Mitochondrial DNA Replication in the Sea Urchin Strongylocentrotus purpuratus

In animal development, the normal regulation of mitochondrial DNA (mtDNA) replication, which ensures a doubling of the amount of mtDNA per cell division, is altered. In oogenesis there is extensive mtDNA synthesis, but no nuclear DNA replication, whereas the situation is reversed in embryogenesis. The aim of this project was to provide basic information about the molecular mechanism of mtDNA replication in the sea urchin Strongylocentrotus purpuratus, so that the question of what regulates mtDNA replication can then be addressed. The two-dimensional (2D) agarose gel electrophoresis system developed by Brewer and Fangman (1987) was used to analyse the structures of the replicating mtDNA molecules. This gel system exploits the fact that non-linear DNA molecules are retarded under gel electrophoresis compared to linear DNA molecules. The DNA moleules are subjected to restriction enzyme digestion before electrophoresis, and curves of different, characteristic shapes are generated by replication intermediates (RIs) of different structures. Different restriction enzyme digestions allow the pattern of replication fork movement to be related to the positions of the restriction sites within the genome, which enables the position of the replication origin to be located in the DNA molecules. The gel electrophoretic analyses provided a complex, but internally consistent set of results, giving a picture of the replication process in S. purpuratus mtDNA molecules. The analyses revealed that the replication origin for leading-strand synthesis was located within the non-coding region of the genome, and that replication was unidirectional, by a strand-displacement mechanism, and occurred towards the 12S rRNA gene. The 2D gel experiments also revealed that pause sites for leading-strand synthesis were located at several sites in the mitochondrial genome. The most prominent of these pause sites occurred close to a prominent lagging-strand replication origin, in the region of the genome near the boundary between the genes for subunit 6 of ATP synthase (A6), and subunit HI of cytochrome c oxidase (COIII). RNase protection experiments, performed using probes covering the noncoding region of the mtDNA, mapped the 5' end of the nascent DNA strands to nt 1150 +/-10, and suggested that the 3' end of lagging-strand molecules also mapped to the same location, implying a pause site for lagging-strand synthesis at the leading-strand origin. The ends of the DNA molecules corresponding to the leading-strand replication pause site, and the origin of lagging-strand synthesis in the A6/COIII region of the genome were, however, below the level of detection of the RNase protection experiments. The ends of the transcripts for the A6 and COIII genes were mapped, which revealed one major and one minor 3' end for the A6 RNA, and a single discrete 5' end for the COIII RNA. From the sizes of the molecules identified by the RNase protection experiments, it appeared that the ends of the 2 transcripts were very close together. An in vitro transcription system was used to investigate whether there is a link between transcriptional control and DNA replication in sea urchin mitochondria. A possible link between the 2 processes was suggested by the location of some of the replication pause sites close to the 3' ends of genes: sites where transcription termination could be involved in the production of transcripts. The in vitro transcription system used bacteriophage RNA polymerase, because the enzyme had not been isolated from S. purpuratus mitochondria at the time the experiments were performed. Bacteriophages have single subunit RNA polymerases, which show considerable sequence similarity at the amino acid (aa) level to the mtRNA polymerase isolated from yeast (Masters et al, 1987). Transcription run-off assays were carried out using the region of the mtDNA containing the A6/COIII gene boundary, because this portion of the genome contains both a replication pause site and a lagging-strand replication origin, as well as being a possible site for transcription termination. When transcription of the mtDNA was carried out in the same direction as RNA synthesis in vivo, 6 sites for transcription termination were detected. However, no sites were detected when transcription through the mtDNA occurred in the opposite direction. The termination sites did not correspond with the 3' end of the A6 transcript detected in vivo by the RNase protection experiments. This would, therefore, imply that if these sites were acting to terminate transcription in vivo, then further processing of the transcript would have to occur to generate the mature mRNA. Although 2 DNA-binding proteins which act close to the A6/COIII gene boundary have been identified in mitochondrial protein extracts (S.A. Qureshi & H.T. Jacobs, unpublished data), the addition of these protein extracts (prepared by SAQ) to the in vitro transcription reactions did not appear to affect the pattern of transcription termination. On the basis of the gel electrophoretic data, I propose a model for mtDNA replication in S. purpuratus (Mayhook et al, 1992). The origin of leading-strand replication is located in the non-coding region of the genome at nt 1150 + 10. DNA synthesis is initially unidirectional, by strand displacement (as proposed for vertebrate mtDNA; Clayton, 1982), towards the 12S rRNA gene. The gel electrophoretic data are consistent with the electron microscopy data of Matsumoto et al (1974), which imply that multiple origins exist for lagging-strand synthesis. A prominent lagging-strand origin was detected near the A6/COIII gene boundary, and its occurrence in the same region of the genome as a pause site for leading-strand synthesis suggests that the pausing of leading-strand replication may have a role in the initiation of lagging-strand synthesis

Cloning and Analysis of a Surface Antigen Gene From Procyclic Trypanosoma congolense

The presence of the variant surface glycoprotein coat, and its modulation by the process of antigenic variation have made it unlikely that a vaccine could be developed against mammalian stages of African trypanosomes. However, except for the metacyclic stage which is preadapted to life in the mammalian host, insect stages of these parasites do not possess the variant surface glycoprotein coat and display a common set of antigens on their surface dominated, in T.brucei, by a surface coat composed of the glycoprotein procyclin. If animals could be vaccinated against these common antigens then tsetse flies feeding on such vaccinated animals would ingest antibody with their bloodmeal which might inhibit development of trypanosome infections in the fly and therefore block transmission. Of a set of monoclonal antibodies which had been raised against living procyclic Trypanosoma congolense, one group appeared to be directed against molecules on the trypanosome surface and were strongly agglutinating and trypanocidal. Each monoclonal antibody in that group detected the same diffuse bands on western blots of SDS-PAGE of trypanosome lysates, indicating that they were against the same immunodominant antigen. The antigen has several unusual properties in common with procyclin and the work presented here was directed towards identifying the gene for this antigen. Heterologous probing of a genomic library with the procyclin cDNA from T.brucei indicated that T.congolense has sequences which share a degree of homology to the repetitive elements in the procyclin gene but these sequences are not expressed and there is no true homologue of this gene in T.congolense. The monoclonal antibodies did not detect the antigen gene from a cDNA expression library, probably because at least one of them appears to be directed against a carbohydrate epitope. However, differential screening of a cDNA library with first strand cDNA from procyclic and bloodstream stages detected several cDNAs, one of which contained an open reading frame with a high degree of homology to two cyanogen bromide peptide sequences derived from a Kilifi-type T.congolense surface antigen isolated by Beecroft et al (manuscript in preparation). Apart from a size difference defined by migration on SDS-PAGE, this antigen has identical properties to that detected by the set of monoclonal antibodies. The cDNA has an open reading frame coding for a protein of 256 amino acids which is rich in alanine and acidic residues. There is no N-glycosylation signal and no obvious signal peptide but the amino terminus is hydrophobic and there is a potential signal at the carboxy terminus for the addition of a glycosyl phosphatidylinositol tail which could anchor the protein in the membrane. This protein, which has been called ARP (for Alanine Rich Protein), has been expressed as a fusion protein in E.coli and used to raise antisera in rats and mice. These antisera label procyclic and epimastigote stages of T.congolense in indirect immunofluorescence and label the surface of procyclic cells in immunogold electron microscopy. In western blots of procyclic trypanosome lysates enriched for membranes they identify diffuse bands similar to those detected by the monoclonal antibodies. Several other cDNA clones were isolated in the differential screen but none of these, except perhaps cDNAPl, proved to be genuinely stage-specific. cDNAPl appears to be transcribed from telomeres but in which direction is not evident because it does not possess a poly (A) tail, although probing of northern blots suggests that some of the homologous transcripts are polyadenylated. cDNAs P7 and P8 appear to be single copy sequences in the trypanosome genome but no function can be ascribed to them as they do not contain open reading frames. cDNAPE encodes the ribosomal protein L29, is highly homologous to the the sequence of L29 from other species and may encode a cycloheximide resistant form of this protein in the stock of trypanosomes from which it was isolated. In conclusion, the apparent lack of stage-specific control at the level of transcription in trypanosomes has pushed the differential screening process to its limits and yielded sequences which are largely artifactual. (Abstract shortened by ProQuest.)

Identification and characterisation of an extrachromosomal element from a multidrug-resistant isolate of Trypanosoma brucei brucei

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Drug resistance together with difficulties involved in the development of new trypanocides are a major problem in the present control of African trypanosomiasis. DNA based diagnostics for drug resistance would overcome problems in the identification of drug-resistant populations and contribute to effective control measures. However, this requires a detailed knowledge of the mode of action and the mechanisms by which trypanosomes can overcome the toxic effects of trypanocides. In this study, a search for molecular differences between a multidrug-resistant isolate of Trypanosoma brucei brucei, CP 547, and a reference drug-sensitive population, ILTat 1.4, led to the identification of a 6.6 kbp extrachromosomal element in the multidrug-resistant population. In light of the involvement of extrachromosomal elements in drug resistance in Leishmana spp. and cancer cells, the identification of the 6.6 kbp element warranted its characterisation. Several different approaches sere attempted before a sequence which hybridised to the 6.6 kbp element its eventually isolated. This sequence is represented by a 108 bp repeat sequence which forms long arrays of tandem repeats. Since N/a III is the sole restriction enzyme that cuts within the repeat, it has been referred to as an N/a III repeal The repeat is flanked by a 5 bp spacer sequence. However, a unique 5 bp direct repeat flanking two complete, and one partial copy of the N/a III repeat may signify the transposition of these sequences. Hybridisation with the N/a III repeat revealed the presence of 'higher' hybridising elements which also appear to be predominantly composed of long tandem arrays of the N/a Ill repeal Through exploitation of the p01) merase chain reaction using arbitrary primers (AP-PCR), additional sequences were identified which are associated with some of the 6.6 kbp and 'higher' hybridising elements. The 6.6 kbp element and some of the 'higher' hybridising elements display features of circular DNA molecules. The 6.6 kbp element also displays some level of size and sequence heterogeneity within different populations of the same trypanosome isolate. The copy number of the 6.6 kbp element is also not stable and appears to be directly affected by the application of selective drug pressure, but a direct association between the presence of the element and the expression of multidrug resistance could not be determined. The N/a III repeat family represents a newly identified repetitive family specific to members of the Trypanozoon subgenus. This repeat family, representing about 5% of the parasite genome, is dispersed through all size classes of chromosomes, in addition to its presence on the extrachromosomal elements. Transcriptional studies of the N/a III repeats have revealed that their transcription is developmentally regulated, since heterogeneous transcripts ranging from greater than 10 kb to smaller than 300 bp are present in the actively dividing long slender bloodstream and insect stage procyclic forms of the parasite but not nondividing, stumpy bloodstream forms. Lastly, the N/a III repeat lacks an open reading frame and transcripts do not appear to have a spliced leader sequence at the 5' end. Furthermore, there is almost an equal representation of polyadenylatcd and non-polyadenlyated transcripts