Anopheles gambiae: A genetic approach Karin Eiglmeier Unité de Biochimie et Biologie Moléculaire...
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Transcript of Anopheles gambiae: A genetic approach Karin Eiglmeier Unité de Biochimie et Biologie Moléculaire...
Anopheles gambiae:
A genetic approach
Karin EiglmeierUnité de Biochimie et Biologie Moléculaire des Insectes
Institut Pasteur
MosquitoesOrder : Diptera
Toxorhynchitinae
Anophelinae Culicinae
Family: Culicidae
Aedes CulexAnopheles
Since when .......????
Anopheline fossile (20 Myr)
http://www.uky.edu/AS/Geology/webdogs/amber/critters/skeeter-b.html
(Amber from the Dominican Republic)Dr. David Grimaldi
Cretaceous carnivorous dinosaures (145-65Myr)
Oldest Culicidae-like fossile: 76-79 Myr
(canadian amber)
FROM: www.dinozaury.ovh.org/ index.php?id=galeria
Generalized mosquito life cycle
The larvae feed on microorganisms and organic matter in the water. 4 -14 days
The pupal stage:non-feeding stage of development, reacts to stimuli-> metamorphosis1- 4 days
Eggs hatch into larvaewithin 48 H.
Larvae
Pupa
Eggs
Adult
modified from: www.edmonton.cal
Mosquito facts
CO2, temperature, humidity, odor, colour (infra-red) mouvement
MalariaWest Nile virusfilarial diseasesdengue encephalitisyellow fever
1 -1.5 miles/hour 75 - 100 miles20 - 35 meters
Detection Transmission
Immunity
Blood-meal
endophage4x weight
Mosquito facts
CO2, temperature, humidity, odor, colour (infra-red) mouvement
MalariaWest Nile virusfilarial diseasesdengue encephalitisyellow fever
1 -1.5 miles/hour 75 - 100 miles20 - 35 meters
Detection Transmission
Immunity
Blood-meal
endophage4x weight
Malaria (“Paludisme”)
- Vector : Anopheles gambiae
- Parasite : protozoans of the genus
Plasmodium
> 90 countries (40% of the world’s population)
- 90% malaria fatalities in sub-saharan Africa
- 300 - 500 millions clinical cases/ year
- 1.5 - 2.7 millions deaths
Plasmodium life cycle
Adapted from Waters, Science,301 (2003)
~13-18 days
Anopheles mosquitoes
Genus: Anopheles
Africa Asia America
An. gambiaeAn. arabiensisAn. funestus
An. stephensiAn. farautiAn. sinensisAn. tellessarusAn. minimus
An. albimanusAn. quadrimaculatusAn. darlingiAn. freeborni
Principal disease transmitting species:
about 70 transmit malaria to humans about 20 are important vectors
- An. gambiae M et S- An. arabiensis- An. melas- An. merus- An. bwambae- An. quadriannulatus- An. quadriannulatus B
Anopheles gambiae complex
Adapted from J.Mouchet & D.Fontenille
Golden Path length ~ 273 Mb
( from: http://www2.ncid.cdc.gov/vector/vector.html)
Anopheles gambiae chromosomes
Whole genome assembly
Genome size: - 278 Mb ( 273 Mb )
- 8987 scaffolds
- 303 scaffolds => 91% of the genome
- 18634 contigs
Inter-scaffold gaps: - sequence gaps, no clones
- repeat sequences
- smaller scaffolds ?
Y chromosome: - no assembly - ongoing ? 0.18Mb
- high repeat content
Current release AgamP3 last update: 2/2006First draft: March 2002
49 61 42 53 24 Mb
Anopheles gambiae chromosomes
+ +
adapted from: http://www.ensembl.org
Immediate results
20 genes (1999) 14700 genes (2005)
Cross disciplinary research:- Drosophila community- Bioinformaticiens- other domains- BLAST
Fieldwork:- Entomologists- Identification follow-up of mutations
adapt strategy
Gene prediction and annotation
Several evidences: - Gene prediction programs:
- open reading frame- signals: start codon, stop codon, poly-
adenylation site - splice sites- bias in base composition- bias in base frequency
- encoded peptide has similarity with known protein
- encoded peptide has similarity with a protein domain or motif
- « evolutionarily conserved sequences » - Ecores
- cDNA, EST, SAGE biological evidences
Annotation
15189 genes
Celera pipeline (Otto) Ensembl pipeline
“Ab inito gene finding” Homology
9896 transcripts14564 transcripts
24460 transcripts
15189 genes
13757840 5974(identified exclusively) (identified exclusively)
“consensus set”
Does gene prediction correspond to real gene ?
Problems:- real gene? => mono-exonic !
- small exons, intron-exon structure
- first and last exon
- untranscribed regions (“UTRs”)
- genes for atypic or specific proteins
- genes duplicated in tandem
- pseudo-genes
Comparison An. gambiae - D.
melanogaster
Common ancestor
Anopheles gambiae Drosophila melanogaster
250
~ 273 Mb15189 annotated genes(13765 in AgamP3)
~ 130 Mb 14651 annotated genes
Protein similarity
14000
12000
10000
8000
6000
4000
2000
Dm Ag
44,2%
11,0%
15,9%
10,3%
18,6%
47,2%
13,8%
17,9%
10,0%
11,1%species specific
Homologs, best matches: - non-insects
Homologs, best matches: - insects
“Many-to-many” orthologs,duplications
1:1 Orthologs (6089 pairs)average identity: 56%
13885 12981
increased speed of divergence :
Orthologs Human - Fugu:average identity: 61%
adapted from: Zdobnov E. Science (2002), 298, 149-159
Starting point: Publication of the Anopheles gambiae genome 2002
- Genome sequence incomplete
- First characterisation and annotation of genes of variable quality
Problems: Post-genomic analysis difficult
Gene detection
Approach: Full-length enriched cDNA libraries:
- developmental stages
- different tissues
Aims: - Identification of new genes
- Improve description of gene structure (TSS, UTRs, Exon / Intron)
- Alternative splicing recombinant proteins
- Facilitate comparative genomics
Genome, gene expression and annotation
How to get more
information?
How to get more
information?
Experimental evidence:
- Transcriptome
- Proteome
- Biochemistry
- RNAi
- Transgenesis
Modified from Zhang MQ Nat. Rev. Genet.2002(9):698-709 and from Ben-Dor,S
1 2 3 4 5
ATG STOP Poly(A) site
TTSTSS
Promoter
1 2 3 4 5
ATG STOP Poly(A) site
DNA
Pre-mRNA
mRNA 1 3 42
AUG
5
STOP
Poly(A)
5’UTR 3’UTRCDS
CAP
From DNA to mRNA
Modified from Zhang MQ Nat. Rev. Genet.2002(9):698-709 and from Ben-Dor,S
1 2 3 4 5
ATG STOP Poly(A) site
TTSTSS
Promoter
1 2 3 4 5
ATG STOP Poly(A) site
DNA
Pre-mRNA
mRNA 1 3 42
AUG
5
STOP
Poly(A)
5’UTR 3’UTRCDS
CAP
From DNA to mRNA
AAAAAAAAAAAA
Gpppp
OH
mRNA + CAPmRNA sans CAPmRNA sans CAP
AAAAAAAAAAAA
GpppOH
OH
AAAAAAAAAAAA
pOH
OH
AAAAAAAAAAAA
OHOH
BAP treatment
TAP treatment
RNA ligation5’-oligo
« Oligo-capping » ( Maruyama & Sugano )
adapted from:Suzuki et al. Genome Res.(2001)11(5):677-84
AAAATTTT
TTTT
TTTTAAAA
TTTTAAAA
Ligation
First strand synthesis
Alkaline degradation
PCR
SfiI Digestion
TTTT
AAAA
adapted from:Suzuki et al. Genome Res.(2001)11(5):677-84
Banques ADNc « Full-length »
modified from: www.edmonton.cal
More genes to discover ?
67044 reads
~ 3700 clusters
85 % improved
cDNAs - submitted to EMBL :
654 new genes
Pilot project: Adult females
3032 Ensembl genes
Perfect annotation
Gene modelcDNA dataproteins
- Ensembl -
Improvement of annotation
- Ensembl -
600 new genes
- how are they?
”New” genes
- Ensembl -
Predictions and proof
- Ensembl -
Banques ADNc « Full-length »
Clustering results:5664 cluster - 869 new genes
175
21
6
12
256
37
401
Adulte females(4056)
Embryos(1816)
Larvae(1982)
Available or planned cDNA libraries:
Available/sequenced:
• Adult females
• Embryo
• Larves
• Salivary glands
Planned:
• Pupa
Plasmodium life cycle
Adapted from Waters, Science,301 (2003) Adapted from: James, A.A., (2003),206:3817-3821
Sporozoites- invasion, specific receptors- secretory cells- storage- influence normal functions
Saliva- proteins involved in
l’hematophagy- modulation of immune defense
An. gambiae salivary glands
Chromosome X, pos. 11070 kb: Anopheles-specific SG1 family
AgamP3
Moz2a v.34Adapted from: Arca et al. J.ExpBiol(2005), 208:3971
What does genomics offer for malaria control ?
Adapted from Waters, Science,301 (2003)
Vector eradication
Vector eradication
Insecticides
• Monitoring of insecticide resistance genes - pyrethroid resistance
- epidemiology
• Detoxifying enzymes • Detox-chip
• New targets
Adapted from Waters, Science,301 (2003)
Host - vector relationship
Host - vector relationship
Behaviour Candidate genes:
odorant - smell 79 putative odorant receptorsgustatory - taste 76 putative gustatory receptors
Attractant/Repellant - Host location
Vectorial capacity
Mating
Oviposition Traps
Adapted from Waters, Science,301 (2003)
Transmission blocking - SM1
Transmission blocking - SM1
SM1 = salivary gland and midgut peptide 1 (Gosh, et al. 2000)
12 amino acids
transgenic mosquitoes:
- midgut expression - blocking plasmodium
- salivary gland expression - blocking invasion
- selective advantage of transgenic mosquitoes
- proof of principle => Marrelli et al. , PNAS 104, 2007
Adapted from Waters, Science,301 (2003)
Immune system
Immune system
Natural efficient immune system
- resistance against Plasmodium
- melanotic incapsulation - several loci
- identify genes
- multiply/release naturally occuring resistant strains
Adapted from Waters, Science,301 (2003)
Salivary glands
Salivary glands
- cDNAs
- RT-PCR
- protein expression
- SAGE analysis of transcriptome
- proteomic studies of salivary glands and saliva
salivary gland sporozoite relationships
- saliva and humans:
- immunomodulatory
- function?
- vaccine target?
Where to go from now ??
Annotation = continuing process
- new start for Anopheles research
- Improvement of genomic annotation:
- Sequence, gene models, promoters
- genome arrays :
-infected non-infected
- Annotation at the protein level:
- protein interaction networks
- hypothesis experiments
- comparative genomics - more insects (honey
bee, Aedes)
- transgenic mosquitoes , RNAi
experiments
Aedes aegypti
- Start : September 2002
- Fin : Spring 2007 ?
first assembly, version 0.5
- Public:
- First annotations
- cDNA sequences
- Genome size: > 1300 Mb => in 4758 supercontigs
- « Evolutionary distance » Anopheles - Aedes:
~ 27-62 Myr
From: www.bg-sentinel.com
Gene size comparisonAnopheles
Aedes
Gene size comparison4 kb
16 kb
Anopheles
Aedes
Aedes aegypti annotation
- genome size bigger than expected (780 Mb => 1300 Mb)
- sequencing strategy different
- cDNAs early in project
- high content of repeat sequences (~68%)
- gene prediction programs adapted
- long genes, nested genes
- Anopheles - Aedes
- synteny between chromosome arms
Data bases
EnsemblAnopheles gambiae
Aedes aegypti
AnobaseAnopheles gambiae
VectorbaseAnopheles gambiae
Aedes aegyptiIxodes scrapularis
TIGR - The Institut for Genome Research
Traps
Identification
Understanding- behaviour
Genome/proteomecaracterisation
Promoter/expression
Vector control strategies: - Transgenesis
Genetically modified mosquitoes-reduced parasite transmission-functional genomics
New insecticidal targets
Vaccines
Main orientations in mosquito research
Immunity
Collaborations
Institut Pasteur
BBMI - Paul BreyCharles RothInge HolmPierre Dehoux (PF4)Shawn GomezSylvie PerrotMarie-Kim ChaverocheJean Sautereau
Plate-forme Genomique - PF1Christiane BouchierAnthony Lepelletier
Genoscope
Jean Weissenbach
Beatrice SegurensPatrick WinckerGabor GyapayCorinne da SilvaBetina Porcel
AMSUD Network
Sergio Verjovski-AlmeidaUniversidade de Sao Paulo
Hamza el DorrySuely L. GomesCarlos F.M. MenckAna L. Nascimento