7-9 Mobile Genetic elements

Question 1

Give the five classifications of transposable elements found in eukaryotes

Answer 1

Require transcription

• Non-LTR retro(trans)posons
• LTR retrotransposons and retrovirus-like elements (have long terminal repeat for replication by retrotranscriptase).

Don’t require transcription
- Cut and paste transposons (DNA transposons)
- Rolling circle transposons (Helitrons)
Self synthesizing transposons (Polintons)

Question 2

What happens if a transposable element doesn’t have autonomous elements (eg. Polymerase, reverse transcriptase, transposase etc.)?

Answer 2

They contain sequences that define the element and allow proteins (from other autonomous elements) to recognize it and ‘help’ it.

Question 3

What is an overlap between transposable elements and repetitive DNA?

Answer 3

DNA transposons (eg. retrotransposons)

Question 4

Retrotransposons can be LTR or non-LTR type.

Give some general features of all retrotransposons

Answer 4

A retrotransposon is a copy in a region of DNA that is surrounded by a type of element and some protein coding genes (if autonomous)

• Transcribed to RNA
• Translated to produce proteins
• REverse transcriptase will copy RNA into cDNA
• RNA strand is degraded and resulting cDNA strand becomes double stranded
• cDNA is inserted somewhere in genome

The donor site retains the retrotransposon.

The long terminal repeats (LTRs) are able to recognize retrotranscriptase.

Question 5

How are LTR retrotransposons and retroviruses related?

Answer 5

• A retrovirus has code that is flanked by two LTRs and a number of protein coding genes (eg. for assembly of capsid and genome replicatio and evelope)
• A retrotransposon has the same parts, but lacks the envelope part (or has pseudogenes of it)

During the life cycle of a retrotransposon, once the element is transcribed to RNA, it is also translated to produce retrotranscriptase and other proteins. Some of these proteins make a capsid for the element, but not an envelope (can’t leave cell).

Question 6

True or false, all ORFs in a retrotransposon are important for transposition?

Answer 6

No, some may just be cellular genes captured by the element.

Question 7

There are many types of retrovirus and therefore there are many types of LTR retrotransposons. How do LTR retrotransposons compare to non-LTR retrotransposons?

Answer 7

Non LTR RTs are much shorter and propagation is driven by cellular retrotranscriptase.

Question 8

What is the most important property of LTRs in retrotransposons?

Answer 8

They have DNA elements that make them good promoters for transcription. Transcription will only not occur if the element falls into an inactive region of chromosome (eg. heterochromatin)

Question 9

Describe the structure (at sequence level) of the following:

• RNA form of retrovirus
• Linear DNA form of virus
• Integrated DNA form of virus

Answer 9

RNA form of virus
- Ends in direct repeats

Free linear DNA form of virus
- Ends in LTRs

Integrated DNA form of virus (provirus)

• Has LTRs that are shortened by two bases each
• Ends at either end with 4-6 bp target DNA repeats

Question 10

How is minus strand DNA generated during reverse transcription of retrovirus RNA?

Answer 10

Minus strand DNA is generated by switching templates during reverse transcription.

• Retrovirus provides plus strand RNA and primer tRNA anneals to binding site on retroviral RNA
• Reverse transcriptase starts synthesis of minus strand
• Enzyme reaches end of template (end of 5’ repeat on RNA template) generating strong STOP
• 5’ terminal region (including repeat) of RNA strand is degraded
• Single stranded DNA direct repeat region pairs with 3’ terminus in first jump to another retroviral RNA (piece of DNA jumps to the other sided repeat on another molecule and DNA synthesis can proceed)
• Reverse transcriptase resumes synthesis of minus strand

There are great diagrams of this online!

Question 11

There are other proteins important to retrotransposons beyond reverse transcriptase ofr dsDNA. Describe the activity of integrase.

Answer 11

Integrase catalyzes the insertion of retrotransposons into the genome.

Transcription generates RNA that serves as a template for reverse transcription, yielding a DNA copy.

• Integrase cuts this viral DNA to get 3’ and 5’ sticky ends
• It cuts a sequence in the target chromosome and the viral DNA attacks this target DNA
• DNA is inserted and 5’ and 3’ gaps are filled by DNA repair with short direct repeats of target DNA sequence (these are markers of retrotransposon insertion! 4-6 bp repeats of target DNA)

Question 12

What are stress granules and how do they relate to replication of non-LTR retrotransposons?

Answer 12

Stress granules are dense aggregations in the cytosol composed of proteins & RNAs that appear when the cell is under stress. The RNA molecules stored are stalled translation pre-initiation complexes - failed attempts to make protein from mRNA.

Replication of non-LTR retrotransposons associated with cellular stress. Stress granules can go back into the nucleus and insert DNA

Question 13

Describe the distribution of transposable elements in the human genome and how some may have contributed to our evolution.

Answer 13

• Most are non-LTR retrotransposons (eg. L1, Alu, SVA etc.) (about 34%)
• Second most are LTR retrotransposons (8.3%)
• Least are DNA transposons (2.8%), though other animals show the opposite with many DNA transposons.

L1 and Alu are known to have expanded a lot in relatively recent history, which may be important for human evolution.

Question 14

What is L1?

Answer 14

A non-LTR retrotransposon.

• Similar to a protein coding gene. Has one or two ORFs, thoug none code for retrotranscriptase or anything like that.
• It has flanking UTRs and remains of a polyA tail in genome

Question 15

What is an Alu element?

Answer 15

A none-LTR retrotransposon

• Has a left and right monomer flanking a A rich region in the centre
• Contains remains of polyA tail

Question 16

What is a SVA?

Answer 16

A non-LTR retrotransposon

- Known endogenous retrovirus with Alu-like centre.

Question 17

What are LINES and SINES?

Answer 17

Elements of non-LTR retrotransposons

LINES: Long interspersed nuclear elements (eg. in L1)
- retrotransposons that derive from a selfish DNA sequence that encodes reverse transcriptase.

SINES: Short interspersed nuclear elements (eg. in Alu)

• SINES replicate through an RNA intermediate, but do not encode RT themselves. They utilize RT already in cell
• Evolved from normal cellular RNAs, most often tRNAs, but also RNA that forms a component of the signal recognition particle, which is required for protein translocation across the endoplasmic reticulum membrane.

Question 18

What RNAs are subject to reverse transcriptase?

Answer 18

Basically and RNA that is able to form hairpin loops. The hairpin loop acts as a primer for reverse transcriptase

Question 19

What are DNA transposons?

Answer 19

DNA transposons move from one genomic location to another by a cut-and-paste mechanism. They are powerful forces of genetic change and have played a significant role in the evolution of many genomes. As genetic tools, DNA transposons can be used to introduce a piece of foreign DNA into a genome. Indeed, they have been used for transgenesis and insertional mutagenesis in different organisms, since these elements are not generally dependent on host factors to mediate their mobility. Thus, DNA transposons are useful tools to analyze the regulatory genome, study embryonic development, identify genes and pathways implicated in disease or pathogenesis of pathogens, and even contribute to gene therapy.

They have 1 protein coding gene for a transposase, which can excise DNA and integrate it elsewhere in the genome.

Question 20

What are the three types of eukaryotic DNA transposons?

Answer 20

• Cut and paste transposons
• Helitrons (rolling circle)
• Polintons (self synthesizing)

Question 21

True or false? DNA transposons are ubiquitous and occur in all three domains of life.

Answer 21

True

We find all the different subtypes of DNA transposons in all the major lineages of eukaryotes. We can assume or infer that when groups share elements, these elements were present in a common ancestor.

However, we know mobile elements can be transferred horizontally, leading us to a simpler explanation.

Question 22

What are polintons?

Answer 22

Self synthesizing DNA transposons in eukaryotes

• These are pretty variable in structure, which made identifying them difficult.
• Categorized by large size.
• Contain about ten genes.
• Their most conspicuous element is a DNA polymerase gene, making it the only transposon that can copy itself by using its own DNA polymerase.
• Has short inverted repeats that are consistent in size and is present in animals and protists

Question 23

Describe Polinton transposition

How does this give us hints for the origin of Politons?

Answer 23

• Long internal inverted repeats allow for stems in the dsDNA, which are recognized by enzymes that can excise structure from DNA, which becomes double stranded extrachromosomal DNA
• DNAB (polymerase) proteins function as a primer in absence of oligonucleotides and/or primer.
• Integrase integrates it into the genome
• These proteins have features that are reminiscent of viruses that can infect other viruses (virophages)

Politons originated from virophages.

Question 24

What are Helitrons?

Answer 24

A new class of DNA transposons. They are usually large and don’t have many conserved structural features, making them difficulut to find and identify.

Helitrons frequently capture host genes, shuffle exons and induce strutural changes in the host chromosomes, generating great structural variability, especially in several plant species.

Question 25

How do helitrons replicate and transpose?

Answer 25

• The element is excised at DNA replication from the chromosome, turning into an extrachromosomal circular DNA molecule (chromoplasmid)
• It contains a replication origin which induces rolling circle replication, creating many copies of itself.
• The copies are cut and integrated in the genome.

Question 26

How can transposable elements play a part in evolution?

Answer 26

They can cause gene duplication and influence gene expression by capturing genes and putting them somewhere else.

They can also create new proteins by combining different exons/modules (eg. zinc finger domain with alpha helix to make a new protein etc. etc.).

Question 27

What are three methods for capturing genes observed with Helitrons?

Answer 27

• Repeats at ends will make strand structures which are essential for replication. Sometimes that will fail, formation of structure at one side will fail and just by chance there is a region in the genome two genes downstream that can form a similar structure. Transposon will be excised and replicated along with the side (it will be longer = composite transposon that will include two genes now part of Helitron).
• Two Helitrons, one has an issue with forming structure and will use upstream side of second helitron to define downstream limit of transposon, capturing everything inbetween.
• When helitron is being transposed, recombination with other pieces of genome may result in combo of new elements. Results in helitron being surrounded by random pieces of genome.

Question 28

Excessive transposon activity is harmful for the host. Explain some mechanisms that limit the ability of transposons to replicate and spread

Answer 28

Think of an arms race.

• DNA methylation within repetitive elements. Inactivation of cytosine methylation in Arabidopsis thaliana causes a burst of retrotransposon and DNA transposon activity, resulting in substantial increases in transposon copy number.
• Small RNAs inhibit transposable elements. endo-siRNAs (post transcriptional disruption of transposon mRNA) and piRNAs (endonuclease activity through complementary sequences) act to defend eukaryotic cells against TEs.

Question 29

Give details on two types of defense by degradation of transposon mRNA by RNAi

Answer 29

Endogenous siRNAs and RISC

• Dicer mediated generation of small double strand siRNAs and incorporation of these single strand siRNA (antisense to the transposable element mRNA) into RISC results in RISC binding to the complementary TE and cleaving it for degradation by cellular enzymes.
• Less specific, seen in many species.
• Quicker response to TEs

Piwi interacting RNAs (piRNAs)

• piRNA clusters are processed and bind to PIWI clade protein to form a mature piRNA that can recognize complementary transposable element mRNA and pick it up
• It can then cleave other copies of that TE as a secondary piRNA
• This is specific and doesn’t occur in some species because many eukaryotic lineages don’t have the machinery for it.
• If you introduce a new type of transposon, this is catastrophic in the first generation because they haven’t developed ways to defend and after generations descendants are weak and have many mutations.

Question 30

Give the type(s) of evolution associated with the following

Repeated DNA sequences
Tandem Repeats
Microsatellites

Paralogues
Retro(pseudo-)genes
LINEs
DNA transposons

Answer 30

Repeated DNA sequences: Segmental duplications, whole genome duplication

Tandem Repeats: replication slippage, gene conversion

Microsatellites: mismatch repair mechanism

Paralogues: reverse transcription

Retro(pseudo-)genes: reverse transcription into heterochromatin region

LINEs: reverse transcription
DNA transposons: transposition

Question 31

Tandemly repeated DNA includes functional clustered genes and satellite DNA. What is a functional reason for this clustering?

Give an eukaryotic example of this.

Answer 31

Regulation of expression. It’s also very efficient for providing lots of output (eg. the subunits manufactured one after the other). The maximum rate is dependent on the number of RNA polymerases that can bind and the number of genes present. So really, the only way to increase this speed is to make more genes.

Ribosomal RNA genes are arranged in clusters in most eukaryotic genomes. They are present at a single genomic location and the rDNA locus consists of 150-200 tandemly repeated transcription units (in yeast at least)

We expect to see these clusters for proteins which are needed in high abundance.

Question 32

What is satellite DNA? Give the three subclassifications of it explain how they act within the genome.

Answer 32

Non-coding DNA sequences that are highly repeated in a tandem fashion.

• Microsatellites (2 to 5 bases)
• Minisatellites (6 - 100 bases)
• Satellites (over 100 bases)

These kind of repeated DNAs are extremely dynamic. Because of the genetic mechanisms involved in their generation, expansion and contraction, their abundance in a given species can change quickly.

New variants of a particular sequence can arise and ‘wipe out’ the original in relatively short time (evolutionarily speaking)

Question 33

How do microsatellites arise?

Answer 33

The arise when short repeats appear as a consequence of mistakes during DNA replication (eg. slippage) or at meiosis (especially in regions that already have repeats).

• Replication machinery can pause on lagging strand due to secondary structure or other lesions.
• Partial unwinding of lagging strand may lead to replication slippage when replication restarts, giving rise to expansion or contraction of the repeat tract, depending on what strand slippage occurred.
• Homologous recombination with sister chromatid after partial unwinding may lead to lesion bypass and may also cause contraction or expansion of the repeat

The presence of short repeats promotes the occurrence of more mistakes, resulting in increasing numbers of repeated units after successive generations.

Satellites over 100 bases long can arise from existing gens or transposons, and not so much by ‘slippage.’

Question 34

How do repeats promote genomic rearrangements?

Answer 34

These increase the chance for recombination, which in turn can bring about rearrangement.

Repeats can pair and recombine. The intrachromosomal recombination can lead to:

• Deletion, where a hypothetical circular fragment is lost
• Causes deletion AND duplication
• Intrachromosomal recombination between two inverted repeats leads to inversion of the intervening DNA sequence (can be silent to lethal)
• Tandem repeat polymorphisms can arise by unequal crossing over.

Question 35

How can tiny microsatellites quickly (relatively speaking) become large noncoding sequences?

Answer 35

the primary unit can multiplicate and form a perfect monotone repeat. The sequence can multiplicate again as one unit, leading to larger secondary unit.

Also, noncoding sequences are subject to neutral selection and accumulation of random mutations. This can lead to processes which may further result in accumulation of rounds of change leading to long secondary units.

Question 36

How can complex satellite DNA arise?

Answer 36

From relicts of transposable elements. Eg. old LTRs from retroviruses/retrotransposons can become the monomeric units of satellite DNA (seen in subterranean rodent Ctenomys)

Question 37

Explain the amplification model of RPCS (a complex satellite that originated from a retrovirus in Ctenomys)

Answer 37

• The proto-monomer was a transposon that underwent mutation, then unequal cross over, and then resulted in an intrastrand exchange which gave rise to a circular plasmid capable of rolling circle replication. Replicates then inserted back into the original tandem.

This rolling circle amplification could result in drastic increase in copy number!

We can test this by using a western blot with restriction endonucleases (eg. EcoRI or PvuII). We will see spots equidistantly apart to indicate the monomeric unit in the repeat

Question 38

The range of copy numbers for RPCS (a complex satellite that originated from a retrovirus in Ctenomys) is 2000 to 6 million, thought to have occurred over a 2 million year period. What is the consequence of this rapid amplification?

Answer 38

Drastic changes in copy number (amplifications and reductions) are associated to extreme levels of chromosomal rearrangement.

Question 39

What is the structural and functional impact of repetitive DNA on their hosts?

Answer 39

• Centromeres and telomeres composed by highly repetitive DNA
• Promote chromosomal rearrangement (hot spots for intra and interchromosomal homologous recombination tha can indu e large scale inversions, translocations, deletions etc. and small scare rearrangements of the DNA
• Transposable elements can act as insertional mutagens (TEs can knock out or alter gene function via insertion into the coding sequence or regulatory environment)

TEs and satellite DNA involved with epigenetic regulation
- TEs that are inactivated by epigenetic mechanisms are said to be silenced. Host’s attempts to protect its genetic info from insertional mutagenesis by silencing TEs may result in epigenetic alterations that affect the activity of the genes it is trying to protect (Eg. satellite DNA is heavily methylated and contributes to the create vase regions of heterochromatic DNA that can affect the transcriptional status of neighbouring genes)

Question 40

Describe the impact of exonization of Alu elements on the human genome

Answer 40

Wherever the Alu sequence lands in an intron, it is bringing sets of nucleotides that resemble exonic elements, causing part of the Alu to be incorporated in the mRNA

This can result in new protein isoforms through alternative splicing (in some cases).

Question 41

Describe the example in drosophila where two transposable elements acted to alter gene regulation.

Answer 41

• Galileo causes an inversion

- And then Kepler initiated antisense RNA transcription after being inserted upstream of Galileo

Question 42

How can LTR elements actually play a large role in the evolution of de novo genes?

Answer 42

LTR elements can provide raw material for new genes.

- They contain promotor elements and other regulatory stuff that is needed.

Question 43

Briefly list the six consequences of transposon mobilization and satellite DNA and how they can lead to evolutionary changes that includes adaptation of populations to new environments, generation of resistance to existing or new threates and even promote speciation by creating reproductive barriers between populations (eg. through chromosomal incompatability)

Answer 43

• Inversions as a result of recombination between inverted repeats
• Deletions as a result of recombinations between direct repeats
• Drastic chromosomal rearrangements including loss of large heterochromatic blocks
• Unequal crossing over between repeats (regions of micro-homology) leads to translocations, deletions and gene duplications
• Gene inactivation as a result of transposon insertion
• Gene activation as a result of a transposon (or retrovirus) inserting near a gene, and driving the transcription of that gene from a transposon promoter