RB Eukaryotic Genome Architecture Flashcards
What are interspersed repeats?
A sequence which is repeated at different locations throughout the genome
What is the C-value?
The amount of DNA in a haploid nucleus for a given species
Define the C-value paradox
The “complexity” of an organism does not correspond to the genome size
Why do human genes contain a considerable amount of intron sequence?
Humans spend a longer time in cell division so there is less selective pressure to get rid of long introns and “junk DNA”
What are satellite DNAs and what are the three categories?
Tandemly repeated sequences of 1-500 bp DNA
- Microsatellites: defined as 1-13 bp
- Minisatellites: defined as 14-100 bp
- Satellite DNA: 100-500 bp, especially important at mammalian centromeres
Why are there differences in the satellite DNA between individuals?
- Replication slippage
- Unequal crossing over during meiosis
How does replication slippage occur?
- Dissociation of the DNA polymerase during replication
- Nascent DNA strand can rehybridise with another repeat in the array, in a misaligned way; i.e. with an identical repeat earlier or later in the array
- Replication continues, yielding a daughter strand that is longer or shorter than the template
How does unequal crossing over during meiosis affect satellite DNA length?
Crossover between misaligned repeats on sister chromatids results in one gamete with more copies and one gamete with less copies of the repeat
Give an example of a problem caused by satellite DNA
- Trinucleotide repeat (CAG) in the Hungtintin gene (HTT): polyglutamine tract
- Proteins with expanded CAG repeats are degraded into toxic fragments that accumulate in neurones and stop them working properly
- Very nasty neurodegenerative disorder with onset in adulthood
- Dominant inheritance
How is DNA fingerprinting carried out?
- Extract DNA
- Digest with a convenient restriction enzyme
- Separate fragments on an agarose gel
- Southern blot using minisatellite sequence as a probe
- Observe characteristic bands for each individual (two alleles
for each locus in a diploid individual) - Do this for a number of minisatellite sequences
What are the two families of transposons?
DNA transposons (cut and paste) and retrotransposons (copy and paste)
What is a direct repeat?
The same sequence repeated in two places
What is an inverted repeat?
A sequence plus its reverse complement
What is the difference between Ac and Ds transposons?
- Ac: “activator”; autonomous - has its own transposase gene
- Ds: “dissociation”; non-autonomous - needs to use transposase enzyme from Ac, therefore has same inverted repeat sequences as Ac
What is the difference between Ac and Ds transposons?
- Ac: “activator”; autonomous - has its own transposase gene
- Ds: “dissociation”; nonautonomous - needs to use transposase enzyme from Ac, therefore has same inverted repeat sequences as Ac
What are P elements?
- DNA transposons found in many modern wild Drosophila (P+), but not in lab strains, many of which are descendants of flies from TH Morgan’s lab (P-)
- Therefore, P elements have arisen in the wild populations since the early 20th century
- When left unchecked, these elements transpose at very high rates and lead to severe problems in the offspring: very high mutation rates, infertility
- A silencing mechanism in the cytoplasm limits transposon movement in P+ strains but not P- strains
- In the new embryo, this cytoplasm comes from the egg, i.e. the mother
What are the three main families of retrotransposons?
- LTR (long terminal repeat) transposons
- LINES (long interspersed elements)
- SINES (short interspersed elements)
What are the components of LTR transposons?
- pol: encodes three protein activities required for transposition: reverse transcriptase, RNAse H, integrase
- LTRs: important for the reverse transcription mechanism
- Direct repeats: generated upon integration
Describe the mechanism of LTR transposon transposition
- Generation of an RNA molecule and of protein products by the host polymerase machinery
- Complex reverse transcriptase mechanism involving the retrotransposon reverse transcriptase (creates cDNA from the mRNA template) and RNAseH (degrades the RNA template)
- A double stranded DNA molecule is formed
- Transport of the dsDNA into the nucleus – guided by integrase bound to the LTRs
- Insertion of the dsDNA into the genome, with creation of target-site direct repeats
What are the components of LINEs?
- A/T rich region
- ORF1: RNA-binding protein
- ORF2: reverse transcriptase and DNA endonuclease
- Target site direct repeat
Describe the mechanism of LINE transposition
- Transposon is transcribed and translated by host machinery, polyadenylated
- ORF1 protein binds LINE RNA, ORF2 protein binds LINE polyA (in the cytoplasm)
- RNA is transported into the nucleus
- ORF2 / polyA RNA binds to complementary polyT DNA sequence somewhere in the genome
- Endonuclease activity from ORF2 nicks the DNA
- ORF2 reverse transcriptase activity, primed by host DNA sequence
- ORF2 continues synthesis, now using host DNA as a template
- A second strand of DNA is made by host enzymes
- Direct repeats are generated due to staggered cut at the insertion site
Why are LINE transposons often truncated?
Often, reverse transcription does not reach the end so the transposon is truncated
Why do SINEs require enzymes from LINEs to function?
- They are non-autonomous
- They contain A/T-rich sequences that bind to ORF1/2 from LINEs
What are the evolutionary consequences of transposition?
- Exon shuffling: due to crossing over between transposons in different parts of the genome or mistakes in transposition
- Gene duplication via replication slippage, unequal crossing over or retrotransposition of an mRNA
Define orthologues
The same gene in two different species, e.g. mouse and human alpha-tubulin; these genes have been evolving separately since the divergence of mouse and human evolution
Define paralogues
In the same species, when a gene has been duplicated and then differences accumulate in the two copies, e.g. mouse alpha- and beta-tubulin; they arose from a duplication of an ancestral tubulin gene
What are the possible fates of a duplicated gene?
- Degradation: accumulation of mutations result in a pseudogene (lost the ability to code for a functional protein)
- Neofunctionalisation: one copy gains a new function
- Subfunctionalisation: each copy specialises
What is the difference between conventional and processed pseudogenes?
Conventional pseudogenes are generated through the accumulation of mutations, whereas processed pseudogenes are generated via LINE proteins
Why does foetal haemoglobin have a higher affinity for oxygen than adult haemoglobin?
To allow oxygen to be passed from maternal blood to foetal blood
How has the globin gene family evolved?
- Unequal crossing over between two transposons
- Chromosome with two beta-globin genes passed on in the germline
- The two copies evolve independently to generate paralogues
Define meiotic nondisjunction
The failure of sister chromatids to separate during meiosis I or II
How can polyploid organisms arise?
Through meiotic nondisjunction, sometimes followed by combination of different gametes
What is a centromere?
The specialised chromosomal region upon which kinetochores assemble and direct the equal segregation of chromosomes during mitosis and meiosis
Describe the properties of yeast “point” centromeres
- Around 120 bp
- Consist of three regions: I, II and III
- Region II is very AT rich
