Lecture 4: Eukaryotic Genomes

Question 1

What makes our genomes so large?

Answer 1

• Gene duplication
  
  • Families of genes n pseudogenes w coordinated regulation
• Large introns
  
  • Often containing retrotransposons
• Transposons
  
  • LINES, SINES, retroviruses, retrotransposons
• Repetitive DNA
  
  • Simple sequence repeats, segmental duplications
• Non-repetitive DNA

Question 2

What is gene duplication?

Answer 2

Copy of a gene is made within a genome, resulting in multiple copies of the same gene.

Question 3

What are some characteristics of gene duplication and its implications for genetic diversity and functional coordination within genomes?

Answer 3

• 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.

Question 4

What are 2 main differences of eukaryotic and prokaryotic genomes?

Answer 4

• Genome size
  
  • Larger in eukaryotes
  • Reflects the complexity of eukaryotes (factor of variability not the cause)
  • Larger genome = greater proportion of ncDNA n repeats
• Number of ncDNA
  
  • More in eukaryotes

Question 5

What does eukaryotic DNA code for?

Answer 5

• Genes that encode proteins
• ncDNA
• Repetitive DNA

Question 6

How are new genes created? Give an example

Answer 6

• Duplication of genes during evolution
• EXAMPLE: globin gene family
  
  • 1 ancestral gene
  • Duplication of that gene
  • Accumulation of mutations in both genes (original n duplication)
  • Accumulation of differences b/w genes
  • Further duplication n divergence result in a different functional protein

Question 7

Describe the developmental changes in globin expression

Answer 7

• Adult Hb = α2β2
• Fetal Hb = β2ϒ2
  
  • ϒ2 chain has higher affinity for O2
  • O2 transferred from mother to fetus
• The proportion of chains change over time
  
  • Loss of gamma chains during adulthood

Question 8

Describe the human genome’s composition and its function

Answer 8

• 40% of our genome is composed of sequences derived from retrotransposons.
• LINEs: long interspersed nuclear elements, generally intergenic
• SINEs: short interspersed nuclear elements, often in gene-dense regions
• FUNCTION: gene expression regulation by affecting chromatin structure, gene transcription and pre-mRNA processing.

Question 9

Describe the structure of chromatin

Answer 9

• Complex of DNA + proteins
• Organized into chromosomes
• Most of the proteins are histones

Question 10

Describe the function of chromatin

Answer 10

• Packaging DNA
• Reinforcing macromolecule structures for mitosis
• Preventing DNA damage
• Regulating gene expression

Question 11

What are 2 forms of chromatin?

Answer 11

• Heterochromatin
  
  • Condensed
  • Inactive for transcription
  • 10% interphase: chromosome stability
• Euchromatin
  
  • Loose
  • Active for transcription
  • 10% active in interphase: transcribed
  • 80% inactive in interphase: not transcribed

Question 12

If we can dissect nucleosomes, can we re-build them?

Answer 12

• 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
  
  • Histones separate according to a combination of size and charge.
• They are often modified, appearing as doublet/triplets/smears rather than singlet proteins

Question 13

Why did early attempts at crystal structures only produce low-resolution structures?

Answer 13

Histone modifications n uneven nucleosome spacing

Question 14

Describe the process of obtaining a high-resolution structure of nucleosomes

Answer 14

• Express each histone separately in Escherichia coli (bacteria do not make their own histones)
• Purify each histone; solubilise and re-fold
• Combine in different combinations pairwise to explore how they interact
• Purified H3 and H4 interact to make a dimer → dimer dimerises to make a tetramer → tetramer can bind DNA
• The DNA chosen was a palindromic repetitive sequence of 147 bp [to force even nucleosome spacing for later crystallisation] on a recombinant plasmid maintained in E. coli
• H3-H4 tetramer: DNA complex binds two separate H2A:H2B dimers → reconstituting nucleosomes in vitro → they pack evenly during crystallisation
• RESULT: high-resolution structure

Question 15

How do dynamic processes involving nucleosomes contribute to the regulation of transcription?

Answer 15

• Nucleosomes are not static
• They can be
  
  • Removed
  • Re-positioned
  • Replaced
  • Covalently modified
• Remodeling makes DNA more/less accessible to further activator proteins, general transcription factors, mediator proteins n RNA pol II

Question 16

How can histone modifications be maintained during transcription?

Answer 16

• In vitro, RNA Polymerase II (Pol II) transcribes naked DNA rapidly but encounters slower transcription when DNA is wrapped around nucleosomes
  
  • Forms a “beads on a string” structure.
• RNA Pol II stalls at nucleosome A → hinders transcription progress
• Pol II unwinds approximately half of the DNA from nucleosome A [to overcome the nucleosomal barrier]
• Transcribed DNA loops and begins winding around nucleosome A, while unwinding of the remaining DNA continues.
• Nucleosomes in the promoter region may undergo modification or remodeling to open chromatin → allowing access for the transcriptional machinery.
• As transcription proceeds, nucleosomes are transferred from downstream to upstream regions, ensuring that the chromatin remains accessible for transcription.

Question 17

What are the 3 functional elements required by chromosome replication/stability?

Answer 17

• Telomere
• Replication origin
• Centromere

Question 18

Describe what happens when you transform yeast on plate w histidine

Answer 18

• Expectation:no colonies.
• Reality: Rare integration events where His is now expressed chromosomally

Question 19

How do you identify a eukaryotic origin and replication?

Answer 19

• Work was first done in yeast
• Clone bacterial plasmid that cannot be maintained in yeast n clone into yeast histidine gene
• If expressed, yeast can grow on medium w/o histidine
• Transform yeast n plate on medium w/o histidine
• Expectation: nothing will grow
• Reality: rare recombination that this gene gets into the yeast
• If you clone random fragments of yeast DNA into the initial plasmid, some of them will contain origins of replication
  
  • Allows plasmid maintenance

Question 20

Describe the events of G1

Answer 20

• An origin of replication has a pre-bound complex of six proteins: ORC 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).

Question 21

Describe the events of the S phase

Answer 21

• Cdk2 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 → recruitment of primase, clamp loader and DNA polymerases

Question 22

How are histone modifications maintained during the S phase?

Answer 22

• Large increase in expression of histones in S phase + new subunits are rapidly acetylated → promotes open structure
• NAP-1 (nucleosome assembly protein 1) chaperones H2A-H2B dimers
• CAF-1 (chromatin assembly factor) chaperones H3-H4 tetramers assemble nucleosomes in an open chromatin structure
• Fresh acetylations are removed.
• Reader-writer complex copies epigenetic information from neighbouring nucleosomes.
• RESULT: daughter cells maintain parental cell histone modifications.

Question 23

How do SINEs regulate in both epigenetic and non-epigenetic gene expression?

Answer 23

• Epigenetic
  
  • SINES are GC-rich and so are hotspots of DNA methylation
  • Can silence nearby genes by stimulating chromatin condensation
• Non-epigenetic
  
  • SINEs can act as enhancers or alternative promoters
  • They can recruit transcription factors and promote expression of nearby genes