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

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. SYLVATICCYCLEDOMESTICCYCLE 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) Variable region T. cruzi minicircle Minicircle population + Restriction Enzyme (Schizodeme analysis) Conserved region Agarose gel

16. Macedo, Martins, Chiari & Pena Mol. Biochem. Parasitol. 55, 147-154 (1992) DNA FINGERPRINTING OF GENOMIC DNA 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 as target 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. CL B167 CA1 B147 SC43 cl1 Bug2149 cl10 NR cl3 SO3 cl5 Esmeraldo cl3 Y Basileu A138 1023 115 226 G Dm28 Tulahuen SilvioX10cl1 YuYu 1017 1001 1004 1009 1018 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 ME rDNA 1 1 1 1/2 2 1 1 1 1 2 2 2 2 2 2 2 2 2 2 0.65 0.40 0.20 0.00 T. cruzi II T. cruzi I Ribosomal RNA and Mini-Exon gene sequences and RAPD analysis define two major phylogenetic lineages of T. cruzi Souto, Fernandes, Macedo, Campbell and Zingales Mol. Biochem. Parasitol. (1996)

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

22. SILVATIC CYCLE DOMESTIC CYCLE T. cruzi II T. cruzi I (and 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 ANALYSIS OF 160 STRAINS FROM 12 STATES OF BRAZIL T. cruzi II

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

24. 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

25. 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

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

27. 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

28. 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

29. 3.5- 1.9- 1.6- 1.1- 0.6- 0.4- Mbp | T. cruzi II ||Group1/2|| T. cruzi I | Molecular Karyotype of T. cruzi strains Chromosome separation by PFGE

30. Molecular Karyotype PFGE of chromosomal DNA Transfer of DNA to nylon membranes - Southern blot Labeling of DNA probes (ESTs) with alpha P 32 d-ATP Hybridization Autoradiography Methodological Approach

31. Tc IITcI1/2 MOLECULAR KARYOTYPE OF STRAINS AND CLONES OF Trypanosoma cruzi

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

33. 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

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

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

36. 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 !!!!!