©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intergenic Regions between Tandem gp63 Genes Influence the Differential Expression of gp63 RNAs in Leishmania chagasi Promastigotes (*)

Ramesh Ramamoorthy (1), Kristin G. Swihart (1), James J. McCoy (2), Mary E. Wilson (2), John E. Donelson (1)(§)

From the (1) Departments of Biochemistry, (2) Internal Medicine, and (3) Microbiology, University of Iowa, the (4) Veterans Administration Medical Center, and the (5) Howard Hughes Medical Institute, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

All Leishmania species possess on their surface a glycolipid-anchored zinc protease, called gp63,() 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) .

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.


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.

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 -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.


EXPERIMENTAL PROCEDURES

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 -Galactosidase Assays

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, 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 -Gal Gene Expression

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 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) .

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 -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.

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 -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.

In the next set of experiments, the 3`-UTRs plus their associated downstream IRs were cloned behind the -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.

shows the results of four independent sets of assays utilizing promastigotes stably transfected with pX-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.

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 -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).

-Gal RNA Levels in the Presence of the gp63 Gene 3`-UTRs Plus Associated IRs

To see if the differences in -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.

When the 3`-UTR + IR of mspC was present (lanes 5), little or no difference in the abundance of the -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.

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 -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).

To summarize the results from a large number of -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.

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 -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.

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 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.

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.

  
Table: -Galactosidase activity assays



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants AI32135 (to M. E. W. and J. E. D.) and AI30126 (to M. E. W.), a Veteran's Administration merit review grant (to M. E. W.), and American Heart Association Established Investigator Award 93002660 (to M. E. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, University of Iowa, Iowa City, IA 52242. Tel.: 319-335-7889; Fax: 319-335-6764.

The abbreviations used are: gp63, glycoprotein of 63 kDa; UTR, untranslated region; IR, intergenic region; mspL, logarithmic gp63 gene; mspS, stationary gp63 gene; mspC, constitutive gp63 gene; -gal, -galactosidase; SL, spliced leader; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s); Pipes, 1,4-piperazinediethanesulfonic acid.

R. Ramamoorthy, unpublished data.


ACKNOWLEDGEMENTS

We thank Steve Beverley for providing plasmid pX-gal and for useful discussions.


REFERENCES
  1. Bordier, C.(1987) Parasitol. Today 3, 151-153 [Medline] [Order article via Infotrieve]
  2. Bordier, C., Etges, R. J., Ward, J., Turner, M. J., and Cardosa de Almeida, M. L.(1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5988-5991 [Abstract]
  3. Chang, K.-P., and Chaudhuri, G.(1990) Annu. Rev. Microbiol. 44, 499-529 [CrossRef][Medline] [Order article via Infotrieve]
  4. Russell, D. G., and Talamas-Rohana, P.(1989) Immunol. Today 10, 328-333 [Medline] [Order article via Infotrieve]
  5. Wilson, M. E., and Hardin, K. K.(1988) J. Immunol. 141, 265-272 [Abstract/Free Full Text]
  6. Wilson, M. E., Hardin, K. K., and Donelson, J. E.(1989) J. Immunol. 143, 678-684 [Abstract/Free Full Text]
  7. Ramamoorthy, R., Donelson, J. E., Paetz, K. E., Maybodi, M., Roberts, S. C., and Wilson, M. E.(1992) J. Biol. Chem. 267, 1888-1895 [Abstract/Free Full Text]
  8. Sacks, D. L., and Perkins, P. V.(1984) Science 223, 1417-1419 [Medline] [Order article via Infotrieve]
  9. Da Silva, R., and Sacks, D. L.(1987) Infect. Immun. 55, 2802-2806 [Medline] [Order article via Infotrieve]
  10. Zarley, J. H., Britigan, B. E., and Wilson, M. E.(1991) J. Clin. Invest. 88, 1511-1521 [Medline] [Order article via Infotrieve]
  11. Roberts, S. C., Swihart, K. G., Agey, M. W., Ramamoorthy, R., Wilson, M. E., and Donelson, J. E.(1993) Mol. Biochem. Parasitol. 62, 157-172 [CrossRef][Medline] [Order article via Infotrieve]
  12. Wilson, M. E., Paetz, K. E., Ramamoorthy, R., and Donelson, J. E. (1993) J. Biol. Chem. 268, 15731-15736 [Abstract/Free Full Text]
  13. Muhich, M. L., and Boothroyd, J. C.(1988) Mol. Cell. Biol. 8, 3837-3846 [Medline] [Order article via Infotrieve]
  14. Kapotas, N., and Bellofatto, V.(1993) Nucleic Acids Res. 21, 4067-4072 [Abstract]
  15. Gonzalez, A., Lerner, T. J., Huecas, M., Sosa-Pineda, B., Nogueira, N., and Lizardi, P. M.(1985) Nucleic Acids Res. 13, 5789-5804 [Abstract]
  16. Wong, S., Morales, T. H., Neigel, J. E., and Campbell, D. A.(1993) Mol. Cell. Biol. 13, 207-216 [Abstract]
  17. Chung, S. H., and Swindle, J.(1990) Nucleic Acids Res. 18, 4561-4569 [Abstract]
  18. Erondu, N. E., and Donelson, J. E.(1991) Mol. Biochem. Parasitol. 49, 303-314 [CrossRef][Medline] [Order article via Infotrieve]
  19. LeBowitz, J. H., Smith, H. Q., Rushe, L., and Beverley, S. M.(1993) Genes & Dev. 7, 996-1007
  20. Walder, J. A., Eder, P. S., Engman, D. M., Brentano, S. T., Walder, R. Y., Knutzon, D. S., Dorfman, D. M., and Donelson, J. E.(1986) Science 233, 569-571 [Medline] [Order article via Infotrieve]
  21. Ullu, E., Matthews, K. R., and Tschudi, C.(1993) Mol. Cell. Biol. 13, 720-725 [Abstract]
  22. Huang, J., and Van der Ploeg, L. H. T.(1991) EMBO J. 10, 3877-3885 [Abstract]
  23. Layden, R. E., and Eisen, H.(1988) Mol. Cell. Biol. 8, 1352-1360 [Medline] [Order article via Infotrieve]
  24. Pearson, R. D., and Steigbigel, R. T.(1980) J. Immunol. 125, 2195-2201 [Abstract/Free Full Text]
  25. LeBowitz, J. H., Coburn, C. M., McMahon-Pratt, D., and Beverley, S. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9736-9740 [Abstract]
  26. Coburn, C. M., Otteman, K. M., McNeely, T., Turco, S. J., and Beverley, S. M.(1991) Mol. Biochem. Parasitol. 46, 169-180 [CrossRef][Medline] [Order article via Infotrieve]
  27. Kapler, G. M., Coburn, C. M., and Beverley, S. M.(1990) Mol. Cell. Biol. 10, 1084-1094 [Medline] [Order article via Infotrieve]
  28. Kooter, J. M., and Borst, P.(1984) Nucleic Acids Res. 12, 9457-9472 [Abstract]
  29. Rudenko, G., Le Blanco, S., Smith, J., Gwo Shu Lee, M., Rattray, A., and Van der Ploeg, L. H. T.(1990) Mol. Cell. Biol. 10, 3492-3504 [Medline] [Order article via Infotrieve]
  30. Mallinson, D. J., and Coombs, G. H.(1989) Parasitology 98, 7-15 [Medline] [Order article via Infotrieve]
  31. Franke, E. D., McGreevy, P. B., Katz, S. P., and Sacks, D. L.(1985) J. Immunol. 134, 2713-2718 [Abstract/Free Full Text]
  32. Sacks, D. L., Brodin, T. N., and Turco, S. J.(1990) Mol. Biochem. Parasitol. 42, 225-234 [CrossRef][Medline] [Order article via Infotrieve]
  33. Coulson, R. M. R., and Smith, D. F.(1990) Mol. Biochem. Parasitol. 40, 63-76 [CrossRef][Medline] [Order article via Infotrieve]
  34. Brodin, T. N., Heath, S., and Sacks, D. L.(1992) Mol. Biochem. Parasitol. 52, 241-250 [Medline] [Order article via Infotrieve]
  35. Casey, J. L., Hentze, M. W., Koeller, D. M., Caughman, S. W., Rouault, T. A., Klausner, R. D., and Harford, J. B.(1988) Science 240, 924-928 [Medline] [Order article via Infotrieve]
  36. Gillis, P., and Malter, J. S.(1991) J. Biol. Chem. 266, 3172-3177 [Abstract/Free Full Text]
  37. Cleveland, D. W.(1989) Curr. Opin. Cell Biol. 1, 1148-1153 [Medline] [Order article via Infotrieve]
  38. Gottlieb, E.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7164-7168 [Abstract]
  39. Rastinejad, F., and Blau, H. M.(1993) Cell 72, 903-917 [Medline] [Order article via Infotrieve]
  40. Rastinejad, F., Conboy, M. J., Rando, T. A., and Blau, H. M.(1993) Cell 75, 1107-1117 [Medline] [Order article via Infotrieve]
  41. Huang, J., and Van der Ploeg, L. H. T.(1991) Mol. Cell. Biol. 11, 3180-3190 [Medline] [Order article via Infotrieve]

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