Maxicircle Transcripts Research Articles

Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei

Trypanosoma brucei is a kinetoplastid flagellate, the agent of human sleeping sickness and ruminant nagana in Africa. Kinetoplastid flagellates contain their eponym kinetoplast DNA (kDNA), consisting of two types of interlocked circular DNA molecules: scores of maxicircles and thousands of minicircles. Maxicircles have typical mitochondrial genes, most of which are translatable only after RNA editing. Minicircles encode guide RNAs, required for decrypting the maxicircle transcripts. The life cycle of T. brucei involves a bloodstream stage (BS) in vertebrates and a procyclic stage (PS) in the tsetse fly vector. Partial [dyskinetoplastidy (Dk)] or total [akinetoplastidy (Ak)] loss of kDNA locks the trypanosome in the BS form. Transmission between vertebrates becomes mechanical without PS and tsetse mediation, allowing the parasite to spread outside the African tsetse belt. Trypanosoma equiperdum and Trypanosoma evansi are agents of dourine and surra, diseases of horses, camels, and water buffaloes. We have characterized representative strains of T. equiperdum and T. evansi by numerous molecular and classical parasitological approaches. We show that both species are actually strains of T. brucei, which lost part (Dk) or all (Ak) of their kDNA. These trypanosomes are not monophyletic clades and do not qualify for species status. They should be considered two subspecies, respectively T. brucei equiperdum and T. brucei evansi, which spontaneously arose recently. Dk/Ak trypanosomes may potentially emerge repeatedly from T. brucei.

P166, a link between the trypanosome mitochondrial DNA and flagellum, mediates genome segregation

Kinetoplast DNA (kDNA), the trypanosome mitochondrial genome, is a giant network containing several thousand interlocked DNA rings. Within the mitochondrion, kDNA is condensed into a disk-shaped structure positioned near the flagellar basal body. The disk is linked to the basal body by a remarkable transmembrane filament system named the tripartite attachment complex (TAC). Following kDNA replication, the TAC mediates network segregation, pulling the progeny networks into the daughter cells by their linkage to the basal bodies. So far TAC has been characterized only morphologically with no known protein components. By screening an RNAi library, we discovered p166, a protein localizing between the kDNA and basal body in intact cells and in isolated flagellum-kDNA complexes. RNAi of p166 has only small effects on kDNA replication, but it causes profound defects in network segregation. For example, kDNA replication without segregation causes the networks to grow to enormous size. Thus, p166 is the first reported molecular component of the TAC, and its discovery will facilitate study of kDNA segregation machinery at the molecular level.

Expression profiling using random genomic DNA microarrays identifies differentially expressed genes associated with three major developmental stages of the protozoan parasite Leishmania major.

To complete its life cycle, protozoan parasites of the genus Leishmania undergo at least three major developmental transitions. However, previous efforts to identify genes showing stage regulated changes in transcript abundance have yielded relatively few. Here we used expression profiling to assess changes in transcript abundance in three stages: replicating promastigotes and infective non-replicating metacyclics, which occur in the sand fly vector, and in the amastigote stage residing with macrophage phagolysosomes in mammals. Microarrays were developed containing 11,484 PCR products that included a number of known genes and 10,464 random 1 kb genomic DNA fragments. Arrays were hybridized in triplicate and genes showing two-fold or greater changes in 2/3 experiments were scored as differentially expressed. Remarkably, only about one percent of the DNAs expression varied by this criteria, in either stage comparison. Northern blot analysis confirmed the predicted change in mRNA abundance for most of these (68%). This set of genes included most of those previously identified in the literature as differentially regulated as well as a number of novel genes. Notably, Leishmania maxicircle transcripts showed strong up-regulation in metacyclic and amastigote parasites, probably associated with changes in parasite energy metabolism. However, current data suggest that expression profiling using shotgun DNA libraries significantly underestimates the extent of regulated transcripts.

Closing the gaps in kinetoplast DNA network replication

Trypanosomatids are protozoan parasites responsible for important tropical diseases. One example, Trypanosoma brucei , causes African sleeping sickness, and related parasites cause Chagas disease and leishmaniasis. Because they are among the earliest-branching eukaryotes, trypanosomatids have unusual biological properties. One of their most curious features is a unique mitochondrial DNA network known as kinetoplast DNA (kDNA) (1). The kDNA network is composed of several thousand minicircles that are interlocked like the links in medieval chain mail. Also intertwined in the network are a few dozen maxicircles. See Fig. 1 for an electron micrograph of a segment of an isolated kDNA network from Crithidia fasciculata , a trypanosomatid often studied because it is nonpathogenic and easy to cultivate. The function of kDNA maxicircles, like mitochondrial DNA in conventional eukaryotes, is to encode a few gene products such as rRNA and subunits of respiratory complexes. However, the mechanism of gene expression is highly unconventional in that maxicircle transcripts must be edited to form a functional mRNA. Editing is an amazing form of RNA processing in which uridine residues are inserted or deleted at precise internal sites within the maxicircle transcripts, generating ORFs. Minicircles encode guide RNAs that are templates for editing of maxicircle transcripts. See ref. 2 for a review of editing and ref. 3 for a discussion of the evolution of kDNA and the significance of the network structure. Fig. 1. Electron micrograph of a segment of a kDNA network from C. fasciculata . Small loops are minicircles. [Reproduced with permission from ref. 1 (Copyright 2001, Elsevier Science).] In this issue of PNAS, Sinha et al. (4 …

The structure and replication of kinetoplast DNA.

The mitochondrial DNA of trypanosomatid protozoa, termed kinetoplast DNA (kDNA), is unique in its structure, function, and mode of replication. kDNA is a massive network, composed of thousands of topologically interlocked DNA circles, which resembles the chain mail of medieval armor. Each cell contains one network condensed into a disk-shaped structure within the matrix of its single mitochondrion. The kDNA circles are of two types, maxicircles present in a few dozen copies and minicircles present in several thousand copies. The maxicircles, which encode ribosomal RNAs and a few mitochondrial proteins, are similar in structure and genetic function to the mitochondrial DNA of other eukaryotes. Many maxicircle transcripts undergo editing, a remarkable process involving the insertion or deletion of uridine residues at specific sites. The minicircles encode small guide RNAs that control the specificity of editing. During kDNA replication, covalently closed minicircles are released from the network by a topoisomerase II. The free minicircles replicate as theta-structures within one of two complexes of replication proteins that are positioned on opposite sides of the kinetoplast disk. The progeny minicircles, which contain nicks or gaps, are attached to the network periphery. Maxicircles also replicate as theta-structures, but they remain linked to the network. As replication proceeds, the number of minicircles and maxicircles increases. When the network has doubled in size, all of the minicircle nicks and gaps are repaired, and the network splits in two. The two progeny networks then segregate into the daughter cells.

Extensive editing of CR2 maxicircle transcripts of Trypanosoma brucei predicts a protein with homology to a subunit of NADH dehydrogenase.

Several genes of the Trypanosoma brucei mitochondrial genome (the maxicircle) encode mRNAs that are so extensively altered by RNA editing that the gene cannot be identified by analysis of the DNA sequence. The 322-nucleotide preedited RNA of one of these genes, CR2, is converted into a 647-nucleotide transcript by the addition of 345 uridines and the deletion of 20 genomically encoded uridines. The fully edited transcript has an open reading frame that predicts a 194-amino-acid protein. This protein, which we name ND9 (NADH dehydrogenase subunit 9), has homology to a subunit of NADH dehydrogenase (respiratory complex I). Seven guide RNAs that can specify edited CR2 sequence have been identified. Steady-state levels of unedited ND9 transcripts are greater in bloodstream than in procyclic forms, but edited ND9 mRNA is present in similar abundance in both life cycle stages.

Conservation of gRNA gene cassette structure in African trypanosomes despite divergence in the defining flanking repeats

The mitochondrial (kinetoplast) DNA of kinetoplastid parasites is composed of two types of unrelated circular molecules: maxicircles, which are the equivalent of mitochondrial DNA of other organisms, and minicircles, which are 0.9-2.5 kb (depending on species) circular molecules present in 5000-10000 copies per kinetoplast [1]. Minicircles encode guide RNAs (gRNAs), small RNAs that contain sequence information for editing of maxicircle transcripts [2-4]. In Trypanosoma brucei, minicircle gRNA genes are almost always present within cassettes bounded by 18-bp inverted repeats, each minicircle containing 3 or 4 such cassettes (refs. 3-5; and R.A. Corell, P.J. Myler, and K. Stuart, unpublished). T. congolense minicircles lack the 18-bp inverted repeats found in T. brucei [6], but we have identified a related sequence that occurs as pairs of 19-bp inverted repeats in these minicircles. These repeats define a minicircle cassette organization which is similar to that in T. brucei, and a putative gRNA gene is encoded within one of these cassettes. Both reported T. congolense minicircles contain 3 pairs of imperfect 19-bp repeats. The location of the repeats relative to the

Trypanosoma equiperdum minicircles encode three distinct primary transcripts which exhibit guide RNA characteristics.

The mitochondrial DNA of trypanosomes is composed of maxicircle and minicircle DNAs catenated into a network, called the kinetoplast. Maxicircles encode proteins and RNAs necessary for mitochondrial assembly. Minicircles encode small transcripts which are believed to serve as guide RNAs in the process of RNA editing of maxicircle transcripts. Trypanosoma equiperdum minicircles contain three transcription units which produce three distinct transcripts. The genes for these transcripts are flanked by imperfect 18-bp repeats separated by approximately 110 bp. The transcripts have a 5' triphosphate, indicating that they are primary transcripts. Minicircle transcription initiates at a purine within a conserved sequence, 5'-AYAYA-3', where Y is a pyrimidine, 32 bp from the upstream inverted repeat, suggesting that the repeats may function in transcript initiation. Transcripts from a single minicircle transcription unit range in size from 55 to 70 nucleotides. This size heterogeneity within a single sequence class is due to the variable length of nontemplated uridine residues composing a 3' tail. The size range and heterogeneous polyuridylate 3' end of the minicircle transcripts appear to be conserved features and may be related to transcript function.

Trypanosoma equiperdum minicircles encode three distinct primary transcripts which exhibit guide RNA characteristics.

The mitochondrial DNA of trypanosomes is composed of maxicircle and minicircle DNAs catenated into a network, called the kinetoplast. Maxicircles encode proteins and RNAs necessary for mitochondrial assembly. Minicircles encode small transcripts which are believed to serve as guide RNAs in the process of RNA editing of maxicircle transcripts. Trypanosoma equiperdum minicircles contain three transcription units which produce three distinct transcripts. The genes for these transcripts are flanked by imperfect 18-bp repeats separated by approximately 110 bp. The transcripts have a 5' triphosphate, indicating that they are primary transcripts. Minicircle transcription initiates at a purine within a conserved sequence, 5'-AYAYA-3', where Y is a pyrimidine, 32 bp from the upstream inverted repeat, suggesting that the repeats may function in transcript initiation. Transcripts from a single minicircle transcription unit range in size from 55 to 70 nucleotides. This size heterogeneity within a single sequence class is due to the variable length of nontemplated uridine residues composing a 3' tail. The size range and heterogeneous polyuridylate 3' end of the minicircle transcripts appear to be conserved features and may be related to transcript function.

Kinetoplastid mitochondria contain functional tRNAs which are encoded in nuclear DNA and also contain small mlnltircJe and maxicircle transcripts of unknown function

The mitochondrion of Leishmania tarentolae contains approximately 35-40 tRNAs many of which comigrate with cytoplasmic tRNAs. Both mitochondrial (KtRNA) and cytoplasmic (CtRNA) tRNAs are functional, as they could be acylated either by mitochondrial or cytoplasmic synthetase extracts. There are two methionyl tRNA species in the cytoplasmic and mitochondrial fractions, one of which is unique to each fraction, indicating that the KtRNA fraction is free of CtRNA contamination. Leucyl and glycyl tRNAs were identified by hybridization with a genomic clone from Trypanosoma brucei. KtRNA hybridizes with nuclear chromosomes, but not with minicircle or maxicircle DNA. KtRNA isolated by DEAE chromatography or agarose gel electrophoresis contains additional small RNAs which hybridize with both minicircle and maxicircle DNA. These transcripts do not migrate like tRNAs in acrylamide gels and their functions is unknown. We suggest that most if not all mitochondrial tRNAs in L. tarentolae are nuclear-encoded and imported into the mitochondrion.

Variation of G-rich mitochondrial transcripts among stocks of Trypanosoma brucei

We have compared maxicircle transcripts from eight stocks of subspecies of Trypanosoma brucei. Transcripts from the rRNA and protein genes have a constant size among stocks and exhibit only minor variation in abundance. In contrast, four of the G + C rich sequences encode multiple transcripts that vary markedly in size or abundance. Maxicircle nucleotide sequence comparison of three stocks shows very limited sequence divergence suggesting that sequence divergence may not explain the transcript variability. These results suggest that the G-rich transcripts do not encode proteins and that their variability among stocks may result from posttranscriptional processing events.

Further characterization of the extremely small mitocbondrial ribosomal RNAs from trypanosomes: a detailed comparison of the 9S and 12S RNAs fromCrithidia fasciculateandTrypanosoma bruceiwith rRNAs from other organisms

We have determined the nucleotide sequence of a maxi-circle segment from the insect trypanosome Crithidia fasciculata mitochondrial DNA, on which the genes for the major maxicircle transcripts of 9S and 12S are localized. The 5'-terminal sequences of these RNAs were determined by wandering spot analysis. The map coordinates of the 9S and 12S RNAs from Trypanosoma brucei were adjusted with respect to a previous report with the aid of primer extension analysis with reverse transcriptase. This approach allowed us to align the corresponding genes from both organisms which show an overall sequence homology of 77%. The 9S and 12S RNA genes from the two trypanosome species contain sequences, closely related to some of the regions that are universally conserved among ribosomal RNAs from members of the three primary kingdoms and their organelles, even though the overall level of sequence homology is extremely low. These universal sequences occur at positions in the 9S and 12S RNAs that are analogous to those occupied by their counterparts in authentic ribosomal RNAs. The characteristic secondary structure elements flanking these universal sequences in genuine ribosomal RNAs can also be formed in the trypanosomal 9S and 12S RNAs. These results provide unequivocal evidence for a ribosomal function of the 9S and 12S RNAs of trypanosomal mitochondria, notwithstanding their extremely small size (estimated to be 612 and 1141 nucleotides in C. fasciculata, 611 and 1150 nucleotides in T. brucei) and their unusual base composition (83% A+U).

Localization of Kinetoplast DNA Maxicircle Transcripts in Bloodstream and Procyclic Form Trypanosoma brucei

Over 80% of the maxicircle and numerous minicircles of Trypanosoma brucei kinetoplast DNA have been cloned. The uncloned maxicircle segment contains few restriction endonuclease cleavage sites, varies in size among strains, and may be unstable in conventional cloning systems. cDNA prepared to bloodstream or procyclic trypomastigote RNA hybridized to all but one maxicircle segment, but did not hybridize to minicircles. Fourteen maxicircle transcripts were detected in RNA from both bloodstream and procyclic trypomastigotes. The coding sequences for these transcripts were localized and account for most of the maxicircle. One region of the maxicircle, which borders the variable region, was not found to be transcribed. We conclude that the maxicircle is largely but not completely transcribed in both bloodstream and procyclic trypomastigotes, whereas minicircle transcription is minimal or absent in these stages. Qualitative transcriptional differences which could account for mitochondrial respiratory differences between the bloodstream and procyclic trypomastigotes were not observed.

Localization of kinetoplast DNA maxicircle transcripts in bloodstream and procyclic form Trypanosoma brucei.

Over 80% of the maxicircle and numerous minicircles of Trypanosoma brucei kinetoplast DNA have been cloned. The uncloned maxicircle segment contains few restriction endonuclease cleavage sites, varies in size among strains, and may be unstable in conventional cloning systems. cDNA prepared to bloodstream or procyclic trypomastigote RNA hybridized to all but one maxicircle segment, but did not hybridize to minicircles. Fourteen maxicircle transcripts were detected in RNA from both bloodstream and procyclic trypomastigotes. The coding sequences for these transcripts were localized and account for most of the maxicircle. One region of the maxicircle, which borders the variable region, was not found to be transcribed. We conclude that the maxicircle is largely but not completely transcribed in both bloodstream and procyclic trypomastigotes, whereas minicircle transcription is minimal or absent in these stages. Qualitative transcriptional differences which could account for mitochondrial respiratory differences between the bloodstream and procyclic trypomastigotes were not observed.

The kinetoplast DNA of the hemoflagellate protozoa.

The kinetoplast DNA of Leishmania tarentolae and Trypanosoma brucei was studied in terms of genetic organization and transcriptional activity. Several minor sequence classes of minicircles from L. tarentolae were cloned in a bacterial plasmid and were compared in terms of sequence organization. The cloned minicircles were characterized by the possession of a constant region of at least 91 nucleotides and a variable region. Minicircles from Trypanosoma brucei strain 366D were also cloned. Fragments of the maxicircle DNA from both species were also cloned in pBR322. No homology with the cloned minicircles was apparent. Several maxicircle transcripts, in addition to the 9 and 12s presumptive ribosomal RNAs, were observed in L. tarentolae. The 9 and 12s RNA genes were also mapped on the T. brucei maxicircle. Sequence homology between the L. tarentolae 9 and 12s RNA genes and the T. brucei 9 and 12s RNA genes was observed. A culture system was developed to study the developmental change of cultured bloodstream forms of T. brucei into procyclic forms. This developmental system is amenable for the study of the role of the kinetoplast DNA in the extensive mitochondrial biogenesis that occurs at this time.