Genome Genome Gene expression Gene expression signal transduction signal transduction Plant microbial interaction Plant microbial interaction

Genome Organization

Genome Complete set of instructions for making an organism Complete set of instructions for making an organism (master blueprints for all enzymes, cellular structures & activities) an organism‘s complete set of DNA an organism‘s complete set of DNA The total genetic information carried by a single set of chromosomes in a haploid nucleus The total genetic information carried by a single set of chromosomes in a haploid nucleus Located in every nucleus of trillions of cells Consists of tightly coiled threads of DNA organized into chromosomes

Typical viral genome DNA or RNA genes

Viral genomes Viral 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 genomes Generally 1 circular chromosome (dsDNA) Generally 1 circular chromosome (dsDNA) Usually without introns Usually without introns Relatively high gene density Relatively high gene density (~2500 genes per mm of E. coli DNA) Often indigenous plasmids are present Often indigenous plasmids are present 1.Eschericia coli 2.Agrobacterium tumefaciens one circular double- stranded DNA chromosome often plasmid(s)

Bacterial genomes: E. coli 4288 protein coding genes: 4288 protein coding genes: Average ORF: 317 amino acids Very compact: average distance between genes 118bp Numerous paralogous gene families: 38 – 45% of genes arisen through duplication Numerous paralogous gene families: 38 – 45% of genes arisen through duplication Short intergenic regions Short intergenic regions Uninterrupted ORFs Uninterrupted ORFs Conserved signals Conserved signals Abundant comparative information Abundant comparative information

Plasmids Extra chromosomal circular DNAs Found in bacteria, yeast and other fungi Found 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 genes May contain resistance genes May be transferred from one bacterium to another May be transferred from one bacterium to another May be transferred across kingdoms May 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 other Plasmids may be incompatible with each other ori

Agrobacterium tumefaciens Plant parasite that causes Crown Gall Disease Plant parasite that causes Crown Gall Disease Lives in intercellular spaces of the plant Lives 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 disease Plasmid contains genes responsible for the disease Wound = entry point  days later, tumor forms Portion of the Ti plasmid is transferred between bacterial cells and plant cells  T-DNA (Transfer DNA) Portion of the Ti plasmid is transferred between bacterial cells and plant cells  T-DNA (Transfer DNA) T-DNA integrates stably into plant genome T-DNA integrates stably into plant genome Single stranded T-DNA fragment is converted to dsDNA fragment by plant cell Single stranded T-DNA fragment is converted to dsDNA fragment by plant cell Then integrated into plant genome Then integrated into plant genome 2 x 23bp direct repeats play an important role in the excision and integration process 2 x 23bp direct repeats play an important role in the excision and integration process

Ti plasmid of A. 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 chromosomes Located on several chromosomes Relatively low gene density Relatively low gene density (50 genes per mm of DNA in humans) (50 genes per mm of DNA in humans) 5, ,000 genes Carry organellar genome Carry organellar genome

Fungal genomes: S. cerevisiae First completely sequenced eukaryote genome First completely sequenced eukaryote genome Very compact genome: Very compact genome: Short intergenic regions Scarcity of introns Lack of repetitive sequences Strong evidence of duplication: Strong evidence of duplication: Chromosome segments Single genes Redundancy: non-essential genes provide selective advantage Redundancy: non-essential genes provide selective advantage

Plant genomes Plant contains three genomes Plant contains three genomes Genetic information is divided in the chromosome. Genetic information is divided in the chromosome. The size of genomes is species dependent The size of genomes is species dependent The difference in the size of genome is mainly due to a different number of identical sequence of various size arranged in sequence The difference in the size of genome is mainly due to a different number of identical sequence of various size arranged in sequence The gene for ribosomal RNAs occur as repetitive sequence and together with the genes for some transfer RNAs in several thousand of copies The gene for ribosomal RNAs occur as repetitive sequence and together with the genes for some transfer RNAs in several thousand of copies Structural genes are present in only a few copies, sometimes just single copy. Structural genes encoding for structurally and functionally related proteins often form a gene family Structural genes are present in only a few copies, sometimes just single copy. Structural genes encoding for structurally and functionally related proteins often form a gene family The DNA in the genome is replicated during the interphase of mitosis The DNA in the genome is replicated during the interphase of mitosis

Plant genomes: Arabidopsis thaliana A weed growing at the roadside of central Europe A weed growing at the roadside of central Europe It has only 2 x 5 chromosomes It has only 2 x 5 chromosomes It is just 70 Mbp It is just 70 Mbp It has a life cycle of only 6 weeks It has a life cycle of only 6 weeks It contains 25,498 structural genes from 11,000 families It contains 25,498 structural genes from 11,000 families The structural genes are present in only few copies sometimes just one protein The structural genes are present in only few copies sometimes just one protein Structural genes encoding for structurally and functionally related proteins often form a gene family Structural genes encoding for structurally and functionally related proteins often form a gene family

Peculiarities of plant genomes Huge genomes reaching tens of billions of base pairs Huge genomes reaching tens of billions of base pairs Numerous polyploid forms Numerous polyploid forms Abundant (up to 99%) non coding DNA which seriously hinders sequencing, gene mapping Abundant (up to 99%) non coding DNA which seriously hinders sequencing, gene mapping Poor morphological, genetics, and physical mapping of chromosomes Poor morphological, genetics, and physical mapping of chromosomes A large number of “small-chromosome” in which the chromosome length does not exceed 3 μm A large number of “small-chromosome” in which the chromosome length does not exceed 3 μm The difficulty of chromosomal mapping of individual genes using in situ hybridization The difficulty of chromosomal mapping of individual genes using in situ hybridization The number of chromosomes and DNA content in many species is still unknown The number of chromosomes and DNA content in many species is still unknown

Size of the genome in plants and in human Genome Arabidopsis thaliana Zea mays Vicia faba Human Nucleus 70 Millions 3900 Millions Millions 2800 Millions Plastid Millions Millions Millions Mitochondrion Millions.570 Millions.290 Millions.017 Millions

What we learned from plant genome project? The number of genes in plants is similar to other higher eukaryots, including humans The number of genes in plants is similar to other higher eukaryots, including humans Most differences between plant species are due to different expression level and different timing of expression of a common or core set of genes, not due to different genes Most differences between plant species are due to different expression level and different timing of expression of a common or core set of genes, not due to different genes Plant evolution has in large part proceeded through changes in transcriptional and other regularly control (arabidopsis has > 1500 transcription factors) Plant evolution has in large part proceeded through changes in transcriptional and other regularly control (arabidopsis has > 1500 transcription factors)

Global Increase in Genome Size Polyploidization (whole genome duplication): Polyploidization (whole genome duplication): Allopolyploidy: combination of genetically distinct chromosome sets. Autopolyploidy: multiplication of one basic set of chromosomes Regional duplication Regional duplication

Repetitive Structure of Eukaryotic Genome Eukaryotic genomes contain various degrees of repetitive structure: satellites, micro/mini-satellites, retrotransposons, retrovirus Eukaryotic genomes contain various degrees of repetitive structure: satellites, micro/mini-satellites, retrotransposons, retrovirus Repetitive sequence size correlates with genome size Repetitive sequence size correlates with genome size Genome size (*10 9 bp) Heterochromatin (*10 9 bp) Hylobates muelleri Homo sapiens Pan troglodites Symphalangus syndactylus Gorrila gorilla

Mechanisms for Regional Increase in Genome Size Duplicative transposition Duplicative transposition Unequal crossing-over Unequal crossing-over Replication slippage Replication slippage Gene amplification (rolling circle replication) Gene amplification (rolling circle replication)

Gene Duplication duplication of a part of the gene: duplication of a part of the gene: domain/internal sequence duplication  enhance function, novel function by new combination duplication of a complete gene (gene family) duplication of a complete gene (gene family) invariant duplication: dose repetitions, variant duplication: new functions. duplication of a cluster of genes duplication of a cluster of genes

Internal Gene Duplication ’3’ Ancestral trypsinogen gene 16’ 5’3’ Thr Ala Ala Gly 16’ 5’3’ Deletion 4 fold duplication + addition of spacer sequence Internal duplications + addition of intron sequence 1 5’ ’ 6’ … Antifreeze glycoprotein gene Spacer: Gly

Complete Gene Duplication Invariant duplication: Invariant duplication: RNA specifying genes: Number of tRNA and rRNA correlates with genome size. Variant duplication: Variant duplication:

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 classification coding genes non-coding genes Messenger RNA Proteins Structural RNA Structural proteinsEnzymes transfer RNA ribosomal RNA other RNA Chromosome (simplified) intergenic region

Prokaryotic genes Most do not have introns Most do not have introns Many are organized in operons: contiguous genes, transcribed as a single polycistronic mRNA, that encode proteins with related functions Many are organized in operons: contiguous genes, transcribed as a single polycistronic mRNA, that encode proteins with related functions Polycistronic mRNA encodes several proteins

Bacterial operon

Eukaryotic coding genes Most have introns Most have introns Produce monocistronic mRNA: only one encoded protein Produce monocistronic mRNA: only one encoded protein Large Large

Protein Coding Genes Segment of DNA which can be transcribed and translated to amino acid

Protein Coding Genes Plant contains about – structural genes Plant contains about – 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 family They often form a gene family The transcription of most structural genes is subject to very complex and specific regulation The transcription of most structural genes is subject to very complex and specific regulation The gene for enzymes of metabolism or protein biosynthesis which proceed in all cells are transcribed more often The gene for enzymes of metabolism or protein biosynthesis which proceed in all cells are transcribed more often Most of the genes are switched off and are activated only in certain organ and then often only in certain cells Most of the genes are switched off and are activated only in certain organ and then often only in certain cells Many genes are only switched on at specific times Many 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) Number of mitochondria in plants can be between Number of mitochondria in plants can be between One mitochondria consists of 1 – 100 genomes (multiple identical circular chromosomes). One mitochondria consists of 1 – 100 genomes (multiple identical circular chromosomes). They are one large and several smaller They are one large and several smaller Size ~15 Kb in animals Size ~15 Kb in animals Size ~ 200 kb to 2,500 kb in plants Size ~ 200 kb to 2,500 kb in plants Mt DNA is replicated before or during mitosis Mt DNA is replicated before or during mitosis 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 mitochondria 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 mitochondria 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 genomes Often A+T rich genomes

Chloroplast genome (ctDNA) Multiple circular molecules, similar to procaryotic cyanobacteria, although much smaller ( %of the size of nuclear genomes) Multiple circular molecules, similar to procaryotic cyanobacteria, although much smaller ( %of the size of nuclear genomes) Cells contain many copies of plastids and each plastid contains many genome copies Cells contain many copies of plastids and each plastid contains many genome copies Size ranges from 120 kb to 160 kb Size ranges from 120 kb to 160 kb Plastid genome has changed very little during evolution. Though two plants are very distantly related, their genomes are rather similar in gene composition and arrangement Plastid genome has changed very little during evolution. Though two plants are very distantly related, their genomes are rather similar 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 signal sequence) Many chloroplast proteins are encoded in the nucleus (separate signal sequence)

The family of plastids Buchannan et al. Fig. 1.44

Endosymbiosis Well accepted that chloroplasts and mitochondria were once free living bacteria Well accepted that chloroplasts and mitochondria were once free living 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 nucleus Most genes transferred from symbiont to nucleus Requires protein targetingRequires protein targeting

DNA for chloroplast proteins can be come from the nucleus or chloroplast genome Buchannan et al. Fig. 4.4

Import of proteins into chloroplasts Buchannan et al. Fig. 4.6

Biochemistry inside plastids Photosynthesis – reduction of C, N, and S Photosynthesis – reduction of C, N, and S Amino acids, essential amino acid synthesis restricted to plastids Amino acids, essential amino acid synthesis restricted to plastids Phenylpropanoid amino acids and secondary compounds start in the plastids (shikimic acid pathway)Phenylpropanoid amino acids and secondary compounds start in the 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 plastids Fatty acids – all fatty acids in plants made in plastids

Exploring metabolism by genetic methods Antisense – what happens when the amount of an enzyme is reduced Antisense – what happens when the amount of an enzyme is reduced not clear how antisense worksnot clear how antisense works Knockouts Knockouts 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 Overexpression Overexpression 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 interference RNA interference 21 to 26 mers seem very effective in regulating translation21 to 26 mers seem very effective in regulating translation