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DO REFERENCE ORGANISMS OF GENOME PROJECTS
COVER THE GENETIC DIVERSITY OF PARASITES ?
Bianca Zingales
Dep. of Biochemistry
Institute of Chemistry
University of São Paulo
[email protected]
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
• 1 - Characteristics of Kinetoplatida
(Leishmania, African and American Trypanosomes)
•Parasites have an asexual reproduction
•Parasites are diploid
• Presence of Kinetoplast
Kinetoplast of T. cruzi
•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
Processing of polycistronic mRNAs
Mini-exon genes (100-200 copies)
Coding genes
A
B
C
Exon - 39 bp
Transcription
Intron ~70 bp
Transcription
A
Intergenic
region
medRNA
C
B
Polycistronic mRNA
Trans-splicing
A
A
AAA
C
B
B
AAA
Mature mRNAs
C
AAA
•
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
Populations of T. cruzi circulate in two cycles
SYLVATIC
CYCLE
DOMESTIC
CYCLE
-
-
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
HETEROGENEITY OF BIOLOGICAL
CHARACTERISTICS OF TRYPANOSOMA CRUZI
STRAINS
• MORPHOLOGY
• COURSE OF INFECTION IN MICE
•TISSUE TROPISM
• SUSCEPTIBILITY TO CHEMOTHERAPEUTIC
AGENTS
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%
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
DNA AMOUNT VARIES IN STRAINS OF
TRYPANOSOMA CRUZI
Dvorak and co-workers, 80’
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)
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
T. cruzi minicircle
Minicircle population + Restriction Enzyme
(Schizodeme analysis)
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
Our group decided to investigate the genetic diversity of T. cruzi
using as target the ribosomal RNA genes (markers for phylogeny)
RIBOSOMAL RNA GENES
prokaryotes
eukaryotes
trypanosomatids
•Definition of three groups of strains:
group 1, 125 bp;
group 2, 110 bp;
group 1/2, 125 and 110 bp
Ribosomal RNA and Mini-Exon gene sequences and
RAPD analysis define two major phylogenetic lineages of
T. cruzi
ME rDNA
T. cruzi II
T. cruzi I
0.65
0.40
0.20
0.00
CL
B167
CA1
B147
SC43 cl1
Bug2149 cl10
NR cl3
SO3 cl5
Esmeraldo cl3
Y
Basileu
A138
1023
115
226
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1/2
2
1/2
1/2
1/2
1
1
1
1
1/2
1/2
1/2
G
Dm28
Tulahuen
SilvioX10cl1
YuYu
1017
1001
1004
1009
1018
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Souto, Fernandes, Macedo,
Campbell and Zingales
Mol. Biochem. Parasitol. (1996)
Two Lineages of
Trypanosoma cruzi
• Biological Meaning?
• Epidemiological distribution?
• Pathogenesis?
Molecular epidemiology of
Chagas disease in Brazil
Amazonas
Pará
Piauí
Paraíba
Goiás
Bahia
Minas Gerais
Espírito Santo
Rio de Janeiro
Santa Catarina
São Paulo
Rio Grande do Sul
ANALYSIS OF 160 STRAINS FROM 12 STATES OF BRAZIL
SILVATIC CYCLE
T. cruzi I (and II)
DOMESTIC CYCLE
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
Parasite Genome Projects
Launched by TDR/WHO at FIOCRUZ (Rio de Janeiro)
1994
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
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
EST (EXPRESSED SEQUENCE TAGS)
mRNA POPULATION
cDNA LIBRARY CONSTRUCTION
(LIBRARY NORMALIZATION)
RANDOM SELECTION OF CLONES
PARTIAL SEQUENCING OF 5´ ENDS
AUTOMATIC SEQUENCING
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
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
Molecular Karyotype of T. cruzi strains
Chromosome separation by PFGE
|T. cruzi II | Group1/2| |T. cruzi I |
Mbp
3.51.91.61.10.60.4-
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
MOLECULAR KARYOTYPE OF STRAINS AND CLONES OF Trypanosoma cruzi
Tc II
1/2
TcI
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…..
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
Microarray Technology
Differential gene expression in T. cruzi strains isolated from
patients with different manifestations of Chagas disease
(ESTs and cloned genes)
*Pop 1
*Pop 2
Preparation of target DNAs
Application on the glass slides
• PCR amplification of ESTs
• Purification of the amplified products
Hybridization with Cy5 and Cy3
cDNA populations
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 !!!!!