Genome Structure and Plasticity Flashcards
1
Q
how frequent are protein coding regions in eukaryotes?
A
they are rare, ~1.5% in humans
2
Q
are genes scattered or conserved throughout the genome?
A
they are scattered throughout, flanked and interrupted by non-coding DNA
3
Q
are coding regions or non-coding regions conserved across species?
A
coding regions are conserved across (related) species, but non-coding regions are not.
-> this suggests there is no constraint on changing the sequence - no function
4
Q
the majority of eukaryotic genomes have no known function. they are made up of two types of sequences, what are they?
A
- unique sequences like introns and non-repeated, non-coding intergenic DNA (including cis-regulatory elements)
- repetitive sequences
5
Q
exons are the ______
A
coding sequence and are broken up by introns that are non-coding and are spliced out at RNA level
6
Q
what is an example of a unique (non-repetitive) sequence within a genome?
A
introns or non-repeated, non-coding intergenic DNA (including cis-regulatory elements)
7
Q
what are the three types of repetitive sequences found in a genome?
A
- long, but low copy (duplications of parts of the genome)
- short, but many local repeats (microsatellites)
- intermediate in length, many copies scattered through genome (mobile DNA elements - transposons)
8
Q
80% of the bases of the human genome are…
A
- transcribed
- associated with modified histones
- found in open-chromatin areas
- binding transcription factors
9
Q
what % of the human genome is functional?
A
80%
10
Q
does the majority of eukaryotic genomes actually have no known function?
A
most of the genome is functional but we just have not studied it hard enough yet, but this is a controversial conclusion
- does not account for evolutional changes in the genome
11
Q
what was the original idea of how DNA is organized prior to mobile DNA elements?
A
all loci are placed in the genome in a linear and stable manner. this is true for most loci and explained classical (mendelian) inheritance
12
Q
Recently what addition has been made to the original idea that all loci are placed in the genome in a linear and stable manner?
A
some loci can excise themselves and re-integrate into a different genome location, these are called ‘jumping genes’, ‘transposons’ or ‘mobile elements’
13
Q
jumping genes, transposons, or mobile elements, make up what percent of the human genome?
A
approx 50%
14
Q
T/F: genome size and transposable elements correlate
A
true, larger genome size = larger TEs
- drives difference in genome size among organisms for the most part.
15
Q
what does difference in genome size depend on?
A
transposons not on how much protein coding genes there are.
16
Q
genetic elements that can move from one place in the genome to another undergo excision and reintegration. what can this lead to?
A
can lead to duplication of transposons in very large copy numbers which make up large fractions in genomes
17
Q
mobile DNA elements do not have an immediate physiological function and due to this have been considered what?
A
to be ‘endo-symbionts’, ‘endo-parasites’, ‘selfish genes’, or ‘junk DNA’
18
Q
despite mobile DNA elements being sometimes considered as junk DNA they can have huge implications in what?
A
genome evolution
19
Q
what are the two major categories of mobile DNA elements?
A
- transpose as DNA (DNA transposons)
- transpose via RNA intermediate (retro-transposons)
20
Q
what type of mechanism does DNa transposons use to move places in the genome?
A
cut and paste mechanism using transposase
21
Q
what type of mechanism does retro-transposons use to move places in the genome?
A
copy and paste mechanism involving reverse transcriptase and integrase
22
Q
how were transposons discovered?
A
colour studies on two maize mutants in the 1940s by barbara mcclintock
23
Q
what is gene C necessary for in maize?
A
for kernel colour formation
- c/c = white
- C/c or C/C = purple (wt)
24
Q
what does gene Ac (activator) do to white kernel maize mutants?
A
if AC is present in c/c mutants a higher frequency revert back to C/c and restore purple colour, this reversal can affect whole kernels or only sectors (the earlier in development reversion happens, the larger the purple sector)
25
what is the segregation pattern of white kernels in maize and what does it indicate about what causes the mutant?
1:3 segregation, indicating it is a single recessive gene mutation
26
what is the reversion of the mutation in gene C in maze caused by?
by dissociation of another element (called Ds - dissociator) from the C locus. Ds is no longer linked to C-locus in revertants and dissociation leads to mapping of Ds to another chromosomal location
27
what activates the re-location (or transposition) of Ds, creating a reversion of mutant colour variant maize back to the wt?
Ac activates re-locations. Ac also changes genetic location within the genome during a reversion
28
how is the Ac element related to the Dc element in maize colour mutants?
Ac element is autonomous where as Ds element is non-autonomous. The Ac element is a functional DNA-transposon that encodes transposase, this mediates Ds transposition. when Ds is transposed, the white mutation it creates in the C gene is reverted
29
what is the difference between the Ac and Ds elements?
Ds is a internally truncated version of Ac that lacks a functional transposase
30
why didnt barbara mcclintock receive a nobel prize for the discovery of transposons until 43 yrs after her work on maize?
she interpreted her results as a controlled gene regulatory mechanism, not a random process like it is
31
what was her hypothesis of how the colour mutation in maize was controlled, a hypothesis that turned out to be wrong...?
a gene control mechanism where if Ds is located away from the target gene, there is no repression but Ac activates Ds translocation into target gene to create repression. Ds translocation is reversible by Ac action leading to dissociation and no repression
32
do DNA transposons occur in proks or euks?
both, they have similar structure and mode of transposition
33
what does a DNa transposon encode?
encodes transposase and site specific endonuclease/DNA ligase to cut transposon out and integrate it into a new position
34
what is the three step process involved in the jumping of DNA transposons?
1) Transposase cuts the donor DNA blunt-end at ends of inverted repeats as well as cutting target DNA staggered creating sticky ends
2) Transposase ligates transposon into the target site
3) Cellular DNA polymerase and ligase fill in overhands and joint the ends.
35
what is the only amplification mechanism of DNA transposons?
amplification can only occur during replication of host DNA when transposition to an already replicated recipient site or to an un replicated recipient site
36
what was the first transposons discovered?
the Ac/Ds system affecting colour development in maize
37
what defines the freq of transposition for transposons?
the expression level of transposase
38
what are the two major classes of retro transposons?
1- retroviral like (contain long direct terminal repeats, LTR)
2. non-retroviral like (contain AT-rich regions at flanking sites, non LTR)
39
LTR containing retrotransposons resemble what?
retroviruses, they encode all proteins of common retroviruses except envelope proteins
40
what is the only protein that is not encoded in retrotranposons but is by retroviruses?
envelope proteins - these allow the virus to have the cell and enter a new host
41
what do RNA intermediates of retrotransposons lack following transcription from the LTR?
The promoter at 5' end (U3 part) and the U5 part at the 3' end
42
What is the LTR solution that corrects the loss of the promoter at 5' end (U3 part) and the U5 part at the 3' end during LTR replications?
A tRNA binds the 3' of U5, reverse transcriptase (RT) extends the 5' end of RNA. RNAse H (RH) degrades RNA in RNA/DNA duplex. R region of cDNA binds complementary R region at 3' end of RNA. RT extends to the new 5' end . RH degrades most of hybrid RNA. RT extends from remaining RNA to and forms dsDNA of 3' end. RH degreages remaining RNA hybrids. Primer binding site (PBS) region of second strand cDNA binds to the other PBS region on first strand. RT extends to form a complete dsDNA of the retro-transposon.
43
What is the most abundant type of retro-transposons in mammals?
non-LTR retro-transposon
44
What are LINE non-LTR Retro-Transposons?
Long interspersed elements (~6kb)
45
What are SINE non-LTR Retro-Transposons?
Short interspersed elements (~0.3kb)
46
what appox % do SINE non-LTR retro-transposons contribute to total genomic DNA?
~13%
47
what do SINEs contain?
have no coding regions but only flanking regions of LINEs. they use the LINE encoded machinery to amplify themselves
48
where is the promotor for non LTR's?
they are expressed from promotors outside of the actual transposon (they depend on the integration close to an endogenous promoter)
49
where are non LTR transposons found within the genome?
scattered throughout but hugely concentrated in a few genomic regions
50
where are non LTR's expressed from?
- promoters outside the actual transposon
- or from internal promoter (SINE)
51
What is the transcriptional start site for non-LTR transposons that is recognized by retro-transposition machinery?
a triple T sequence, any TTT sequence will be recognized
52
How does non-LTR transposition occur?
Chromosomal DNA and LINE NA are arranged in the Reverse transcriptase (RT), two nick sites are made and priming of RT by chromosomal DNA occurs. Reverse transcription of LINE RNA by RT begins until the staggard end is reached where RT switches from RNA template to DNA template. The insertion is completed by cellular enzymes.
53
Are the vast majority of transposons in eukaryotes complete?
No, the vast majority are non-autonomous or are complete relics and can't move any more.
54
What are two major disadvantages of having tons of 'Junk' DNA transposons?
1) Constitutes huge metabolic costs
2) Extends time needed for replication
55
What is a possible advantage of transposons?
They may act as a buffer against mutagens by making important regions less likely to incur deleterious mutations (dilute the genome)
56
Transposable elements are considered toys of evolution. What three things may they lead to?
1) Inactivation of target genes
2) Gene duplication
3) Mix-and-match of genes
57
Transposable elements are considered toys of biotech. What three ways can they be used for?
1) Tagged mutagen
2) Tool to make transgenic organisms
3) Gene therapy
58
What is exon shuffling?
Protein domains with distinct functions are frequently encoded on different exons, exon shuffling occurs via transposition and can combine exons from distinct proteins possibly creating new functions.
59
What are the three duplication types in transposons and gene duplication?
1) Whole genome, segmental, local duplications
2) Duplication as a passenger of a DNA-transposon or retro-transposon
3) Duplication as retroposon
60
What are the three possible ways that transposable elements can be located in gene duplication via transposition of two integrated DNA transposons and what are they termed?
1) On either side of the gene -> gene translocation or duplication
2) On either side of an exon -> exon shuffling
3) On either side of a regulatory element -> enhancer translocation/duplication
61
What are the two possible transpoasbale element (TE) organizations in gene duplication that can occur during transposition of an retro-transposons and what does it lead to?
1) TE located upstream of the gene -> gene duplication
2) TE located in an intron -> exon shuffling
62
Gene and Exon shuffling can occur via homologous recombination between transposable elements (TE). This involves double cross-overs between identical TE sequences. What are the two possible arrangements of the TE and what do they produce in this mechanism?
1) On either side of an intron -> exon shuffling
2) On either side of the gene -> gene translocation
63
How does gene duplication via retroposition occur using normal cellular gene transcripts?
reverse transcriptase of non-LTR transposons may act on any RNA with a polyA tail, 'hijacking' protein coding mRNAs.
64
What are the three ways that retroposons differ from other gene-duplicates?
1) Direct repeats and A-tail remainder
2) No introns
3) No regulatory elements duplicated
65
1) Direct repeats and A-tail remainder
2) No introns
3) No regulatory elements duplicated
1) Needs to be inserted completely
2) Must originate from a gene expressed in germ line
3) Gain regulatory elements by being integrated close to or into an existing gene
4) New regulatory modules must be advantageous
66
What is the most likely fate of duplicated genes?
Loss of function becoming a pseudogene
67
After duplication there may be degeneration of different modules in each copy, can function be maintained?
Duplicated genes together fulfill function via maintaining (purifying) selection acting on both copies
68
How can gene duplication lead to neo-functionalization?
Acquisition of new module(s) in one copy may provide novel function under positive selection while the original gene maintains original function via purifying selection.
69
What are the three hallmarks of retroposons?
1) No introns
2) Have a homolog in the same genome with introns (the 'parent')
3) Different expression pattern that 'parent'
70
How was the human genome screened for putative retroposons (retrocopies)?
All protein coding sequences were similarity searched against the complete human genome, hits without gaps were considered candidate retroposons. These were then similarity searched against all protein coding sequences and hits with >50% similarity with gaps are considered to have a putative parent and so considered a putative retroposon.
71
How can researchers determine how old retroposons are, ie how long ago did they duplicate from their 'parent'?
As mutation rates at silent sites (areas that don't code amino acids) should be constant, the sequence can be compared to the parent and estimate when the two genes diverged.
72
Why is it harder to trace divergence of duplicate genes the further you go back in time?
Because the constant mutations at the silent sites will eventually diverge so much that any similarity is not detectable.
73
By estimating the age of candidate retroposons found in the human genome, what approximate time did a burst of transposition occur within the primate linage?
Burst of transposition occurred 38-50 million years ago just after primates diverged from other mammals.
74
What four (a bit twisted) assumptions were made by researchers when trying to determine how many of the candidate retroposons they found in the human genome gained an 'important' function?
1) The new regulator elements the retroposon gained drive new expression patter
2) If the duplicate is not important the coding region should evolve neutrally
3) If the functionality of the original protein is adaptively important in both the old and the new then the coding region of both should be under purifying selection.
4) If the duplicate in addition to its new expression also changes its protein function then the coding region of the retroposon should be under positive selection and the coding region of the parent should be under purifying selection.
75
What are the three assumptions when trying to test for purifying, neutral, or positive selection?
1) Natural selection acts on proteins, not on genes
2) Silent mutations cause no change in protein sequence and their frequency should be the same regardless of selection pressure on the protein
3) Amino acid changing mutations likely change property of proteins either being under purifying or positive selection for functionality.
76
How can researchers test for purifying, neutral or positive selection?
By comparing the number of silent mutations with the number of amino acid changing mutations (both need to be normalized for the possible number of their occurrence)
77
What is the ratio used to test for purifying, neutral, or positive selection?
Ratio of Ka/Ks (also called ω). ~1 = neutral evolution, <1 = purifying selection, >1 = positive selection.
78
what does ~1 = neutral evolution mean?
it does not matter if a mutation does or does
not change the coding region, both silent and aa changing mutations should be equally frequent
79
what does <1 = purifying selection mean?
silent mutations are more frequent than aa changing mutations, bcuz aa changing mutations have been selected against
80
what does >1 = positive selection mean?
aa changing mutations are more frequent than silent mutations, bcuz aa changes have been selected for
81
How can extinct gene sequences be reconstructed in order to understand how two retroposons are related?
Extinct sequences may be reconstructed based on phylogeny.
82
How can relatedness of two genes be determined easily without the need to reconstruct extinct sequences using phylogeny?
Gene one and two can be compared directly, but only if the ratio of Ka/Ks between them is <0.5 (the average of Ka/Ks for both genes against a extinct gene)
83
What type of selection is over-represented in modern human intact retroposons?
purifying selection
84
How many retroposons are fixed under purifying selection in primate lineage in the past 63 million years?
76
85
Why are most of the 76 retroposons found to be fixed under purifying selection in primate lineage predominantly expressed in male germ line tissues (testis)?
Genes on the X chromosome are silenced during haploid stages of spermatogenesis, the retroposons on autosomes take over functions during spermatogensis of X-chromosome encoded genes.
86
What are the four things that transposons and/or transposition may lead to?
1) Gene inactivation
2) Gene translocation
3) Exon shuffling
4) Gene duplication
87
What are the three things that may happen to duplicated genes?
1) Loose their function
2) Divide function with parental gene (subfunctionalization)
3) Gain a new function (neofunctionalization)
88
what are retro-copies?
Copies of 'normal' transcripts generated by retrotransposon machinery
89
What are the three ways that ancient whole genome duplication can be identified?
1) Analysis of complete genomes
2) Comparative genome mapping
3) Micro-colinearity of genes (synteny)
90
When do whole genome duplication most often occur?
Right at or soon after a large divergence in lineages.
91
What is the only specific phenotypic trait that is caused by whole genome duplication?
body size
92
What evolutionary advantage does whole genome duplication provide?
It may allow rapid evolution of a whole genome while a 'back-up' genome is still present.
93
What is sentimental duplication?
Duplications that involves only a small portion of the genome, up to 50kb frequent, that mainly result from replication errors and inexact DNA-break repair frequently arranged in tandem in genome.
94
The antarctic fish Notothenia coriceps can survive freezing. It does this by producing antifreeze proteins in the pancreas that is rich in what two amino acids?
alanine and threonine
95
To find the antifreeze gene in antarctic fish, researchers used a labelled DNA probe to look for the previously sequence Ala Ala Thr repeat regions in the gene. What special nucleotide substitute did they used?
Inosine which pairs with A, C, or G.
96
After finding the antifreeze gene, researchers did a similarity search for the parent gene that may have given rise to it. What did they find?
No overall similarity with other genes but a small portion was very similar to pancreatic trypsinogen (precursor of an enzyme used for protein digestion).
97
What are the major differences between AFP(antifreeze protein) and trypsinogen?
AFP has two exons only and the second is mostly repetitive DNA that encodes AAT over and over where as trypsinogen has six exon, that only the UTRs and exon 1 and 6 similar to AFP. But Trypsinogen has the same repetitive DNA as AFP just upstream of and into exon 2.
98
How did AFP (antifreeze protein) evolve from the trypsinogen gene?
The new gene 'used' formerly intronic DNA to encode a new protein.
99
What created a premature STOP codon in the AFP (antifreeze protein)?
'incorrect' fusion of exon 6 to tandem repeats created a frameshift mutation and resulted in a premature STOP.
100
Despite the remarkable differences between AFP (antifreeze protein) and trypsinogen, the authors of the paper believe the gene duplication event was quite recent. What data supports this hypothesis?
The UTR region is very similar between the two genes, as these regions are not under selective pressure mutations happen at a constant rate and will diverge relatively rapidly.
