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DO REFERENCE ORGANISMS OF GENOME PROJECTS COVER THE GENETIC DIVERSITY OF PARASITES ?

DO REFERENCE ORGANISMS OF GENOME PROJECTS COVER THE GENETIC DIVERSITY OF PARASITES ? Bianca Zingales Dep. of Biochemistry Institute of Chemistry University of São Paulo zingales@iq.usp.br. This talk has three main goals:

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DO REFERENCE ORGANISMS OF GENOME PROJECTS COVER THE GENETIC DIVERSITY OF PARASITES ?

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  1. DO REFERENCE ORGANISMS OF GENOME PROJECTS COVER THE GENETIC DIVERSITY OF PARASITES ? Bianca Zingales Dep. of Biochemistry Institute of Chemistry University of São Paulo zingales@iq.usp.br

  2. This talk has three main goals: • 1- To describe some characteristics of the genome and molecular biology of parasitic protozoa • 2 - To show you that most parasites present great genetic diversity • 3 -To discuss how the information generated in Parasite Genome Projects - that employ Reference Organisms - can be used to approach specific problems of parasite isolates

  3. 1 - Characteristics of Kinetoplatida • (Leishmania, African and American Trypanosomes) • Parasites have an asexual reproduction • Parasites are diploid • Presence of Kinetoplast

  4. Kinetoplast of T. cruzi

  5. 1 - Characteristics of Kinetoplatida • (Leishmania and African and American Trypanosomes) • Most of the expressed genes have NO introns • Promoters of RNA polymerase II have not been found • Transcription occurs in polycistronic pre-mRNAs • The conversion of these mRNAs into individual messages occurs by the addition of a mini-exon sequence (spliced leader) to the 5´end o mRNA (trans-splice process) • Trans-splicing and addition of a poliA tail to the 3´ end of the mRNA are concomitant processes

  6. Processing of polycistronic mRNAs Mini-exon genes (100-200 copies) Coding genes A B C Exon - 39 bp Intron~70 bp Transcription Transcription A C Intergenic region B medRNA Trans-splicing A C B A C B AAA AAA AAA Mature mRNAs Polycistronic mRNA

  7. 2 - Genetic diversity of Parasites: • Trypanosoma cruzi as a model • Protozoan causative of Chagas disease • Affects 16 million people in Latin America • From Mexico to Chile and Argentina • There are no available drugs for treatment • There is no vaccine to prevent infection

  8. SYLVATIC CYCLE DOMESTIC CYCLE Populations of T. cruzi circulate in two cycles

  9. STRAIN OR STOCK Any parasite population isolated from the blood of a mammalian host or digestive tract of the insect The strains are propagated in the laboratory in liquid medium, in experimental animals, or in tissue culture Biological, biochemical, immunological and genetic parameters are defined for each strain

  10. HETEROGENEITY OF BIOLOGICAL CHARACTERISTICS OF TRYPANOSOMA CRUZI STRAINS • MORPHOLOGY • COURSE OF INFECTION IN MICE • TISSUE TROPISM • SUSCEPTIBILITY TO CHEMOTHERAPEUTIC AGENTS

  11. CLINICAL CHARACTERISTICS OF CHAGAS DISEASE CHRONIC AND INCURABLE DISEASE CLINICAL MANIFESTATIONS IN THE CHRONIC PHASE: INDETERMINATE FORM - 70 - 80% CARDIAC - 20 - 30% DIGESTIVE - 10% NEUROLOGICAL AND MIXED - 1%

  12. Central question: Is there a correlation between the biological variability of the parasite and the clinical manifestations of Chagas Disease ? Trypanosoma cruzi invading a mammalian cell Carlos Chagas - 1909

  13. DNA AMOUNT VARIES IN STRAINS OF TRYPANOSOMA CRUZI Dvorak and co-workers, 80’

  14. MOLECULAR TYPING OF KINETOPLASTIDA BASED ON GENOMIC OR MITOCHONDRIAL DNA (kDNA) • RFLP - Restriction fragment length polymorphism • DNA FINGERPRINTING • RAPD - Randomly amplified polymorphic DNA • PCR of specific sequences (rRNA genes, mini-exon genes)

  15. STRAINS OF T.cruzi CAN BE CHARACTERIZED BY THE PATTERN OF RESTRICTION ENDONUCLEASE PRODUCTS OF KINETOPLAST DNA Morel, Chiari, Camargo, Mattei, Romanha & Simpson. PNAS 77, 6810-6814 (1980) Agarose gel Conserved region Variable region Minicircle population + Restriction Enzyme (Schizodeme analysis) T. cruzi minicircle

  16. DNA FINGERPRINTING OF GENOMIC DNA Macedo, Martins, Chiari & Pena Mol. Biochem. Parasitol. 55, 147-154 (1992) • Approach • Genomic DNA digested with restriction enzymes • Southern blot • Hybridization with microsatellite labeled probe

  17. Our group decided to investigate the genetic diversity of T. cruzi using astarget the ribosomal RNA genes (markers for phylogeny)

  18. Definition of three groups of strains: group 1, 125 bp; • group 2, 110 bp; • group 1/2, 125 and 110 bp

  19. Ribosomal RNA and Mini-Exon gene sequences and RAPD analysis define two major phylogenetic lineages of T. cruzi ME rDNA CL 1 1 B167 1 1 T. cruzi II CA1 1 1 B147 1 1/2 SC43 cl1 1 2 Bug2149 cl10 1 1/2 NR cl3 1 1/2 SO3 cl5 1 1/2 Esmeraldo cl3 1 1 Y 1 1 Basileu 1 1 A138 1 1 1023 1 1/2 115 1 1/2 226 1 1/2 G 2 2 Dm28 2 2 T. cruziI Tulahuen 2 2 SilvioX10cl1 2 2 YuYu 2 2 1017 2 2 1001 2 2 1004 2 2 1009 2 2 1018 2 2 Souto, Fernandes, Macedo, Campbell and Zingales Mol. Biochem. Parasitol. (1996) 0.65 0.40 0.20 0.00

  20. Two Lineages of Trypanosoma cruzi • Biological Meaning? • Epidemiological distribution? • Pathogenesis?

  21. ANALYSIS OF 160 STRAINS FROM 12 STATES OF BRAZIL SILVATIC CYCLE DOMESTIC CYCLE T. cruzi I (and II) T. cruzi II T. cruzi II Chagas Disease Fernandes et al., Am. J. Trop. Med Hyg. 58: 807-811, 1998 Zingales et al., Int. J. Parasitol. 28: 105-112, 1998

  22. Parasite Genome Projects Launched by TDR/WHO at FIOCRUZ (Rio de Janeiro) 1994

  23. Reference Organism: T. cruzi CL-Brener • Origin : • Isolated from Triatoma infestans • Characteristics : • belongs to T. cruzi II (domestic cycle) • shows clear acute phase in mice (and accidentally infected humans) • shows chronic phase in mice, with preference for heart and muscle cells • is highly susceptible to drugs used against Chagas disease • differentiates efficiently to metacyclics in-vitro • isoenzyme profile, schizodeme and RAPD patterns, and karyotype are stable for at least 100 generations • haploid genome size: 43.5 Mb

  24. Sequencing of T. cruzi Genome -(CL Brener) • (October 2000) • 10,000 ESTs • 12,000 GSS • 900 other sequences • partial sequence of chromosome 3 • Approximately 50% of the genes of unknown function

  25. EST (EXPRESSED SEQUENCE TAGS) AUTOMATIC SEQUENCING mRNA POPULATION cDNA LIBRARY CONSTRUCTION (LIBRARY NORMALIZATION) RANDOM SELECTION OF CLONES PARTIAL SEQUENCING OF 5´ ENDS

  26. Sequencing of Chromosome 3 of Trypanosoma cruzi (93.4 kb) • 20 - 30 novel genes and several repeat elements • Two long clusters , transcribed in opposite directions • Separated by an ~20-kb long, GC-rich sequence • Analogous situation was found for chromosome 1 of Leishmania major (257 kb) • 79 protein coding genes • 29 genes encoded on one strand; 50 genes on the opposite strand

  27. Molecular Karyotype of T. cruzi strains • Verify chromosome polymorphism among strains • Establish gene linkage groups • Compare molecular karyotype of T. cruzi lineages • Establish molecular markers for chromosome sequencing of CL Brener

  28. 3.5- |T. cruzi II| |Group1/2| |T. cruzi I| 1.9- Mbp 1.6- 1.1- 0.6- 0.4- Molecular Karyotype of T. cruzi strains Chromosome separation by PFGE

  29. Molecular Karyotype Methodological Approach • PFGE of chromosomal DNA • Transfer of DNA to nylon membranes - Southern blot • Labeling of DNA probes (ESTs) with alpha P32 d-ATP • Hybridization • Autoradiography

  30. MOLECULAR KARYOTYPE OF STRAINS AND CLONES OF Trypanosoma cruzi Tc II 1/2 TcI

  31. Conclusions • Polymorphism in the molecular karyotype of the strains • Homologous chromosomes may have different sizes • Definition of chromosome markers for genome sequencing • Other non-published observations…..

  32. Central question: Definition of genetic markers of the strains causative of different clinical manifestations of Chagas Disease Application: Prognosis and potential targets for treatment Trypanosoma cruzi invading a mammalian cell Carlos Chagas - 1909

  33. *Pop 1 *Pop 2 Microarray Technology Differential gene expression in T. cruzi strains isolated from patients with different manifestations of Chagas disease (ESTs and cloned genes)

  34. Application on the glass slides • Preparation of target DNAs • PCR amplification of ESTs • Purification of the amplified products Hybridization with Cy5 and Cy3 cDNA populations

  35. Microarray technology can also be employed to investigate the representativeness of genes in the genome of two populations of strains (presence, absence, copy number, etc.) We hope to have interesting results in the near future !!!!!

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