Jizhong Zhou, Dorothea K. Thompson, Ying XuJizhong Zhou, Dorothea K. Thompson, Ying Xu

Prepared by: Merced M. GutierrezNano Biomaterials Science Laboratory

Supervisor: Prof. Sung Chul YoonGyeongsang National University

Chapter 11Chapter 11

The Functional Genomics ofThe Functional Genomics of

Model Organisms:Model Organisms:

Addressing Old QuestionsAddressing Old Questions

from a New Perspectivefrom a New PerspectiveDorothea K. Thompson and Jizhong ZhouDorothea K. Thompson and Jizhong Zhou

Table of ContentsTable of Contents11.1 Introduction11.1 Introduction

11.2 11.2 Escherichia coliEscherichia coli: A Model Eubacterium: A Model Eubacterium

11.2.1 11.2.1 E. coli E. coli genome genome

11.2.2 11.2.2 E. coli E. coli transcriptomics transcriptomics

11.2.3 11.2.3 E. coli E. coli proteomics proteomics

11.2.4 Modeling 11.2.4 Modeling E. coli E. coli metabolism: in silico metabolomics metabolism: in silico metabolomics

11.3 11.3 Bacillus subtilisBacillus subtilis: A Paradigm for Gram-Positive Bacteria: A Paradigm for Gram-Positive Bacteria

11.3.1 11.3.1 B. subtilis B. subtilis genomegenome

11.3.2 11.3.2 B. subtilis B. subtilis transcriptomics transcriptomics

11.3.3 11.3.3 B. subtilis B. subtilis proteomics proteomics

11.4 11.4 Saccharomyces cerevisiaeSaccharomyces cerevisiae: A Model for Higher Eukaryotes : A Model for Higher Eukaryotes

11.4.1 Yeast genome 11.4.1 Yeast genome

11.4.2 Yeast transcriptomics 11.4.2 Yeast transcriptomics

11.4.3 Yeast proteomics11.4.3 Yeast proteomics

11.4.4 Yeast interactome: mapping protein–protein interaction11.4.4 Yeast interactome: mapping protein–protein interaction

11.5 Comparative Genomics of Model Eukaryotic Organisms11.5 Comparative Genomics of Model Eukaryotic Organisms

Introduction

E. coli , Bacillus subtilis and S. cerevisiae serves as a model organisms because of their reduced structural and functional complexity and their intrinsic advantages as experimental systems.

In this chapter, we focused on the impact of structural and functional genomics on elucidating the dynamics of the transcriptome, proteome, metabolome and interactome of the well-studied model organisms.

Model Organisms:

The Genome Sequence

E.coli

B. subtilis

S. cerevisiae

TRANSCRIPTOMETRANSCRIPTOME

METABOLOMEMETABOLOME

PROTEOMEPROTEOME

INTERACTOMEINTERACTOME

Dna MicroarrayDna Microarray

BioinformaticsBioinformatics

Computer modelingComputer modeling

2D-PAGE2D-PAGE

Mass Mass SpectrometrySpectrometry

Protein chipsProtein chips

Two-hybrid Two-hybrid systemsystem

Mass Mass spectrometryspectrometry

Phage Phage displaydisplay

Fig 11.1 Elucidation of Fig 11.1 Elucidation of the cellular domains of the cellular domains of model organisms using model organisms using a functional genomics a functional genomics

approach and approach and comprehensive comprehensive technologies.technologies.

best-characterized free-living, single-celled organism, served best-characterized free-living, single-celled organism, served as a biological model for cellular processes.as a biological model for cellular processes.38% 38% of the of the 4,2884,288 protein-coding genes ( no attributed biological protein-coding genes ( no attributed biological role) and role) and 1,8531,853 previously described previously described (Blatter et.al, 1997)(Blatter et.al, 1997)

DNA replication and repair, DNA transcription, Metabolic DNA replication and repair, DNA transcription, Metabolic pathways, Adaptive stress responses, Signal transduction, pathways, Adaptive stress responses, Signal transduction,

Genetic regulationGenetic regulation

Escherichia coliEscherichia coli: A MODEL : A MODEL EUBACTERIUMEUBACTERIUM

Escherichia coliEscherichia coli: GENOME: GENOME

Structural Analysis Structural Analysis - - Genome-Wide Parallel Functional AnalysisGenome-Wide Parallel Functional Analysis

valZvalZ asnW

6 newly proposed genes

lysZ ileYlysYlysY lysQ

Transcriptional units

unit

lyst operon

Bioinformatic analysisBioinformatic analysis- - Structural and Regulatory Components of Structural and Regulatory Components of Various Biochemical pathways or Cellular Machineries Various Biochemical pathways or Cellular Machineries

Pathway for degradation Pathway for degradation

of aromatic compoundsof aromatic compounds Transcription regulator for encoding the aromatic

degradation

Operon for the degradation of aromatic compounds

A. The Heat Shock ResponseA. The Heat Shock Response

(i) Correlation of Gene Expression & Function (i) Correlation of Gene Expression & Function

* alternate sigma (* alternate sigma (δδ) factor ) factor rpoHrpoH ( (δδ32) and 32) and rpoE rpoE ((δδE)E)

- - heat shock proteins, homeostatic mechanism exhibited by living heat shock proteins, homeostatic mechanism exhibited by living

cells when exposed to suboptimal elevated temperaturecells when exposed to suboptimal elevated temperature

(ii) Connection between Gene Expression & Physiological State(ii) Connection between Gene Expression & Physiological State

* * When complexed with the core RNA polymerase, the When complexed with the core RNA polymerase, the E. coli E. coli δδ32 32 transcription factor permits the transcription machinery to initiate heat transcription factor permits the transcription machinery to initiate heat shock-regulated promoters fro both steady state and stress-activated shock-regulated promoters fro both steady state and stress-activated levels of heat shocks gene expression.levels of heat shocks gene expression.

..

E. Coli E. Coli TranscriptomicsTranscriptomics

A. The Heat Shock ResponseA. The Heat Shock Response

(iii) Technological Advantages(iii) Technological Advantages

* DNA microarray technology * DNA microarray technology accurately detect alternatives in accurately detect alternatives in bacterial transcript abundance & illustrated how functional genomics bacterial transcript abundance & illustrated how functional genomics allow well-characterized cellular processes to be examined from a new allow well-characterized cellular processes to be examined from a new & global perspective.& global perspective.

E. Coli E. Coli TranscriptomicsTranscriptomics

Large scale cultureLarge scale cultureTransformationTransformation

50

100

Re

lativ

e I

nte

ns i

ty [

%]

1000 1500 2000 2500 3000 m/z

M

*

*

l M

ll

l l

l

l

l

ll

ll

ll

l

l

Separation Separation Mass spectrometryMass spectrometry

An integrated workflowAn integrated workflow

B. Transcriptome Analysis of Cellular Metabolism and GrowthB. Transcriptome Analysis of Cellular Metabolism and Growth

(i) Correlation of Gene Expression & Function (i) Correlation of Gene Expression & Function

* * trptrp operon ( operon ( trpE, trpD, trpC, trpB trpE, trpD, trpC, trpB andand trpA trpA)) - - enzymes required for the conversion of chorismate, a enzymes required for the conversion of chorismate, a branch-point intermediate in the aromatic amino acid pathway branch-point intermediate in the aromatic amino acid pathway to tryptophan.to tryptophan.

(ii) Importance of Transcriptional Regulation(ii) Importance of Transcriptional Regulation

* * Transcription of the Transcription of the trp otrp operon is governed by repression peron is governed by repression control via the repressor protein control via the repressor protein TrpRTrpR and an entirely different and an entirely different mode of regulation termed transcriptional attenuation.mode of regulation termed transcriptional attenuation.

E. Coli E. Coli TranscriptomicsTranscriptomics

B. Transcriptome Analysis of Cellular Metabolism and GrowthB. Transcriptome Analysis of Cellular Metabolism and Growth

(iii) Technological advantages(iii) Technological advantages

1. DNA microarray technology 1. DNA microarray technology monitor global change in mRNA monitor global change in mRNA abundance connected with tryptophan biosynthesis, transport and abundance connected with tryptophan biosynthesis, transport and regulation.regulation.

E. Coli E. Coli TranscriptomicsTranscriptomics

trptrp – tryptophan biosynthesis– tryptophan biosynthesis

aroHaroH – aromatic amino acid biosynthesis – aromatic amino acid biosynthesis

mtrmtr- tyrptophan-specific permease- tyrptophan-specific permease

trpRtrpR- tryptophan repressor regulon- tryptophan repressor regulon

B. Transcriptome Analysis of Cellular Metabolism and GrowthB. Transcriptome Analysis of Cellular Metabolism and Growth

(iii) Technological advantages(iii) Technological advantages

2. DNA microarray technology 2. DNA microarray technology provides genomic expression provides genomic expression for cell growth particularly in the differences in rRNA and tRNA.for cell growth particularly in the differences in rRNA and tRNA.

E. Coli E. Coli TranscriptomicsTranscriptomics

1. 1. translational apparatustranslational apparatus

2. nitrogen metabolism 2. nitrogen metabolism

3. amino acid biosynthesis 3. amino acid biosynthesis

4. biosynthesis of vitamins, cofactors, 4. biosynthesis of vitamins, cofactors,

prosthetic groups and carriers prosthetic groups and carriers

5. nucleotide biosynthesis5. nucleotide biosynthesis

6. fatty acid biosynthesis and degradation6. fatty acid biosynthesis and degradation

7. carbon and energy metabolism7. carbon and energy metabolism

8. cellular processes and global regulators8. cellular processes and global regulators

B. Transcriptome Analysis of Cellular Metabolism and GrowthB. Transcriptome Analysis of Cellular Metabolism and Growth

(iii) Technological advantages(iii) Technological advantages

3. DNA microarray technology 3. DNA microarray technology provides genomic expression for cell provides genomic expression for cell growth particularly in the differences protein synthesis and ribosome growth particularly in the differences protein synthesis and ribosome abundanceabundance. .

E. Coli E. Coli TranscriptomicsTranscriptomics

ilv GMEDA operon ilv GMEDA operon – isoleucine and valine synthesis– isoleucine and valine synthesis

leuABCD operonleuABCD operon – leucine synthesis– leucine synthesis

trpEDCBA operontrpEDCBA operon - tryptophan synthesis- tryptophan synthesis

The step required in a microarray experimentThe step required in a microarray experiment

B. Transcriptome Analysis of Cellular Metabolism and GrowthB. Transcriptome Analysis of Cellular Metabolism and Growth

(iii) Technological advantages(iii) Technological advantages

4. DNA microarray technology 4. DNA microarray technology provides genomic expression for cell provides genomic expression for cell growth particularly in the number of carbon and energy metabolism genes.growth particularly in the number of carbon and energy metabolism genes.

dlddld – D-lactate utilization– D-lactate utilization tpoStpoS – regulation of – regulation of poxBpoxB expression expression

poxBpoxB- acetate formation- acetate formation aceA, aceB, gltA, lcd, & mdhaceA, aceB, gltA, lcd, & mdh- acetate utilization- acetate utilization

uspAuspA- (universal stress protein) - (universal stress protein)

coupling of glucose and acetate cometabolismcoupling of glucose and acetate cometabolism

E. Coli E. Coli TranscriptomicsTranscriptomics

B. The NtrC regulonB. The NtrC regulon

(i) Correlation of Gene Expression & Function (i) Correlation of Gene Expression & Function

* * NTr NTr (nitrogen-regulated) system & (nitrogen-regulated) system & NtrC(NtrC(nitrogen regulatory protein)nitrogen regulatory protein)

- - perform a cellular function in response to information processes about perform a cellular function in response to information processes about the physiological state of the cell nd NtrC transcriptional genes response to the physiological state of the cell nd NtrC transcriptional genes response to external nitrogen limitation.external nitrogen limitation.

(ii) Connection between Gene Expression & Physiological States(ii) Connection between Gene Expression & Physiological States

* * NtrC protein activates transcription of sigma 54-dependent genes and NtrC protein activates transcription of sigma 54-dependent genes and Nac (Nac (nitrogen assimilation controlnitrogen assimilation control) protein serves as an adapter to activate ) protein serves as an adapter to activate transcription of sigma 70-dependent genes.transcription of sigma 70-dependent genes.

E. Coli E. Coli TranscriptomicsTranscriptomics

B. The NtrC regulonB. The NtrC regulon

(iii) Technological advantages(iii) Technological advantages

* DNA microarray technology * DNA microarray technology shows operons specified ATP-binding shows operons specified ATP-binding cassette transporters.cassette transporters.

E. Coli E. Coli TranscriptomicsTranscriptomics

potFGHIpotFGHI – putrescine– putrescine

dppABCDFdppABCDF – dipeptides – dipeptides

oppABCDFoppABCDF- oligopeptides- oligopeptides

nupCnupC- secondary ion-coupled transporter for nucleosides- secondary ion-coupled transporter for nucleosides

cycAcycA- D-alaline/D-serine/glycine- D-alaline/D-serine/glycine

A. Protein identification and analysisA. Protein identification and analysis

Proteome analysis concern the authencity of the ORF ( Proteome analysis concern the authencity of the ORF ( sequence sequence annotationannotation) and physical properties () and physical properties (isoelectric point (pI) isoelectric point (pI) and and molecular massmolecular mass))

* * vivo protein abundancevivo protein abundance , , post translational modification and post translational modification and proteolysisproteolysis

B. Proteome-wide differential display of protein in B. Proteome-wide differential display of protein in various conditionsvarious conditions

2-DE2-DE - 60% of the 223 identified - 60% of the 223 identified

loci encoded proteinsloci encoded proteins

364 2-DE spots using364 2-DE spots using - 18% of 2DE-spots, represented - 18% of 2DE-spots, represented

amino acid (N) terminalamino acid (N) terminal isoforms in a narrow pI range 4 to 7 isoforms in a narrow pI range 4 to 7 Edman Edman sequence analysissequence analysis molecular mass 10 to 100KDa molecular mass 10 to 100KDa

E. Coli E. Coli ProteomicsProteomics

C. Protein-protein interactionC. Protein-protein interaction

E. Coli E. Coli ProteomicsProteomics

a.a. TryptophanaseTryptophanase ( (TnaATnaA), which catalyzes both the ), which catalyzes both the degradation and synthesis of tryptophan.degradation and synthesis of tryptophan.

b. b. Galactose –binding protein Galactose –binding protein ((MglBMglB), which is ), which is involved in the transport of galatose into the cell.involved in the transport of galatose into the cell.

c. c. Starvation- inducible protein Starvation- inducible protein ((DpsDps), which froms ), which froms stable complexes with DNA and thus protects DNA stable complexes with DNA and thus protects DNA from oxidative damage.from oxidative damage.

C. Protein-protein interactionC. Protein-protein interaction

E. Coli E. Coli ProteomicsProteomics

Figure 11.2 Figure 11.2 : : Proteomic analysis Proteomic analysis of the outer membrane of of the outer membrane of E coliE coli. .

(a)(a)Proteins in carbonate-treated Proteins in carbonate-treated membranes of membranes of E. coli E. coli were were separated by two-dimensional gel separated by two-dimensional gel electrophoresis and then identified electrophoresis and then identified using mass spectrometry. using mass spectrometry.

(b) (b) E. coli E. coli cells grown under cells grown under conditions of iron limitation were conditions of iron limitation were subjected to carbonate treatment subjected to carbonate treatment and then separated by two-and then separated by two-dimensional gel electrophoresis. dimensional gel electrophoresis.

Functional analysis by coresponses : Metabolite analysis Functional analysis by coresponses : Metabolite analysis

ModelingModeling E. Coli E. Coli Metabolism: Metabolism: In silico In silico MetabolomicsMetabolomics

Figure 11.3 Figure 11.3 Reconstruction Reconstruction

of microbial of microbial metabolic metabolic

networks from networks from annotated annotated genome genome

sequence, sequence, biochemical biochemical

data, and data, and physiology physiology

data.data.

B. subtilis B. subtilis : GENOME: GENOME

Structural Analysis Structural Analysis – – Genome Mapping Sequence analysisGenome Mapping Sequence analysis

10 of 11 AraC 10 of 11 AraC family family

Helix-turn-helix (HTH) family transcription factor

Two-component regulatory system

5 of 12 LacI 5 of 12 LacI family family

15of 19 LysR 15of 19 LysR family family

18 of 20 GntR 18 of 20 GntR family family

Physiological analysis Physiological analysis – – Adaptation of B. subtilisAdaptation of B. subtilis

77 ABC transporter – gene duplication

77 ABC transporter – gene duplication

4% codes for multi functional enzymes

4% codes for multi functional enzymes

43 temperature shock/ stress proteins

43 temperature shock/ stress proteins

4.2Mb resembles E. coli (25%) in terms of size

4.2Mb resembles E. coli (25%) in terms of size

A. Global characterization of Heat ShocksA. Global characterization of Heat Shocks

(i) Correlation of Gene Expression & Function (i) Correlation of Gene Expression & Function

* alternate sigma transcription factor* alternate sigma transcription factor

- - δδBB stress regulon, stress regulon, HrcAHrcA, heat-inducible genes and , heat-inducible genes and CtsRCtsR, , transcriptional regulatorstranscriptional regulators

(ii) Connection between Gene Expression & Physiological State(ii) Connection between Gene Expression & Physiological State

* * transcription of many general stress genes occurs at a transcription of many general stress genes occurs at a basal level from vegetative basal level from vegetative δδA A –dependent promoters, but is –dependent promoters, but is increased dramatically in a increased dramatically in a δδB -B -dependent manner in response dependent manner in response to stress and starvation.to stress and starvation.

B. subtilis B. subtilis TranscriptomicsTranscriptomics

B. Two-Component Regulatory SystemsB. Two-Component Regulatory Systems

(a) (a) DegS/DegU two component systemDegS/DegU two component system

- - controls exoprotease production, competence controls exoprotease production, competence development and motilitydevelopment and motility

(b) (b) ComP/ComA two component systemComP/ComA two component system

- - cell density signal activatorcell density signal activator

(i) (i) PhoP/PhoR two component systemPhoP/PhoR two component system

- - induction of the Pho regulon in response to phosphate induction of the Pho regulon in response to phosphate starvationstarvation

B. subtilis B. subtilis TranscriptomicsTranscriptomics

C. DNA Microarray AnalysisC. DNA Microarray Analysis

(a) (a) aprE, nprE and ispAaprE, nprE and ispA

- - 116 target gene DegU overproduction116 target gene DegU overproduction

(b) (b) bpr, yukl, ycdA and murDbpr, yukl, ycdA and murD

- - newly DegU operonnewly DegU operon

(c.) (c.) YdbG/YdbF two component systemYdbG/YdbF two component system

- - overproduction of YdbF chemostaxisoverproduction of YdbF chemostaxis

(d.) (d.) YufL/ YufM two component systemYufL/ YufM two component system

- - regulating competence controlled by ComKregulating competence controlled by ComK

(e.) (e.) YvrG/YvrH two component systemYvrG/YvrH two component system

- - cell membrane related proteinscell membrane related proteins

B. subtilis B. subtilis TranscriptomicsTranscriptomics

Stages of Sporulation in Stages of Sporulation in Bacillus Bacillus subtilissubtilis

Stage Stage 00

Stage Stage IIII11Asymmetric Asymmetric

septationseptation

Stage Stage IIII33EngulfmentEngulfment

Stage Stage IIIIII

Prespore Prespore protoplastprotoplast

Stage Stage IVIVCortexCortex

formationformation

Stage Stage VVCoatCoat

formationformation

Stage Stage VIVI

MaturationMaturation

Stage Stage VIIVII

GerminationGermination

Stages of Sporulation in Stages of Sporulation in Bacillus Bacillus subtilissubtilis

Stage Stage 00

Stage Stage IIII11

Stage Stage IIII33

Stage Stage IIIIII

Stage Stage IVIV

Stage Stage VV

Stage Stage VIVI

Stage Stage VIIVII

How is the process How is the process regulated?regulated?

spspo0o0

spoIIspoIIII

spospoVIVI

spoIspoIVV

spospoVV

spoIspoIII

Stages of Sporulation in Stages of Sporulation in Bacillus Bacillus subtilissubtilis

Stage Stage 00

Stage Stage IIII11

Stage Stage IIII33

Stage Stage IIIIII

Stage Stage IVIV

Stage Stage VV

Stage Stage VIVI

Stage Stage VIIVII

spspo0o0

spoIIspoIIII

spospoVIVI

spoIspoIVV

spospoVV

spoIspoIII

Golden age of Golden age of Bacillus Bacillus geneticsgenetics

• Identify Identify spospo genes genes• Clone and characterize Clone and characterize spospo genes genes• Determine their interdependenciesDetermine their interdependencies

Stages of Sporulation in Stages of Sporulation in Bacillus Bacillus subtilissubtilis

Stage Stage 00

Stage Stage IIII11

Stage Stage IIII33

Stage Stage IIIIII

Stage Stage IVIV

Stage Stage VV

Stage Stage VIVI

Stage Stage VIIVII

spspo0o0

spoIIspoIIII

spospoVIVI

spoIspoIVV

spospoVV

spoIspoIII

• Care in using reporter gene (Care in using reporter gene (lacZlacZ))• How is regulation exerted?How is regulation exerted?• Dependence of Dependence of spoIIA spoIIA expression on other expression on other genesgenes

spoIIspoIIAA

FiguFigure 11.4 re 11.4 Hierarchical cluster analysis Hierarchical cluster analysis of microarray-derived transcription of microarray-derived transcription profiles of 586 Bacillus subtilis genes profiles of 586 Bacillus subtilis genes whose expression levels depended on whose expression levels depended on Spo0A.Spo0A.

(I)(I)genes whose expression was genes whose expression was dependent on Spo0A but not on sF; dependent on Spo0A but not on sF;

(II) genes whose expression was (II) genes whose expression was

inhibited by Spo0A; and inhibited by Spo0A; and

(III) genes whose expression was under (III) genes whose expression was under

the control of sF or some the control of sF or some

downstream transcription factor in downstream transcription factor in

sporulation.sporulation.

Global Gene Expression During Global Gene Expression During Growth and Sporulation of Growth and Sporulation of

B. subtilisB. subtilis

A. The Extracellular ProteomeA. The Extracellular Proteome

Two-dimensional map of cytosolic proteins of B. subtilis indicated that Two-dimensional map of cytosolic proteins of B. subtilis indicated that most abundant proteins perfomed mainly housekeeping functions in most abundant proteins perfomed mainly housekeeping functions in glycolysis, TCA cycle, amino acid biosynthesis, translation and glycolysis, TCA cycle, amino acid biosynthesis, translation and protein quality control.protein quality control.

B. Heat Stress Proteome during SporulationB. Heat Stress Proteome during Sporulation

60 stress proteins were synthesized de novo and/or 60 stress proteins were synthesized de novo and/or overexpressed in B. subtilis during sporulation concurrent to overexpressed in B. subtilis during sporulation concurrent to acquired thermotolerance in spores.acquired thermotolerance in spores.

B. subtilis B. subtilis ProteomicsProteomics

Saccharomyces cerevisiae :Saccharomyces cerevisiae :A MODEL FOR A MODEL FOR HIGHER EUKARYOTESHIGHER EUKARYOTES

A yeast cells is about 4-7A yeast cells is about 4-7m large, the ”eyes” at the bottom are bud scarsm large, the ”eyes” at the bottom are bud scars

simple unicellular eukaryote, valuable for studying the basic mechanisms simple unicellular eukaryote, valuable for studying the basic mechanisms of cell life particularly in human genetic diseases. Very manipulable, capable of cell life particularly in human genetic diseases. Very manipulable, capable of being easily deleted, mutated, reintroduced, overexpressed, tagged and of being easily deleted, mutated, reintroduced, overexpressed, tagged and comprehensively analyzed.comprehensively analyzed.

Yeast Yeast GENOMEGENOME

The The S. cerevisiaeS. cerevisiae nuclear genome has 16 nuclear genome has 16 chromosomes chromosomes

6, 274 potential ORFs6, 274 potential ORFs Genetical genomesGenetical genomes

(1) a much lower gene density and largely (1) a much lower gene density and largely untranscribeed DNAuntranscribeed DNA

(2) the presence of several apparent (2) the presence of several apparent pseudogenes and a 15-kbp redundant pseudogenes and a 15-kbp redundant sequencesequence

(3) the absence of genes essential for (3) the absence of genes essential for vegetative growthvegetative growth

A.A. Genetic Basis of Metal HomeostasisGenetic Basis of Metal Homeostasis

(i) Correlation of Gene Expression & Function (i) Correlation of Gene Expression & Function

* * Zap1p transcription factorZap1p transcription factor - - senses the status of cellular zinc levels and stimulates expression of its senses the status of cellular zinc levels and stimulates expression of its target genes in response to zinc limitationtarget genes in response to zinc limitation

be examined from a new & global perspective.be examined from a new & global perspective.

Yeast Yeast TranscriptomicsTranscriptomics

A.A. Genetic Basis of Metal HomeostasisGenetic Basis of Metal Homeostasis

(ii) Connection between Gene Expression and Physiological State(ii) Connection between Gene Expression and Physiological State

* copper ions 3 functional cofactors key enzymes* copper ions 3 functional cofactors key enzymes

Yeast Yeast TranscriptomicsTranscriptomics

(1) (1) an active cytochrome oxidase complex, which an active cytochrome oxidase complex, which enables yeast cells to grow on nonfermentable carbon enables yeast cells to grow on nonfermentable carbon sourcessources

(2) the copper-metalloenzyme superoxide dismutase, (2) the copper-metalloenzyme superoxide dismutase, which protects the cell against the detrimental effects which protects the cell against the detrimental effects of reactive superoxide dismutaseof reactive superoxide dismutase

(3) the copper-metalloenzyme Fet3, a ferro-oxidase that (3) the copper-metalloenzyme Fet3, a ferro-oxidase that is critical for fe(II) uptakeis critical for fe(II) uptake

B. Functional Genomics of Metabolic ReprogrammingB. Functional Genomics of Metabolic Reprogramming

(i) Connection between Gene Expression and Physiological State(i) Connection between Gene Expression and Physiological State

* * Genome-wide transcription patterns under aerobic and anaerobicGenome-wide transcription patterns under aerobic and anaerobic

Yeast Yeast TranscriptomicsTranscriptomics

1)1) 219 genes displayed a greater 3-fold higher transcription 219 genes displayed a greater 3-fold higher transcription level, while 140 genes showed a greater than 3-fold level, while 140 genes showed a greater than 3-fold increase in transcript level in response to anaerobosis.increase in transcript level in response to anaerobosis.

2) 2) 34 genes whose expression at the diauxin shift is 34 genes whose expression at the diauxin shift is dependent on a functional Cat8p, a zinc cluster-dependent on a functional Cat8p, a zinc cluster-containing transcriptional activator that is essential for containing transcriptional activator that is essential for growth on nonfermentable carbon source. growth on nonfermentable carbon source.

C. Nucleosome Remodeling Complexes in Gene RegulationC. Nucleosome Remodeling Complexes in Gene Regulation

(i) Connection between (i) Connection between Gene Expression and Gene Expression and Physiological StatePhysiological State

* Nucleosomal * Nucleosomal inhibition of gene inhibition of gene transcription can occur transcription can occur at the stages of at the stages of transcription factor transcription factor binding, preinitiation binding, preinitiation complex formation, or complex formation, or transcription transcription elongation.elongation.

Yeast Yeast TranscriptomicsTranscriptomics

C. Nucleosome Remodeling Complexes in Gene RegulationC. Nucleosome Remodeling Complexes in Gene Regulation

(i) Connection between (i) Connection between Gene Expression and Gene Expression and Physiological StatePhysiological State

* * Hierarchical Hierarchical cluster analysis of cluster analysis of whole genome whole genome microarray data from microarray data from a study assessing a study assessing the genome-wide the genome-wide effects of Rvb effects of Rvb inactivation.inactivation.

Yeast Yeast TranscriptomicsTranscriptomics

a. Green and red indicates a. Green and red indicates a decrease or an a decrease or an increase in mRNA increase in mRNA abundanceabundance

b. b. Rvb1p and Rvb2p Rvb1p and Rvb2p are are essential components essential components of a chromatin of a chromatin remodeling complex remodeling complex that regulates that regulates transcription of over transcription of over 5% of yeast genes.5% of yeast genes.

A. The Extracellular ProteomeA. The Extracellular Proteome

2,774 yeast proteins was determined by high-throughput 2,774 yeast proteins was determined by high-throughput immunolocalization of epitope-tagged gene productsimmunolocalization of epitope-tagged gene products

Yeast Yeast ProteomicsProteomics

B. Proteome MicroarrayB. Proteome Microarray

A.) A.) Protein chip fabrication and analysis of yeast protein kinasesProtein chip fabrication and analysis of yeast protein kinases. .

A protein chips were constructed by pouring PDMS over an acrylic A protein chips were constructed by pouring PDMS over an acrylic mold, curing, and mounting the wells on a glass slides. The surface mold, curing, and mounting the wells on a glass slides. The surface of the wells was then modified, followed by protein attachment.of the wells was then modified, followed by protein attachment.

Yeast Yeast ProteomicsProteomics

B. Proteome MicroarrayB. Proteome Microarray

B.) B.) Protein chip fabrication and analysis of yeast protein kinasesProtein chip fabrication and analysis of yeast protein kinases. . Kinase activities were detected using protein chips. Images of Kinase activities were detected using protein chips. Images of phosphorylation signals in the presence of 12 substrates are shown.phosphorylation signals in the presence of 12 substrates are shown.

Yeast Yeast ProteomicsProteomics

Yeast Yeast Interactome: Mapping Protein-Protein Interactome: Mapping Protein-Protein InteractionsInteractions

A. Genomic Two-hybrid ScreensA. Genomic Two-hybrid Screens

A.) A.) GAL4, protein, a transcriptional activator that control expression GAL4, protein, a transcriptional activator that control expression of genes involved in galatose utilization. of genes involved in galatose utilization. It contain two separable It contain two separable and functionally distinct domains that are both essential for and functionally distinct domains that are both essential for activation of target gene expression:activation of target gene expression:

1. 1. N-terminalN-terminal domain domain –responsible for specific DNA-binding activity–responsible for specific DNA-binding activity

2. 2. C-terminal C-terminal domaindomain –contains acidic regions that are required for –contains acidic regions that are required for

activation of transcription.activation of transcription.

Yeast Yeast Interactome: Mapping Protein-Protein Interactome: Mapping Protein-Protein InteractionsInteractions

A. Genomic Two-hybrid ScreensA. Genomic Two-hybrid Screens

B.) B.) Ydr016 protein, has potential involvement in spindle pole body Ydr016 protein, has potential involvement in spindle pole body function. function. Three hypothetical proteins, Three hypothetical proteins, Ydr016c, Ykr083c Ydr016c, Ykr083c and and Ylr423cYlr423c, are implicated in spindle pole body function based on their , are implicated in spindle pole body function based on their interactions with proteins of known function.interactions with proteins of known function.

Yeast Yeast Interactome: Mapping Protein-Protein Interactome: Mapping Protein-Protein InteractionsInteractions

B. Visualizing Protein-Protein Interaction Network in B. Visualizing Protein-Protein Interaction Network in silicossilicos

1.) 1.) 63% 63% of the assembled connections occurring between proteins of the assembled connections occurring between proteins assigned a common functional role and assigned a common functional role and 76% 76% occuring between occuring between proteins residing in the same subcellular compartment.proteins residing in the same subcellular compartment.

2.) 2.) 2121 proteins involved in membrane fusion and the proteins involved in membrane fusion and the 141 141 proteins proteins involved in vesicular transport.involved in vesicular transport.

Yeast Yeast Interactome: Mapping Protein-Protein Interactome: Mapping Protein-Protein InteractionsInteractions

B. Visualizing Protein-Protein Interaction Network in B. Visualizing Protein-Protein Interaction Network in silicossilicos

Protein–protein interaction map for Cdc28 and Fkh1/2 complexes Protein–protein interaction map for Cdc28 and Fkh1/2 complexes involved in signaling pathways.involved in signaling pathways. Gray dotted arrows indicate new Gray dotted arrows indicate new interactions determined by high-throughput mass spectrometric interactions determined by high-throughput mass spectrometric protein complex identification (HMS-PCI).protein complex identification (HMS-PCI).

Yeast Yeast Interactome: Mapping Protein-Protein Interactome: Mapping Protein-Protein InteractionsInteractions

C. Analysis of Yeast Multiprotein Complexes by Mass C. Analysis of Yeast Multiprotein Complexes by Mass SpectrometrySpectrometry

1.) 1.) High-throughput mass spectrometric protein complex High-throughput mass spectrometric protein complex identification (HMS-PCI) identification (HMS-PCI) detects 3, 617 associated proteins , detects 3, 617 associated proteins , representing 25% of the yeast proteome. representing 25% of the yeast proteome.

2.) 2.) Immuno-affinity purification based on the Flag epitope tagImmuno-affinity purification based on the Flag epitope tag was was used to capture 100 protein kinases, 36 phosphatases and used to capture 100 protein kinases, 36 phosphatases and regulatory subunits, and 86 proteins functionally implicated in the regulatory subunits, and 86 proteins functionally implicated in the cellular response to DNA damage.cellular response to DNA damage.

COMPARATIVE GENOMICS OF MODEL COMPARATIVE GENOMICS OF MODEL EUKARYOTIC ORGANISMEUKARYOTIC ORGANISM

Distribution of Distribution of core biological core biological

functions functions conserved in conserved in both yeast both yeast and wormand worm

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Large scale cultureLarge scale cultureTransformationTransformation

Separation Separation

Mass spectrometryMass spectrometry

LIMS systemLIMS system

An integrated An integrated workflowworkflow