The biology of Leishmania beyond the Genome Project

1
The biology of Leishmania beyond the Genome
Project
2
(No Transcript)
3
Leishmania some features

• No chromosome condensation
• Generally accepted as a diploid, asexual organism
• No mutants available

• A Friendly parasite for genome research
• 35 Mb genome - 36 size-polymorphic chromosomes
• High GC content (60)
• High gene density
• Low frequency of repetitive sequences
• Transcription is polycistronic
• RNA processing by trans-splicing (no introns)

4
Transcription in Leishmania
Trans-splicing polyA addition
5
Leishmania some features

• No introns (virtually)
• Unclear rules for the regulation of gene
  expression
• Few genes detected as stage specific messages
• No mutants available

• A Friendly parasite for genome genetic
  manipulation
• Reverse genetics is well established
• overexpression of genes/variety of shuttle
  vectors
• gene knockout through homologous recombination
• genetic complementation

6
Leishmania Genome Network LGN

• Genome Project of Parasites
• WHO/TDR initiative launched in 1994
• International laboratories network established
  (LGN)
• Funding expansion 96-98 NIH, WT, BW, EC

• http//www.sanger.ac.uk/Projects/L_major/
• http//www.genedb.org/genedb/leish/index.jsp

7
Leishmania Genome Project LGN

• Main activities
• Molecular karyotype definition of syntenic
  groups
• A physical map for the entire L.major Friedlin
  genome
• (cosmid - cLHYG - library)
• Sequencing
• Gene discovery/expressed sequences
• Genomic sequencing
• Functional genomics
• systematic gene knockout
• microarray
• proteomics, metabolomics

• http//www.sanger.ac.uk/Projects/L_major/
• http//www.genedb.org/genedb/leish/index.jsp

8
Molecular Karyotype
L.donovani
L.major
C
kb
630
194
97
9
Definition of syntenic groups
P.Bastien coworkers
10
Syntenic groups a molecular karyotype
for L.major -Friedlin
LGN laboratories
11
Leishmania Genome Project LGN

• Main activities
• Molecular karyotype definition of syntenic
  groups
• A physical map for the entire L.major Friedlin
  genome
• (cosmid - cLHYG - library)
• Sequencing
• Gene discovery/expressed sequences
• Genomic sequencing

• http//www.sanger.ac.uk/Projects/L_major/
• http//www.genedb.org/genedb/leish/index.jsp

12
(No Transcript)
13
Minimal tiling set of overlapping clones
14
(No Transcript)
15
(No Transcript)
16
Leishmanias e leishmanioses
Leishmaniose tegumentar L.(L.) amazonensis L.(V.)
braziliensis L.(V.) guyanensis L.(V.) lainsoni
Leishmaniose visceral L.(L.) chagasi
17
Comparative Genomics L.braziliensis X L.major

• Choosing an approach
• Complete genome sequencing
• Expressed genome
• ESTs
• microarrays
• Genome survey sequences

• GSS (genome survey sequences)
• gene discovery
• comparative genomics

18
Leishmania braziliensis GSS library
Genomic DNA
Mechanical fragmentation
Fragment size selection 1-2 kb
Genomic Library in pUC18
Sequencing of 12000 clones randomly picked
Assembly clusterization
Data Bank non redundant GSS
19
Leishmania braziliensis GSS Analysis in DataBanks

• 2.309 hits BlastX
• 9.128 no hits DB

20
Leishmania braziliensis GSS Analysis of
similarity
21
Leishmania braziliensis GSS on going analysis

• Use of different data banks for the search of
  similarity
• Definition of functional classes
• Definition of repetitive elements and classes
  present in L.braziliensis
• Construction of maps of sinteny

22
Tackling mapping and sequencing information in
Leishmania

• Functional organization of chromosomal ends

• Leishmania unusual transcripts

23
The organization of the chromosomal ends

• Repetitive sequences and organisms specific
  genes
• Sequence shuffling and genetic diversity
• In protozoan parasites
• Genes involved with Antigenic Variation
• Surface proteins

• Tools
• A sheared genomic library
• cLHYG LV39 L.major
• The telomeric hexamere repeat from T.brucei

24
Characterization of three chromosomal ends

• Presence and distribution of reiterated sequences

PFGE Reference strains
1
2
3
B1
Chr3
B2
Chr7
E8
Chr20
Pedrosa et al, MBP, 2001
25
Sequence and annotation of one end of chr. 20
Tel
PGKB
PGKC
ORF1
ORF2
ORF3
ORF4
ORF5
RNA Pol III
ORF6
ORF7
1,0 kb
Tel
LST-R378

• Are annotated ORFs real genes?
• Are they transcribed? When?
• Is sequence shuffling (gene truncation) a common
  event?

LCTAS
ORF
Pedrosa et al, MBP 2001
26
Transfection of episomes for transcript detection
at the chromosomal ends
Tel
cL-Hyg
Telomeric clones
27
Overexpression of episomal genes mapping
transcripts
Total RNA from Leishmania promastigotes
B2-HH-3.2
DHFRTS
Chr3
B1
Tel
H
H
H
H
H
B
B
B
G
G
G
G
E
E
E
E
H
B2
Chr20
4.5
chr7
E8
28
Transcriptional silencing, Sequence shuffling and
gene truncation at chromosomal ends
chr3
6.4
chr7
chr20
DHFRTS
29
At the chromosome extremities findings and
perspectives
30
A tool from the Leishmania genome project 2500
ESTs from cDNA libraries

• Genes of unknown function - 50 of the ESTs
• Do they have any unexpected features?
• Could they be functional molecules?
• proteins
• non coding RNAs

• gt100 ESTs classified as genes of unknown
  function
• Re-sequenced, re-blasted
• GC content analysed
• length of the transcripts (ORFs?)

31
Some features of a sample of orphan Genes (ESTs)

• Identified proteins

• Identified ESTs

• Unidentified ESTs

GC content ()
ORF length (kb)
32
Transcripts with odd characteristics

• Possible explanations
• polycistronic transcription is promiscuous
• high level of junk transcripts
• regulatory RNA molecules/unusual small proteins
• how to sort them out?

33
ODD1 a spurious or a functional transcript ?

• single copy sequence (chr. 6)
• conserved in several species
• the transcript is 448 bases long
• GC content of 53.8
• two internal stop codons

34
ODD1 Transcript is processed

• RNA is processed and transported to the cytoplasm
• Higher levels of the transcript are present in
  promastigotes
• Transcript is present in many Old and New World
  species

35
ODD1 a spurious or a functional transcript ?

• Two internal stop codons conserved in several
  species
• selenoprotein
• selenocystein
• read-through mechanism
• Tryptophan (AT rich genomes)
• tRNA suppressor
• non coding RNAs (ncRNAs)

36
Is ODD1 translated?

• Selenium incorporation assays did not identify
  ODD1 as a selenoprotein gene
• 7 selenoproteins were detected in Leishmania
• Anti-ODD1 (rabbit) failed to localize a protein
  in Western blots
• No overexpressed protein was detected in ODD1
  Leishmania transfectants

37
ODD 1 TRANSCRIPT HAS A POTENTIAL HAIRPIN AT THE
3END
that seems to be no SECIS...

• ncRNAs involved in control of gene expression
• short transcripts
• no poly A tails
• presence of hairpins
• 20-25 nucleotides complementary to an organisms
  gene

38
Screening for Potential ncRNA genes

• 12 Odd transcripts were selected for further
  investigation
• chromosomal assignment
• conservation among species
• search for similarities in databanks
• search for a transcript in pro- and amastigotes
  (of different species)
• investigation of RNA secondary structure

39
André L. Pedrosa Eliane C. Laurentino Jeronimo C.
Ruiz M. Pilar Iribar Simone A. Antoniazi
University of São Paulo at Ribeirão Preto
Financial Support by FAPESP
Luiz R. O. Tosi, Faculdade de Medicina Ribeirão
Preto, USP, Brasil Marla Berry, Harvard Medical
School, Boston, USA