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Genomics and Biotechnology
DNA structure
Replication
Gene expression
Genome size
Genome structure:
Coding sequences
Transposable elements
Genome technologies:
¾sequencing
¾PCR
¾DNA fingerprinting
¾EST databases
¾Microarrays
¾Comparative and functional genomics
Model Systems for Plant Genomics
Regulation of gene expression by methylation and
RNA interference (Time?)
Nucleotides
DNA structure
Linear and circular DNA molecules
DNA packaging in cell
Structure of nucleosome
DNA packaging in cell
Chromatin organization
DNA replication: semi-conservative model
Replicative complex
The structure of eukaryotic gene. mRNA synthesis.
What is genome?
The genome of an organism is a complete DNA sequence of
one set of chromosomes
Genome of plants:
Nuclear
Mitochondrial
Chloroplast
Nuclear genome size
Genome complexity in plants
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Arabidopsis thaliana * : 120 Mbp (120,000,000 bp)
Poplar * : 550 Mbp
Rice * : 450 Mbp
Maize 2,500 Mbp,
5,000 Mbp barley,
Hexaploid wheat:16,000 Mbp
Fritillaria assyriaca, (Lilliaceae ): >87,000 Mbp
• * sequenced genomes
Genome size/number of genes
Gene density in genomes
C-value paradox
The total amount of DNA in the haploid genome is called its C value.
Psilotum nudum ("whisk fern") is a far simpler plant than Arabidopsis
(it has no true leaves, flowers, or fruit). Nevertherless, it has 3000 times as much DNA
as Arabidopsis. 80% or more of Psilotum DNA is repetitive DNA containing
no genetic information.
Some amphibians contain 30 times as much DNA as humans.
The lack of a consistent relationship between the C value and the complexity of
an organism is called the C-value paradox.
Eukaryotic genome organization
Genome instability: transposable elements
Transposons
are segments of DNA that can move to different positions
in the genome of a single cell.
Class II Transposons consist of DNA that moves directly from place to place.
Key components required for DNA transposition: flanking inverted repeats and
enzyme transposase encoded by transposon
DNA mutations caused by Class II transposon movement:
- insertions
- deletions
- translocations
Maize transposable elements Ac/Ds:
Class III Transposons
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Class III Transposons or MITEs (Miniature Inverted-repeats Transposable
Elements).
Structure of C. elegans and rice MITEs:
• 5' GGCCAGTCACAATGG..~ 400 nt..CCATTGTGACTGGCC 3'
3' CCGGTCAGTGTTACC..~ 400 nt..GGTAACACTGACCGG 5'
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- too small to encode any protein
- the mechanism of transposition is not known (possibly depends on
proteins of Class II transposons that recognize MITEs inverted
repeats
-100,000 MITEs represent 6% of the total rice genome.
Retrotransposons
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Class I Retrotransposons transcribe the DNA into RNA and then
use reverse transcriptase to make a DNA copy of the RNA
template to insert in a new location.
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At least 50% of the nuclear DNA of maize consists of
retrotransposons
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Retrotransposons represent about 40% of the entire human genome
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Retrotransposons in corn and other plants appear to be retroviruslike parasites, an unexpected finding because other organisms with
such matter in their genomes, such as humans, are susceptible to
retroviral diseases. Active retroviruses have never been seen in
plants.
Structure of the Adh1-F region of maize.
Retrotransposons accounted for over 60% of the Adhl-F
region
A 280 kb region containing the maize Adhl-F and u22 genes is composed primarily of
retrotransposons inserted within each other. Ten retroelement families were discovered, with
reiteration frequencies ranging from 10 copies to 30,000 copies per haploid genome.
Are transposons just genomic “parasites” or
active participants in genome evolution?
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Transposons have been called "junk" DNA and "selfish" DNA. "selfish" because their only function
seems to make more copies of themselves and "junk" because there is no obvious benefit to their host.
Retrotransposons cannot be so selfish that they reduce the survival of their host. Perhaps, they even
confer some benefit.
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Transposons can destroy or alter the gene's activity by disrupting it’s functional sequence.
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Retrotransposons often carry some additional sequences at their 3' end as they insert into a new
location. Perhaps these occasionally create new combinations of exons, promoters, and enhancers that
benefit the host. Example:
- Thousands of our Alu elements occur in the introns of structural genes
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-Some of these contain sequences that when transcribed into the primary transcript are
recognized by the spliceosome.
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These can then be spliced into the mature mRNA creating a
new exon, which will be transcribed into a new protein product.
Alternative splicing can provide not only the new mRNA (and thus protein) but also the old.
In this way, nature can try out new proteins without the risk of abandoning the tried-and-true old
one.
Finally, transposons can cause duplications as a result of unequal crossover during
recombination.
Transposons in genome studies
and biotechnology
Class II transposons are frequenly used for:
- mutagenesis to obtain loss-of-function
insertions into the gene of interest
- gene discovery systems based on Ds
transposon (gene trap and enhancer trap)
Genome technologies:
¾Sequencing
¾PCR
¾DNA fingerprinting
¾Gene expression profiling: microarrays
¾EST databases
¾Comparative and functional genomics
Dideoxysequencing
Sequenced genomes
The genomes of more than 150 organisms have been sequenced since
1995
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Arabidopsis thaliana
Oryza sativa (rice)
Populus trichocarpa (poplar)
Saccharomyces cerevisiae (yeast)
Caenorhabditis elegans
Anopheles gambiae (mosquito )
Apis mellifera (bee)
Mus musculus (mouse)
Rattus norvegicus (rat)
Pan troglodytes (chimpanzee)
Homo sapiens (human)
http://www.genomenewsnetwork.org/resources/sequenced_genomes/genome_guide_p1.shtml
Polymerase Chain Reaction
PCR-based DNA fingerprinting. SSR markers
PCR primer
5’-[ATTT]x-3’
PCR primer
Microsatellites (short tandem repeats) contain 2-5 bp repeats.
Microarray technology
DNA hybridization
Microarray chip technology
Detail:
Detail:
Detail:
Size: 12cm x 8cm
Size: 5,4cm x 0,9cm
Size: 1,28cm x 1,28cm
•2400 clones by membrane
•radioactive labelling
•1 experimental condition by
membrane
•10000 clones by slide
•fluorescent labelling
•2 experimental conditions by
slide
•300000 oligonucleotides by slide
•fluorescent labelling
•1 experimental condition by
slide
Microarray image analysis
Microarray technique applications
• Gene expression profiling
• Differential expression of genes at the
whole genome scale.
• Tissue-specific gene expression
Functional and comparative plant genomics
¾What is functional genomics?
Understanding the function of genes and other parts of the genome
¾What is comparative genomics?
Comparative genomics is the analysis and comparison of genomes from different species.
¾ What makes Arabidopsis
a model plant system for functional genomics?
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Small genome size (1.2x108 bp)
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High gene density throughout most of the Arabidopsis genome (approximately one gene per 4
to 5 kb)
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Repetitive DNAs are relatively rare, comprising ~10% of the Arabidopsis genome.
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Short life cycle (~ 6 weeks)
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NSF: The Project 2010: to establish function of all 25,498 Arabidopsis genes.
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Populus is a model system for tree genomics. Poplar genome is sequenced. 95,000 ESTs from
20 different cDNA libraries from a range of tissues and developmental stages are available.
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Plants exhibit extensive conservation of both
gene content and gene order
Loblolly pine and Arabidopsis thaliana differ greatly in form, ecological niche,
evolutionary history, and genome size. Nevertherless, for contigs 1,100 bp or
longer, 90% have an apparent Arabidopsis gene homolog.
Kirst et. al. 2003, PNAS,100 (12),| 7383-7388.
Regulation of gene expression by methylation
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The vast majority of methylation is related to the sequence 5'CpG-3'
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% 5'-mC: animals 2-7%; plants >25%
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CpG islands exist that are often associated with genes
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the methylation pattern is heritable from generation to generation.
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Low 5'-mC-----------> high levels of gene expression
High 5'-mC ------------> low levels of gene expression
Regulation of gene expression by RNA
interference and micro RNA
• RNAi is a highly potent and specific process which is
actively carried out by special mechanisms in the cell,
known as the RNA interference machinery.
• the presence of small fragments of double-stranded RNA
(dsRNA) whose sequence matches a target
gene interferes with the expression of that gene
• miRNA - short RNA molecules that fold back on
themselves in a hairpin shape to create a double strand
• RNAi has been applied as gene“knockout “ tool
Summary
DNA is built from deoxyribonucleotides, RNA contains ribonucleotides. In RNA deoxythymidine is substitutetd by uracil.
The strands in DNA double helix are anti-parallel.
DNA molecules could be linear or circular.
Nucleosome is a structural unit of DNA packaging in chromosomes of eukaryotic cell.
DNA replicates in a semi-conservative manner. Replicative complex machinery replicates DNA with high accuracy and processivity.
Eukaryotic genes consist from exons (coding regions) interrupted by non-coding introns. During RNA splicing in nucleus
introns are removed from pre-mRNA. The main regulatiry elements of eukaryotic gene: promoter, terminator, poly-adenilation signal,
translation initiation stop and codons.
The genome of an organism is a complete DNA sequence of one set of chromosomes. Genome of plants: nuclear, mitochondrial,
and chloroplast.
Eukaryotic genome organization: unique coding DNA (unique coding genes), repetitive DNA (functional dispersed and tandemly
repeated gene families; transposons) and spacer DNA. Gene density is higher in prokaryotic organisms than in eukaryotic.
The total amount of DNA in the haploid genome is called its C value.
The lack of a consistent relationship between the C value and the complexity of an organism is called the C-value paradox.
Transposons are segments of DNA that can move to different positions in the genome of a single cell.
Class II transposons consist of DNA that moves directly from place to place. Key components required for DNA transposition:
flanking inverted repeats and enzyme transposase encoded by transposon
Class III Transposons or MITEs are similar to Class II but too small to encode any protein, the mechanism of MITEs
transposition is not known.
Class I Retrotransposons transcribe the DNA into RNA and then use reverse transcriptase to make a DNA copy of the RNA
template to insert in a new location.
The large portions of genome consist of transposons and other repetitive DNA. Transposons can cause DNA mutations
including insertions, deletions and chromosomal translocations that could be beneficial or detrimental for the evolution.
Transposon mutagenesis is a valuable tool in molecular genetics and biotechnology.
Summary (continuation)
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Automated high-throughput dideoxysequencing allows to obtain sequence of the entire genome. More than 150
genomes have been sequenced by date.
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Microsatellites or short tandem repeats containing 2-5 bp repeats are molecular markers used for genetic mapping
and DNA fingerprinting using PCR.
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PCR (polymerase chain reaction) allows amplification of defined fragment from genomic DNA. PCR sensitivity is
so great that it allows to amplify DNA from of a single cell.
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Microarray technology allows to monitor differential expression of genes at the whole genome scale.
Functional genomics is understanding the function of genes. Comparative genomics is the analysis and comparison
of genomes from different species.
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Plants exhibit extensive conservation of both gene content and gene order among species.
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Gene expression regulation in plants.
Methylation: High content of methylated cytosin correlates with low levels of gene expression. The methylation
pattern is heritable from generation to generation.
RNA inerference is triggered by the presence of small fragments of double-stranded RNA (dsRNA) whose sequence
matches a target gene. RNAi is is a highly potent and specific process developed to control viral replication,
retrotransposon movement and to recognize and to destroy aberrant dsRNAs.
RNAi that is applied in biotechnology as gene“knockout “ tool.
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Questions
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What are the differences in the structure of DNA and RNA?
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What is C-value paradox?
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Are transposons just genomic “parasites” or active participants in genome evolution?
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What are the subjects of studies in functional and comparative genomics?
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Are gene content and gene order conserved among plants?
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Is it possible to amplify DNA from a single cell using polymerase chain reaction?
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What is the major application of microarray technique in functional genomics?
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How DNA methylation affects gene expression?
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Can RNAi be used as gene“knockout “ tool?