From the
The major surface protease, gp63, of Leishmania chagasi is encoded by 18 or more tandem msp genes that can be
grouped into three classes on the basis of their unique 3`-untranslated
sequences (3`-UTRs) and their differential expression. RNAs from the
mspLs occur predominantly during the logarithmic phase of
promastigote growth in vitro, RNAs from the mspSs are
present mainly in stationary phase, and RNAs from mspCs occur
throughout growth in culture. All three classes of gp63 genes are
constitutively transcribed during all growth phases, indicating that
their expression is post-transcriptionally regulated. Chimeric plasmids
containing the three different 3`-UTRs and downstream intergenic
regions (IRs) fused downstream of the
All Leishmania species possess on their surface a
glycolipid-anchored zinc protease, called gp63,
The haploid
genome of L. chagasi contains 18 or more genes encoding gp63,
named msp for genes of the major surface protease
(11) .
These genes (1.8 kb coding region) are clustered in tandem on a single
chromosome (Fig. 1) and are differentially expressed by
promastigotes during growth in vitro(7, 11) .
At least five genes in the cluster (mspS1-S5 in
Fig. 1
) encode a 3.0-kb mRNA species that occurs predominantly in
stationary phase promastigotes. A minimum of 12 mspLs encode a
2.7-kb gp63 mRNA expressed predominantly in logarithmic phase
promastigotes, and one mspC specifies two gp63 mRNA species of
2.6 and 3.1 kb that are expressed constitutively throughout
promastigote growth in vitro. The major differences among the
three classes of gp63 genes and their mRNAs are the unique sequences
and lengths of their 3`-untranslated regions (3`-UTRs) and downstream
intergenic regions (IRs)
(7) . A few clustered point differences
also occur in the coding regions of
mspS1-S5
(11) . In addition to changes in these
same clustered regions, mspC encodes a gp63 with an altered
and longer carboxyl terminus in which the signal for attachment of a
glycolipid anchor is replaced by a sequence of 41 amino acids that
could be a transmembrane region with a short cytoplasmic tail.
In Leishmania and other
trypanosomatid protozoa, tandem copies of the same gene, or adjacent
genes specifying different proteins, are often transcribed into a
polycistronic precursor RNA (13-18). These precursor transcripts
are processed into individual mRNAs by intergenic cleavages followed by
addition of a 39-nucleotide spliced leader (SL) to the 5` ends in a
trans-splicing reaction and addition of a poly(A) to the 3` ends via
polyadenylation
(19, 20, 21) . The mRNA abundance
could be regulated at one or both of these addition reactions. For
example, the efficiency of trans-splicing has been shown to be affected
by the pyrimidine richness of the sequence near the splice acceptor
site which varies for different genes
(22, 23) . However,
it has yet to be demonstrated that these intergenic pre-mRNA processing
sites participate in the mechanisms of differential expression.
Here
we show that all three classes of the tandemly arrayed gp63 genes are
constitutively transcribed throughout promastigote growth in
vitro. The three different 3`-UTRs and their IRs were cloned
immediately downstream of the
In the first series of experiments, the 3`-UTRs
alone of mspL, mspS, and mspC (extending
from the stop codon through the 3`-poly(A)) were isolated by
restriction enzyme digestion or by PCR amplification of the
corresponding cloned cDNAs. The lengths of these 3`-UTRs are 1.2, 0.9,
and 1.3 kb, respectively, for mspS, mspL, and
mspC (7). They were cloned in both orientations at a
XbaI site immediately downstream of the
It turned out that in all cases the
presence and orientation of the three different 3`-UTRs alone had
little or no effect on the relative amount of
In the next set of experiments, the 3`-UTRs plus
their associated downstream IRs were cloned behind the
shows the
results of four independent sets of assays utilizing promastigotes
stably transfected with pX-
The ratios of the normalized values in
stationary cells versus logarithmic cells are given in the
right-hand column of . This ratio was 0.8 when the
mspL 3`-UTR + IR was downstream of the
When
the 3`-UTR + IR of mspC was present (lanes 5),
little or no difference in the abundance of the
The
nitrocellulose filter was stripped and reprobed with the unique 3`-UTR
of mspC (Fig. 3, right panel) to verify the
presence of the 2.6-kb and 3.1-kb mspC mRNAs in all of the
transfected cells and to confirm the existence of the chimeric
To summarize the results from a
large number of
We previously showed that mspL mRNA increases
16-fold when promastigotes are incubated with cycloheximide, suggesting
the level of this RNA may be regulated by a labile, sequence-specific
protein that targets this RNA for rapid degradation
(12) . Since
cycloheximide had little effect on the mspS and mspC
mRNAs which have 3`-UTRs very different from the mspL mRNA, we
originally thought the three distinct 3`-UTRs might affect expression
of the
This result led us to examine the IRs downstream
of each gp63 gene class. Previous studies of IRs between trypanosomatid
genes have focused on the relationship between 5`-SL addition and
3`-polyadenylation, both events of which are directed by signals in the
IR. In African trypanosomes, SL addition precedes polyadenylation at
the tubulin locus
(21) , and it follows polyadenylation at the
HSP70 locus
(41) . In Leishmania, the temporal order of
the two events is not known, but the polyadenylation site is dictated
by the presence of a SL addition site 200-500 nucleotides
downstream in the IR
(19) . At the L. chagasi gp63
locus, each internal IR ends with a highly conserved 216-bp sequence
that provides the SL addition site for the mRNA of the following gene
(11).
The simplest model consistent with the results
described here is one in which the amounts of mspL and
mspS mRNA are each post-transcriptionally regulated by
different molecular mechanisms. One possibility is that the steady
state level of mspL mRNA is controlled by its own stability
after it has been processed from the precursor RNA, whereas the level
of mspS RNA is determined during the pre-mRNA processing
events themselves. However, the levels of the two RNA species also
could be regulated independently by a combination of
post-transcriptional events such as RNA localization, transport or
translation, as well as stability, 5`-SL addition, or
3`-polyadenylation. Clearly, the growth-specific, differential
expression of the gp63 genes is a complicated process determined
largely by post-transcriptional events.
In summary, many biochemical
changes have been detected during the growth of Leishmania promastigotes in vitro. Our observations indicate that at
least one of these changes is due to different mechanisms of
post-transcriptional processing of the same RNAs by the different
parasite stages. Elucidation of the molecular interactions responsible
for this differential gene expression should provide a more complete
understanding of the molecular events leading to the development of
virulence in this pathogen.
We thank Steve Beverley for providing plasmid
pX-
-galactosidase (
-gal)
coding region were transfected into L. chagasi, and their
effects on
-gal RNA processing and enzymatic activity were
examined. The presence of the 3`-UTRs by themselves had no substantive
effect on
-gal expression. However, the 3`-UTR from a
mspS plus its IR resulted in about 20-fold more
-gal
activity and RNA in stationary phase relative to logarithmic phase
cells. In contrast, the 3`-UTRs plus IRs of mspL and
mspC had either no or little effect, respectively, on
-gal expression. Thus, differential expression of the
mspLs and mspSs is post-transcriptionally controlled
by different mechanisms.
(
)
that participates in attachment of the promastigote stage
of these protozoan parasites to host macrophages (1-6). During
growth in liquid culture, promastigotes of many Leishmania species develop from a less infectious form in the logarithmic
phase of growth to a highly virulent form in stationary
phase
(7, 8, 9, 10) . In Leishmania
chagasi, this increase in virulence is accompanied by an 11-fold
increase in the amount of gp63 per cell
(10) .
Figure 1:
Diagram of the organization of gp63
genes in the L. chagasi genome (11). The large boxes indicate the 1.8-kb coding regions, and the small boxes are the 3`-UTRs. The 3`-UTRs of the tandem mspS2, -S1,
-S3, and -S5 are on 6.0-kb ClaI fragments, the tandem
mspL 3`-UTRs are on 3.0-kb ClaI fragments, and the
mspC 3`-UTR is on a 3.6-kb ClaI fragment. A final
stationary gene, mspS4, follows mspC. The indicated
3`-UTRs plus their downstream IRs, and the 3`-UTRs by themselves, were
cloned at the indicated site immediately after the coding region of the
-gal gene in plasmid pX-
gal. Cross-hatch lines between mspS3 and mspS5, and between two
mspLs, signify that the total number of genes is not known.
The indicated restriction sites are for HindIII (H),
ClaI (C), XbaI (X), and the first
SalI site in the gp63 coding regions
(S).
Previously, we showed that inhibitors of protein synthesis affect
the steady state levels of the three gp63 mRNA classes
differently
(12) . The amount of the 2.7-kb mspL mRNA
increases 16-fold after incubation in cycloheximide or other protein
synthesis inhibitors, even though the transcriptional rate of the
mspLs is unaltered. This increase in the steady state level of
the 2.7-kb mspL mRNA is accompanied by a corresponding
increase in its half-life, suggesting that its abundance is controlled
by a labile regulatory protein that specifically targets this mRNA for
degradation. In contrast, the amounts of the 3.0-kb mspS mRNA
and the two mspC mRNAs increase only 2-fold or less in the
presence of protein synthesis inhibitors. Thus, their steady state
levels are regulated by a different molecular mechanism than is that of
the mspL mRNA.
-galactosidase (
-gal) coding
region to examine the influence of these sequences on
-gal
expression during promastigote growth from logarithmic to stationary
phase. We find that the IR downstream of mspS affects the
levels of
-gal RNA and enzymatic activity, but that the
corresponding IRs downstream of mspL and mspC have no
effect and much less effect, respectively. Thus, the downstream IR
regulates mspS, but not mspL, mRNA abundance in
different promastigote growth phases. In contrast, protein synthesis
inhibitors have the opposite effect; they affect mspL, but not
mspS, mRNA abundance
(12) . These results collectively
demonstrate that expression of both mspLs and mspSs
is regulated post-transcriptionally but that this regulation occurs via
different molecular mechanisms for the two gene classes.
Parasites
A Brazilian isolate of L. chagasi was kindly provided by Richard Pearson at the University of
Virginia. Parasites were maintained in hamsters, and promastigotes were
cultured in vitro in a modified minimal essential medium
(HOMEM) as described previously
(24) , with or without 20 µg
of G418/ml (Geneticin; Life Technologies, Inc.).
Plasmid Constructions
All plasmids were derived
from the parent plasmid pX-gal kindly provided by Steve
Beverley
(25) . The 3`-UTR of a mspS and its downstream
IR, and the 3`-UTR of a mspL and its downstream IR, were
isolated by XbaI/SalI double digestion of recombinant
phage DNAs containing these genomic DNA regions of L. chagasi (11). XbaI cleaves at the stop codon of mspS and
mspL, and SalI cleaves 5 base pairs downstream from
the start codons of all known L. chagasi gp63 genes (see
Fig. 1
). Since a XbaI site does not occur in
mspC, its 3`-UTR and downstream IR were obtained by PCR
amplification using a forward primer whose sequence includes the
mspC stop codon and a reverse primer whose sequence is near
the SalI site of the downstream mspS4 (Fig. 1).
The 3`-UTRs alone of mspL and mspS were excised from
the corresponding cDNAs using XbaI which cleaves at the stop
codon and at a downstream site in the vector. The 3`-UTR of
mspC was obtained by PCR amplification. These fragments were
cloned into an XbaI site at the end of the
-gal coding
region in pX-
gal, as depicted in Fig. 1.
DNA Transfections and
Plasmid DNAs used for transfections were prepared using
Qiagen columns (Qiagen Inc., Chatsworth, CA) according to the
supplier's instructions. The plasmids were transiently
transfected into early logarithmic or stationary phase L. chagasi by electroporation as described previously
(26) . Stable,
G418-resistant, L. chagasi transfectants were obtained by
plating parasites 1 day after transfection on agar plates containing
medium 199 (Life Technologies, Inc.) supplemented as
described
(27) . Plates were incubated at 26 °C under
CO-Galactosidase
Assays
, and colonies were picked 2 weeks later and grown in 1
ml of HOMEM. Parasites were transferred to 5 ml or more of HOMEM and
grown under the selective pressure of 20 µg of G418/ml. Extracts of
logarithmic (7-8
10
cells/ml) or stationary
(4-7
10
cells/ml) phase cells were prepared
and assayed for
-gal enzyme activity as described
(25) .
Nuclear Run-on Assays
Promastigotes were harvested
in logarithmic or stationary phase and centrifuged at 3000 rpm in a
Beckman J-6B centrifuge for 5 min. The cell pellet was resuspended at 4
10
cells/ml in ice-cold hypotonic buffer (0.5
M hexylene glycol, 1 mM Pipes, 1 mM
spermidine, 0.1 mM phenylmethylsulfonyl fluoride, 1
mM EDTA, 1 mM EGTA, and 1 mM
dithiothreitol). Nonidet P-40 and Triton X-100 were added to a final
concentration of 0.5% each, and the cells were lysed by vigorously
vortexing for 30 s. Immediately, 2 volumes of ice-cold 2
nuclei
wash buffer (40 mM Tris-HCl, pH 7.5, 0.64 M sucrose,
1 mM spermidine, 0.1 mM phenylmethylsulfonyl
fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, and 60 mM KCl) were added and mixed by
vortexing. As described previously
(12) , the nuclei were
concentrated and incubated with [
-
P]UTP,
and the labeled RNA was isolated for use in probing filters. The
filters contained 2 µg each of single-stranded M13mp19 DNA without
insert or with insert containing the coding regions of ATPase and gp63,
or the 3`-UTRs of mspL, mspS, and mspC, or a
430-bp sequence from the large subunit rRNA of L. chagasi.
Other Methods and Procedures
RNA and DNA were
isolated from logarithmic or stationary phase promastigotes, and
Northern or Southern blots were conducted as described
(11) . For
Southern blots, DNA probes were P-labeled with a random
primer labeling kit (Boehringer Mannheim). For Northern blots,
P-labeled noncoding strand DNA probes were prepared from
PCR products by a primer extension reaction using an oligonucleotide
specific for the noncoding strand.
RESULTS
The Expression of the Three gp63 Gene Classes Is
Regulated Post-transcriptionally
Long exposures of Northern
blots containing L. chagasi promastigote RNA probed with the
gp63 coding region (not shown) revealed the expected gp63 mRNAs plus a
faint ladder of larger multimeric sized RNA molecules, suggesting that
some, and perhaps all, of the tandem gp63 genes are transcribed into a
polycistronic precursor RNA. To examine which gp63 gene classes are
transcribed during logarithmic and stationary phases of growth, nuclear
run-on experiments were conducted. Nuclei were isolated from
promastigotes in each growth phase, and their nascent RNAs were labeled
by incubation with [-
P]UTP for use as
probes in slot blot hybridizations of the plus and minus strands of
various genes or 3`-UTRs (Fig. 2). The drug
-amanitin (500
µg/ml) was included in some nuclei incubations as a control to
verify the authenticity of the nuclear run-on assays. This drug
inhibits transcription of most protein-coding genes in trypanosomatids,
with the notable exception of genes for the surface proteins of African
trypanosomes (procyclin and the variant surface
glycoprotein)
(28, 29) .
Figure 2:
The three gp63 gene classes are
constitutively transcribed in both logarithmic and stationary phase
promastigotes. P-Labeled, run-on transcripts from nuclei
isolated from logarithmic or stationary phase cells incubated in the
presence (+) or absence (-) of
-amanitin were
hybridized to 2 µg each of the following membrane-bound plus
(+) and minus (-) strand DNAs: DNA from phage M13mp19 alone
(M13mp19), M13 containing the coding sequences for Leishmania ATPase (ATPase), gp63 (gp63 coding) and ribosomal RNA (rRNA), or
M13 containing the unique sequences of the 3`-UTRs of mspL
(gp63 log), mspS (gp63 stationary), and mspC (gp63
constitutive).
As shown in Fig. 2,
transcription of the 3`-UTRs of mspL, mspS, and
mspC occurred to the same extent in both logarithmic and
stationary phase nuclei, despite the fact that mspL
transcripts occur predominantly in logarithmic phase cells and
mspS transcripts appear predominantly in stationary phase
cells
(6, 7) . Thus, the differential expression of
mspL and mspS mRNAs at the different growth stages
must be controlled by regulatory events that occur
post-transcriptionally. The housekeeping genes for an ATPase and a
ribosomal RNA served as control genes in this analysis, and their
transcription occurred equally in both logarithmic and stationary phase
cells, as expected. Transcription of all the genes tested was derived
predominantly from the expected plus strand. In addition, transcription
of the genes for gp63 and ATPase was clearly inhibited by
-amanitin, a feature of genes normally transcribed by RNA
polymerase II, whereas transcription of the ribosomal RNA gene was not
inhibited as expected for genes transcribed by RNA polymerase I. Thus,
unlike African trypanosomes, genes for the major protein on the surface
of Leishmania promastigotes are transcribed by a conventional
RNA polymerase II.
Influence of the gp63 Gene 3`-UTRs and Downstream IRs on
Since the differential expression of
the three gp63 gene classes is controlled post-transcriptionally and
since all of the genes share the same 5`-UTR
(11) , the unique
3`-UTRs of these genes and/or their associated downstream intergenic
regions (IRs) may play a role in this post-transcriptional regulation.
To examine this possibility, the 3`-UTRs alone and the 3`-UTRs plus
their associated IRs were cloned downstream of the -Gal Gene Expression
-gal coding
region in the plasmid pX-
gal
(25) . As shown in
Fig. 1
, this plasmid contains a gene for neomycin resistance
which permits selection of transformed Leishmania cells in the
presence of the drug G418 and also contains the Escherichia coli gene for
-gal whose activity can be assayed readily. Both
genes are transcribed when the plasmid is transfected into
promastigotes, and both have an upstream SL addition
signal
(25) .
-gal coding
region. A total of six recombinant plasmids (containing each of the
three 3`-UTRs inserted in both orientations) and the parent plasmid
pX-
gal were electroporated into promastigotes. In some experiments
the circular plasmids were transiently transfected into either
logarithmic or stationary phase promastigotes, and, after 24 h of
incubation in culture medium (without G418), a protein extract was
prepared for assaying
-gal activity. In other experiments, stably
transfected cells were cloned by plating and then grown in liquid
medium containing G418 for 3 weeks before an extract was prepared for
-gal enzyme assays.
-gal enzyme activity
in either logarithmic or stationary phase cells (not shown). In no case
was the activity altered more than 1.5-fold from that of the parent
plasmid pX-
gal. This lack of a substantive difference in the
-gal activity derived from 3`-UTR-containing plasmids and from the
parent plasmid was observed in both cells transiently transfected with
the plasmids for 1 day and cells stably transfected with the plasmids
for several weeks.
-gal coding
region, as indicated in Fig. 1. These cloned segments extend from
the gp63 termination codon to a SalI site 5 base pairs beyond
the start codon of the downstream gp63 gene. Their lengths are 4.2,
1.2, and 1.7 kb, respectively, for mspS, mspL and
mspC. Again, these segments were inserted in both orientations
relative to the
-gal gene, and the recombinant plasmids were
introduced transiently or stably into promastigotes. When the segments
were in the reverse orientation, only small differences (less than
2-fold) from that of pX-
gal occurred (not shown). When the
segments were inserted in the forward direction, however, the results
summarized in were obtained.
gal or pX-
gal containing the
downstream 3`-UTRs + IRs of mspL, mspS, and
mspC. These promastigotes were grown to logarithmic phase (day
2 or 3) or stationary phase (day 8 or 9), and cell extracts were
prepared for
-gal enzyme assays. The growth phases were defined
according to concentration and morphology as described
previously
(10) . In the pX-
gal transfectants, the amount of
-gal activity was about 11-fold more during logarithmic phase than
stationary phase (15.7 units versus 1.4 units), which likely
reflects the increased metabolic rate at that growth stage. Thus, the
amounts of
-gal activity in the logarithmic or stationary phase
transfectants containing pX-
gal were normalized to one, and the
relative amounts of activity in the other transfectants adjusted to
this normalized value. For example, the normalized
-gal activity
value in logarithmic phase promastigotes containing the mspS
3`-UTR + IR is 0.3.
-gal gene,
indicating that this region does not affect substantively the relative
amounts of
-gal activity in logarithmic and stationary phase
promastigotes. It was 2.7 when the corresponding region of
mspC was present, suggesting that this region causes a
2-3-fold increase in the level of
-gal in stationary phase
cells. The most dramatic change, however, was with mspS 3`-UTR
+ IR, where a 19.7-fold increase in the
-gal activity
occurred in stationary versus logarithmic phase cells. Of this
nearly 20-fold difference, about 3-fold is due to a decrease of
-gal activity in logarithmic phase cells when mspS 3`-UTR
+ IR is present versus when pX-
gal is present (0.3
unit versus 1.0 unit), and the remainder is due to an increase
in stationary phase cells relative to when pX-
gal is present (5.9
units versus 1.0 unit).
To see if the differences in
-Gal RNA Levels in the Presence of the gp63 Gene
3`-UTRs Plus Associated IRs
-gal activity shown in reflect differences in
-gal mRNA levels, Northern blots were conducted on total RNAs
extracted from the same stably transfected logarithmic or stationary
phase promastigotes. Fig. 3shows the results of a Northern blot
probed with the
-gal coding region and with the unique 3`-UTR of
mspC. As expected, no
-gal RNA occurred in untransfected
promastigotes (lanes 1, left panel). In both
logarithmic and stationary phase cells containing pX-
gal
(lanes 2, left panel), the
-gal probe hybridized
predominantly to a 7.5-kb RNA and weakly to smaller RNAs. Since the
5`-UTR of the
-gal RNA is very short due to the presence of a SL
addition site just upstream of the 3-kb
-gal coding
region
(25) , about 4.5 kb of the major 7.5-kb
-gal RNA must
be a 3`-UTR derived from the plasmid sequence. The simplest explanation
of this result is that transcription extended about 4.5 kb beyond the
-gal gene to a sequence fortuitously recognized by Leishmania as a site for polyadenylation (or as a site for SL addition which
in turn directs the location of polyadenylation, see Ref. 19). When the
3`-UTR + IR of mspL was present (lanes 3), a
-gal RNA of 4.4 kb occurred in both logarithmic and stationary
phase cells that was only slightly more abundant than the 7.5-kb
-gal RNA from pX-
gal (compare lanes 2 and
3). Two conclusions can be derived from this result. First,
neither the 0.9-kb 3`-UTR nor the 0.3-kb IR of mspL enhances
the abundance of the
-gal RNA in logarithmic phase cells or
diminishes it in stationary phase cells. Thus, this 1.2-kb sequence by
itself is not sufficient to regulate the different levels of
mspL RNA in logarithmic and stationary phase cells. Secondly,
the 0.3-kb IR provides a signal for polyadenylation (or adjacent
downstream SL addition
(19) ) because insertion of this segment
reduces the size of the
-gal RNA from 7.5 kb to about 4.4 kb. If
one considers the length of the poly(A) tail and the distance between
the SL and the
-gal start codon, it is likely that this signal is
the same one used for polyadenylation and/or SL addition by the
mspLs flanking this IR.
Figure 3:
Northern blots of RNAs isolated from
stable transfectants containing the plasmids indicated in Fig. 1. Total
RNAs were isolated from logarithmic (log) or stationary
(stationary) phase promastigotes that contained no plasmid
(lanes 1), pX-gal alone (lanes 2), pX-
gal
with the 3`-UTR of mspL + its IR (lanes 3),
pX-
gal with the 3`-UTR of mspS1 + its IR (lanes
4), and pX-
gal with the 3`-UTR of mspC + its IR
(lanes 5). The filter was probed with
P-labeled
-gal coding region (
gal probe) and then stripped
and reprobed with the
P-labeled 3`-UTR of mspC
(gp63 constitutive probe).
In cells containing the 3`-UTR
+ IR of mspS (lanes 4), a -gal RNA of 4.6
kb was much more abundant in stationary phase than in logarithmic phase
cells (compare the two lanes 4 in the left panel).
This result is consistent with the finding that stationary phase cells
containing this recombinant plasmid had about 20 times more
-gal
activity than did the corresponding logarithmic phase cells
(). In addition, the 4.6-kb size of the RNA (versus 4.4 kb when the 3`-UTR + IR of mspL was present)
suggests that the mspS polyadenylation site is used.
-gal RNAs in
logarithmic and stationary phase cells was observed despite the
2.7-fold increase in
-gal activity in stationary cells
(), but consistent with the fact that the mspC
RNAs vary in abundance either minimally or not at all during
promastigote growth
(7, 12) . The presence of at least
two
-gal RNA species in these cells (particularly apparent in
lane 5 containing RNA from stationary phase cells) is
consistent with the occurrence of two mspC mRNAs (2.6 and 3.1
kb), as generated by two independent polyadenylation (or SL addition)
signals downstream of mspC (7). Since the relative abundance
of the mspC protein product in logarithmic versus stationary phase promastigotes is not known, we did not examine
further the discrepancy between the 2.7-fold increase in
-gal
activity in stationary phase cells and the lack of a corresponding
increase in
-gal RNA levels. However, one possibility for this
discrepancy is that there is a difference in the translational
efficiency of the mspC mRNAs in the two growth phases.
-gal RNAs containing the 3`-UTR of mspC (lanes 5). As
expected, all lanes had weak bands corresponding to the endogenous 2.6-
and 3.1-kb mspC RNAs, including lanes 1 containing
RNA from untransfected promastigotes. Two additional very strong bands
in lanes 5 correspond to the bands detected with the
-gal
coding region probe, confirming that these chimeric RNAs contain the
-gal coding region fused to the 3`-UTR of mspC. The
presence of the 4.6-kb band in lane 4 of stationary phase
cells is due to residual hybridization of the
-gal probe that was
not washed off. The signal intensities of the 2.6- and 3.1-kb bands
serve as internal standards for the amount of RNA loaded in each lane.
They indicate that in this particular experiment somewhat less RNA from
the logarithmic phase transfectant with the 3`-UTR + IR of
mspS was added than RNA from the same transfectant in
stationary phase growth (compare lanes 4 probed with the
constitutive probe). However, similar Northern blots in which the
signal intensities of the 2.6- and 3.1-kb RNAs from logarithmic and
stationary phase cells containing the 3`-UTR + IR of mspS
were the same yielded similar results, indicating that in these
stationary phase cells the
-gal RNA was at least 20 times more
abundant than in the corresponding logarithmic phase cells. In
addition, when similar filters containing RNAs from the same
transfectants were probed with 3`-UTR sequences unique to mspS
or mspL, the same general hybridization pattern was obtained,
i.e. the mspS 3`-UTR probe hybridized to the
endogenous mspS RNA and to the chimeric
-gal RNA fused to
the mspS 3`-UTR, and the mspL 3`-UTR probe gave a
corresponding result (not shown).
-gal activity assays and Northern blots similar to
those shown in and Fig. 3, the presence of the
3`-UTR + IR of mspS downstream of the
-gal gene
increased the abundance of
-gal RNA and the amount of
-gal
enzymatic activity by a factor of about 20-fold in stationary phase
cells compared to logarithmic phase cells. However, the 3`-UTR +
IR of mspL did not exert a similar influence on the level of
-gal RNA and activity in logarithmic cells. Likewise, and
consistent with the lack of growth phase-associated regulation of
mspC expression
(7) , the 3`-UTR + IR of
mspC also had less effect on
-gal RNA and activity in
logarithmic versus stationary phase cells than did the 3`-UTR
+ IR of mspS.
Plasmid Copy Number in Stably Transfected
Promastigotes
Since the number of plasmid molecules in the
stably transfected promastigotes used in the experiments shown in
and Fig. 3could affect the levels of -gal
enzyme activity and
-gal RNA, the relative plasmid copy number in
each of the transfectants was estimated (Fig. 4). Total DNAs
(genomic and plasmid) from the different stably transfected
promastigotes were cleaved with ClaI and hybridized in a
Southern blot with a mixture of
-gal and gp63 coding region
probes. Prior Southern blots and sequence determinations showed that
ClaI cleaves at the same coding region location within each of
the three msp classes as shown in Fig. 1(11) .
Thus, the unique 3`-UTRs of mspL, mspS, and
mspC occur in ClaI fragments of 3.0, 6.0, and 3.6 kb,
respectively. In addition, most of the coding region of the first
mspS gene in the cluster occurs in a 2.2-kb ClaI
fragment. In lanes 1 of Fig. 4, which contain DNAs of
untransfected logarithmic or stationary phase promastigotes, only
restriction fragments containing these 3`-UTRs are detected. At the
exposure shown in this figure, the 6.0-, 3.6-, and 2.2-kb fragments are
most obvious in lane 3 containing DNA from logarithmic phase
cells because more DNA was added in this lane.
Figure 4:
Estimation of relative plasmid copy number
in the stable transfectants used in the experiments shown in Fig. 3 and
described in Table I. Lanes 1-5 contain
ClaI-digested total DNAs (genomic + plasmid) from the
same stable transfectants in logarithmic and stationary phase growth as
described for lanes 1-5 in Fig. 3. The DNAs were
hybridized in this Southern blot with a mixture of the -gal and
gp63 coding region probes.
ClaI cleaves
once in the pX-gal plasmid and its derivatives. In lanes
2-5, the signals from these linearized plasmids, as detected
with the
-gal gene probe, produce the single bands corresponding
to fragments of 9.2 to 13.4 kb. For example, the 9.2-kb pX-
gal is
the smallest plasmid (lanes 2) and its derivative containing
the 4.2-kb mspS 3`-UTR plus IR (lanes 4) is the
largest plasmid. Since the number of mspL genes in the genome
is constant, the signals from the plasmids can be compared with the
signals from the 3.0-kb genomic fragment in each lane to estimate the
relative number of plasmid molecules in each transfectant (lanes
2-5). When a short exposure of the autoradiograms shown in
Fig. 4
was scanned by densitometry, the plasmid copy number was
found to vary by less than a factor of 2 in each of the recombinant
transfectants. Likewise, the relative number of plasmid molecules in
logarithmic versus stationary phase cells of the same
transfectant did not vary by more than 2-fold. Therefore, variations in
plasmid copy number do not account for the increased levels of
-gal activity and
-gal RNA in stationary phase transfectants
containing the plasmid with the 3`-UTR + IR of mspS.
DISCUSSION
During growth in liquid medium, the promastigote form of
Leishmania protozoa develops from a less infectious form
during logarithmic growth to a highly virulent form (termed the
metacyclic promastigote) at stationary phase
(8, 9) .
Accompanying this change in virulence are changes in parasite
morphology
(8, 30) , increased resistance to complement-
and HO
-mediated
killing
(10, 31) , lower respiratory rate
(30) ,
increased glycosylation of the surface lipophosphoglycan
(32) ,
an increase in hsp70 mRNA
(33) , increased expression of a
protein containing a basic zipper motif
(34) , and an increase in
the amount of gp63 protein
(10) . Given the growth-related
increase in virulence across different Leishmania species, it
is likely that the development of these virulence characteristics in
the sand fly vector are crucial for the parasite's infection of a
mammalian host. Thus, a definition of the molecular events accompanying
these changes should reveal processes required for the parasite's
survival. Based on our prior observation that different gp63 gene
transcripts occur in logarithmic and stationary phase parasites, we
undertook the present series of experiments to identify regions within
the gp63 gene cluster that are responsible for this differential
regulation.
-gal reporter gene differently in logarithmic and
stationary phase cells. Consistent with this possibility are reports
from other experimental systems that 3`-UTRs can participate in
phenomena as diverse as self-regulation of their own
degradation
(35, 36) , rates of translation
(37) ,
mRNA localization
(38) , cellular growth and
differentiation
(39) , and tumor suppression
(40) .
However, when only the 3`-UTRs of the gp63 genes were cloned downstream
of the
-gal gene, they had no substantive differential effect on
-gal expression during the two growth phases. Thus, presence of
the actual 3`-UTR sequences themselves are not the sole determinant of
the different steady state levels, although it is worth noting that the
-gal transcripts from these 3`-UTR-containing plasmids likely
extend beyond the poly(A) derived from the cDNAs, and it is not clear
what influence this additional segment of the transcript has on the
steady state level.
(
)
The sequences and sizes of the IRs
upstream of this conserved sequence differ for each of the three gene
classes. The sequences of the short IRs between the mspLs (273
bp) and downstream of mspC (422 bp) have been determined and
do not share obvious homologies except for the 216 bp. The sequence of
the 3-kb IR between the mspSs has not been determined
completely, but Southern blots indicate that, aside from the 216 bp, it
is different from the IRs following the mspL and mspC
genes. Based on the current results, we hypothesize that this 3-kb
region contains the sequence elements responsible for the increased
amount of mspS mRNA in stationary phase cells. These elements
could influence polyadenylation and/or trans-splicing since the only
known polyadenylation and SL addition sites in this IR are separated by
3 kb, much longer than the 200-500 bp described above (19). Since
the 4.6-kb
-gal transcript seen in lanes 4 of
Fig. 3
includes only about 1.2 kb from the entire 4.2-kb
mspS 3`-UTR + IR, there may be an additional
sequence-driving polyadenylation contained within this region. It also
is possible that events other than 5` and 3` processing are influenced
by the mspS 3`-UTR + IR, accounting for the growth
phase-specific regulation of mspS RNA. To identify the
locations and sequences of these elements, systematic deletion and
mutagenesis of segments within the IR downstream of the mspSs
will be necessary.
Table:
-Galactosidase activity assays
-gal,
-galactosidase; SL, spliced leader; PCR, polymerase chain
reaction; kb, kilobase(s); bp, base pair(s); Pipes,
1,4-piperazinediethanesulfonic acid.
gal and for useful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.