Identification and characterization of a Plasmodium falciparum RNA polymerase gene with similarity to mitochondrial RNA polymerases.

Nearly all mitochondrial RNA polymerase genes identified to date are encoded in the nucleus and have similarities to T3 and T7 bacteriophage RNA polymerases. Some chloroplast genes are also transcribed by T3/T7 phage-like RNA polymerases, raising the possibility that the apicomplexan parasites, which have both a mitochondrion and a plastid, might have two such genes. As part of an investigation of Plasmodium falciparum organelle transcription, we initiated a search for T3/T7 bacteriophage-like RNA polymerase genes. We employed degenerate primers based on highly conserved plant, animal and fungal mitochondrial RNA polymerase sequences to amplify corresponding P. falciparum sequences by polymerase chain reaction (PCR). Less well-conserved flanking sequences were obtained by inverse PCR. The resulting sequence predicts a 1503 amino acid open reading frame with similarity to other T3/T7 phage-like RNA polymerases. Essential amino acids that have been identified in T7 mutant analyses are conserved in the P. falciparum RNA polymerase gene. Comparison of the sequence with preliminary data from the P. falciparum genome sequencing project revealed strain heterogeneity within two regions of the gene. The amino-terminal predicted amino acid sequence of the RNA polymerase gene has similarities to mitochondrial targeting sequences. Taken together, these points suggest that we have identified the P. falciparum mitochondrial RNA polymerase gene.

Peptidomimetic inhibitors of protein farnesyltransferase show potent antimalarial activity.

Malaria continues to represent a very serious health problem in the tropics. The current methods of clinical treatment are showing deficiencies due to the increased incidence of resistance in the parasite. In the present paper we report the design, synthesis, and evaluation of potential antimalarial agents against a novel target, protein farnesyltransferase. We show that the most potent compounds are active against Plasmodium falciparum in vitro at submicromolar concentrations.

Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system.

Apicomplexan parasites possess a plastid-like organelle called the apicoplast. Most proteins in the Toxoplasma gondii apicoplast are encoded in the nucleus and imported post-translationally. T. gondii apicoplast proteins often have a long N-terminal extension that directs the protein to the apicoplast. It can be modeled as a bipartite targeting sequence that contains a signal sequence and a plastid transit peptide. We identified two nuclearly encoded predicted plastid proteins and made fusions with green fluorescent protein to study protein domains required for apicoplast targeting. The N-terminal 42 amino acids of the apicoplast ribosomal protein S9 directs secretion of green fluorescent protein, indicating that targeting to the apicoplast proceeds through the secretory system. Large sections of the S9 predicted transit sequence can be deleted with no apparent impact on the ability to direct green fluorescent protein to the apicoplast. The predicted transit peptide domain of the S9 targeting sequence directs protein to the mitochondrion in vivo. The transit peptide can also direct import of green fluorescent protein into chloroplasts in vitro. These data substantiate the model that protein targeting to the apicoplast involves two distinct mechanisms: the first involving the secretory system and the second sharing features with typical chloroplast protein import.

Mitochondrial genome diversity in parasites.

Mitochondrial genomes have been sequenced from a wide variety of organisms, including an increasing number of parasites. They maintain some characteristics in common across the spectrum of life-a common core of genes related to mitochondrial respiration being most prominent-but have also developed a great diversity of gene content, organisation, and expression machineries. The characteristics of mitochondrial genomes vary widely among the different groups of protozoan parasites, from the minute genomes of the apicomplexans to amoebae with 20 times as many genes. Kinetoplastid protozoa have a similar number of genes to metazoans, but the details of gene organisation and expression in kinetoplastids require extraordinary mechanisms. Mitochondrial genes in nematodes and trematodes appear quite sedate in comparison, but a closer look shows a strong tendency to unusual tRNA structure and alternative initiation codons among these groups. Mitochondrial genes are increasingly coming into play as aids to phylogenetic and epidemiologic analyses, and mitochondrial functions are being recognised as potential drug targets. In addition, examination of mitochondrial genomes is producing further insights into the diversity of the wide-ranging group of organisms comprising the general category of parasites.

Transcriptional mapping and RNA processing of the Plasmodium falciparum mitochondrial mRNAs.

The mitochondrial genome of Plasmodium falciparum encodes three protein coding genes and highly fragmented rRNAs. The genome is polycistronically transcribed and, since gene-size transcripts are much more abundant than the polycistronic transcripts, the latter are presumably cleaved to produce the smaller, mature mRNAs and rRNAs. Mapping the transcripts of the P. falciparum mitochondrial protein coding genes shows that the 3' end of each gene directly abuts the 5' end of the gene located immediately downstream. The 5' ends of the protein coding genes are also closely apposed to adjacent genes, with one directly abutting a gene on the same DNA strand and two others separated by just 13 nt from an rDNA fragment encoded on the opposite strand. These mapping data are consistent with production of the mRNAs by cleavage from a polycistronic precursor transcript. Further processing of the mRNAs comes from addition of oligo(A) tails. Unexpectedly, the presence and length of such tails varies in a gene-specific fashion. In this regard, polyadenylation of the P. falciparum mitochondrial mRNAs is more similar to that seen for the P. falciparum mitochondrial rRNAs than that of mitochondrial mRNAs in other organisms.

The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum have short A tails.

The mitochondrial genome of Plasmodium falciparum encodes highly fragmented rRNAs. Twenty small RNAs which are putative rRNA fragments have been found and 15 of them have been identified as corresponding to specific regions of rRNA sequence. To investigate the possible interactions between the fragmented rRNAs in the ribosome, we have mapped the ends of many of the small transcripts using primer extension and RNase protection analysis. Results obtained from these studies revealed that some of the rRNA transcripts were longer than the sequences which encode them. To investigate these size discrepancies, we performed 3' RACE PCR analysis and RNase H mapping. These analyses revealed non-encoded oligo(A) tails on some but not all of these small rRNAs. The approximate length of the oligo(A) tail appears to be transcript-specific, with some rRNAs consistently showing longer oligo(A) tails than others. The oligoadenylation of the rRNAs may provide a buffer zone against 3' exonucleolytic attack, thereby preserving the encoded sequences necessary for secondary structure interactions in the ribosome.

Identification of additional rRNA fragments encoded by the Plasmodium falciparum 6 kb element.

Sequences similar to mitochondrial large and small subunit rRNAs are found as small scattered fragments on a tandemly reiterated 6 kb element in the human malaria parasite Plasmodium falciparum. The rDNA sequences previously identified include strongly conserved portions of rRNA, suggesting that fragmented rRNAs derived from them are able to associate into functional ribosomes. However, sequences corresponding to other expected rRNA regions were not found. We here report that 10 of the 13 previously described rDNA regions have abundant small transcripts. An additional 10 transcripts were found from regions not previously known to contain genes. Five of the latter have been identified as rRNA fragments, including those corresponding to the 5'end and 790 loop sequences of small subunit rRNA and the sarcin/ ricin loop of large subunit rRNA. Demonstration that most of the previously described rDNA regions have abundant transcripts and the identification of new transcripts with other portions of conventional rRNAs provide support for the hypothesis that these small transcripts comprise functional rRNAs.

The Plasmodium falciparum 6 kb element is polycistronically transcribed.

The Plasmodium falciparum 6 kb element encodes three protein coding genes and highly fragmented large and small subunit rRNAs; its gene content makes it the probable mitochondrial genome. Many of the genes are encoded so close to each other that there is insufficient room for specific promoters upstream of each gene. RNase protection analysis of two rRNA fragments whose genes are adjacent provided evidence for a polycistronic transcript containing sequences from both, as well as separate small RNAs. To evaluate the possibility of further polycistronic transcription, several sets of oligonucleotide primers located in different regions of the 6 kb element were employed to amplify cDNAs. These analyses have revealed the existence of 6 kb element transcripts as long as 5.9 kb. Both mRNA and rRNA sequences are included on these putative precursor transcripts. Since these types of RNA are known to have different patterns of abundance changes during the erythrocytic portion of the parasite life cycle, RNA stability is presumably an important feature in regulating mitochondrial transcript abundance.

Plasmodium falciparum: alterations in organelle transcript abundance during the erythrocytic cycle.

The malaria parasite Plasmodium falciparum has two extrachromosomal DNAs, a 6 kb reiterated element which appears to be the mitochondrial DNA and a 35 kb circular DNA of unknown function. Examination of relative steady-state transcript abundance during parasite development in the erythrocyte shows that transcripts of 6 kb element protein-coding genes are least abundant in the ring and early trophozoite stages and most abundant in late trophozoites and schizonts, while transcripts from the RNA polymerase subunits of the 35 kb DNA, also least abundant in ring stage, are relatively similar in abundance in succeeding stages. The fragmented rRNAs of the 6 kb element appear to be constitutively abundant except for an increase in the schizont stage, while rRNAs from the 35 kb DNA are least abundant in early trophozoites and most abundant in schizonts. Thus the relative abundance of organelle transcripts alters during the erythrocytic portion of the P. falciparum developmental cycle. These alterations may reflect the relative importance of the roles played by organelle gene products in different life cycle stages.

Nine duplicated tRNA genes on the plastid-like DNA of the malaria parasite Plasmodium falciparum.

A major feature of the plastid-like circular DNA of Plasmodium falciparum is an inverted repeat comprising duplicated genes for rRNA (rrn) and tRNA (trn). We have identified nine putative trn genes in each arm of the repeat on the basis of their potential clover-leaf structures and conserved residues. Northern blots indicate that these trn genes are expressed.

The extrachromosomal DNAs of apicomplexan parasites.

Molecular analyses in recent years have begun to elucidate the identity and role of two extrachromosomal DNAs found in apicomplexan parasites. One of these is a small tandemly repeated DNA that encodes three classical mitochondrial protein coding genes, attesting to its identity. This molecule also encodes mitochondrial rRNAs as small fragments in scattered locations. Despite their unusual nature, evidence suggests that these rRNAs are functional. They offer an opportunity to evaluate structure-function correlations in the absence of much of the more variable sequences found in other rRNAs. The second extrachromosomal DNA has characteristics reminiscent of chloroplast DNAs and thus points to an unexpected ancestry for the apicomplexans. Both DNAs are inherited maternally, as is usual for organelle DNAs, but their subcellular locations have not been demonstrated unequivocally. Although the majority of studies with these two DNAs thus far have been with Plasmodium species, evidence from other apicomplexans suggests that these unusual organelle genomes are common to the phylum.

Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences.

Small guide RNAs (gRNAs) may direct RNA editing in kinetoplastid mitochondria. We have characterized multiple gRNA genes from Trypanosoma brucei (EATRO 164), that can specify up to 30% of the editing of the COIII, ND7, ND8, and A6 mRNAs and we have also found that the non-translated region of edited COIII mRNA of strain (EATRO 164) differs from that of another strain. Several of the gRNAs specify overlapping regions of the same mRNA often specifying sequence beyond that required for an anchor duplex with the next gRNA. Some gRNAs have different sequence but specify identical editing of the same region of mRNA. These data indicate a complex gRNA population and consequent complex pattern of editing in T. brucei.

Sequence and organization of large subunit rRNA genes from the extrachromosomal 35 kb circular DNA of the malaria parasite Plasmodium falciparum.

The malaria parasite Plasmodium falciparum carries an extrachromosomal 35 kb circular DNA molecule of unknown provenance. A striking feature of the circle is a palindromic sequence of genes for subunit rRNAs and several tRNAs, spanning ca. 10.5 kb. The palindrome has an intriguing resemblance to the inverted repeat of plastid genomes, and the sequence and putative secondary structure of the malarial large subunit (LSU) rRNA described in this report were used as the basis of a phylogenetic study. The malarial rRNA was found to be highly divergent in comparison with a selected group of chloroplast LSU rRNAs but was more closely related to them than to mitochondrial LSU rRNA genes.

Structural organization of the maxicircle variable region of Trypanosoma brucei: identification of potential replication origins and topoisomerase II binding sites.

The maxicircle of the parasitic protozoan Trypanosoma brucei, one component of the mitochondrial genome, has size differences among isolates that localize to the variable region (VR) between the ND5 and 12S rRNA genes. We present here the nucleotide sequence of this entire region, thus completing the sequence of the maxicircle genome. We also find heterogeneously sized transcripts from throughout most of the VR. The VR has three distinct sections, each with characteristic repeated sequences. The repeated sequences in two sections are short and highly reiterated; the intraspecies size variation occurs within this region. The third section contains non-repetitive sequences and a large duplication immediately upstream of the 12S rRNA gene. Two repeat units within section I contain a sequence that has homology to the DNA replication origin of minicircles. This region also contains sequences with homology to topoisomerase II binding and cleavage sites. These findings suggest a role for the VR in DNA replication of the maxicircle.

Transcript-specific developmental regulation of polyadenylation in Trypanosoma brucei mitochondria.

Transcripts from many mitochondrial genes in kinetoplastids are heterogeneous in size, often occurring as 2 distinct size classes, but this cannot be accounted for by RNA editing alone. Analyses of transcripts from 6 mitochondrial genes of Trypanosoma brucei indicates that the size variation is due to poly(A) tail length. A larger fraction of CYb, COI and COII transcripts have longer poly(A) tails in procyclic than in bloodstream forms. These transcripts are also more abundant in the procyclic forms. In contrast, a more substantial fraction of CR1 transcripts have longer poly(A) tails in bloodstream than in procyclic forms and these transcripts tend to be more abundant in bloodstream forms. Both ND4 and MURF1 transcripts show a similar size distribution of poly(A) tail lengths in these life cycle states although both transcripts are more abundant in bloodstream forms. Furthermore, genes with edited transcripts tend to have longer poly(A) tails than unedited transcripts. Transcript abundance is not strictly correlated with longer poly(A) tails. Thus, poly(A) length variation appears to be developmentally regulated in a transcript-specific fashion in T. brucei. This regulation of polyadenylation may influence mitochondrial gene expression as polyadenylation can regulate cytoplasmic gene expression in eukaryotes.

Guide RNAs for transcripts with developmentally regulated RNA editing are present in both life cycle stages of Trypanosoma brucei.

RNA editing of several mitochondrial transcripts in Trypanosoma brucei is developmentally regulated. The cytochrome b and cytochrome oxidase II mRNAs are edited in procyclic-form parasites but are primarily unedited in bloodstream forms. The latter forms lack the mitochondrial respiratory system present in procyclic forms. Editing of the NADH dehydrogenase 7 (ND7) and ND8 transcripts is also developmentally regulated but occurs preferentially in bloodstream forms. Other transcripts, cytochrome oxidase III and ATPase 6, are edited in both life forms. We have identified many minicircle-encoded guide RNAs (gRNAs) for ATPase 6, ND7, and ND8. The characteristics of these gRNAs reveal how extensively edited RNA can be edited in the 3'-to-5' direction. Northern (RNA) blot and primer extension analyses indicate that gRNAs for transcripts whose editing is developmentally regulated are present in both procyclic and bloodstream form parasites. These results suggest that the developmental regulation of editing in these transcripts is not controlled by the presence or absence of gRNAs.

Subcellular fractionation of the two organelle DNAs of malaria parasites.

Malaria parasites contain two extrachromosomal DNAs, a 6 kb repetitive linear molecule which is assigned on the basis of its genetic content to the mitochondria, and a 35 kb transcriptionally active circular molecule whose intracellular location is not known. We used the polymerase chain reaction to detect and estimate the numbers of both molecules in sub-cellular fractions derived from the rodent parasite Plasmodium yoelii. The two DNA molecules were not coordinately partitioned by the fractionation process, the 6 kb molecule being more abundant, relative to the 35 kb circle, in a fraction enriched for mitochondria, the converse being true for a less dense fraction of unknown identity. This implies that the two molecules are located in different cellular compartments, and is consistent with other evidence suggesting they have different evolutionary origins.

Homologies between the contiguous and fragmented rRNAs of the two Plasmodium falciparum extrachromosomal DNAs are limited to core sequences.

Plasmodium falciparum contains two extrachromosomal DNAs, a 6 kb linear element and a 35 kb circular DNA; both encode rDNA sequences. The 6 kb element rDNAs comprise fragments of both large and small subunit rRNAs. Comparison of these with corresponding rDNA sequences from the 35 kb DNA and E. coli show that sequences conserved between the three are largely confined to highly conserved core regions; in fact, most of the 6 kb rDNA sequences correspond to core regions. Both the 6 kb element and 35 kb rDNAs show less conservation to each other than to E. coli sequences, suggesting that the two extrachromosomal DNAs of P. falciparum are not closely related. The characteristics of the fragmented rRNAs from the 6 kb element suggest they are functional, possibly in mitochondrial ribosomes.