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Comparative Genomics of Aspergilli. William Nierman TIGR. Electrophoretic Karyotyping 5 day run. Af. S p. Sc. 5.7. 5.0. 4.6. 1x 4.0. 3.5. 3.5. 1.8. CHEF DRII 1.2% CGA, 1x TAE, 14 C, 1.8 V/cm: 2200 s, 48 h; 2200-1800 s, 68 h sizes in Mb. A. fumigatus Chromosomes.

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slide1

Comparative Genomics

of Aspergilli

William Nierman

TIGR

electrophoretic karyotyping 5 day run
Electrophoretic Karyotyping 5 day run

Af

Sp

Sc

5.7

5.0

4.6

1x 4.0

3.5

3.5

1.8

CHEF DRII 1.2% CGA, 1x TAE, 14C, 1.8 V/cm: 2200 s, 48 h; 2200-1800 s, 68 h sizes in Mb

slide3

A. fumigatus Chromosomes

Size (MB)

1

4.891

4.834

4.018

3.933

3.922

3.779

2.021

1.789

2

3

~35 copies rDNA

4

5

6

7

8

Centromeric area

Telomere

centromeres and telomeres
Telomere repeat TTAGGG, 7-21 repeat units

Subtelomeric regions- identical sequences for several kb, helicase pseudogenes, 7 secondary metabolite clusters, niche adaption role? (Mark Farman)

Centromeres

Uncloned in shotgun libraries; 36.2 - 55.9kb

Flanked on each side by low complexity AT rich repeat region

Chromosome 2 centromere 12 kb PCR product 75% AT, overall centromeric AT of 63%, 40kb.

Centromeres and Telomeres
slide5

Annotation Pipeline

Finished chromosome sequences

Masked genomic sequence

EST alignments

Gene prediction

Protein alignments

Optimize Predictions

Eukaryotic Genome Control (EGC) is the annotation pipeline responsible for processing genomic sequence
training data
Full Length cDNAs (625) and 42 partials from 589 loci in 19 Aspergillus species

2,633 A. fumigatus ESTs from UK and Spanish collaborators

Training Data

Gene and splicing site predictions including Glimmer,Exonomy, Unveil, Phat and GeneSplicer were trained with following experimental data:

optimize predictions
Optimize Predictions
  • Combiner combines gene model evidence from:
    • Gene prediction programs
    • Splice site prediction programs
    • Alignments from protein, cDNA and EST databases
    • Generates final gene model.

All the genes were manual reviewed and the observed

splits and merges were corrected.

annotation station screenshot
Annotation Station Screenshot

Scytalone dehydratase

Yellowish-green

1,3,6,8-tetrahydroxynaphthalene reductase

Polyketide synthetase

Brown 1

Brown 2

slide14

Overview – Comparative Statistics

The ortholog was computed by performing an all vs. all BlastP of the three

proteomes with a cut-off of 1 x e-15 (no length requirement).  The mutual best

hits were then organized into clusters based on shared protein nodes.

tigr autoannotation vs sanger curated annotation
Status Count

Total Sanger Genes analyzed 360

Same gene structure 137

Different gene structure 177

Sanger missing in TIGR annotation 37

Sanger matches multiple TIGR annotations 2

Sanger, TIGR annotations opposite strands 7

TIGR missing in Sanger annotation 12

TIGR matches multiple Sanger annotations 9

TIGR Autoannotation vs Sanger Curated Annotation
slide17

Using Ortholog Clusters

to Identify Potential Annotation Problems

slide18

Using Ortholog Clusters

to Identify Potential Annotation Problems

Different exon number due to annotation discrepancy

slide19

In some cases, differences in exon number are real

We need to be able to distinguish annotation inconsistencies from real, interesting phenomena

apoptosis in fungi
Apoptosis-like process detected in S. cerevisiae, S. pombe, and Aspergilli.

Fungal genomes lack metazoan upstream machinery.

Metacaspase-dependent phenotype observed in A. fumigatus and A. nidulans.

Analysis by Goeff Robson

Apoptosis in Fungi
aspergillus fumigatus secondary metabolites

Aspergillus fumigatus Secondary Metabolites

Heterogeneous group of low molecular weight products.

Toxic, antibiotic, and immunosuppressant activities.

– fumagillin, gliotoxin (apoptosis and phagocyte dysfunction), fumitremorgin, verruculogen, fumigaclavine, helvolic acid, phthioc acid (granulomas when injected into mice) and sphingofungins

Virulence properties may be augmented by the A. fumigatus numerous secondary metabolites.

slide23

Gene type

A. oryzae

A. fumigatus

A. nidulans

PKS

30

14

27

NRPS

18

14

14

FAS

5

1

6

Sesquiterpene cyclase

1

(1)

(1)

DMATS

2

7

2

Secondary Metabolite Genes

Analysis by G. Turner, N. Keller, Dr. Kitamoto, and R. Kulkarni

slide24

Serine

Phenylalanine

2 module NRPS?

Gliotoxin

Terpene

Sesquiterpene cyclase

Fumagillin

Tryptophan

DMAT synthetase (X2)

Fumigaclavines

Tryptophan

Proline

NRPS?

DMAT synthetase

Fumitremorgens

slide25

Gene type

A. oryzae

A. fumigatus

A. nidulans

PKS2

30

14

27

NRPS

18

14

14

FAS

5

1

6

Sesquiterpene cyclase

1

(1)

(1)

DMATS

2

7

2

Five 2-module NRPS

a fumigatus secondary metabolite genes
Few true orthologues across the genus Aspergillus. Each species has its own repertoire.

Gene/product relationship requires functional analysis in most cases

Indole alkaloid pathway in A. fumigatus only. Closely related to Claviceps purpurea ergotamine pathway

Penicillin and aflatoxin pathways are absent.

A hybrid PKS/monomodular NRPS seems to be present in several fungi.

A. fumigatus Secondary Metabolite Genes
slide27

Identify A.fumigatus specific genes

A.fumigatus genes

(9746)

All vs. all BlastP of the AFU1,ANA1, AOAN proteomes

cut-off E value: 1 x e-15, filtering the results for mutual best hits

between genomes.

A. fumigatus singletons

(2075)

BLASTP vs ANA1 and AOA1 proteomes

A. fumigatus singletons E-value > e-10

(1081)

Extend 50bp on both ends of the gene in the genome, Tblastx

the genomic seq of the gene vs ana and aoa genomic seq

A. fumigatus specific gene candidates E-value > e-50

(1011)

BLASTP vs ANA1 and AOA1 proteomes

e-5>E-value>e-10

E-value > e-5

(203)

(808)

Extend 50bp on both ends of the gene in the genome, Tblastx

the genomic seq of the gene vs ana and aoa genomic seq

e-50<E-value < e-10

e-5>E-value>e-10

E-value > e-5

(552)

(181)

(75)

aspergillus fumigatus unique genes
Vast majority are hypothetical

Includes

Several transcriptional regulators

A chaperonin

An hsp 70 related protein

Aspergillus fumigatus Unique Genes
arsenic fungi
19th century poisonings associated with green pigments.

1892 B. Gosio, certain fungi could metabolize arsenic pigments producing toxic trimethylarsine (Gosio gas).

Screen in the 1930s (Thom & Raper) found A. fumigatus to be an arsenic fungus.

Napoleon, imperial colors green and gold, copper arsenite (Jones 1982).

Analysis of history and genome by J. Bennett, N. Hall, J. Wortman, C. Lu.

Arsenic Fungi
a fumigatus arsenate genes
Arsenite efflux pump

Arsenite translocating ATPase

Two possibly duplicated clusters

arsC – arsenate reductase (A. fumigatus unique)

arsB – arsenite symporter

arsH

Methyltransferase

A. fumigatus Arsenate Genes
slide31

Chromosome 5

Chromosome 1

slide32

arsB

Methytrasferase

arsH

arsC

arsB

Methyltransferase

arsH

arsC

a fumigatus teichoic acid biosynthesis protein
Good homology to a the full length of the Streptomyces griseus protein.

Secretion signal peptide may direct for cell wall.

Teichoic acids demonstrated to be a virulence factor for Staphylococcus aureus.

No intervening sequences in gene.

A. Fumigatus Teichoic Acid Biosynthesis Protein

Analysis by Neil Hall

slide34

More highly expressed at 48oC

More highly expressed at 37oC

A. Fumigatus Thermotolerance

a fumigatus thermotolerance
Relatively few genes altered

Some HSPs transiently or stably induced (weakly) and repressed at 37oC.

HSPs induced throughout 180 min 48oC period

Transposases induced at 48oC (Mariner 4).

Stress related genes up regulated at 48oC.

Metabolic proteins down regulated at 48oC

A. fumigatus Thermotolerance

“This fungus likes it hot.”

J. Bennett

aspergillus fumigatus af293 project participants

Aspergillus fumigatus AF293 Project Participants

The University of Manchester, UK

The Wellcome Trust Sanger Centre, UK

The Institute for Genomic Research, USA

The University of Salamaca, Spain

Complutense University, Spain

Centro de Investigaciones Biológicas, Spain

slide38

Aspergillus fumigatus AF293

Joan Bennett

Matt Berriman

Jean Paul Latge

Paul Dyer

Paul Bowyer

Neil Hall

David Denning

Michael Anderson

Arnab Pain

Goeff Robson

Javier Arroyo

Goeff Turner

David Archer

Aspergillus nidulans – James Galagan

Aspergillus oryzae – Masayuki Machida

slide39

TIGR

Sequencing and Closure

Tamara Feldblyum

Hoda Khouri

Annotation

Jennifer Wortman

Jiaqi Huang

Resham Kulkarni

Natalie Fedorova

Charles Lu

Lab Group

Heenam Kim

Dan Chen

Claire Fraser

NIAID and Dennis Dixon