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lf206: molecular cell biology > Lecture 4: Eukaryotic Genomes > Flashcards

Lecture 4: Eukaryotic Genomes Flashcards

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1
Q

What makes our genomes so large?

A
  • 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
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2
Q

What is gene duplication?

A

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

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3
Q

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

A
  • 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.
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4
Q

What are 2 main differences of eukaryotic and prokaryotic genomes?

A
  • 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
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5
Q

What does eukaryotic DNA code for?

A
  • Genes that encode proteins
  • ncDNA
  • Repetitive DNA
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6
Q

How are new genes created? Give an example

A
  • 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
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7
Q

Describe the developmental changes in globin expression

A
  • 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
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8
Q

Describe the human genome’s composition and its function

A
  • 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.
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9
Q

Describe the structure of chromatin

A
  • Complex of DNA + proteins
  • Organized into chromosomes
  • Most of the proteins are histones
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10
Q

Describe the function of chromatin

A
  • Packaging DNA
  • Reinforcing macromolecule structures for mitosis
  • Preventing DNA damage
  • Regulating gene expression
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11
Q

What are 2 forms of chromatin?

A
  • 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
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12
Q

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

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
    • Histones separate according to a combination of size and charge.
  • They are often modified, appearing as doublet/triplets/smears rather than singlet proteins
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13
Q

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

A

Histone modifications n uneven nucleosome spacing

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14
Q

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

A
  • 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
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15
Q

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

A
  • 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
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16
Q

How can histone modifications be maintained during transcription?

A
  • 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.
17
Q

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

A
  • Telomere
  • Replication origin
  • Centromere
18
Q

Describe what happens when you transform yeast on plate w histidine

A
  • Expectation:no colonies.
  • Reality: Rare integration events where His is now expressed chromosomally
19
Q

How do you identify a eukaryotic origin and replication?

A
  • 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
20
Q

Describe the events of G1

A
  • 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).
21
Q

Describe the events of the S phase

A
  • 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
22
Q

How are histone modifications maintained during the S phase?

A
  • 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.
23
Q

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

A
  • 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

lf206: molecular cell biology (16 decks)

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