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Y2 Genomes > Eukaryotic Organisation > Flashcards

Eukaryotic Organisation Flashcards

1
Q

What is the main paradox in genome size?

A

Complexity of an organism does not correspond to genome size

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2
Q

What is the C-value?

A

amount of DNA contained in a haploid nucleus

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3
Q

How does human genome density compare to yeast genomes?

A

Human genome is less densely packed due to more space devoted to introns and repeat sequences.

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4
Q

How large is satellite DNA usually?

A

100-500bp. Important at mammalian centromeres

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5
Q

How large are microsatellites?

A

1-13bp

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6
Q

What is satellite DNA?

A

Tandem repeats of 1-500bp which make up ~6% of the human genome. Mainly phenotypically neutral, but some exceptions such as CAG repeats in huntington’s disease?

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7
Q

How large are minisatellites?

A

14-100bp. Can form 1-5kb tandem arrays around the genome.

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8
Q

How does replication slippage cause satellite DNA to differ between individuals?

A

DNA polymerse dissociates during replication and the nascent DNA strand rehybridises with another repeat in the array. This can cause satellite DNAin the daughter strand to be shorter or longer than the template

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9
Q

How does unequal crossing over in meiosis cause satellite DNA to differ?

A

Crossing over between misaligned repeats on sister chromatids results in less copies of a repeat on one strand. One of the microsatellite tracts shrinks

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10
Q

How can satellite probes be used in DNA fingerprinting?

A

Minisatellite sequences as can be used as a probe on DNA fragments which are separated with southern blotting. This usually gives two bands of the satellite DNA, then other bands from other satellites from different regions.

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11
Q

How does transposase work?

A

Binds to inverted repeats on a transposon and cuts the DNA. A cut is made somewhere else to allow the sequence to insert itself (non-replicative).

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12
Q

What is the difference between Ac and Ds transposons in maize?

A

Ac transposon- autonomous
Ds transposon- non-autonomous. Uses the transposon from Ac as it has the same inverted repeats

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13
Q

What types of eukaryotic reterotransposons are there?

A

LTR
LINE
SINE

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14
Q

How does the pol gene integrate into a host genome?

A
  1. Host polymerase generates an RNA molecule in the nucleus
  2. Retrotransposon reverse transcriptase converts RNA to cDNA in the cytosol
    RNAse H degrades the RNA template
  3. dsDNA is guided to the nucleus by integrase bound to LTRs
  4. dsDNA moves to the nucleus and inserts into the genome, creating direct repeats as a target site
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15
Q

What types of LTR reterotransposons are there?

A

pol gene
ERV elements (endogenous retrovirus). Makes up 8% of the human genome
Yeast Ty elements

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16
Q

What is the only functional LINE?

A

L1 in the human genome. L2 and L3 are non=functional.

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17
Q

What do the open reading frames in long interspersed elements (LTRs) do?

A

ORF1 encodes RNA binding protein.
ORF2 encodes reverse transcriptase and DNA endonuclease

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18
Q

How are LINEs transposed?

A
  1. The transposon is expressed using host machinery and polyadenylated.
  2. ORF1 proteins binds LINE RNA and ORF2 protein binds LINE polyA
  3. RNA is transported into the nucleus
  4. ORF2/polyA RNA binds to a polyT somewhere in the genome
  5. Endonuclease activity from ORF2 nicks the DNA
  6. ORF2 reverse transcriptase uses host DNA as a template
  7. If it continues to the end of the strand, a second DNA strand is made by host enzymes
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19
Q

When does replicative transposition take place?

A

S-phase of mitosis

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20
Q

Outline the mechanism of transposase

A
  1. Binds to inverted repeats at the end of the transposon sequence
  2. The enzyme cuts DNA to remove the transposon
  3. A cut is made somewhere else in DNA and the sequence inserts there
21
Q

What is a pseudogene?

A

a conserved gene that has lost the ability to encode a functional protein

22
Q

What consequences do transposition have?

A

Exon shuffling where exons flanked by a transposon cross over
Mistakes from LINE transposition using polyA signals from neighbouring genes

23
Q

What are conventional pseudogenes?

A

Pseudogenes that cannot function. Caused by frame shifts and point mutations. If there is a copy of the functional gene, pseudogene function can be lost altogether

24
Q

Outline how processed pseudogenes come about

A
  1. Functional mRNA is reverse transcribed into cDNA
  2. cDNA is inserted into the genome by LINE proteins.
  3. Inserted genes don’t have proper processing signals so aren’t functional
    e.g actin and ferritin pseudogenes
25
Q

What are orthologue genes?

A

The same gene in different species due to speciation
e.g α-tubulin

26
Q

What are paralogue genes?

A

Different genes that have arisen from duplication events followed by differences occurring on the genes.
e.g α and β tubulin

27
Q

Why do amounts of satellite DNA vary across individuals

A

Replication slippage
Unequal crossing over
Retrotransposition of DNA

28
Q

Tell me about centromeric nucleosomes

A

146bp DNA
8 histones
2 Histones are H3
CENP-A replaces H3 at eukaryotic centromeres
The CENP-A nucleosome is recognised by kinetochore proteins

29
Q

Tell me about the human “regional centromere”

A

High order structure with several repeats
Alphoid satellite DNA has AT-rich sequences, each 171bp
These form tandem arrays
A conserved 17bp sequence in this binds CENP-B which targets CENP-A to this region
Additional modified nucleosomes e.g with methylated H3

30
Q

What sort of chromatin is around the human centromere?

A

Pericentric heterochromatin which prevents the regions around the centromere being transcribed. The centromere itself is highly transcribed

31
Q

Outline the mechanism of the kinetochore

A
  1. Recognises centromeric epigenetic markers such as epigenetic markers and alternative histones.
  2. The Ndc80 complex on the kinetochore grabs the centromere. Microtubules then attach , allowing segregation at mitosis
32
Q

What are the holocentric histones in C. elegans?

A

CenH3 histones are CENP-A homologues which attach to the kinetochore
Holocentric means these attachments are along the whole chromosome
Shows how chromatin structure is well conserved

33
Q

Outline how early eukaryotic replication is initiated

A
  1. The origin recognition complex binds to DNA
  2. The pre-replication complex is assembled with MCM proteins and CDC6
  3. The pre-initiation complex assembles and polymerases turn this to the initiation complex
34
Q

Tell me about budding yeast autonomous replication sequences

A

Only some ARSs initiate replication
11bp autonomous consensus sequence
Transcriptionally silent proteins are more likely to be bound by replication proteins

35
Q

How can autonomous replication sequences be found?

A
  1. ARS plasmids are purified and sequenced
  2. The ARS inserts are amplified
  3. ARS sub-fragments are sheared and closed up
  4. After being added to a vector, the mini ARS plasmids are isolated
36
Q

Outline how an RNA-primed nascent strand can be obtained

A
  1. DNA fragments are isolated from a population undergoing replication
  2. These are treated with λ-exonuclease which degrades DNA, but not okazaki fragments, because they contain an RNA primer
  3. RNA is amplified and sequenced
37
Q

What are origin G-rich repeated elements (OGREs)?

A

G-rich octamers found at <60% of human origins
These form nucleosome-free G-quadruplex structures upstream of the initiation site. These promote origin activity.
Mispairing forms a rectangle structure between the nucleotides

38
Q

What are telomeres?

A

Regions at the ends of chromosome consisting of repeats and a 3’ overhang.
TT-GGG is very well conserved between mammals and Tetrahymena thermophila

39
Q

How long are telomeres in humans?

A

10-15kb

40
Q

How did telomeres arise in T. thermophila?

A

It amplified its genome to generate lots of short chromosomes. This resulted in ~400bp repeats

41
Q

What does the shelterin complex do?

A

Forms a cap with differentiates the end of the chromosome from DNA breaks. This avoids chromosomes being repaired and stuck together
Promotes a t-loop tertiary structure in DNA
recruits telomerase
protects telomeres from nucleases

42
Q

What types of telomerase are there?

A

TERT (telomeric reverse transcriptase) is a protein component
TERC (telomerase RNA component) is the reverse transcriptase template

43
Q

Outline how telomerase extends telomeres

A
  1. TERC (RNA component) base-pairs with the 3’ overhang
  2. Telomerase translocates along the 3’ overhang elongates using RNA as a template
  3. RNA is removed and DNA polymerase synthesises a second strand
44
Q

Why are single celled eukaryotes considered immortal?

A

They have high telomerase activity

45
Q

What happens when telomeres are too short

A

Shelterin binds to the end of the chromosome triggering: SCAG
Senscence (loss of a cell’s ability for division and growth)
Cycle arrest
Apoptosis
Genome instability

46
Q

There is evidence that telomere shortening is sped up by…?

A

Oxidative stress, building up single strand breaks

47
Q

What are shorter telomeres in humans associated with?

A

Age related diseases

48
Q

Why are 90% of cancer cell considered immortal?

A

Telomerase activity is raised, often due to mutations at the telomerase promotor

Y2 Genomes (7 decks)

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