard
to obtain enough parasites to generate the levels of labeled
transcripts required for hybridizations. Furthermore, the purification
of amastigotes from macrophages requires several hours, and the
transcription levels of developmentally regulated genes could be
significantly altered once the amastigotes are released from the
environment of the parasitophorous vacuole. For similar reasons,
measurements of mRNA stability in intracellular amastigotes are
also problematic. In principle, these technical problems can be
overcome using axenically cultured amastigotes, so that nuclear
transcription and mRNA decay experiments can be performed in the
same way they are done with cultured promastigotes.
However, to establish the validity of axenic amastigotes as a model for
studying regulation of gene expression, it was first essential to
demonstrate that axenic amastigotes regulate the expression of genes,
especially the LmGT genes, in essentially the same way that
intracellular amastigotes regulate their expression. We have determined
the pattern of expression of a range of genes in promastigotes and
purified macrophage-derived amastigotes and have shown that axenic
amastigotes regulate expression all of these genes in a pattern that is
very similar to intracellular amastigotes. This is true both for
transcripts that are up-regulated or down-regulated in amastigotes.
Thus, axenically cultured amastigotes represent an excellent model for
studies of amastigote gene regulation and provide a compelling reason
to undertake these experiments in L. mexicana.
Structure and Multiplicity of Glucose Transporter Genes in L. mexicana--
To begin studies on glucose transporter gene expression
in L. mexicana, we cloned the LmGT genes from
this parasite and determined their sequence and genomic arrangement.
Three LmGT genes are present at a single locus and encode
closely related isoforms that differ from one another mainly at their
predicted NH2 and COOH termini. The existence of three
related but distinct glucose transporter isoforms in L. mexicana suggests that each of these three permeases may subserve
a unique or specialized physiological function. However, the
potentially distinct biological roles of these transporters remain to
be determined.
The pronounced sequence similarity between the LmGT genes
from L. mexicana and the Pro-1 genes from
L. enriettii confirms that these are homologous genes from
these two species of Leishmania parasite. Nonetheless, there
are notable differences between the glucose transporter isoforms of
L. mexicana and L. enriettii (42). Isoform 1 and
isoform 2 of L. enriettii Pro-1 are targeted to different
locations at the cell surface (43), and the structural information that
directs this targeting is contained within the divergent
NH2 termini (26). The rest of the coding region of the two
L. enriettii isoforms, from the first transmembrane segment to the COOH terminus, is identical. Each of the three LmGT isoforms has
unique NH2-terminal hydrophilic domains, but, in contrast to the L. enriettii Pro-1 isoforms, LmGT3 has a divergent
COOH terminus compared with LmGT1 and LmGT2, and LmGT1 has a divergent cluster of amino acids at the cytosolic end of predicted transmembrane domain IV compared with LmGT2 and LmGT3. There is also a number of
single amino acid differences that distinguish these isoforms from one
another. It is possible that the distinct NH2 termini of
the three LmGT isoforms are involved in differential subcellular targeting, as in L. enriettii, and that the other
polymorphic amino acids might confer some additional biochemical or
kinetic differences between the two isoforms. Alternatively, it is
possible that divergent sequences in both the NH2 terminus
and elsewhere are involved in differential subcellular targeting of the
LmGT isoforms. Understanding the functional differences between the three isoforms of LmGT transporters will require further
experimentation, including functional expression of both isoforms in a
heterologous system as well as subcellular targeting studies similar to
those performed on isoforms 1 and 2 of the L. enriettii
Pro-1 transporters (43). Despite the divergence of the NH2
and COOH termini, LmGT2 is nonetheless more closely related to the
L. enriettii isoform 2 within both these domains than is
either LmGT1 or LmGT3. This observation suggests the possibility that
LmGT2 might be the functional homolog of isoform 2.
It is noteworthy that the LmGT2 and LmGT3
transcripts have identical 5'-UTRs, even though there are significant
differences within the protein-coding regions. It is currently not
clear why these two mRNAs are identical in this region, whereas the
5'-UTR of LmGT1 is divergent. In contrast, the limited
sequence we have obtained indicates that the 3'-UTRs of all three
LmGT mRNAs are divergent. It is possible that the
different 3'-UTRs are involved in the differential regulation of these
transcripts during the life cycle.
Regulation of Glucose Transporter Genes in L. mexicana--
The
LmGT2 transcripts in L. mexicana are
down-regulated ~15-fold in both isolated in vivo
amastigotes and in axenically cultured amastigotes. Like
LmGT2, both Pro-1 transcripts in L. enriettii (5)
and the D2 hexose transporter transcript in L. donovani (6)
are down-regulated in amastigotes compared with promastigotes. In
contradistinction, the LmGT1 and LmGT3
transcripts are expressed at similar levels in both life cycle stages
and thus represent a unique example of unregulated glucose transporter
genes. A detailed kinetic analysis of transport by all three isoforms
might suggest a physiological rationale for the differential regulation
of the three genes.
Analysis of the regulation of LmGT2 gene expression was
greatly facilitated by the availability of an axenic culture system for
amastigotes. The nuclear run-on transcription experiments clearly show
that the level of transcription of LmGTs (relative to
constitutively transcribed ribosomal RNA) is very similar in both life
cycle stages. Since the cloned
Down-regulation of mature mRNA might be effected at the level of
post-transcriptional processing or via modulation of message stability.
We utilized a transcription inhibitor, actinomycin D, to investigate a
potential role for differential message stability in LmGT2
regulation. Since actinomycin D shuts off all RNA synthesis and can
also affect other metabolic processes, such as translation, these
experiments are potentially subject to artifacts, and the rates of
mRNA decay measured in the presence of drug could differ from those
operating in the absence of drug. The level of actinomycin D used in
these experiments is not in excess of that required to efficiently
inhibit transcription, and cells did not seem to be affected
morphologically nor was their motility reduced during the 5-h
incubation in drug. Nonetheless, we have attempted to measure mRNA
decay more directly by in vivo pulse-chase experiments involving radioactive labeling of RNA with [3H]uridine
(44), but we have not been able to achieve sufficient labeling of the
moderately abundant LmGT2 transcript to perform quantitative
decay experiments.
Although the biphasic shape of the decay curves (Fig. 6) precludes the
determination of a quantitative half-life characteristic of simple
exponential decay, the data point to a clear qualitative difference in
LmGT2 mRNA stability between these two life cycle stages, at least in the presence of drug. This complexity in the decay
curves makes it difficult to definitively determine the relative role
of mRNA half-life in LmGT2 gene regulation during the
parasite life cycle. Thus, if the true half lives are represented by
the 0-2-h region of the decay curves, there is a dramatic difference in stability of the LmGT2 mRNA between promastigotes and
amastigotes. Nonetheless, even if the difference in mRNA half lives
is not as pronounced as suggested by the early region of the decay
curves, the more modest difference suggested by following mRNA
decay over the entire 5-h time course could still effect a very large
difference in steady-state mRNA levels. Thus, Ross (45) has
mathematically demonstrated that 2-4-fold differences in mRNA half
lives can result in differences of at least two orders of magnitude in
mRNA steady-state levels.
In contrast, the stability of total
This life cycle-dependent difference in transcript
stability may be explained by either stabilization of LmGT
mRNA in promastigotes or destabilization in amastigotes and
predicts a role for cis-acting sequences in the control of
mRNA stability. Cis-acting sequences in both the 5'- and
3'-UTRs have been implicated in differential decay of hsp83 mRNA
during heat shock in L. amazonensis (39, 40), and sequences
within the 3'-UTR have been demonstrated to play a role in the
post-transcriptional regulation of the A2 amastigote-specific gene from
L. donovani (46), establishing a precedent for this type of
regulation among Leishmania species in response to changing
environmental conditions. It should now be possible to define specific
elements involved in the regulation of the LmGT2 gene during
the parasite life cycle by systematic deletion and modification of
discrete regions of sequence.
Expression of LmGT Proteins during the Parasite Life
Cycle--
Immunoblots of lysates from the two life-cycle stages
confirm that the total LmGT protein is significantly less abundant in amastigotes compared with promastigotes (Fig. 7). Although the antiserum used in these experiments recognizes all glucose transporter isoforms, the band intensity in the promastigote lane probably represents primarily the LmGT2 isoform and confirms that the level of
this protein is down-regulated concommitantly with the down-regulation of the LmGT1 mRNA. Although we cannot directly determine
whether or not the LmGT1 and LmGT3 polypeptides, encoded by the
significantly less abundant mRNAs, are regulated during the life
cycle, the constitutive expression of these RNAs during the life cycle
suggests that the corresponding permeases are likely to be expressed at similar levels in both life cycle stages. This hypothesis is consistent with the presence of an LmGT band in the amastigote lane in exposures of the immunoblot that are longer than that shown in Fig. 7 (data not
shown).
Additional Advantages of Studying Glucose Transporters in L. mexicana--
The cloning and structural analysis of glucose
transporter genes in L. mexicana offers several advantages
for functional studies on these transporters. L. mexicana
parasites can establish infections in laboratory-reared sandflies (47)
and in J774.G8 tissue culture macrophages (16), unlike some other
species of Leishmania such as L. enriettii.
Hence, generation of glucose transporter null mutant (48, 49) strains
in L. mexicana will allow phenotypic testing of these
mutants within the insect vector and within the macrophage and will
greatly enhance our ability to assess the biological functions of
glucose transporters in intact Leishmania parasites.
We thank David Russell for providing the
L. mexicana strain M379 and for advice on the axenic culture
of amastigotes, Jonathan LeBowitz for a gift of the cloned PRF-1 gene,
Diane McMahon-Pratt for a gift of the cloned cysteine protease-2 gene,
and David Weston for advice concerning nuclear run-on transcriptions.
We also thank Andreas Seyfang for help with the oocyte expression and
Marco Sanchez, Andreas Seyfang, and Buddy Ullman for critical comments on the manuscript.
*
This work was supported by Grant AI25920 and Career
Development Award AI01162 (to S. M. L.) from the National Institutes
of Health and by an American Heart Association Fellowship (to
R. J. S. B.).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) AF071217, AF039221, and AF039222.
The abbreviations used are:
LmGT1, LmGT2, and
LmGT3, L. mexicana glucose transporter proteins 1, 2, and 3; LmGT1, LmGT2, and LmGT3, glucose
transporter genes 1, 2, and 3 from L. mexicana; UTR, untranslated region; PCR, polymerase chain reaction; kb, kilobase
pairs; GST, glutathione S-transferase.
Differential Regulation of Multiple Glucose Transporter Genes
in Leishmania mexicana*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1), glucose (2.5 g·liter1), trypticase (5 g·liter1),
L-glutamine (0.75 g·liter1), HEPES (5.96 g·liter1), NaHCO3 (2.2 g·liter1), and hemin (20 mg·liter1).
The pH was adjusted to 6.0 with HCl. Axenic amastigotes were transformed from mid-log phase promastigote cultures by dilution into
an equal volume of JH-30A medium, containing either 20%
heat-inactivated fetal bovine serum or 20% bovine embryonic fluid,
followed by incubation at 32.5 °C. Continuous cultures were
maintained at 32.5 °C by periodic dilution with JH-30A containing
serum or embryonic fluid.
-32P]rUTP at 3000 Ci
mol1 (NEN Life Science Products), 10 mM
creatine phosphate, 20 µg·µl1 creatine
phosphokinase. Nuclei were kept on ice until all reaction components
were added, and then promastigote nuclei were incubated at 26 °C and
amastigote nuclei at 32 °C, both for 20 min. As appropriate, -amanitin was added to nuclei 10 min prior to other reaction components. Samples were extracted with 1 volume of phenol:chloroform (1:1), and the aqueous phase was extracted with 1 volume of chloroform. Nucleic acids were precipitated with 2 M ammonium acetate
(pH 7) and 3 volumes of ethanol and centrifuged at full speed for 20 min in a microcentrifuge, with 10 µg of yeast tRNA as a carrier, rinsed twice with 70% ethanol, and resuspended in 200 µl of water. Incorporation of [-32P]rUTP was determined by counting
a 2-µl aliquot.
1 yeast tRNA, 5 × Denhardt's solution, 5 mM EDTA (19). Run-on transcripts were denatured by heating
to 95 °C for 5 min and then added to the slot blot in
prehybridization solution. Hybridization was allowed to proceed with
gentle agitation for ~48 h. Blots were washed at 55 °C, three
times for 30 min each in 0.1 × SSC, 0.1% SDS, 5 mM
EDTA, dried, and exposed to film or phosphorimage screen for a period
between 12 h and 1 week. Analysis of intensity of hybridization to
individual slot blots was performed using a Phosphorimager SI
(Molecular Dynamics) and IP Lab Gel software for Apple Macintosh
(Signal Analysis Corp.).
1 was the IC90 for actinomycin D
(Boehringer Mannheim) inhibition of [3H]uracil (NEN
Life Science Products) incorporation in L. mexicana. Promastigote and amastigote cultures, at approximately 2 × 107 cells·ml1, were incubated with 10 µg·ml1 actinomycin D under normal culture conditions.
Aliquots were removed at various time points, and nucleic acids were
extracted and analyzed by Northern blot and exposure to a phosphorimage screen, as described above.
1 and heating immediately to 65 °C for 5 min. Aliquots of the lysate were assayed in triplicate for protein
content by the Bradford method (25). Samples (10 µl) were separated
on a 10% SDS-polyacrylamide gel and electroblotted onto
nitrocellulose, and the blot was incubated overnight in TBST buffer (20 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20, 3% (w/v) powdered milk) containing the affinity-purified P1L antiserum
directed against the large extracellular loop of the L. enriettii Pro-1 glucose transporter (26) (1:1000 dilution). Prior
to application to the blot, the P1L antiserum was preincubated for
1 h in a 200-µl volume of TBST buffer containing 50 µg of either glutathione S-transferase (GST) or the GST-fusion
protein used to raise the P1L antibody. Blots were developed with a
horseradish peroxidase-conjugated secondary goat anti-rabbit (1:40,000)
IgG and the Supersignal chemiluminescence kit (Pierce) using the
manufacturer's instructions and then exposed to XAR-5 film (Eastman
Kodak Co.).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Structure of the LmGT gene locus
in L. mexicana. A, Southern blot analysis.
Genomic DNA restriction fragments were separated by electrophoresis,
transferred to a nylon membrane, and hybridized with a protein-coding
region of L. enriettii Pro-1 glucose transporter gene.
Lane 1, BglII; lane 2,
NotI; lane 3, EcoRI; lane
4, HindIII; lane 5, BamHI;
lane 6, EcoRV. B, restriction map of
~14-kb EcoRI genomic clone. Southern blot analysis
indicated that glucose transporter homologs mapped to three fragments
in a KpnI-EcoRI digest of this genomic clone.
These three fragments, an EcoRI-KpnI fragment of
~5.2 kb, a KpnI fragment of ~3.8 kb, and a
KpnI-EcoRI fragment of ~2.7-kb, were subcloned
and partially sequenced. Protein-coding regions, indicated by
solid boxes, were deduced and revealed that the single
L. mexicana glucose transporter locus contains three
clustered genes encoding three glucose transporter isoforms, LmGT1,
LmGT2, and LmGT3. Coding and 3'-UTR sequences downstream of the
~14-kb EcoRI fragment were obtained from cDNA using
primers at positions indicated by the small
arrows marked 1, 2, and 3 (see text). R,
EcoRI; K, KpnI; S,
ScaI; B, BglII; P,
PstI; C, ClaI.
BglII/ClaI (BC) and PstI
(P) restriction fragments, indicated by the solid
lines below the map, are discussed in the text.
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Fig. 2.
Alignment of the predicted amino acid
sequences of the L. mexicana glucose transporters LmGT1,
LmGT2, and LmGT3. Amino acids that are non-identical in all three
proteins are indicated by a white background, whereas those
that are identical in two out of three sequences are indicated by a
gray background. The numbers at the left indicate
the amino acid positions in each sequence, and the numbers
at the end of each sequence indicate the total number of constituent
amino acids. Spaces introduced to optimize the alignment are indicated
by a period. Predicted transmembrane domains are indicated
by numbered bars above the aligned sequences.
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Fig. 3.
Uptake of radiolabeled
2-deoxy-D-[3H]glucose in Xenopus
oocytes injected with LmGT2 RNA. Oocytes were
incubated in the presence of 50 µM
2-deoxy-D-[3H]glucose for the indicated
periods of time. For each time point, uptake into three oocytes was
measured and averaged; error bars represent standard
deviations of these three values. Closed circles represent
oocytes injected with LmGT2 RNA, and open circles
represent oocytes injected with water.
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Fig. 4.
Expression of RNAs in promastigotes, axenic
amastigotes, and macrophage-derived amastigotes. Northern blots of
RNA from promastigotes (1), axenically cultured amastigotes
(2), and macrophage-derived amastigotes (3) were
hybridized with the radiolabeled probes described in "Experimental
Procedures" (indicated below each panel) representing the
LmGT genes (A); the 3'-UTR of LmGT1
(B); the 3'-UTR of LmGT2 (C); the
3'-UTR of LmGT3 (D); a gene encoding the L. mexicana paraflagellar rod protein, PFR-1
(E); the L. enriettii -tubulin
(-tub) gene (F); the L. pifanoi
cysteine protease-2 gene, Lpcys-2 (G); and the
L. enriettii 
-tubulin (-tub) gene
(H). The panels below each Northern blot represent
rehybridizations of each blot with a probe for the L. enriettii
rRNA. A ~6-kb band marked "X" in panel
A and a ~5-kb banded marked "Y" in panel
H are discussed in the text. The lane marked 2+
in panel A is a longer exposure of lane
2 in panel A. The numbers to the
left of each panel indicate the size (in kb) and mobility of RNA
molecular weight standards.
-tubulin
(30) was expressed at similar levels in all three L. mexicana cell types studied here (Fig. 4E). The RNA for
the developmentally regulated cysteine protease gene Lpcys2 (31) was quantitatively up-regulated in both macrophage-derived and
axenic amastigotes compared with promastigotes (Fig. 4F). Finally, a 2.4-kb -tubulin transcript decreased in abundance in both
macrophage-derived and axenic amastigotes compared with promastigotes,
and a 2.8-kb transcript increased in abundance in both types of
amastigote (Fig. 4H). A -tubulin transcript of ~5 kb
(marked "Y" in Fig. 4H) increased in abundance in
intracellular amastigotes but not in axenic amastigotes. A similar
pattern of differentially regulated -tubulin transcript sizes has
been reported for Leishmania amazonensis (30). In summary,
three constitutively expressed RNAs (-tubulin, LmGT1,
LmGT3), three RNAs that are down-regulated in amastigotes
(LmGT2, PFR-1, -tubulin 2.4-kb transcript), and two RNAs
that are up-regulated in amastigotes (cysteine protease-2 and
-tubulin 2.8-kb transcript) are correctly regulated in axenic
amastigotes. Although a quantitative difference in the expression of
the ~5-kb -tubulin transcript was observed between intracellular
and axenic amastigotes, this latter transcript is larger in size than
the mature -tubulin mRNAs and may represent unprocessed mRNA
precursors. However, mature mRNAs are expressed at very similar
levels in macrophage-derived and axenic amastigotes, confirming the
utility of this in vitro system for analysis of gene
expression.
-tubulin, L. mexicana PFR-1, and L. enriettii
rRNA. The results (Fig. 5, A
and C) show that transcription of all genes occurred at
approximately the same rates, relative to rRNA transcription, in both
promastigotes and axenic amastigotes. No hybridization was detected to
pBluescript, a plasmid that contains no DNA of leishmanial origin. The
results of three independent run-on transcription experiments were
quantitated and are reported in Table I.
The hybridization signal to the heterologous L. enriettii rRNA clone was arbitrarily chosen as 1.0, and the other signals were
reported relative to this value. Although there is considerable scatter
for each normalized hybridization signal from one experiment to
another, it is clear that the relative rates of transcription for all
genes examined do not vary significantly between promastigotes and
axenic amastigotes. Since all the genes investigated here are
transcribed at similar rates in both life cycle stages, the developmentally regulated LmGT2 and PFR-1 genes must be
post-transcriptionally controlled. The absolute rate of transcription
per nucleus was approximately 10-fold lower in axenic amastigotes than
in promastigotes, as determined by quantitation of radioactivity
incorporated into total RNA during the run-on transcriptions (3.6 ± 1 × 107 cpm per 109 promastigote
nuclei (n = 3) compared with 3.9 ± 0.8 × 106 cpm per 109 axenic amastigote nuclei
(n = 3)). Transcription of the LmGT, PFR-1,
and -tubulin genes, but not the rRNA genes, was inhibited by 50 µg·ml1 -amanitin (Fig. 5B), a level of
drug similar to that which inhibited transcription of other
mRNA-encoding genes in Leishmania tarentolae (32).
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Fig. 5.
Nuclear run-on transcriptions from
promastigotes and axenic amastigotes hybridized to various cloned
DNAs. Run-on transcriptions were performed with nuclei from
promastigotes in the absence (A) or the presence
of 50 µg·ml 1 -amanitin (B) and with
nuclei from axenic amastigotes (C). The products of each
nuclear run-on transcription were hybridized to linearized plasmid DNA
immobilized on a nylon filter. Plasmids contain inserts for the
L. enriettii -tubulin (-tubulin), L. mexicana glucose transporters (fragment P, Fig.
1B) (LmGT), L. mexicana paraflagellar
rod protein-1 (PFR-1), and L. enriettii rRNA
(rRNA). The slot marked pBluescript contains the
Bluescript SK+ plasmid.
Quantitation of relative transcription rates for various genes in L. mexicana promastigotes and axenic amastigotes
-tubulin and
rRNA).
1 actinomycin D, a drug concentration that
inhibits RNA synthesis by greater than 90% (data not shown). Total RNA
was isolated from cultures from 0 to 5 h following addition of
drug, and the levels of LmGT2 and -tubulin mRNAs were
measured in each sample by quantitation of Northern blots with a
phosphorimager. The results of three independent experiments revealed
that LmGT2 mRNA was more stable in promastigotes than in
amastigotes (Fig. 6A).
Although LmGT2 mRNA decayed rapidly, with a half-life of
approximately 1 h in amastigotes, this mRNA was quite stable
in promastigotes for the first 2 h following drug addition and
then decayed more rapidly, producing a biphasic decay curve in this
life cycle stage. Although it is not possible to assign a unique
half-life to LmGT2 mRNA in drug-treated promastigotes,
the substantial difference in mRNA decay rates between
promastigotes and amastigotes, especially during the initial 2 h
of drug treatment, suggests that LmGT2 mRNA levels may
be regulated during the life cycle, at least in part, by differential
mRNA stability. In contrast, the decay of -tubulin mRNAs
(sum of the hybridization signals of the 2.4- and 2.8-kb transcripts)
was essentially the same in promastigotes and axenic amastigotes (Fig.
6B). When the 2.4- and 2.8-kb -tubulin transcripts were
analyzed individually, the 2.4-kb transcript was found to be more
stable in promastigotes, and the 2.8-kb transcript was more stable in
axenic amastigotes (data not shown). Thus, differential message
stability may further modulate expression of various -tubulin
mRNAs.
View larger version (14K):
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Fig. 6.
RNA decay in promastigotes and axenic
amastigotes treated with actinomycin D. RNA isolated from
promastigotes ( 
) and axenic amastigotes (
), after incubation with
actinomycin D (9 µg·ml1) for between 0 and 5 h,
was separated on agarose-formaldehyde gels, blotted, and hybridized
with probes for the LmGT (fragment P, Fig.
1B) (A) and the -tubulin (B) genes.
Transcript abundance was quantitated using a phosphorimager and was
normalized by reprobing each blot with a cloned rRNA gene. The data
from three independent experiments are expressed as the means and
standard deviations (error bars) of the percentage of
hybridization signal present at each time following actinomycin D
addition, compared with the signal present at 0 h of drug
treatment.
View larger version (74K):
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Fig. 7.
Immunoblots of lysates from promastigotes and
axenic amastigotes. Lysates containing 1.3 µg of protein from
2.5 × 106 promastigotes (lanes
1 and 3) or 1.1 µg of protein from 5 × 106 axenic amastigotes (lanes 2 and
4) were separated by SDS-polyacrylamide gel electrophoresis,
electroblotted onto nitrocellulose filters, and incubated with the P1L
antibody (26) directed against the Pro-1 glucose transporters from
L. enriettii. For lanes 1 and 2, the
P1L antibody was preincubated with GST, and for lanes 3 and
4, the P1L antibody was preincubated with the GST-fusion
protein used to generate the P1L antibody. The solid arrow
indicates the band that is competed by GST-fusion protein but not by
GST and hence represents the LmGT transporters. The open
arrow indicates a background band whose intensity does not change
significantly between promastigotes and axenic amastigotes. The
numbers at the left indicate the mobility and relative
molecular weights (kDa) of protein molecular weight standards.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-tubulin and ribosomal RNA genes are
from another Leishmania species, it is not possible to
quantitate absolute rates of transcription in these experiments. However, it is clear that the ~15-fold down-regulation of
steady-state LmGT2 mRNA in amastigotes cannot be
explained by transcriptional regulation. Thus, LmGT2
expression is regulated primarily at the post-transcriptional
level.
-tubulin mRNA seems to be
similar in both promastigotes and amastigotes, a finding that is
consistent with our observation and a previous report (30) that total
-tubulin mRNA is constitutively expressed in L. mexicana. It is interesting, however, that the most abundant
-tubulin transcript shifts from 2.4 to 2.8 kb when promastigotes are
compared with amastigotes and that these major transcripts are also
more stable in promastigotes and axenic amastigotes, respectively.
ACKNOWLEDGEMENTS
FOOTNOTES
A recipient of a Molecular Parasitology Scholar Award from the
Burroughs Wellcome Fund. To whom reprint requests should be addressed:
Dept. of Molecular Microbiology and Immunology, Oregon Health Sciences
University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-2426; Fax: 503-494-6862.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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D. R. Brooks, H. Denise, G. D. Westrop, G. H. Coombs, and J. C. Mottram The Stage-regulated Expression of Leishmania mexicana CPB Cysteine Proteases Is Mediated by an Intercistronic Sequence Element J. Biol. Chem., December 7, 2001; 276(50): 47061 - 47069. [Abstract] [Full Text] [PDF]
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A. Seyfang and S. M. Landfear Four Conserved Cytoplasmic Sequence Motifs Are Important for Transport Function of the Leishmania Inositol/H+ Symporter J. Biol. Chem., February 25, 2000; 275(8): 5687 - 5693. [Abstract] [Full Text] [PDF]
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M. Yamage, A. Debrabant, and D. M. Dwyer Molecular Characterization of a Hyperinducible, Surface Membrane-anchored, Class I Nuclease of a Trypanosomatid Parasite J. Biol. Chem., November 10, 2000; 275(46): 36369 - 36379. [Abstract] [Full Text] [PDF]
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