Genome Features and Transposition Flashcards
What makes up a genome?
- DNA or RNA
- Genes- encode biological information
- Genome replication/segregation – to allow genome to be inherited
What is c-value?
• Amount of DNA/number of kilo-bases per haploid cell
What types of organisms have the smallest genomes?
• Viruses
How does polarity of virus genomes affect transcription?
- +ve can be translated immediately by host machinery
- –ve requires synthesis of +ve RNA using viral encoded RdRp
What can make up the viral genome?
- Genes for genome replication (e.g. polymerase)
- Genes for coat proteins
- Genes that reflect different life history (lytic vs lysogenic)
What characterises prokaryotic genomes?
- Uni or multi-chromosome number (in nucleoid)
- Circular mostly, sometimes linear
- Small
- No histones
- High gene density
- Most non coding regions for gene regulation (e.g. promoters)
- Not much repetitive DNA
- Operon structure
- Genome size varies but gen density relatively constant
- Chromosome type and number varies
- Sometimes plasmids part of genome
What other type of genetic material can exist in prokaryotes? What does it encode?
- Plasmids
- Extra chromosomal
- Coexist with main chromosome
- Range of functions encoded
- Dispensable for host function (usually) – not essential to live
Are all plasmids dispensable?
• No, some are considered part of genome
How can the genome be arranged in prokaryotes?
• Loops anchored to protein core then supercoiled to release tension stress
What are non-coding regions associated with in prokaryotes?
• Gene regulation
How does gene density differ among bacteria?
• Relatively constant
What makes up the eukaryotic genome?
• Nuclear and organelle genomes
What are features of the chloroplast genome?
- All circular
- Ds supercoiled DNA
- Similar size and gene content to land plants
- Most genes for photosynthesis
- Chloroplast enzyme subunits encoded for by nucleus
- Operon-like clusters of photosynthetic and ribosomal genes
- Use standard genetic code
What are features of the mitochondria genome?
- Most genomes circular
- Ds supercoiled DNA
- Bigger variation in size but similar gene content to metazoans
- Human mtDNA has codes for oxidative phosphorylation
- Non-standard genetic code
- Yeast mtDNA less compact because of introns and inter-genic spaces
What are nucleoids?
- In organelles, house the genomes
- Variable number of chromosomes inside
- Variable number of nucleoids per organelle
What are features of the nuclear genome?
- Multi chromosome (one two or more copies)
- Linear DNA molecules
- Chromosome number varies but no link to complexity of organisms
- Genome size doesn’t link to complexity
Does genome size correlate with chromosome number in eukaryotes?
• No
How are euchromatin and heterochromatin different?
- Euchromatin (stain light, less condensed, easy access)
- Heterochromatin (stain dark, very condensed, hard access)
How are Facultative heterochromatin and Constitutive heterochromatin different?
- Facultative heterochromatin for different cells expressing different genetic material
- Constitutive heterochromatin for same genetic material in each cell
What is the C value paradox and what does it suggest about eukaryotes?
- Amount of DNA in haploid cell not precisely related to evolutionary complexity
- Similar organisms can differ greatly in genome size
- Suggests variable gene density in eukaryotes and other elements in genome
What are the types of inter-genic sequences?
• Unique and repeated
What are the types of repeated inter-genic sequences?
• Tandem repeats and interspersed
What constitutes the nuclear genome of a human?
- 46% repetitive DNA
- 35% Unique DNA
- 5% introns
- 1.5% Protein-coding DNA
How can genomes vary? How can this be measured?
- By base composition (melt curve analysis, density gradient centrifugation)
- By complexity (re-association kinetics)
- By gene composition (nucleic acid hybridisation)
What is Tm?
• Temperature at which half DNA is ss
What is the relationship between Tm and GC content?
- More GC content = larger Tm
- Stronger bonds so harder to melt
How can melt curves differ between species?
- GC composition differs between species
- Even if same GC composition, nucleotide distribution differs so still different curves
What is the relationship between GC content and density?
• High GC content = higher mass/density (an extra nitrogen makes it heavier)
What happens in density gradient centrifugation?
- High speeds (centrifuge), different density molecules separate into discrete bands in salt gradient
- Distinguish DNA conformation/base composition
- Supercoiled DNA (plasmid, organelle) more dense than linear so lower down
What is satellite DNA?
- Low density bands = satellite DNA
- Highly repetitive DNA, low GC content (AT rich)
What is special about density gradient centrifugation on animals?
• Animals can have distinct bands (satellite DNA)
Does GC content vary more in Eukaryotes or Prokaryotes? Why might this be?
• Varies most in prokaryotes (environments vary a lot) • Doesn’t vary much in eukaryotes
What happens in re-association analysis?
- Cool fragmented DNA slowly
- Complimentary sequences eventually reassociate/re-nature by random collisions
What is re-association dependent on?
• Dependent on concentration of DNA and follows second order kinetics and on complexity of genome
What is a C0t curve? How is it constructed?
- C0t = DNA concentration (C0) multiplied by incubation time (t)
- Smooth curve = gradual re-annealing
What role does repetitive DNA have in C0t curves?
- Lots of copies of sequence = faster re-nature time because more likely to find each other (smaller C0t)
- Not many copies of sequence = slower re-nature time because harder to find each other (higher C0t)
- Presence of repeated DNA sequences alters shape of curve
Why can c0t curves vary between species?
- Differs between species (larger genome = longer re-association time)
- Shape same though
What do uneven C0t curves indicate?
• Uneven curve suggests presence of different classes of DNA (highly repetitive, moderately and single copy DNA. Common for eukaryotes)
How can repetitive DNA be arranged?
- Tandem- units next to each other
- Interspersed- units randomly distributed in genome
What composes most non-functional DNA? What can it account for?
- Majority of non-functional DNA = transposons (account for c-value paradox) What is a probe?
- Purified labelled small ss RNA or DNA for visualisation
What happens in nucleic acid hybridisation?
- Have complex mix of nucleic acid molecules (denature, ss DNA/RNA, attached to matrix)
- Have labelled probe (purified nucleic acid, ss DNA/RNA)
- Hybridise and wash and have conditions for annealing of complimentary sequences
- Detect nucleic acid duplex
How can nucleic acid hybridisation be applied to chromosomes?
- Chromosome in situ hybridisation
- Make chromosomes ss DNA, hybridise with labelled probe, anneal
- E.g. use mini-satellite DNA as probe, telomeres fluoresces
How can nucleic acid hybridisation be applied to DNA fragments?
• Southern blot hybridisation
What are the steps of southern blot hybridisation?
- Cleave purified DNA, separate by size on gel (restriction enzymes and gel electrophoresis)
- Transfer ss DNA to membrane (capillary blotting), anneal to probe
- When incubated with ss probe and washed several times, can show distribution of labelled elements (e.g. transposons)
Where in the eukaryotic genome are tandem repeats found? Where are interspersed repeats found? What types of chromatin are involved?
Tandem repeats at telomeres, centromere
- Constitutive heterochromatin
Between telomeres and centromere
- Interspersed repeats -Transposable elements
- Unique sequences (introns/exons)
- Tandem repeats
- Facultative heterochromatin
- Euchromatin
Why is selfish DNA named so?
- Mechanism to maintain non-functional DNA (no selective pressure)
- Molecular parasites- replicate and increase number at cost of host genome
What can act as transposable elements in eukaryotes and prokaryotes?
Prokaryotic TE
- Insertion sequences, transposons
Eukaryotic TE
- DNA transposons, retro-transposons
What are two possible requirements for replicative transposons?
- DNA replication
- RNA intermediate
How do the two types of transposition vary?
Replicative
- Copy and paste
- Copy TE to new location, original remains
- Rapidly increase in number
- Possible Requirements
- DNA replication
- RNA intermediate
Conservative
- TE moves to new location
- No DNA replication
- No net increase in number
How can conservative transposons increase in numbers?
- Most active during DNA replication
- Can increase in numbers by jumping ahead of replication fork (increase copy number)
What molecular events occur during conservative transposition?
- Transposase cleaves at IR (4 cuts needed to excise the TE)
- Transposase carries transposon to target site
- Transposase cleaves target site with staggered cuts (the overhang is repaired at the end and this forms the DR)
- Transposon inserted to gap and DNA gap repair occurs
What molecular events occur during replicative transposition?
- Donor plasmid cut by Transposase at the IR (2 Cuts)
- Transposase cuts target plasmid target site using staggered cuts (lead to DR later)
- Cleaved strands of each plasmid link up and co-integrate is formed by DNA replication fixing single stranded bits
- Resolvase resolves the co-integrate to form and original and new plasmid, both with copies of the transposon
How do direct repeats form?
• When staggered cuts are made by Transposase, DNA replication fills in the gaps later
What are consequences of transposition on gene activity?
- Can be altered because of random integration
- Inserted to non-regulatory region: no effect
- Inserted to regulatory region: alter expression, reduce activity
- Inserted into gene itself: inactive gene, truncated transcript
What are prokaryotic insertion sequence elements?
- Terminal inverted repeats (TIRs)
- Only encode genes for mobilisation and insertion to new place (TRANSPOSASE)
- In bacterial chromosome and integrative plasmids
What are the features of prokaryotic transposons? • More complex than IS elements
- Terminal inverted repeats
- Genes are for mobilisation and other unrelated functions
- Some encode RESOLVASE for replicative transposition
What types of transposons are there and how do they differ?
Simple Tn
- The small flanking IRs are not IS elements (TIR)
Composite Tn
- The flanking IRs are IS elements
What are eukaryotic Class II TE? What is an example?
- DNA transposons
- Example are P elements from drosophila (P in m cytotype)
- Perfect TIRs and encode Transposase
How can Ds move if it is non-autonomous?
- Activator/Ac is functional Transposase and autonomous element
- Disassociation/Ds is non-functional Transposase and non-autonomous element (can’t move by itself)
- Ac encodes Transposase to recognise IRs of Ac and Ds, so Ds can move around
- Transposase sequence specific and act on elements from same family
What are eukaryotic class I TE?
- Retro-transposons
- Like retrovirus
- Suggests transposition occurs via RNA intermediate
How are viral like retro-elements different to a retrovirus?
- Retrovirus has Long Terminal Direct Repeats (LTRs), gag (integrase), pol (reverse transcriptase), env (coating proteins)
- Viral like retro-elements have LTRs, pol, gag, but no env
How are retro-transposons transposed?
- RNA intermediate copied into dsDNA by pol (RT)
- dsDNA inserts randomly into the genome (copy paste)
- Original element
- Transcription to RNA transcript and translation of pol and gag
- Reverse transcription to ssDNA copy using pol to form dsDNA copy
- Insertion using gag
How can retro-transposons be eliminated?
- Retro-transposon insertions often unstable
- Reversion can occur due to recombination between LTRs
- LTRs match up, form loop, eliminate retro-transposon in-between
What are the Mammalian retro-elements?
- Long Interspersed Elements (LINE)
- Short Interspersed Elements (SINE)
- E.g. Alu
What are non-viral like retro-elements?
- Less like retrovirus
- No LTR, may have reverse transcriptase
- Can be derived from eukaryotic genes
What is special about SINEs?
• Non-autonomous, require pol from LINEs
What kind of organism have a lot of TE in their genome?
• Eukaryotes, but the TE are often stationary and don’t increase in copy number
What makes up the eukaryotic genome? What characterises these DNAs?
Functional DNA
- Under selective pressure
- Scales to organism complexity (gene and regulatory elements)
Non Functional DNA
- Not under selective pressure
- Varies between organisms (repetitive elements 50% of human genome)
Junk DNA
- Not much adaptive advantage
- But shape genome (rearrangements, gene shuffling, gene expression patterns)
How can transposons shape the genome? How does it vary between the same or different chromosomes?
- Homologous recombination between transposons can cause genome rearrangement
- Same chromosome: deletion, inversion
- Different chromosomes: deletion, duplication, translocation
