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LF206: Molecular Cell Biology > Lecture 4. Eukaryotic Genome Organisation > Flashcards

Lecture 4. Eukaryotic Genome Organisation Flashcards

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1
Q

As genomes get larger, what does an increasing proportion of DNA become?

A

Non-coding (non protein coding)
The stuff we think of as important is the minor part of the genome

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2
Q

What makes our genome so large?

A

Gene duplication: families of genes and pseudogenes with (often) co-ordinated regulation - multiple copies of genome
Large introns: often containing retrotransposons
Transposons: LINES (long interspersed nuclear elements), SINES (short INES), retroviruses, retrotransposons
Repetitive DNA: simple sequence repeats, segmental duplications
Non-repetitive DNA

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3
Q

What is gene duplication?

A

Protein-coding genes have relatives with which they share common ancestry
Some genes exist in families and super-families
Within a genome, families can be dispersed or clustered
Maintenance of clusters implies functional co-ordination/regulation
e.g. Globin genes (oxygen binding proteins coded by Globin genes)

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4
Q

Where did the ancestral globin gene originate from?

A

Archaea

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5
Q

How were the haemoglobin and myoglobin genes formed from the ancestral globin gene?

A

Ancestral globin gene suplicated into two idential genes
These two genes diverged due to mutations in different places (including control sequences) into the haemoglobin and myoglobin genes
Haemoglobin gene underwent further duplication and divergence to sprout families of α and β subunits with different regulatory sequences (and mutational inactivation and decay - pseudogenes where not every copy works but still kept which impies some kind of function)

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6
Q

What are the roles of haemoglobin and myoglobin?

A

Haemoglobin: oxygen transporter in the blood
Myoglobin: oxygen store in muscle cells

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7
Q

What is the structure of the α-like subunits haemaglobin?

A

On chromosome 16
ζ (zeta) - Ψ1 (pseudogene) - ζΨ1 - α2 - α1 (α1 and 2 are duplicated of each other)

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8
Q

What is the structure of the β-like subunits?

A

On chromosome 11
ε - γG- γA - Ψβ1 - δ - β
[ε - γG- γA] = foetal
[δ - β] = adult

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9
Q

What is the largest contributor to maternal blood (major haemoglobin)?

A

Tetramer made up of 2 α and 2 β subunits

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10
Q

What is the largest contributor to foetal blood (major haemoglobin)?

A

Tetramer made up of 2 α and 2 γ subunits

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11
Q

What happens in the process of oxygen transfer fro the maternal blood to the foetal blood?

A

Oxygen transfer triggers a regulated developmental switch that is developmentally controlled changes the conformation

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12
Q

What does developmental control allow in haemoglobins?

A

Developmental control allows the gradual change from foetal to adult haemoglobins –requires temporal transcriptional regulation

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13
Q

What is the difference between a human Huntingtin gene and a fugu Huntingtin gene?

A

Human Huntingtin gene has large introns containing retrotansposons whilst the Huntingtin gene in fugu has small introns and no obvious retrotransposons
Human Huntingtin gene is 9x longer than the fugu equivalent

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14
Q

What remains the same between the human Huntingtin gene and the fugu Huntingtin gene?

A

Both genes have 67 exons that align in 1:1 correspondence to one another
Intron positions are often conserved between species

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15
Q

What makes up ~50% of the human haploid genome?

A

High-copy repetitive elements

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16
Q

What makes up ~3.5% of the human haploid genome?

A

Highly conserved sequences

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17
Q

What makes up ~1.5% of the human haploid genome?

A

Protein exons

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18
Q

What is >40% of our genome composed of?

A

Sequences derived from retrotransposons

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19
Q

What are LINEs?

A

Long interspersed nuclear elements, generally intergenic

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20
Q

What are SINEs?

A

Short interspersed nuclear elements, often in gene-dense regions

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21
Q

What is the function of the LINEs and SINEs?

A

Gene expression regulation by affecting chromatin structure, gene transcription and pre-mRNA processing

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22
Q

What is the nucleosome?

A

The set of histones in which the DNA is coiled round solenoid-like (some definitions include the linker DNA to the next nucleosome)

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23
Q

How can the nucleosome core particle be dissected to release histones?

A

Linker DNA is digested with DNase
Left with singular nucleosome core particles (11 nm thick)
Placed in highly concentrated salt, dissociating the core particle into the octameric histone core and 147bp dsDNA
Octameric histone core dissociates twice into eight histone subunits (per nucleosome core molecules)

24
Q

What are the four histone subunits that make up the octameric histone core?

A

2 copies of each
H2A, H2B, H3, H4

25
Q

Why is it not straightforward to re-build nucleosomes?

A

Histones are all small basic (positively charged, rich in R and K) proteins
High resolution separation requires addition of urea and acid to the gels: the histones separate according to a combination of size and charge
They are often modified, appearing as doublet/triplet/smears rather than singlet proteins

26
Q

Why did early attempts at crystal structures of nucleosomes give only low-resolution structures?

A

Because of histone modifications and uneven nucleosome spacing

27
Q

What was the big clue that allowed for nucleosome recombination?

A

Order of addition is important
Purified H3 and H4 interact to make a dimer and the dimer dimerises to make a tetramer and the tetramer can bind DNA
The H3-H4 tetramer:DNA complex binds two separate H2A:H2B dimers reconstituting nucleosomes in vitro and they pack evenly during crystallisation giving a high resolution structure

28
Q

How can the original 30nm fibres be reconstructed from the 11mm fibre?

A

Add HI (H1) (linker histone) and raise salt concentration

29
Q

How is each DNA molecule packaged?

A

Into a mitotic chromosome that is 10,000-fold shorter than its extended length
Beads on a string

30
Q

How densely packed is sperm DNA?

A

Sperm DNA is packed even more densely, wrapped around protamines. Approx 85% protamines, 15% histones

31
Q

What must happen to chromosomes to allow transcription and replication to occur?

A

Chromoeomes must be moved or remodelled to allow transcription and replication

32
Q

What is the role of the histone ‘code’?

A

Regulates genetic code

33
Q

What happens when some specific lysine residues (K) are modified by acetylation (Ac)?

A

A code for ‘relax the chromatin structure’

34
Q

What are histone modifying complexes recruited by?

A

Gene activator proteins
Chromatin remodelling complexes bind to the gene activator protein and can remodel nucleosomes, as well as remove and replace histone chaperones
Histone modifying enzymes can also copy changes made to histones and spread them to other histones

35
Q

Because nucleosomes are not static, what can happen to them?

A

They can be: removed, re-positioned, replaced or covalently modified

36
Q

What does remodelling of the DNA allow?

A

Remodelling makes the DNA more/less accessible to further activator proteins, general transcription factors, mediator proteins and RNA polymerase II
Thus histones regulate transcription

37
Q

How can a gene be silenced?

A

Some specific lysine residues (K) and arginine residues (R) can be modified by methylation (Me)
On lysyls, acetylation and methylation are competitive (inset)

38
Q

What effect can methylation have on chromatin and what does this effect depend on?

A

Methylation can relax or compact chromatin depending on:
a) which residue is methylated and b) the degree of methylation (mono-, di-, tri-)

39
Q

What does phosphorylation (P) of some serine (S) and threonine (T) residues promote?

A

Transcription (phosphorylation is normally a relax message)

40
Q

What does ubiquitylation (Ub) of some lysine (K) on H2A and H2B lead to?

A

On 2A recruitment sites for DNA repair, and some are activating and on H2B are repressive (closes chromatin)

41
Q

What is epigenetics?

A

Heritable changes in gene expression that are not mediated at the DNA sequence level

42
Q

What are the two ways epigenetic changes can take place?

A

DNA methylation and Histone modifications

43
Q

How was it proven that nucleosomes are a barrier to transcription?

A

When chromatin is open and add general transcription factors and RNA pol to the open chromatin, it will transcribe “beads on a string” slowly with pauses (stop-start)

44
Q

How is the histone code maintained during transcription?

A

RNA Pol II stalls at nucleosome A
RNA Pol II unwinds about half of the DNA from nucleosome A - nucleosome has two coils of DNA around it
The transcribed DNA is looped and starts to wind around nucleosome A, whilst unwinding continues
And so on… (same nucleosome is transferred from in front of the polymerase to behind the polymerase)

45
Q

In summary, what happens to nucleosomes during transcription?

A

Nucleosomes in the promoter region are modified/re-modelled to open the chromatin to allow access of the transcriptional apparatus
Nucleosomes are transferred from downstream to upstream as transcription proceeds

46
Q

What are the three structure functional elements in chromosomes?

A

Telomeres
Replication origin
Centromere

47
Q

How do you identify a eukaryotic origin of replication (in yeast)?

A

Take a bacterial plasmid that cannot be maintained in yeast and clone into it a Yeast His gene
Transform yeast and plate on media without histidine
Rare integration (recombination) events where His is now expressed chromosomally
Now clone random fragments of yeast into that DNA’s plasmid and transform them into yeast, some will contain origins of replication (Ars regions) allowing maintenance of plasmids

48
Q

Because multiple origins of replications (Ars in yeast) are fired at different times in the S phase (some fire early whilst some fire later), what does this tell us?

A

There is regulated control of the origin of replication
Multiple origins of replication per chromosome
Our origins of replication are larger and less well-defined than those in yeast: but the replication process is the same

49
Q

What happens to origins of replication during the G₁ phase of the cell division?

A

An origin of replication has a pre- bound complex of six proteins: ORC (origin recognition complex) 1-6. ORC remains associated throughout the cell cycle
Cdc6 and Cdt1 associate with ORC
Mcm helicases are recruited to form the prereplicative complex (pre-RC)
In G₁, the pre-RC is formed but is not activated

50
Q

What happens to origins of replication during the S phase of the cell division?

A

Cdk2 (cyclin dependent kinase 2) phosphorylates Cdc6
Proteasomal degradation of phosphorylated Cdc6
Phosphorylation of ORC. The RC is activated but new ones cannot form (no Cdc6)
Mrm helicases open the DNA (separates the strands) allowing recruitment of primase, clamp loader and DNA polymerases

51
Q

What happens to the histones during the early events of cell division?

A

The RNA polymerase complex unwinds the DNA, displacing the H2A-H2B dimers, but the H3-H4 tetramers remain associated (some on leading strands some on lagging strands) - both new strands inherit them

52
Q

What happens to the histones during the S phase of cell division?

A

There is a large increase in expression of histones in S phase, and the new subunits are rapidly acetylated (to promote open structure). NAP-1 (nucleosome assembly protein 1) chaperones H2A-H2B dimers and CAF-1 (chromatin assembly factor) chaperones H3-H4 tetramers assembling nucleosomes in an open chromatin structure
The fresh acetylations are removed. A ‘reader-writer complex’ copies epigenetic information from neighbouring nucleosomes. Thus, daughter cells maintain parental cell histone modifications

53
Q

What is methylated within DNA and what does this methylation do?

A

Cytosines in DNA can be methylated: methylated DNA gives a simple signal - do not express
Methylated histones can recruit DNA methylases that methylate C residues Methylated DNA can recruit histone methylases: causes silencing by condensing chromatin
The histone code and DNA methylation work together (histone doe changes methylation of cytokines to drive epigenetic phenomena)

54
Q

What is the non-epigenetic role of SINEs?

A

SINEs can act as enhancers or alternative promoters: they can recruit transcription factors and promote expression of nearby genes

55
Q

What is the epigenetic role of SINEs?

A

SINES are GC-rich and so are hotspots of DNA methylation: and this can silence nearby genes by stimulating chromatin condensation

56
Q

How is the ova effected by epigenetic alterations?

A

Some epigenetic marks (histone and DNA) occur in the ova
Part of the environmental history of the mother is inherited by her children

57
Q

How is the sperm effected by epigenetic alterations?

A

Sperm chromatin has 15% histones, 85% protamines. The protamines are replaced with acetylated histones from the ovum’s cytoplasm and male DNA is then systematically demethylated
But some male epigenetic marks remain

LF206: Molecular Cell Biology (18 decks)

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