Vl 18 (George Soultoukis) Flashcards
Important Key terms
Transposable element (TE):
a mobile genetic element capable of self duplication and migration
Transposase:
a protein complex catalysing the transposition of a transposable element
Role of TEs in the genome evolution context (e.g. homologous recombination and allelic polymorphism)
Class I TE:
self-replicating transposons
Class II TE:
self-excision and migration transposons
TE mutagenesis and disease caused by transposition events
Repetitive DNA sequences: tandem repeats in a genomic region or dispersed elements across genome
Satellites, microsatellites, and minisatellites are tandem nucleic acid repeated elements
Transposable Elemts
- TEs (transposons) are discrete sequences in the genome that are mobile
–> TEs are capable of transporting themselves to other locations in the genome without an independent intermediate (plasmid or virus) but directly from one site to another
- Sequences of DNA coding for DNA-manipulating proteins and related regulatory sequences
–> can result in mutations e.g. when not in triplet transfer: insertions, inversions, deletions, translocations.
–> generate “portable regions of homology” thereby providing sites for reciprocal recombination - Transposition intermediates are „transposomes“:
complexes of proteins/nucleic acids/Mg2+ (active) - Found in all organisms, might have originated from LUCA or spread across life soon after
- Inactive TEs found as introns across genome
–> can therefore change the structure of genes, by changing the gaps between exons, and increasing or decreasing the probability of homologous recombination, and therefore the evolution of new gene products.
How to classify transposons?
- TE classification by mechansim
–> replicative vs. conservative
Class I TE = Retrotransposons
–> characterised by “copy and paste” mechanism
–> such DNA duplications or transpositions can result in gene duplication, which plays an important role in genomic evolution
Class II TE = DNA transposons
–> utilising a “cut and paste” mechanism
Explain Class I (Retrotransposons) in detail
- unfolding the DNA double strand and transcribing the TE in both strands to generate ssRNA.
- synthesis of reverse transcriptase (in the transposon) and its binding to the newly synthesised ssRNA results in reverse transcription of ssRNA into ssDNA
- ssDNA is converted to dsDNA with DNA polymerase. The complex of the two copies of the transposon are crossed over by transposase, and along with the original DNA replicons they form the cointegrate.
- final integration step involves reactions that “nick” or cut the dsDNA in the target locus and proteins called “integrases” that are responsible for catalysing the ligation of the copy TE into the new host DNA in the target locus.
–> The process results into a new copy of transposed DNA, with the original templated DNA sequence of the transposed element intact (“copy and paste” mechanism)
44% of human genome
Explain Class II (DNA Transposons) in detail
- nicking of the parent dsDNA sequence that is targeted for transposition, producing sticky ends (staggered cut).
- Transposases then remove the cleaved dsDNA from the parent double strand and transfer it to the new target locus which is cleaved to produce sticky ends.
- The transposed DNA is integrated into the new target DNA locus by enzymes called ligases, which join the transposed dsDNA to the new target dsDNA.
- insertion sites of DNA transposons may be identified by short direct repeats followed by inverted repeats.
2% of human genome
What are autonomous TEs
- Autonomous TEs can move by themselves
—> independent transposition
Commonly grouped into 2 main gropus:
1. Retrotransposons with long terminal repeats (LTRs)
–> have LTR sequences at ther 5´and 3´ends sequenced. (green box with yellow triangle) and are further classified according to the order of their coding proteins: capsid protein (CP), protease (PR), integrase (IN), reverse transcriptase (RT), and RNaseH (RH)
–> Ty1-copia
–> Ty3-gypsy classes
2. Retrotransposons without LTRs but with long interspersed nuclear elements (LINEs, LINE-1s, or L1s)
–> Non-LTR autonomous retrotransposons lack LTR sequences and contain long interspersed nuclear elements (LINEs)
–> have a terminal inverted repeat (TIR) at both ends (represented as a blue triangle), and a transposase sequence
Helitrons are replicative DNA transposons that contain a helicase and replicase sequence
What are non-autonomes TEs?
Non-autonomous TEs cannot transpose alone, but require additional autonomous elements
Class I
–> lacks reverse transcriptase
class II l
–> lacks functional transposase (red line is a break in the transposase sequence, or MITE)
- Include terminal repeat retrotransposons in miniature (TRIMs) and large retrotransposon derivatives (LARDs)
–> Class II transposases can be either
(i) targeting specific DNA sequences or
(ii) non-specific or indiscriminate in the DNA sequences they transpose
- The process results into a single copy of transposed DNA removed from the parent location, and integrated into a new target location (“cut and paste” mechanism)
Transposable elemets are genome function
** TEs affect gene transcription via insertion into promoter regions and change gene structure by altering splicing (transcription affected) and polyadenylation (transcription and translation) patterns**
- TEs can provide an important mechanism for gene duplication („jumping“ genes), mutation, chromosome recombination, and genomic rearrangements, and are therefore considered „mutagenic“
- Can cause codon (triplet code) mutations and exon damage, or exon shuffling (inversions)
- A transposition event can be beneficial or detrimental to the host genome and organism: inactivate a necessary gene or confer a selective advantage by a new duplicated gene function
- TEs can also alter TF-binding DNA sequences, e.g. promoters, thereby having subtler gene expression effects, or affect expression of downstream genes by inducing higher expression through their own promoters
- TEs can result in DNA breakage that is left unrepaired, results in chromosome breakage and chromatin failure
- Multiple sequence copies (homologous regions) can result in unequal crossovers in chromosomal recombination during mitosis or meiosis
- Genome-residing parasitic „selfish DNA“ that promoted natural selection of host organisms in rare cases
- Duplication of detrimental alleles can exacerbate a disease or cause new disease phenotypes in the host
Give an Example for TE disease
Porphyria also know as “vampire disease”
- Liver disorders that result in accumulation of red-purple pigments called porphyrins in the skin and nervous system
- Acute or mild forms, with an incidence of around 1:50,000 individuals
- Insertion of TE (Alu) element found to be responsible in cases of acute intermittent porphyria
–> An Alu element is a short stretch of DNA originally characterized by the action of the Arthrobacter luteus (Alu) restriction endonuclease.
–> Alu elements are the most abundant transposable elements, containing over one million copies dispersed throughout the human genome.
–> Alu elements were thought to be selfish or parasitic DNA, because their sole known function is self reproduction.
–> However, functionally and evolutionary important. - Symptoms and severity vary a lot, in extreme cases.
Defence mechanisms against TEs
- Cells can defend against the proliferation of TEs using DNA methylation, which silences majority of TEs in the human genome
- piRNAs (26-31nt piwi-interacting RNAs) target retrotransposons and silence them by translational repression, or transcriptional silencing complexes, but can also lead them to RNA degradation pathways
- siRNAs (20-24nt) can also silence TEs after they have been transcribed, but mostly lead retrotransposons to RNA degradation
- miRNAs (21-24nt) can also participate in TE silencing and degradation
- Although small RNA molecules can silence or degrade TEs, TEs also result in new small RNA molecules including miRNAs and siRNAs, thereby resulting in changes in gene expression regulation
What are DNA repeats?
Repeated DNA sequences are patterns of nucleic acid bases that occur in multiple copies throughout the genome.
- When multiple occurences of a nuclei acid sequence is observed in a genome
- Repetitive elements found in genomes fall into different classes, depending on their structure and/or the mode of multiplication
- Such repetitive elements can be found in a tandem array or in a dispersed (interspersed) random pattern
- Used to be referred to as “junk DNA” but now there is evidence for regulatory roles, for example it is thought that tandem DNA repeats positions can be used by the cell as a genome folding map for chromatin condensation during chromosome separation in mitosis or meiosis, and can be enriched in heterochromatin (the condensed chromatin).
- TEs can result in mutations e.g. when transferred not in triplet-code within a protein coding DNA sequence or near-by : insertions, inversions, deletions, translocations.
What is satellite DNA detection?
Repetitive satellite DNA is small (low mass and density) and produces bias in the frequency of the 4 bases
–> A+T-rich satellite DNA has lower density while G+C-rich satellite DNA has higher density
(AT base pairs have a lower molecular weight and therefore lower density than GC base pairs, which have a higher molecular mass and density.)
- Density gradient centrifugation separates DNA fragments with significantly different base compositions
- The main band representes the bulk DNA and the „satellite“ bands originate from tandem repeats
- DNA concentration across the gradient is analysed by differential fraction analysis of spectrophotometric absorbance
- The main band absorbs at a higher density fraction, while the two satellite bands absorb at lower fractions
Today satellite DNA can be derived computationally through NGS genome data
Picture:
* Sample fractionation is a common technique for separating a liquid phase (sample) into smaller fractions, whereby individual aliquots are generated sequentially by taking up a small volume multiple times until the entire centrifuged (gradient-positive) sample has been separated in different sample tubes each one containing a different gradient fraction.
* Here this is done without disturbing the density gradient, often these days by automatic sample fractionation.
* These fractions can then be analysed separately by spectrophotometry.
–> x-axis shows grams per ml,
–> y-axis shows wavelength absorbance units
* Here bulk DNA has the highest mass, migration down the gradient, and spectrophotometric absorbance.
* G+C-rich satellite has lower mass, migration down the gradient, and absorbance seen as the second smaller peak.
* T+A-rich satellite has the lowest mass, migration down the gradient and absorbance seen here as the third smallest peak.
What is Satellite DNA?
- Large arrays of pericentromeric, tandemly repeating, non-coding DNA
- the main structural constituent of heterochromatin, also found in most telomeres, and occassionally in other loci
- Each unit has 1-3000 bp depending on species, and is repeated 102-106 times
Example: centromeric „alpha“ satellite sequence
has 171 bp with multiple repeats in all chromosome centromeres
Binds the kinetochore proteines which anchor the spindle fibers
–> Many different types of satellite DNAs combine in unique patterns between chromosomes to direct mitotic events
What is Minisatellite DNA?
- A small type of satellite DNA
- Tandem repeats that consist of 10 to 100 base pair-long monomer repeating sequences
- Rich in Cytosine and Guanine bases with higher density (w/v)
- Vertebrate telomeres: Guanine-rich hexanucleotide repeat motif (TTAGGG) named TRS (telomere repeat seq.)
- Some minisatellites contain a central sequence (or “core unit”) of nucleobases “GGGCAGGANG”
- Synonyms:
Variable number tandem repeats (VNTRs – this term also refers to microsatellite DNA)
Formular: Cn(A/T)m where n>1 and m≤4
What are Microastellites?
- The smallest type of satellite DNA
- Short tandem repeats that consist of 1 to 5 base pair-long monomer repeating sequences
- They form a range of polymers from pentamers (pentanucleotides with x5 base units, e.g. “TATCC”) to 50-mers (x50 units), repeated thousands of times in a genome
- Rich in Adenine and Thymine bases with lower density (w/v)
- Synonyms:
Simple sequence repeats (SSRs)
Short tandem retears (STRs) - Have high mutation rate, e.g. triplet codon expansion in Huntington‘s neurodegenerative disease on the short arm of chromosome 4
Repeat DNA: sequence type and structure
sequence type
* Directly repeated DNA element
* Inverted repeats (also known as palindromes or palindromic repeat sequences)
* Everted DNA sequences: contain a centre of symmetry, or mirror repeats (MRs) that can be imperfect with <100% symmetry (IMRs)
Sequence structure
* Repeat sequences used to be thought of as „junk“ or „selfish“
* Evidence shows they play important roles in genetic variation and gene expression regulation
* Structural 3D conformations of repeat DNAs and co-localisation within the nuclear space suggest genome folding roles
* Ubiquitous palindromes are self-complementary and can form hairpin and cruciform (cross-shaped) structures
* Multiple tandem copies, in combination to contortional stress (e.g. from DNA twisting during chromatic supercoiling) can fold into comlpex structures comprised of multiple hairpins and/or cruciforms
Picture
* hydrogen bonds are reversible and can be relatively easily rearranged
* referred to as DNA zipper, so it can be unzipped under stress e.g. during negative or positive twisting
* alpha bar symbol denotes complementary repeat sequences, same for any letter with a bar symbol.
- (a) Direct repeats.
- (b) Inverted repeats.
- (c) Mirror repeats.
- (d) Everted repeats.
* E, f, and g are a consequence of inverted repeats arranged in specific patterns in a single strand.
- (e) Recursive secondary structure.
- (f) Cruciform structure.
- (g) Nested pseudo-knot structure.
* Many more conformations are possible, and secondary and tertiary structures of nuclei acid repeats are the subject of structural biology and computational structural biology
