GenomeGenome Gene expressionGene expression signal transductionsignal transduction Plant microbial interactionPlant microbial interaction

Genome OrganizationGenome Organization

GenomeGenome

Complete set of instructions for making an organismComplete set of instructions for making an organism(master blueprints for all enzymes, cellular structures & activities) (master blueprints for all enzymes, cellular structures & activities)

an organism‘s complete set of DNAan organism‘s complete set of DNA The total genetic information carried by a single set of The total genetic information carried by a single set of

chromosomes in a haploid nucleuschromosomes in a haploid nucleusLocated in every nucleus of trillions of cells Located in every nucleus of trillions of cells

Consists of tightly coiled threads of DNA organized into Consists of tightly coiled threads of DNA organized into chromosomeschromosomes

Typical viral genome

DNAor

RNA

4-200 genes

Viral genomesViral genomesViral genomes: ssRNA, dsRNA, ssDNA, dsDNA, linear or circular

Viruses with RNA genomes • Almost all plant viruses and some bacterial and animal viruses

• Genomes are rather small (a few thousand nucleotides)

Viruses with DNA genomes (e.g. lambda = 48,502 bp):• Often a circular genome.

Replication of viral genomes all ssRNA viruses produce dsRNA molecules many linear DNA molecules become circular

Procaryotic genomesProcaryotic genomes Generally 1 circular Generally 1 circular

chromosome (dsDNA)chromosome (dsDNA) Usually without intronsUsually without introns

Relatively high gene density Relatively high gene density (~2500 genes per mm of (~2500 genes per mm of E. E.

colicoli DNA) DNA) Often indigenous Often indigenous plasmidsplasmids

are presentare present

1. Eschericia coli

2. Agrobacterium tumefaciens

one circular double-

stranded DNA

chromosome

oftenplasmid(s)

Bacterial genomes: Bacterial genomes: E. coliE. coli

4288 protein coding genes:4288 protein coding genes:• Average ORF: 317 amino acidsAverage ORF: 317 amino acids• Very compact: average distance Very compact: average distance

between genes 118bpbetween genes 118bp Numerous paralogous gene Numerous paralogous gene

families: 38 – 45% of genes arisen families: 38 – 45% of genes arisen through duplicationthrough duplication

Short intergenic regionsShort intergenic regions Uninterrupted ORFsUninterrupted ORFs Conserved signalsConserved signals Abundant comparative informationAbundant comparative information

PlasmidsPlasmids

Extra chromosomal circular DNAsExtra chromosomal circular DNAs

Found in bacteria, yeast and other fungiFound in bacteria, yeast and other fungi Size varies form ~ 3,000 bp to 100,000 bp.Size varies form ~ 3,000 bp to 100,000 bp.

Replicate autonomously (origin of replication)Replicate autonomously (origin of replication) May contain resistance genesMay contain resistance genes

May be transferred from one bacterium to anotherMay be transferred from one bacterium to another May be transferred across kingdomsMay be transferred across kingdoms

Low - Multicopy plasmids (1 to 400 plasmids/per cell)Low - Multicopy plasmids (1 to 400 plasmids/per cell) Plasmids may be incompatible with each otherPlasmids may be incompatible with each other

ori

Agrobacterium tumefaciensAgrobacterium tumefaciens

Plant parasite that causes Crown Gall DiseasePlant parasite that causes Crown Gall DiseaseLives in intercellular spaces of the plantLives in intercellular spaces of the plant

Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid)Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid)Plasmid contains genes responsible for the diseasePlasmid contains genes responsible for the disease

Wound = entry point 10-14 days later, tumor formsPortion of the Ti plasmid is transferred between bacterial cells and plant Portion of the Ti plasmid is transferred between bacterial cells and plant

cells cells T-DNA (Transfer DNA) T-DNA (Transfer DNA)T-DNA integrates stably into plant genomeT-DNA integrates stably into plant genomeSingle stranded T-DNA fragment is converted to dsDNA fragment by Single stranded T-DNA fragment is converted to dsDNA fragment by plant cellplant cell

Then integrated into plant genomeThen integrated into plant genome2 x 23bp direct repeats play an important role in the excision and integration 2 x 23bp direct repeats play an important role in the excision and integration processprocess

Ti plasmid of Ti plasmid of A. tumefaciensA. tumefaciens

1. Auxin, cytokinin, opine synthetic genes transferred to plant

2. Plant makes all 3 compounds

3. Auxins and cytokines cause gall formation

4. Opines provide unique carbon/nitrogen source that only A. tumefaciens can use!

Typical eukaryotic genome

Located on several Located on several chromosomeschromosomes

Relatively low gene densityRelatively low gene density (50 genes per mm of DNA (50 genes per mm of DNA

in humans)in humans) 5,000 - 125,000 genes

Carry organellar genomeCarry organellar genome

Fungal genomes: Fungal genomes: S. cerevisiaeS. cerevisiae

First completely sequenced eukaryote First completely sequenced eukaryote genomegenome

Very compact genome:Very compact genome:• Short intergenic regionsShort intergenic regions• Scarcity of intronsScarcity of introns• Lack of repetitive sequencesLack of repetitive sequences

Strong evidence of duplication:Strong evidence of duplication:• Chromosome segmentsChromosome segments• Single genesSingle genes

RedundancyRedundancy: non-essential genes provide : non-essential genes provide selective advantageselective advantage

Plant genomesPlant genomes Plant contains three genomesPlant contains three genomes Genetic information is divided in the chromosome.Genetic information is divided in the chromosome. The size of genomes is species dependentThe size of genomes is species dependent The difference in the size of genome is mainly due to a different number The difference in the size of genome is mainly due to a different number

of identical sequence of various size arranged in sequenceof identical sequence of various size arranged in sequence The gene for ribosomal RNAs occur as repetitive sequence and together The gene for ribosomal RNAs occur as repetitive sequence and together

with the genes for some transfer RNAs in several thousand of copieswith the genes for some transfer RNAs in several thousand of copies Structural genes are present in only a few copies, sometimes just single Structural genes are present in only a few copies, sometimes just single

copy. Structural genes encoding for structurally and functionally related copy. Structural genes encoding for structurally and functionally related proteins often form a gene familyproteins often form a gene family

The DNA in the genome is replicated during the interphase of mitosisThe DNA in the genome is replicated during the interphase of mitosis

Plant genomes: Plant genomes: Arabidopsis thalianaArabidopsis thaliana A weed growing at the roadside of central EuropeA weed growing at the roadside of central Europe It has only 2 x 5 chromosomesIt has only 2 x 5 chromosomes It is just 70 MbpIt is just 70 Mbp It has a life cycle of only 6 weeksIt has a life cycle of only 6 weeks It contains 25,498 structural genes from 11,000 It contains 25,498 structural genes from 11,000

familiesfamilies The structural genes are present in only few The structural genes are present in only few

copies sometimes just one proteincopies sometimes just one protein Structural genes encoding for structurally and Structural genes encoding for structurally and

functionally related proteins often form a gene functionally related proteins often form a gene familyfamily

Peculiarities of plant genomesPeculiarities of plant genomes Huge genomes reaching tens of billions of base pairsHuge genomes reaching tens of billions of base pairs Numerous polyploid formsNumerous polyploid forms Abundant (up to 99%) non coding DNA which seriously hinders Abundant (up to 99%) non coding DNA which seriously hinders

sequencing, gene mappingsequencing, gene mapping Poor morphological, genetics, and physical mapping of chromosomesPoor morphological, genetics, and physical mapping of chromosomes A large number of “small-chromosome” in which the chromosome length A large number of “small-chromosome” in which the chromosome length

does not exceed 3 does not exceed 3 μμmm The difficulty of chromosomal mapping of individual genes using in situ The difficulty of chromosomal mapping of individual genes using in situ

hybridizationhybridization The number of chromosomes and DNA content in many species is still The number of chromosomes and DNA content in many species is still

unknownunknown

Size of the genome in plants and in humanSize of the genome in plants and in human

GenomeGenome Arabidopsis Arabidopsis thalianathaliana

Zea maysZea mays Vicia fabaVicia faba HumanHuman

NucleusNucleus 70 Millions70 Millions 3900 Millions3900 Millions 14500 Millions14500 Millions 2800 Millions2800 Millions

PlastidPlastid 0.156 Millions0.156 Millions 0.136 Millions0.136 Millions 0.120 Millions0.120 Millions

MitochondrioMitochondrionn

0.370 Millions0.370 Millions .570 Millions.570 Millions .290 Millions.290 Millions .017 Millions.017 Millions

What we learned from plant What we learned from plant genome project?genome project?

The number of genes in plants is similar to other higher The number of genes in plants is similar to other higher eukaryots, including humanseukaryots, including humans

Most differences between plant species are due to Most differences between plant species are due to different expression level and different timing of expression different expression level and different timing of expression of a common or core set of genes, not due to different of a common or core set of genes, not due to different genesgenes

Plant evolution has in large part proceeded through Plant evolution has in large part proceeded through changes in transcriptional and other regularly control changes in transcriptional and other regularly control (arabidopsis has > 1500 transcription factors)(arabidopsis has > 1500 transcription factors)

Global Increase in Genome SizeGlobal Increase in Genome Size

Polyploidization (whole genome duplication):Polyploidization (whole genome duplication):

Allopolyploidy:Allopolyploidy: combination of genetically distinct combination of genetically distinct chromosome sets. chromosome sets. Autopolyploidy:Autopolyploidy: multiplication of one basic set of multiplication of one basic set of chromosomeschromosomes

Regional duplicationRegional duplication

Repetitive Structure of Repetitive Structure of Eukaryotic GenomeEukaryotic Genome

Eukaryotic genomes contain various degrees of repetitive structure:Eukaryotic genomes contain various degrees of repetitive structure: satellites, micro/mini-satellites, retrotransposons, retrovirussatellites, micro/mini-satellites, retrotransposons, retrovirus

Repetitive sequence size correlates with genome sizeRepetitive sequence size correlates with genome size

Genome size (*109bp)Het

eroc

hro

mat

in (

*109 b

p)

Hylobates muelleri

Homo sapiens

Pan troglodites

Symphalangus syndactylus

Gorrila gorilla

Mechanisms for Regional Mechanisms for Regional Increase in Genome SizeIncrease in Genome Size

Duplicative transpositionDuplicative transposition Unequal crossing-overUnequal crossing-over

Replication slippageReplication slippage Gene amplification (rolling circle replication)Gene amplification (rolling circle replication)

Gene DuplicationGene Duplication

duplication of a part of the gene:duplication of a part of the gene:

domain/internal sequence duplication domain/internal sequence duplication enhance function, novel function by enhance function, novel function by new combination new combination

duplication of a complete gene (gene family)duplication of a complete gene (gene family)

invariant duplication: dose repetitions,invariant duplication: dose repetitions,

variant duplication: new functions.variant duplication: new functions. duplication of a cluster of genesduplication of a cluster of genes

Internal Gene DuplicationInternal Gene Duplication

1 2 3 4 5 65’ 3’

Ancestral trypsinogen gene

1 6’5’ 3’

Thr Ala Ala Gly

1 6’5’ 3’

Deletion

4 fold duplication + addition of spacer sequence

Internal duplications + addition of intron sequence

15’ 1 2 3 4 5 6 7 37 38 39 40 41 3’6’…

Antifreeze glycoprotein gene

Spacer: Gly

Complete Gene DuplicationComplete Gene Duplication Invariant duplication:Invariant duplication:

RNA specifying genes: Number of tRNA and rRNA RNA specifying genes: Number of tRNA and rRNA correlates with genome size.correlates with genome size.

Variant duplication:Variant duplication:X-linked autosomal

TrichromaticHuman female

TrichromaticHuman male

DichromaticHuman male(color blind) or

DichromaticorNew world monkeyfemale

TrichromaticNew world monkeyfemale

DichromaticNew world monkeymale or

What do the genes encode?What do the genes encode?

Genes for basic cellular functions such as translation, transcription, replication and repair share similarity

among all organisms

Basic functions

Yeast – simplest eukaryote

Worm –programmed development

Fly – complex development

Arabidopsis – plant life cycle

+

Microbes highly specialized

Gene families expand to meet biological needs.

Gene classificationGene classification

coding genesnon-coding genes

Messenger RNA

Proteins

Structural RNA

Structural proteins Enzymes

transfer RNA

ribosomal RNA

otherRNA

Chromosome(simplified)

intergenic region

Prokaryotic genesProkaryotic genes

Most do Most do notnot have introns have introns Many are organized in operons: contiguous genes, Many are organized in operons: contiguous genes,

transcribed as a single polycistronic mRNA, that transcribed as a single polycistronic mRNA, that encode proteins with related functionsencode proteins with related functions

Polycistronic mRNA encodes several proteinsPolycistronic mRNA encodes several proteins

Bacterial operonBacterial operon

Eukaryotic coding genesEukaryotic coding genes

Most have intronsMost have introns Produce monocistronic mRNA: only one encoded proteinProduce monocistronic mRNA: only one encoded protein

LargeLarge

Protein Coding GenesProtein Coding GenesSegment of DNA which can be transcribed and translated to amino acid

Protein Coding GenesProtein Coding Genes Plant contains about 10 000 – 30 000 structural genesPlant contains about 10 000 – 30 000 structural genes They are present in only a few copies, sometimes just one (single copy gene)They are present in only a few copies, sometimes just one (single copy gene) They often form a gene familyThey often form a gene family The transcription of most structural genes is subject to very complex and The transcription of most structural genes is subject to very complex and

specific regulationspecific regulation The gene for enzymes of metabolism or protein biosynthesis which proceed in The gene for enzymes of metabolism or protein biosynthesis which proceed in

all cells are transcribed more oftenall cells are transcribed more often Most of the genes are switched off and are activated only in certain organ and Most of the genes are switched off and are activated only in certain organ and

then often only in certain cellsthen often only in certain cells Many genes are only switched on at specific timesMany genes are only switched on at specific times

House keeping gene:

The genes which every cell needs for such basic functions independent of its specialization

Mitochondrial genome (mtDNA)Mitochondrial genome (mtDNA)

Number of mitochondria in plants can be between 50-2000Number of mitochondria in plants can be between 50-2000 One mitochondria consists of 1 – 100 genomes (multiple identical circular One mitochondria consists of 1 – 100 genomes (multiple identical circular

chromosomes). chromosomes). They are one large and several smaller They are one large and several smaller

Size ~15 Kb in animalsSize ~15 Kb in animals Size ~ 200 kb to 2,500 kb in plantsSize ~ 200 kb to 2,500 kb in plants

Mt DNA is replicated before or during mitosisMt DNA is replicated before or during mitosis Transcription of mtDNA yielded an mRNA which did not contain the correct Transcription of mtDNA yielded an mRNA which did not contain the correct

information for the protein to be synthesized. RNA editing is existed in plant information for the protein to be synthesized. RNA editing is existed in plant mitochondriamitochondria

Over 95% of mitochondrial proteins are encoded in the nuclear genome.Over 95% of mitochondrial proteins are encoded in the nuclear genome. Often A+T rich genomesOften A+T rich genomes

Chloroplast genome (ctDNA)Chloroplast genome (ctDNA) Multiple circular molecules, similar to procaryotic cyanobacteria, Multiple circular molecules, similar to procaryotic cyanobacteria, although much smaller (0.001-0.1%of the size of nuclear genomes)although much smaller (0.001-0.1%of the size of nuclear genomes)

Cells contain many copies of plastids and each plastid contains Cells contain many copies of plastids and each plastid contains many genome copies many genome copies

Size ranges from 120 kb to 160 kbSize ranges from 120 kb to 160 kb Plastid genome has changed very little during evolution. Though Plastid genome has changed very little during evolution. Though

two plants are very distantly related, their genomes are rather two plants are very distantly related, their genomes are rather similar in gene composition and arrangementsimilar in gene composition and arrangement

Some of plastid genomes contain introns Some of plastid genomes contain introns Many chloroplast proteins are encoded in the nucleus (separate Many chloroplast proteins are encoded in the nucleus (separate

signal sequence)signal sequence)

The family of plastidsThe family of plastids

Buchannan et al. Fig. 1.44

EndosymbiosisEndosymbiosis

Well accepted that chloroplasts and mitochondria were once free Well accepted that chloroplasts and mitochondria were once free living bacterialiving bacteria

Their metabolism is bacterial (e.g. photosynthesis)Their metabolism is bacterial (e.g. photosynthesis) Retain some DNA (circular chromosome)Retain some DNA (circular chromosome)

• Protein synthesis sensitive to chloramphenicolProtein synthesis sensitive to chloramphenicol• Cytosolic P synthesis sensitive to cycloheximideCytosolic P synthesis sensitive to cycloheximide

Most genes transferred from symbiont to nucleusMost genes transferred from symbiont to nucleus• Requires protein targetingRequires protein targeting

DNA for chloroplast proteins can be come from DNA for chloroplast proteins can be come from the nucleus or chloroplast genomethe nucleus or chloroplast genome

Buchannan et al. Fig. 4.4

Import of proteins into chloroplastsImport of proteins into chloroplasts

Buchannan et al. Fig. 4.6

Biochemistry inside plastidsBiochemistry inside plastids

Photosynthesis – reduction of C, N, and SPhotosynthesis – reduction of C, N, and S Amino acids, essential amino acid synthesis restricted to plastidsAmino acids, essential amino acid synthesis restricted to plastids

• Phenylpropanoid amino acids and secondary compounds start in the Phenylpropanoid amino acids and secondary compounds start in the plastids (shikimic acid pathway)plastids (shikimic acid pathway)

• Site of action of several herbicides, including glyphosateSite of action of several herbicides, including glyphosate• Branched-chain amino acidsBranched-chain amino acids• Sulfur amino acidsSulfur amino acids

Fatty acids – all fatty acids in plants made in plastidsFatty acids – all fatty acids in plants made in plastids

Exploring metabolism by genetic methodsExploring metabolism by genetic methods

Antisense – what happens when the amount of an enzyme is Antisense – what happens when the amount of an enzyme is reducedreduced• not clear how antisense worksnot clear how antisense works

KnockoutsKnockouts• Often more clear-cut since all of the enzyme is goneOften more clear-cut since all of the enzyme is gone• Use of t-DNA, Salk linesUse of t-DNA, Salk lines

OverexpressionOverexpression• Use an unregulated version of the protein or express on a strong promoterUse an unregulated version of the protein or express on a strong promoter• Sometimes leads to cosuppressionSometimes leads to cosuppression

RNA interferenceRNA interference• 21 to 26 mers seem very effective in regulating translation21 to 26 mers seem very effective in regulating translation