Glycoprotein 46 mRNA Abundance Is Post-transcriptionally Regulated during Development of Leishmania chagasiPromastigotes to an Infectious Form*

GP46 is an abundant glycoprotein of 46 kDa on the surface of the promastigote form of most Leishmaniaspecies. We show that the steady state level of GP46 mRNA increases >30-fold as Leishmania chagasi promastigotes developin vitro from a less infectious form during logarithmic growth to a highly infectious form in the stationary phase of cultivation. Nuclear run-on experiments demonstrate that this increase in GP46 mRNA abundance is regulated post-transcriptionally. Plasmids containing the 3′-untranslated regions (UTRs) and downstream intergenic regions (IRs) of two different GP46 genes fused immediately downstream of the β-galactosidase coding region were transfected intoL. chagasi, and β-galactosidase activity and mRNA levels were examined. The presence of the 3′-UTR + IR of one GP46 gene (gp46A) resulted in a steady increase in β-galactosidase activity and mRNA level as the transfected promastigotes developed from logarithmic to stationary phase. This differential effect parallels that of the 3′-UTRs + IRs of a family of genes for an unrelated Leishmania surface glycoprotein, GP63. Thus, post-transcriptional regulation of the genes for two different surface glycoproteins of Leishmania occurs via a similar mechanism.

Protozoan parasites of the genus Leishmania cause a diverse group of diseases collectively called leishmaniasis, which range in severity from spontaneously healing cutaneous ulcers to potentially fatal visceral disease. These parasites have a digenetic life cycle, passing from the infected sandfly vector to the mammalian host as the female fly takes a blood meal. In the fly, Leishmania exist as extracellular flagellated promastigotes within the alimentary canal, and in mammals, they exist as intracellular aflagellate amastigotes within phagolysosomes of macrophages.
While multiplying in the sandfly gut, promastigotes progress through a series of morphologically distinct developmental stages culminating in the highly infectious metacyclic stage (1).
Some aspects of this development are mirrored in vitro during the growth of promastigotes from logarithmic (less infectious) phase to stationary (highly infectious) phase in liquid culture medium (2). For example, in several Leishmania species, the glycosyl side chain of the abundant lipophosphoglycan on the surface of promastigotes elongates as parasites grow to their infectious metacyclic form in stationary phase (3,4). This increase in metacyclic lipophosphoglycan size has been correlated with enhanced virulence of the parasite (5,6). Another major surface constituent of promastigotes is a well characterized 63-kDa glycoprotein called GP63, 1 a zinc protease with broad substrate specificity and a wide pH optimum (7,8). In Leishmania chagasi, the cause of South American visceral leishmaniasis, the amount of GP63 increases 11-fold as the promastigotes grow from logarithmic to stationary phase (9). Biological functions ascribed to GP63 include evasion of complement-mediated lysis, attachment to macrophages, cleavage of various substrates including C3b, and elicitation of both humoral and cellular immune responses that are protective in several mouse models (10 -14).
In earlier studies of GP63 expression, we found that L. chagasi has Ͼ18 tandemly arrayed GP63 genes divisible into three classes on the basis of their developmental expression (9,12). Genes of the class encoding 2.7-kb mRNAs occurring predominantly in logarithmic phase promastigotes are called mspL, where msp and L refer to the genes encoding major surface protease (GP63) and logarithmic phase, respectively. Genes of the class encoding 3.0-kb mRNAs found predominantly in stationary phase promastigotes are called mspS. Promastigotes at an intermediate growth phase possess both RNAs. Class mspC is comprised of a single gene that is constitutively expressed at a low level throughout promastigote growth as 2.6-and 3.1-kb mRNAs. The 3Ј-UTR and downstream intergenic region (IR) of the mspS genes play an important role in the stationary phase expression of these genes, whereas the corresponding 3Ј-UTR ϩ IR sequence of the mspL genes does not seem to be responsible for their differential RNA expression (15).
Another abundant protein present on the surface of promastigotes is a 46-kDa glycoprotein called GP46 or promastigote surface antigen 2 (16,17). Genes encoding GP46 have been detected in Crithidia fasciculata and in all Leishmania sp. examined except for members of the Leishmania braziliensis complex (18,19). The organization of this gene family has not been fully characterized in any Leishmania species, but in those that have been investigated, multiple nonidentical copies of GP46 genes are arranged in clusters (18). Although the biological function(s) of GP46 are not known, immunization with GP46 partially protects experimental mice against challenge with Leishmania amazonensis (20,21). Recent experiments have demonstrated that Leishmania major amastigotes and promastigotes express different GP46 mRNAs and proteins (22), indicating that these genes undergo developmental regulation.
Since GP63 genes are differentially expressed during L. chagasi promastigote growth, we questioned whether GP46 gene expression also varies during promastigote growth. We found that the abundance of GP46 RNA in L. chagasi promastigotes increases dramatically as they grow from logarithmic to stationary phase, paralleling the increase in mspS mRNA. Stationary phase promastigotes have approximately equivalent steady state levels of GP46 and mspS mRNAs. Two GP46 genes were isolated from the L. chagasi genome, and a plasmid-borne ␤-galactosidase (␤GAL) reporter gene was used to show that the 3Ј-UTR ϩ IR of one GP46 gene is responsible for its growthregulated expression.

EXPERIMENTAL PROCEDURES
Parasites-An isolate of L. chagasi derived from a Brazilian patient with visceral leishmaniasis was maintained in hamsters. Promastigotes were cultured in vitro at 26°C as described (9) in a modified minimal essential medium (HOMEM) supplemented with 10% fetal bovine serum and 5.6 g/ml hemin (23). Logarithmic and stationary phase promastigotes were defined by morphology and concentration criteria (24). Medium for stably transfected promastigotes contained 40 g/ml G418 (Life Technologies, Inc.).
Genomic and cDNA Libraries-A L. chagasi genomic DNA library and a L. chagasi cDNA library made from RNA isolated from stationary phase promastigotes were described earlier (9,12). Clones hybridizing to radiolabeled DNA probes were purified and processed as described (Stratagene, La Jolla, CA).
PCR Amplification of Partial GP46 cDNA Clones-A series of primers designed from the coding sequence for L. amazonensis GP46 (accession number M38368) were used in attempts to PCR-amplify a partiallength GP46 coding region from L. chagasi and Leishmania mexicana genomic DNA. The forward primer, 5Ј GGGGACGAGCGACTTCAC 3Ј, and reverse primer, 5Ј CCGGCACAGACCACGAGA 3Ј, successfully amplified a fragment from L. mexicana DNA that was subsequently used to screen the L. chagasi cDNA library.
To obtain a cDNA sequence corresponding to the 5Ј end of GP46 mRNA, reverse transcriptase-PCR was conducted using RNA isolated from stationary phase L. chagasi promastigotes and a forward primer containing a partially spliced leader sequence (5Ј AACTAACGCTA-TATAAGTATCAGTT 3Ј). The reverse primer for this reversed transcriptase-PCR, 5Ј CGACGTTGGTGTAGTCGA 3Ј, was designed from the sequence of a partial length (2.4 kb) GP46 cDNA isolated from the L. chagasi cDNA library.
Southern and Northern Blot Analysis-Total L. chagasi DNA was isolated using DNAzol (Life Technologies), and Southern blots were conducted as described (25). For Northern blots, the L. chagasi RNA was isolated using a guanidinium-based method (26). RNA was separated by electrophoresis on a 1.2% agarose gel (6% formaldehyde buffered with 160 mM NaH 2 PO 4 , pH 6.8), transferred overnight in 6 ϫ sodium saline citrate (25) to positively charged nylon membrane, and fixed by baking 30 min at 80°C. DNA probes used in Southern or Northern blot analysis were 1) a 0.6-kb HpaI fragment from the 3.0-kb ␤GAL coding sequence in plasmid pX-␤gal 2, 2) a 0.98-kb EcoRI-SacII fragment from the 1.3-kb L. chagasi GP46 coding sequence, 3) a 0.89-kb NdeI-ScaI fragment from the 1.8-kb stationary GP63 coding sequence in plasmid pGP63S (9). The tubulin probe used in initial experiments (see Fig. 2, panel C) contained the sequence for Trypanosoma brucei rhodesiense ␣and ␤-tubulin (27). The ␣-tubulin probe used in later experiments was PCR-amplified from L. chagasi genomic DNA using oligonucleotide primers 5Ј ATGCGT-GAGGCTATCTGCAT 3Ј and 5Ј TTAGTACTCCTCGACGTCC 3Ј derived from the coding sequence for ␣-tubulin of Leishmania donovani (accession number U09612).
For determining the relative abundance of GP46 and GP63 mRNAs, purified 0.98-kb GP46 and 0.89-kb GP63 DNA fragments were quanti-fied by A 260 and by the relative intensities of ethidium bromide stains of agarose gel-separated fragments compared with mass standards (Life Technologies). These fragments were serially diluted (in 0.4 N NaOH, 0.01 M EDTA, 0.08 g of plasmid pAcUW21/ml as a carrier) and applied in parallel rows to positively charged nylon membranes via slot blot. The serial dilutions (1:2, 1:4, 1:8, etc.) spanned a range from 1.25 ϫ 10 3 to 0.305 picograms of target DNA/slot. These membranes and Northern blot membranes containing RNA from stationary phase promastigotes were incubated in the same hybridization solution and probed with either the 0.98-kb GP46 or 0.89-kb GP63 DNA fragments. The relative abundance of the GP46 and GP63 RNAs was determined by comparing the intensity of the signals on the Northern blots containing the RNAs with the intensity of the signals on the slot blots containing the GP46and GP63-DNA mass standards.
DNA Constructions-The starting plasmids were pBluescript ® SK (Stratagene) and pX-␤gal 2 (a generous gift from S. Beverley). A fragment containing the 3Ј-UTR and IR of gp46A was isolated after NotI digestion of recombinant phage DNA containing this genomic region of L. chagasi DNA and then ligated into the NotI site of pX-␤gal 2. NotI cleaves 76 bases upstream (5Ј) of the stop codon in gp46A; therefore, this clone contains a small piece of the coding region. A partial 3Ј-UTR and IR of gp46B was similarly isolated from recombinant phage DNA by XhoI digestion, blunt ended, and ligated into pX-␤gal 2 that had previously been digested with NotI, blunt ended, and dephosphorylated. The orientation of the inserts was determined by sequencing across the insert boundaries. The pX-␤gal 2 plasmid constructs containing the 3Ј-UTRs and IRs of the GP63 genes have been described (15).
Nuclear Run-on Assays-Promastigotes were harvested in logarithmic or stationary phase, pelleted by centrifugation for 5 min at 3000 ϫ g, and washed twice in Hanks' balanced salt solution. Disruption of cells and labeling of nuclei were as described previously (28).
DNA Transfections and ␤GAL Assays-L. chagasi promastigotes were transfected with plasmids and plated onto solid medium containing 25 or 40 g/ml G418 for selection and isolation of clonal transfectants as described (29). Clonal isolates were grown in liquid culture, and aliquots of 1.5 ϫ 10 8 promastigotes were removed daily, washed three times (centrifuged for 5 min at 4000 ϫ g then resuspended in 1.4 ml of phosphate-buffered saline), resuspended in 100 l of phosphatebuffered saline, and then stored at Ϫ70°C. For ␤GAL assays, aliquots were thawed quickly with the addition of 2 volumes (200 l) of lysis buffer (100 mM KH 2 PO 4 , pH 7.8, 0.33% Triton X-100), lysed by three freeze-thaw cycles in dry ice/ethanol and a 37°C water bath, and centrifuged (5 min at 10,000 ϫ g). The supernatant was assayed for protein concentration (bicinchoninic acid reagent and assay, Pierce) and for ␤GAL activity (using Galacton-Star chemiluminescent substrate in a fluorometric assay, CLONTECH, Palo Alto, CA). Fluorescence was measured in a Monolight ® 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).
General Methods-Standard techniques of molecular biology were used (25). DNA was sequenced using dye terminator cycle sequencing chemistry and analyzed on a 373A stretch fluorescent automated sequencer (Perkin-Elmer). The radioactivity of the blots was quantified using InstantImager electronic autoradiography (Packard Instrument Co.). DNA and protein sequences were compared using the University of Wisconsin Genetic Computer Group program version 7.0.

RESULTS
Isolation of a cDNA for GP46 of L. chagasi-The cDNA sequences encoding GP46 have been reported for L. major and L. amazonensis (30,31) but not for L. chagasi. Attempts to PCR-amplify a partial GP46 cDNA using degenerate and nondegenerate primers designed from the L. amazonensis sequence yielded a 1.3-kb product with L. mexicana genomic DNA template but no product with L. chagasi genomic DNA. This 1.3-kb fragment was shown by DNA sequencing to encode GP46 and was used to screen 2.7 ϫ 10 4 phage in an L. chagasi cDNA library. Fourteen phage clones hybridized to the probe (0.05%), and 10 were plaque-purified for further characterization. The complete 2.4-kb sequence of the largest of these 10 cDNA inserts was determined and found to be a partial-length cDNA lacking the 39-nucleotide-spliced leader found on the 5Ј ends of all Leishmania mRNAs (32).
To isolate the missing 5Ј segment, a reverse transcriptase-PCR was conducted on total L. chagasi promastigote RNA using a forward primer containing part of the spliced leader sequence and a reverse primer based on the cDNA sequence. The resulting 500-bp PCR product showed complete sequence identity in a 194-bp overlap with the cDNA. The combined GP46 cDNA sequence of 2754 bp (Fig. 1A) is consistent with the 2.8-kb size of the major GP46 mRNA species observed on a Northern blot (Fig. 2A). The deduced nascent protein sequence of 44 kDa (Fig. 1B) is similar to GP46 proteins of other Leishmania species.
The Steady State Level of GP46 mRNA Is Developmentally Regulated by Post-transcriptional Events-Total RNA was isolated from promastigotes at various times during growth in culture and probed in Northern blots with the GP46 cDNA (Fig.  2). Promastigotes in these cultures entered logarithmic phase of growth within 1-2 days after passage and reached stationary phase by day 7, according to morphology and concentration as previously defined (24). Panel A of Fig. 2 shows that two GP46 RNA species that hybridize to the cDNA probe exist in stationary phase L. chagasi promastigotes, a major species of 2.8 kb and a minor one of 4.8 kb. Both species steadily increase in abundance as the cells grow from logarithmic to stationary phase. InstantImager analysis of the blot indicated that the steady state level of the 2.8-kb GP46 RNA is Ͼ30-fold higher at day 7 than at days 3-4. The 4.8-kb RNA increases a similar amount. On the basis of size similarity, the cDNA shown in Fig.  1A is likely derived from the 2.8-kb GP46 RNA.
Panel B shows the same RNAs probed with the GP63 coding region. The hybridization pattern is very similar to the pattern reported previously (15). A GP63 RNA of 2.7 kb occurs in logarithmic phase promastigotes (days 3-4), and a GP63 RNA of 3.0 kb occurs in stationary phase promastigotes (days 6 -7). Promastigotes at an intermediate phase of growth (day 5) have both GP63 RNA species. A comparison of panels A and B demonstrates that the abundance of GP46 RNA closely parallels that of the stationary 3.0-kb GP63 RNA. Both RNAs are very rare in days 3-4, begin to appear in day 5, and increase in abundance during days 6 -7.
Panels C and D show a hybridization with a tubulin probe and an ethidium bromide stain, respectively, to detect variations in RNA loadings to the gel lanes. Both panels indicate that in this particular experiment more RNA was added to lane 5 (day 5) and less to lane 7 (day 7) than to the other lanes. When these differences are taken into account, the increases in GP46 and stationary GP63 RNA in stationary phase promastigotes (day 7) is even more dramatic than those indicated by the relative band intensities in panels A and B. As shown in panel C, the control hybridization with the T. brucei ␣and ␤-tubulin probe resulted in the typical one ␣and three ␤-tubulin band pattern seen in prior studies (28). To eliminate this pattern of The experiment shown represents one of four independent time courses of promastigote growth from which RNAs were isolated for subsequent Northern blots. Panel E, RNAs on a separate blot were probed with the 1.3-kb region within the 3Ј-UTR of gene gp46B that is not in the cDNA or gp46A (see Fig. 4). multiple bands, subsequent blots utilized a probe from an ␣-tubulin gene of L. chagasi (see Fig. 6B).
The similar sizes of the GP46 and GP63 RNAs (2.8 kb and 3.0 kb) precluded a quantification of their relative abundance in a single Northern blot analysis. Thus, Northern blots hybridized separately with each probe were incubated in the hybridization solution with a second filter containing serial dilutions in slot blots of DNA fragments possessing the GP46 and GP63 coding regions. The relative amount of GP46 and GP63 RNA was estimated by comparing the intensities of their signals on the Northern blot filter to the signals of the serially diluted DNAs on the slot blot filter. Using this approach, the abundance of GP46 2.8-kb RNA was found to be about the same as that of GP63 RNA in stationary phase day 7 cells (data not shown).
To determine whether the increase in the steady state level of GP46 mRNA that occurs as promastigotes grow from logarithmic to stationary phase is due to enhanced transcription initiation or to post-transcriptional events, nuclear run-on experiments were conducted using nuclei isolated from logarithmic and stationary phase promastigotes (Fig. 3). The radioactive RNA isolated from logarithmic nuclei consistently had a higher specific activity than that obtained from stationary nuclei, probably indicating that logarithmic phase cells are transcriptionally more active. Thus, the amount of radioactive RNA hybridizing to each of the test DNAs in Fig. 3 was normalized to the amount hybridizing to DNA encoding ␣-tubulin. The normalized ratio of GP46 run-on RNA in stationary versus logarithmic nuclei was found to be about 0.51, approximating the corresponding ratio of GP63 run-on RNA. This result indicates that there is no difference in the transcription rate of the GP46 genes in logarithmic and stationary phase promastigotes relative to ␣-tubulin gene transcription despite the 30-fold increase in the steady state level of GP46 mRNA in stationary phase cells. Thus, this increase in GP46 mRNA must be regulated primarily by post-transcriptional events.
The 3Ј-UTR and Downstream IR of the GP46 Genes Influence Expression of a ␤GAL Reporter Gene-To examine which sequences of the GP46 genes might contribute to the increased level of their mRNAs in stationary promastigotes, we first screened a bacteriophage library of L. chagasi genomic DNA for genomic DNA clones that contain GP46 genes. Two such clones were isolated and characterized by restriction mapping, and several of their restriction fragments were subcloned for DNA sequencing. The genomic DNA segments in these two clones were found to overlap in a region of the genome containing two GP46 genes (gp46A and gp46B) that are separated by 7.2 kb (Fig. 4). The end of one of the genomic clones occurs within codon 163 of gp46A, so the sequence preceding that codon could not be determined. However, the sequence of the rest of the gp46A coding region and its entire 3Ј-UTR of 1.3 kb was determined and found to be identical to the GP46 cDNA sequence (Fig. 1A), indicating that the corresponding mRNA could have arisen from this gene. The complete sequence of gp46B was determined (not shown), and its coding region was found to have only 78% nucleotide identity with the corresponding region of the cDNA sequence (Fig. 4), largely due to short stretches of DNA sequence in gp46B that were not present in gp46A. Most of its 3Ј-UTR has 93% identity with the cDNA, but it also has an internal 1.3-kb segment with no similarity (shaded rectangle in Fig. 4). In Northern analysis (Fig. 2E), this 1.3-kb segment of gp46B predominately hybridized to 4.8-kb RNAs that increased in abundance during promastigote development in a pattern equivalent to that seen for the 2.8-kb RNAs of Fig. 2A. Thus, gp46B may be a source of the 4.8-kb GP46 RNAs. Fragments containing all of the gp46A 3Ј-UTR and about 65% of the gp46B 3Ј-UTR plus their downstream IRs were cloned immediately downstream of the ␤GAL coding region in pX-␤gal 2 (Fig. 4). This parent plasmid is maintained episomally in Leishmania and contains a neomycin resistance gene that allows for the selection of cells that contain the plasmid by Since one of the two genomic DNA clones ends within the gp46A coding sequence, the dashed rectangle at the 5Ј region of gp46A indicates the sequence predicted from the cDNA sequence (see "Results"). Thin rectangles denote 3Ј-UTRs of the GP46 genes. The shaded region in the 3Ј-UTR of gp46B indicates a 1.3-kb sequence that does not occur in the 3Ј-UTR of gp46A. Fragments containing the two genes were subcloned and sequenced. The percentages shown below the map indicate the nucleotide identity of the indicated regions with the corresponding regions of the GP46 cDNA (Fig. 1A). A 3.2-kb NotI fragment containing the 3Ј-UTR and downstream IR of gp46A was cloned downstream of the ␤GAL gene in plasmid pX-␤gal 2 as indicated. A 4.2-kb XhoI fragment containing part of the 3Ј-UTR ϩ IR of gp46B was also cloned at the same site. growth in the presence of the drug G418. In earlier studies, we had already constructed derivatives of pX-␤gal 2 in which the corresponding 3Ј-UTRs and IR regions of the three GP63 gene classes, i.e. mspS (stationary), mspL (logarithmic), and mspC (constitutive), were cloned at the same site downstream of the ␤GAL gene (15). These five plasmids containing the various Leishmania 3Ј-UTRs ϩ IRs and the parent plasmid pX-␤gal 2 were introduced into promastigotes, and clones of stable transfectants were selected (listed in Table I). Fig. 5 shows the ␤GAL enzymatic activities that were determined during growth of the six transfectants from logarithmic to stationary phase and corresponds to experiment 1 of Table I. No change in ␤GAL activity with time occurred in the transfectants containing the parent plasmid and the plasmids with the 3Ј-UTRs ϩ IRs of mspL and mspC, as demonstrated previously (15). Likewise, the presence of the partial 3Ј-UTR ϩ IR of gp46B did not change the ␤GAL activity from that of the parent plasmid, possibly due to the lack of a full-length 3Ј-UTR. However, the 3Ј-UTR ϩ IR of gp46A stimulated a dramatic increase in ␤GAL activity with time in culture, similar to that of the 3Ј-UTR ϩ IR of mspS. In both cases, the ␤GAL activity in logarithmic phase promastigotes was lower on day 3 than in the other transfectants, increased to the level observed in the other transfectants by day 4, and subsequently increased steadily until by day 8 it was about 20 -30-fold higher than on day 3. In two other experiments similar to that shown in Fig. 5, the ratio of ␤GAL activity on day 8 versus day 3 was Ն 12 in transfectants containing the 3Ј-UTRs ϩ IRs of gp46A and mspS, whereas no substantive difference was observed in transfectants bearing the other plasmids (Table I). Thus, the gp46A and mspS 3Ј-UTRs ϩ IRs exert parallel effects on ␤GAL activity when placed downstream of the ␤GAL gene.
The ␤GAL Activity in the Transfectants Reflects the Steady State Level of ␤GAL mRNA-To ensure that the increase in ␤GAL enzymatic activity observed in the transfectants bearing plasmids with the 3Ј-UTRs ϩ IRs of gp46A and mspS was due to increased ␤GAL mRNA, Northern blots were conducted on RNAs extracted from the transfectants. Fig. 6A shows that in transfectants containing the 3Ј-UTRs ϩ IRs of gp46A and mspS, the steady state level of ␤GAL mRNA is much higher in stationary phase (S, day 8) than in logarithmic phase (L, day 3). In addition, the ␤GAL mRNA containing the 3Ј-UTR of gp46A (1.3 kb) is slightly larger than the ␤GAL mRNA with the 3Ј-UTR of mspS (1.1 kb), as expected from the known sizes of these 3Ј-UTRs. In contrast, in the transformant bearing only the parent plasmid (called ␤-gal in Fig. 6), the level of ␤GAL RNA does not vary as much with respect to the phase of growth. In addition, the major ␤GAL RNA species in this transformant is about 7.0 kb, suggesting that its primary poly-adenylation site occurs about 4 kb downstream of the 3-kb ␤GAL coding region. The membrane shown in Fig. 6A was stripped and reprobed with the ␣-tubulin gene to determine the relative amounts of RNA added to each lane (Fig. 6B). The relative increase in ␤GAL mRNA in stationary versus logarithmic phase transfectants was 13-fold for the clone containing gp46A sequence, 12-fold for the clone containing mspS sequence, and Ͻ2-fold for the parent plasmid. These increases in steady state mRNA levels are consistent with the increases observed in the ␤GAL enzymatic activity.
To verify that the plasmid copy number does not change  during growth from logarithmic to stationary phase, DNA was extracted from the transfectants at days 3 and 8 and probed with the ␤GAL coding sequence (Fig. 7A). The same blot was stripped and reprobed with GP46 coding sequence to adjust for DNA loading (Fig. 7B). The relative amount of the plasmid in each transformant was found to vary by Ͻ10% during growth of the transfectants.
In these Southern blots, the ␤GAL probe hybridizes only to a single 3.6-kb AccI fragment of the plasmid, since the probe is contained within this fragment. Conversely, because the plasmid constructs do not contain GP46 coding sequence, the GP46 coding region probe hybridizes only to GP46 genes in the L. chagasi genome. Thus, in panel B, all of the fragments are derived from the genome. The 2.0-kb fragment and one of the Ϸ7.5-kb doublet fragments correspond to expected fragments based on the locations of AccI sites in the genomic and cDNA sequences shown in Fig. 3. The additional 1.6, 2.7, and Ϸ7.5-kb fragments to which the GP46 probe hybridizes with varying intensities indicate the existence of other genes besides gp46A and gp46B. These results are consistent with similar studies of GP46 gene organization in other Leishmania species that showed that GP46 is encoded by a family of nonidentical genes that are organized as a cluster(s) within the genome (18). The weak signals of fragments whose sizes are not indicated in Fig.  7B may suggest either the presence of additional sequences in the genome that have partial identity to the GP46 coding region probe or incomplete digestion of genomic DNA. DISCUSSION Previous work with in vitro cultured L. chagasi has documented differential expression of RNAs from distinct msp genes encoding the surface glycoprotein GP63 (9). The current study was based on the hypothesis that the genes coding for other surface proteins might be similarly regulated. We tested this hypothesis with another Leishmania gene family encoding GP46, which, like GP63, is a glycoprotein that is expressed on the parasite surface membrane.
The deduced amino acid sequence of L. chagasi GP46 shown in Fig. 1B displays 61% identity to GP46 of L. major (30) and 65% identity to GP46 of L. amazonensis (31). All of these GP46 sequences contain hydrophobic amino and carboxyl termini that are probably post-translationally cleaved during the translocation of the protein across the endoplasmic reticulum and its linkage to membrane-anchored glycosylphosphatidylinositol, respectively. Additionally, all contain 3 (L. major) to 7 (L. chagasi) leucine-rich repeats of 24 residues. Leucine-rich repeats of 24 residues are components of many other proteins and are thought to be sites of specific protein-to-protein interactions (33,34), suggesting a similar role for these repeats in GP46. Protozoan parasites of the genus Giardia also have a surface glycoprotein that contains leucine-rich repeats of 24 residues (35), raising the possibility that the surface glycoproteins of these two distantly related genera may have analogous function.
Based upon our work on GP46 gene transcription and mRNA stability, we would predict that GP46 levels vary during promastigote development, as has been shown for GP63. Recent analysis in L. major showed that different GP46 RNAs are expressed in amastigotes and promastigotes, and that the glycosylphosphatidylinositol-anchored GP46 in amastigotes, but not promastigotes, is resistant to hydrolysis by phosphatidylinositol-specific phospholipase C (22). The work presented here establishes the need for a similar study of GP46 expression throughout promastigote development.
Northern blot and nuclear run-on experiments showed that the varied abundance of GP46 mRNA in promastigotes is due to post-transcriptional events ( Fig. 2 and 3). The steady state level of a number of RNAs in the order Kinetoplastidae are also regulated post-transcriptionally (36 -38). Such regulation may be linked to the observation that in this order, many highly expressed proteins are encoded by tandemly repeated genes present in a linked cluster. Some, if not all, of these gene clusters are transcribed as polycistronic precursor RNAs that subsequently undergo processing by the addition of a 39-nucleotide-spliced leader at the 5Ј end and polyadenylation at the 3Ј end of each mRNA. 5Ј processing of a downstream gene is coupled to polyadenylation of the upstream gene product (39). Thus, post-transcriptional regulation would provide the means for controlling levels of specific mRNAs derived from a polycistronic transcript.
The parallel expression profiles of GP46 and mspS RNA in promastigotes as well as the equivalent effects of the 3Ј-UTRs ϩ IRs of gp46A and mspS on ␤GAL activity and mRNA levels suggest that both genes are post-transcriptionally regulated by a similar mechanism. From this observation, one would hypothesize that homologous regions within the 3Ј-UTRs of these two genes may be responsible for the increased abundance of these mRNAs during stationary phase growth. Comparison of the 3Ј-UTR ϩ IR sequences of gp46A and mspS with BESTFIT FIG. 7. Southern blots to estimate the relative plasmid copy number in the logarithmic and stationary phase stable transfectants. DNA was isolated from the same transfectants in logarithmic (L) and stationary (S) phase as indicated in Fig. 6. The DNAs (5 g per lane) were digested with AccI and probed with the ␤GAL coding region (panel A). The membranes were stripped and reprobed with the gp46A coding region (panel B).
indicates several regions of similarity within their 3Ј-UTRs, the highest scoring of which is depicted in Fig. 8. In contrast, there is little identity within their IRs. A test of whether these homologous DNA segments in the 3Ј-UTRs are responsible for the stationary-specific expression pattern will require further transfection experiments with these putative regulatory regions either deleted or inserted downstream of a reporter gene. The 3Ј-UTR of gp46B also contains this region of similarity, leading us to hypothesize that the reason for the lack of growthregulated expression of the Bgal construct containing the partial length gp46B 3Ј-UTR ϩ IR is that all sequences essential for its growth-regulated expression (Fig. 2E) were not contained in this construct.
The actual molecular mechanism by which the GP46 and mspS genes are regulated remains unclear. Our prior work documented differences in the mechanisms regulating expression of the mspL and mspS genes within the same cluster (15,28). In experiments comparable to those reported here, the 3Ј-UTR ϩ IR of mspS, but not of mspL, was responsible for augmenting both the steady state RNA and protein expression levels of an upstream reporter gene during growth of L. chagasi from logarithmic to stationary phase (15). In other experiments, the levels and half-lives of mspL RNAs, but not of mspS RNAs, were dramatically up-regulated by the addition of protein synthesis inhibitors that did not influence the rate of transcription, implying the involvement of a negative regulatory protein factor in targeting mspL RNAs for rapid degradation (28). Similar studies with the GP46 genes hold the promise of revealing further intricacies about gene regulation in this class of protozoan parasites.