The Human Genome 2- Repetitive DNA Flashcards by Emma Valbret

Gene duplication can create tandem arrays of identical genes- examples are genes whose products are needed in large amounts by the cell. Name a specific one and where it is found

Ribosomal RNA genes in humans. This makes up 80-90% of total mass of cellular RNA. There are 200 copies in 10 clusters near the tip of 5 chromosomes- they appear as satellite stalks (this is different to satellite DNA!). These chromosome are all acocentric

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Where does rRNA synthesis take place?

Which part of the nucleolus contains many tandem copies of rDNA genes?

In nucleolus:
Fibrillar core
Granular cortex

Nucleolus organiser region contains tandem copies of rDNA genes.

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Each repeat unit of a ribosomal gene cluster contains a transcribed region together with what?

How many repeat units are there per cluster?

A non-transcribed spacer- there are 30-40 of these tandem repeats per cluster

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What % of the human genome is made of repetitive DNA?

50%

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What two main classes of repeats are there?

Tandem repeats (repeats found clustered together in one chromosomal location)
Interspersed repeats (Scattered throughout the genome also known as transposons and make up 43% of total genome)

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Out of the following, which fall under tandemly repeated DNA and which fall under interspersed repeated DNA?

Satellite DNA
Minisatellite DNA
Microsatellite DNA
LINEs
SINEs
Retrovirus-like (LTR transposons)
DNA transposon fossils

Satellite, minisatellite and microsatellite DNA are all tandemly repeated DNA

LINEs, SINEs, LTR and DNA transposon fossils are all interspersed repeats

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Tandemly repeated DNA is classified as according to length of total array. Which is the largest and what is its composition like?

Satellite DNA (6.5% of human genome). It has a base composition very different from the average base composition of the genome- 41% GC. It therefore has a different bouyant density to the rest of the genome. There are three satellite bands I, II and III

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What sort of chromatin state does satellite DNA constitute and where is it normally found on a chromosome?

Heterochromatin. Found at centromeres, short arms of acrocentric chromosomes and sometimes in regions close to centromeres

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Are satellite DNA regions well or poorly conserved between species?

Poorly conserved

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There are classes of satellite DNA, some being 1, 2 and 3. Name another which is well characterised and is found at the centromeres of all chromosomes.

Alphoid repeats

Remember, there is a hierarchical system where smaller units such as alphoid repeats are repeated to give a higher-order repeat and this is in turn duplicated as one larger unit. The size of the higher-order unit varies between chromosomes

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Minisatellies and microsatellites are smaller repeat units than satellites and their composition is much the same as bulk genomic DNA. What does this mean in terms of detection on CsCl gradients?

They are not detected as satellite bands as oppose to satellites which are

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Are minisatellites and microsatellites polymorphic between individuals? What other name is given to them?

Yes, very variable between individuals. Also called Variable Number Tandem Repeats (VNTRs)

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How big are minisatellites compared to microsatellites? Where are each found on the chromosome?

Minisatellites: 100 bp-20 kb, repeat units: 9-64 bp found at telomeres and subtelomeric regions
Microsatellites: >100 bp and re[eat units are only a few bp long

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What are satellites, minisatellites and microsatellites classification based on?

Satellites, microsatellites and minisatellites are based on total array length not the size of their repeated units

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Pericentromeric and subtelomeric regions are unstable. What may this lead to? Give two examples.

Recombination that can generate duplicated gene segments being distributed to other chromosomal locations

NF1 gene
PKD1

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Originally, transposition is what allowed the dispersal of interspersed repeats throughout the genome. Are all interspersed repeats still able to transpose?

No are not actively transposing

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Transposons can be classified based on their method of transposition. How do retrotransposons do this and how do DNA transposons do it?

Retrotransposon are transported via copying of its own RNA transcript into cDNA (reverse transcription) followed by integration of this cDNA into a new locus (e.g LINEs, SINEs and LTRs)
DNA transposons migrate by cut-and-paste mechanism that does not involved a cDNA intermediate

Transposons can also be classified into autonomous transposable elements and nonautonomous transposable elements. What do these mean?

autonomous means they can encode all info needed for their own transposition
nonautonomous means they cannot transpose independently but instead use proteins made by an autonomous one

What are retrovirus retrotransposons characterised by? What about LINEs and SINEs?

retrovirus retrotransposons have long terminal repeats (LTRs)
LINEs and SINEs have a poly A sequence and are also known as PolyA transposons

How much do human LTR transposons make up of our genome? Are they able to transpose still?

9% and they are defective (not able to transpose anymore)

What % of our human genome is made from DNA transposons? and are these still able to transpose? What are the two main families of these?

3% and they are not able to transpose
MER1 and MER2
Some of our functional genes originated from these

How much of our genome is made of LINE elements (the most important transposable elements in humans) and which chromatin state regions are they found in?

21% and in euchromatic regions located mainly in the dark AT rich bands

Do LINE elements have a long evolutionary history (are they seen in other mammals)? What are they also known as?

Yes they are e.g dogs

autonomous poly-A transposons

Out of the LINE families, which can still transpose?

The LINE 1 family is the only family of LINE elements still capable of transposing and also are responsible for transposing other transposons and cellular genes

In a LINE element, does the 5' UTR contain an internal (downstream) or external (upstream) promoter? What does ORF 2 encode?

Internal- so this means that the transcription initiation sequence lies downstream of the actual transcription start site ORF 2 encodes a critical protein required for transposition (this has reverse transcriptase activity and endonuclease activity)

How does a LINE element transpose?

It is transcribed into an RNA and the LINE encoded proteins are synthesised in the cytosol. These then bind to the mRNA (cis-preference) from which they were translated and this complex migrates back into the nucleus. A cDNA copy of the mRNA is synthesised and inserted at a new location into the genome; this is done by binding to the target genomic DNA smd the LINE-encoded endonuclease cleaves the DNA at this sequence to enable RNA-DNA hybrid.

Does the new copy of the LINE element have the internal promoter after the transposition process?

Yes, as it lies downstream from the transcription start site

Why are short cDNA copies of LINE elements generated during transposition? Can these retranspose?

Because reverse transcription often fails to proceed to the 5' end of the LINE RNA template- this generates truncated nonfunctional insertions that have lost their promoter and can therefore no longer re transpose

What genetic diseases can LiNE element transposition cause by disruption of genes?

Haemophilia

In contrast to humans, LINE elements are still highly active in which species' genome?

Mouse

Where must transposition be located if they want to survive more than one generation?

In the germline- somatic ones do not survive beyond one generatiom

LINE machinary required for transposition (reverse transcription and endonuclease) preferentially bind to the 3' region of the mRNA it was translated from. They can however also bind to any mRNA that has what?

poly A sequence and promoterso can help transpose nonautonomous transposons and cellular transcripts

A cellular protein-coding mRNA can be boind to by LINE machinery. It can then be copied and integrated into another location. What is it then known as?

A processed pseudogene (it has lost all its introns and is non functional because it cannot be expressed as mRNA as it has lost its promoter)- take note that these are different to pseudogenes that are generated through duplication

What is a retrogene?

When cDNA copy of a gene is integrated downstream of a promoter and therefore can be expressed as mRNA (can be expressed unlike processed pseudogenes)

Retrogenes have specific patterns of expression. Use the example of them in the testis to explain this statement.

Several retrogenes are processed copies of genes that originated on the X chromosomes that have evolved a testis specific pattern of expression, thereby mediating the expression of critical x-linked genes in the testis

SINES are non autonomous poly-a retrotransposons. So how can they transpose?

They use LINE machinery as they contain an internal promoter and poly A tail

From where/what did SINEs originate from? Can they re transpose more than once?

cDNA copies of small RNAs transcribed using RNA polymerase III. They are able to re transpose since they carry a promoter with them

What family of repeats are considered the most important SINE sequences in the human genome? How is it transcribed/transposed?

Alu family. More than a million copies of these and many still able to transpose (using LINE machinery of course) Transcribed using Poly III and has an internal promoter.

How was the Alu family of repeats named? Where are these repeats found on the chromosomes? Which species are they found in?

As they contain a recognition site for restriction enzyme AluI. Found in GC-rich regions of euchromatin,however newly transposing ones show a preference for AT-rich regions. These are only found in primates- recent evolutionary origin

What do SINE elements promote when the organism is under stress?

Translation by generating transcripts that bind to a protein kinase usually involved with blocking translation under stress

Has the overall transposition activity in humans increased or decreased over the last 50 million years? Is this different from mice? What about new mutations arising?

Decreased substantially. Mice have too however at a much slower rate. Mutations in germline are occuring much more in mice than humans

Transposition is a major source of mutation and evolutionary change in other mammals but not humans. Maize and drosophila have much higher levels of what type of repeat than humans?

LTR repeats