genome organization & protein synthesis and processing in plants
DESCRIPTION
Genome Organization & Protein Synthesis and Processing in Plants. Viral genomes. Viral genomes: ssRNA, dsRNA, ssDNA, dsDNA, linear or ciruclar Viruses with RNA genomes: Almost all plant viruses and some bacterial and animal viruses Genomes are rather small (a few thousand nucleotides) - PowerPoint PPT PresentationTRANSCRIPT
Genome Organization & Protein Synthesis and Processing in Plants
Viral genomesViral genomes: ssRNA, dsRNA, ssDNA, dsDNA, linear or ciruclar
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.Replicative form of viral genomes•all ssRNA viruses produce dsRNA molecules•many linear DNA molecules become circularMolecular weight and contour length: • duplex length per nucleotide = 3.4 Å• Mol. Weight per base pair = ~ 660
Procaryotic genomes• Generally 1 circular chromosome
(dsDNA)• Usually without introns• Relatively high gene density (~2500
genes per mm of E. coli DNA)• Contour length of E.coli genome: 1.7
mm• Often indigenous plasmids are present
PlasmidsExtra chromosomal circular DNAs• 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• Multicopy plasmids (~ up to 400 plasmids/per cell)• Low copy plasmids (1 –2 copies per cell)• Plasmids may be incompatible with each other• Are used as vectors that could carry a foreign gene
of interest (e.g. insulin)
-lactamase
ori
foreign gene
Eukaryotic genome
• Moderately repetitive– Functional (protein coding, tRNA coding)– Unknown function
• SINEs (short interspersed elements)– 200-300 bp– 100,000 copies
• LINEs (long interspersed elements)– 1-5 kb– 10-10,000 copies
Eukaryotic genome• Highly repetitive
– Minisatellites• Repeats of 14-500 bp• 1-5 kb long• Scattered throughout genome
– Microsatellites• Repeats up to 13 bp• 100s of kb long, 106 copies• Around centromere
– Telomeres• Short repeats (6 bp)• 250-1,000 at ends of chromosomes
Eucaryotic genomes• Located on several chromosomes• Relatively low gene density (50 genes per mm
of DNA in humans)• Contour length of DNA from a single human cell
= 2 meters• Approximately 1011 cells = total length 2 x 1011
km• Distance between sun and earth (1.5 x 108 km)• Human chromosomes vary in length over a 25
fold range • Carry organelles genome as well
Mitochondrial genome (mtDNA)
• Multiple identical circular chromosomes
• Size ~15 Kb in animals• Size ~ 200 kb to 2,500 kb in plants• Over 95% of mitochondrial proteins are
encoded in the nuclear genome.• Often A+T rich genomes. • Mt DNA is replicated before or during
mitosis
Chloroplast genome (cpDNA)• Multiple circular molecules • Size ranges from 120 kb to 160 kb• Similar to mtDNA• Many chloroplast proteins are
encoded in the nucleus (separate signal sequence)
“Cellular” GenomesViruses Procaryotes Eucaryotes
Viral genome Bacterial chromosome
Plasmids
Chromosomes(Nuclear genome)
Mitochondrial genome
Chloroplast genome
Genome: all of an organism’s genes plus intergenic DNA Intergenic DNA = DNA between genes
Capsid
Nucleus
Estimated genome sizes
1e1 1e2 1e3 1e4 1e5 1e6 1e7 1e8 1e9 1e10 1e11 1e12
viruses (1024)
bacteria (>100)fungi
mitochondria (~ 100)
plants
mammals
Size in nucleotides. Number in ( ) = completely sequenced genomes
Size of genomes
Epstein-Barr virus 0.172 x 106
E. coli 4.6 x 106
S. cerevisiae 12.1 x 106
C. elegans 95.5 x 106
A. thaliana 117 x 106
D. melanogaster 180 x 106
H. sapiens 3200 x 106
Chromosome organizationEucaryotic chromosome
Telomere TelomereCentromere
Centromere: • DNA sequence that serve as an attachment for protein during mitosis. • In yeast these sequences (~ 130 nts) are very A+T rich. • In higher eucaryotes centromers are much longer and contain “satellite DNA”Telomeres:• At the end of chromosomes; help stabilize the chromosome• In yeast telomeres are ~ 100 bp long (imperfect repeats)• Repeats are added by a specific telomerase
p-arm q-arm
5’ – (TxGy)n3’ – (AxCy)n
x and y = 1 - 4n = 20 to 100; (1500 in mammals)
Gene classificationcoding genes
non-coding genes
Messenger RNA
Proteins
Structural RNA
Structural proteins Enzymes
transfer RNA
ribosomal RNA
otherRNA
Chromosome(simplified)
intergenic region
What is a gene ?• Definitions
1. Classical definition: Portion of a DNA that determines a single character (phenotype)
2. One gene – one enzyme (Beadle & Tatum 1940): “Every gene encodes the information for one enzyme”
3. One gene – one protein: “One gene contains information for one protein (structural proteins included) one gene – one polypeptide
4. Current definition: A piece of DNA (or in some cases RNA) that contains the primary sequence to produce a functional biological gene product (RNA, protein).
Coding regionNucleotides (open reading frame) encoding
the amino acid sequence of a protein
The molecular definition of gene includes more than just the coding region
Noncoding regions
• Regulatory regions– RNA polymerase binding site– Transcription factor binding sites
• Introns• Polyadenylation [poly(A)] sites
Gene
Molecular definition:Entire nucleic acid sequence necessary for the
synthesis of a functional polypeptide (protein chain) or functional RNA
Anatomy of a gene
• ORF. From start (ATG) to stop (TGA, TAA, TAG)
• Upstream region with binding site. (e.g. TATA box).
• Poly-a ‘tail’• Splices. Bounded by AG and GT splice
signals.
Bacterial 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
What would be the effect of a mutation in the control region (a) compared to a
mutation in a structural gene (b)?
Bacterial operon
Eucaryotic genes
Exon 190 bp
Exon 2222 bp
Exon 3126 bp
Intron A131 bp
Intron B851 bp
Hemoglobin beta subunit gene
Introns: intervening sequences within a gene that are not translatedinto a protein sequence. Collagen has 50 introns.
Exons: sequences within a gene that encode protein sequencesSplicing: Removal of introns from the mRNA molecule.
Splicing
Regulatory mechanisms
• ‘organize expression of genes’ (function calls)
• Promoter region (binding site), usually near coding region
• Binding can block (inhibit) expression• Computational challenges
– Identify binding sites– Correlate sequence to expression
Eukaryotic genes
• Most have introns• Produce monocistronic mRNA: only one
encoded protein• Large
Alternative splicing
• Splicing is the removal of introns• mRNA from some genes can be spliced into
two or more different mRNAs
“Nonfunctional” DNA
• Higher eukaryotes have a lot of noncoding DNA
• Some has no known structural or regulatory function (no genes)
80 kb
Types of eukaryotic DNA
Duplicated genes• Encode closely related (homologous)
proteins• Clustered together in genome• Formed by duplication of an ancestral gene
followed by mutation
Five functional genes and two pseudogenes
Pseudogenes
• Nonfunctional copies of genes• Formed by duplication of ancestral gene, or
reverse transcription (and integration)• Not expressed due to mutations that
produce a stop codon (nonsense or frameshift) or prevent mRNA processing, or due to lack of regulatory sequences
Repetitive DNA• Moderately repeated DNA
– Tandemly repeated rRNA, tRNA and histone genes (gene products needed in high amounts)
– Large duplicated gene families– Mobile DNA
• Simple-sequence DNA– Tandemly repeated short sequences– Found in centromeres and telomeres (and others)– Used in DNA fingerprinting to identify
individuals
Types of DNA repeats
Tandem repeats (e.g. satellite DNA)
Inverted repeats (e.g. in transposons)
5’-CATGTGCTGAAGGCTATGTGCTGCGACG- 3’3’-GTACACGACTTCCGATACACGACGCTGC- 5’
5’-CATGTGCTGAAGGCTCAGCACATCGACG- 3’3’-GTACACGACTTCCGAGTCGTGTAGCTGC- 5’ Stem
Loop
Palindroms = adjacent inverted repeats (e.g. restriction sites)• Form hairpin structures
• Form stem-loop structures
Hairpin
Perfect repeats vs degenerate repeats
Repetitive sequencesChromosomal DNA
Satellite DNA
Caesium chloridedensity gradient
Type No. of Repeats
Size Percent of genome
Highly repetitive
> 1 Mill < 10 bp 10 %
Moderately repetitive
> 1000 ~ 150 - ~300 bp 20 %
Repeats in the mouse genome
DNA repeats and forensics
878 bp556 bp
M F Suspect
Alu sequenceY
X
M F Suspect
528 bp199 bp
X-Y homologous regionsAluSTYa
AluSTXa
AluSTYa
Gender determination1) Standard technique: PCR
amplification of the amelogenin locus (Males = XY => 103 + 109 bp)
2) AluSTXa Alu insertion on X 3) AluSTYa Alu insertion on Y
Mobile DNA
• Move within genomes• Most of moderately repeated DNA sequences
found throughout higher eukaryotic genomes– L1 LINE is ~5% of human DNA (~50,000 copies)– Alu is ~5% of human DNA (>500,000 copies)
• Some encode enzymes that catalyze movement
Transposition
• Movement of mobile DNA• Involves copying of mobile DNA element
and insertion into new site in genome
Why?
• Molecular parasite: “selfish DNA”• Probably have significant effect on
evolution by facilitating gene duplication, which provides the fuel for evolution, and exon shuffling
RNA or DNA intermediate
• Transposon moves using DNA intermediate
• Retrotransposon moves using RNA intermediate
Types of mobile DNA elements
LTR (long terminal repeat)• Flank viral retrotransposons and retroviruses• Contain regulatory sequences
Transcription start site and poly (A) site
LINES and SINES• Non-viral retro-transposons
– RNA intermediate– Lack LTR
• LINES (long interspersed elements)– ~6000 to 7000 base pairs– L1 LINE (~5% of human DNA)– Encode enzymes that catalyze movement
• SINES (short interspersed elements)– ~300 base pairs– Alu (~5% of human DNA)
Proteins
• Most protein sequences (today) are inferred• What’s wrong with this?• Proteins (and nucleic acids) are modified• ‘mature’ Rna• Computational challenges
– Identify (possible) aspects of molecular life cycle– Identify protein-protein and protein-nucleic acid
interactions
Genetic variation
• Variable number tandem repeats (minisatellites). 10-100 bp. Forensic applications.
• Short tandem repeat polymorphisms (microsatellites). 2-5 bp, 10-30 consecutive copies.
• Single nucleotide polymorphisms
Single nucleotide polymorphisms
• 1/2000 bp. • Types
– Silent– Truncating – Shifting
• Significance: much of individual variation.• Challenge: correlation to disease
Yeast genome
• 4.6 x 106 bp. One chromosome. Published 1997.
• 4,285 protein-coding genes• 122 structural RNA genes• Repeats. Regulatory elements. Transposons.• Lateral transfers.
Yeast protein functionsRegulatory 45 1.05%Cell structure 182 4.24Transposons,etc 87 2.03Transport & binding 281 6.55Putative transport 146 3.40Replication, repair 115 2.68Transcription 55 1.28Translation 182 4.24Enzymes 251 5.85Unknown 1632 38.06