Download No Slide Title





Genome
Gene expression
signal transduction
Plant microbial interaction
Genome Organization
Genome
Complete set of instructions for making an organism
(master blueprints for all enzymes, cellular structures & activities)
an organism‘s complete set of DNA
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
4-200 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
one circular
doublestranded
DNA
chromosome
often
plasmid(s)
 Generally 1 circular
chromosome (dsDNA)
 Usually without introns
 Relatively high gene
density
(~2500 genes per mm of E.
coli DNA)
 Often indigenous plasmids
are present
1. Eschericia coli
2. Agrobacterium tumefaciens
Bacterial genomes: E. coli

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
Short intergenic regions
Uninterrupted ORFs
Conserved signals
Abundant comparative information
Plasmids
Extra chromosomal circular DNAs
ori
 Found in bacteria, yeast and other fungi
 Size varies form ~ 3,000 bp to 100,000 bp.
 Replicate autonomously (origin of replication)
 May contain resistance genes
 May be transferred from one bacterium to another
 May be transferred across kingdoms
 Low - Multicopy plasmids (1 to 400 plasmids/per cell)
 Plasmids may be incompatible with each other
Agrobacterium tumefaciens
Plant parasite that causes Crown Gall Disease
Lives in intercellular spaces of the plant
Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid)
Plasmid contains genes responsible for the disease
Wound = entry point  10-14 days later, tumor forms
Portion of the Ti plasmid is transferred between bacterial cells and plant
cells  T-DNA (Transfer DNA)
T-DNA integrates stably into plant genome
Single stranded T-DNA fragment is converted to dsDNA fragment by
plant cell
Then integrated into plant genome
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
Relatively low gene density
 (50 genes per mm of DNA
in humans)
5,000 - 125,000 genes
Carry organellar genome
Fungal genomes: S. cerevisiae




First completely sequenced eukaryote
genome
Very compact genome:
• Short intergenic regions
• Scarcity of introns
• Lack of repetitive sequences
Strong evidence of duplication:
• Chromosome segments
• Single genes
Redundancy: non-essential genes provide
selective advantage
Plant genomes
 Plant contains three genomes
 Genetic information is divided in the chromosome.
 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 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
 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
It has only 2 x 5 chromosomes
It is just 70 Mbp
It has a life cycle of only 6 weeks
It contains 25,498 structural genes from 11,000
families
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
Peculiarities of plant genomes







Huge genomes reaching tens of billions of base pairs
Numerous polyploid forms
Abundant (up to 99%) non coding DNA which seriously hinders
sequencing, gene mapping
Poor morphological, genetics, and physical mapping of chromosomes
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 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
14500 Millions
2800 Millions
Plastid
0.156 Millions
0.136 Millions
0.120 Millions
Mitochondrion
0.370 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
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)
Global Increase in Genome Size
Polyploidization (whole genome duplication):
Allopolyploidy: combination of genetically distinct
chromosome sets.
Autopolyploidy: multiplication of one basic set of
chromosomes
Regional duplication
Repetitive Structure of
Eukaryotic Genome

Eukaryotic genomes contain various degrees of repetitive structure:
satellites, micro/mini-satellites, retrotransposons, retrovirus
Repetitive sequence size correlates with genome size
Heterochromatin (*109bp)

Gorrila gorilla
Symphalangus syndactylus
Pan troglodites
Homo sapiens
Hylobates muelleri
Genome size (*109bp)
Mechanisms for Regional
Increase in Genome Size




Duplicative transposition
Unequal crossing-over
Replication slippage
Gene amplification (rolling circle replication)
Gene Duplication

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)
invariant duplication: dose repetitions,
variant duplication: new functions.

duplication of a cluster of genes
Internal Gene Duplication
5’
1
2
3
4
5
3’
6
Ancestral trypsinogen gene
Deletion
6’
1
5’
3’
Thr Ala Ala Gly
4 fold duplication + addition of spacer sequence
6’
1
5’
Internal duplications + addition of intron sequence
5’
1
1
2
3
4
5
6
3’
Spacer: Gly
7
…
37
38
Antifreeze glycoprotein gene
39
40
41
6’
3’
Complete Gene Duplication

Invariant duplication:
RNA specifying genes: Number of tRNA and rRNA correlates with
genome size.

Variant duplication:
X-linked
autosomal
Trichromatic
Human female
Trichromatic
Human male
Human male
(color blind)
New world monkey
female
or
or
Dichromatic
Trichromatic
New world monkey
female
New world monkey
male
Dichromatic
or
Dichromatic
What do the genes encode?
Microbes
highly
specialized
Basic functions
+
Yeast –
simplest
eukaryote
Fly –
complex
development
Genes for basic cellular functions such as translation,
transcription, replication and repair share similarity
among all organisms
Worm –
programmed
development
Arabidopsis –
plant life cycle
Gene families expand to
meet biological needs.
Gene classification
coding genes
Chromosome
(simplified)
intergenic
non-coding
region
genes
Messenger RNA
Structural RNA
Proteins
transfer
RNA
Structural proteins
Enzymes
ribosomal
RNA
other
RNA
Prokaryotic genes


Most do not have introns
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
Produce monocistronic mRNA: only one encoded protein
 Large


Protein Coding Genes
Segment of DNA which can be transcribed and translated to amino acid
Protein Coding Genes







Plant contains about 10 000 – 30 000 structural genes
They are present in only a few copies, sometimes just one (single copy gene)
They often form a gene family
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
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
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 50-2000
 One mitochondria consists of 1 – 100 genomes (multiple identical circular
chromosomes).
 They are one large and several smaller
 Size ~15 Kb in animals
 Size ~ 200 kb to 2,500 kb in plants
 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
 Over 95% of mitochondrial proteins are encoded in the nuclear genome.
 Often A+T rich genomes
Chloroplast genome (ctDNA)
 Multiple circular molecules, similar to procaryotic cyanobacteria,
although much smaller (0.001-0.1%of the size of nuclear genomes)
 Cells contain many copies of plastids and each plastid contains
many genome copies
 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
 Some of plastid genomes contain introns
 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
Their metabolism is bacterial (e.g. photosynthesis)
Retain some DNA (circular chromosome)
• Protein synthesis sensitive to chloramphenicol
• Cytosolic P synthesis sensitive to cycloheximide
Most genes transferred from symbiont to nucleus
• Requires 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
Amino acids, essential amino acid synthesis restricted to plastids
• Phenylpropanoid amino acids and secondary compounds start in the
plastids (shikimic acid pathway)
• Site of action of several herbicides, including glyphosate
• Branched-chain amino acids
• Sulfur amino acids
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
• not clear how antisense works
 Knockouts
• Often more clear-cut since all of the enzyme is gone
• Use of t-DNA, Salk lines
 Overexpression
• Use an unregulated version of the protein or express on a strong promoter
• Sometimes leads to cosuppression

RNA interference
• 21 to 26 mers seem very effective in regulating translation