Chromosomes

L10

Statistics that prove we have a lot of long DNA in our cells and body in general

• Our genome is about 3 billion base pairs in length

• Distance between nucleotide residues 0.34nm

• Each cell has about 1 meter of DNA

• We have about 37 trillion cells

=> Put together, we have about 37 billion kilometers of DNA in our bodies — Enough to travel from the Earth to Pluto 5 times!!!

• DNA is 2nm indiameter

  • ~25 thousandtimes thinner than a hair

What are the two levels of organization of our chromosomes?

• chromatin

• chromosome

what are the two phases of eukaryotic DNA

• interphase (contains G1, S, G2)

• metaphase (contains M)

Metaphase

• occurs shortly before cell division during mitosis

• DNA highly compacted for transmission to daughter cells

  • this is the X shape of chromosomes we often think of

• no transcription

  • because too compacted

Interphase

• all the other stages of the cell excluding metaphase

  • G1, S, G2

  • where transcription and DNA replication occur

• DNA not as compacted as in M-phase

chromatin

• nucleoprotein complex DNA exists as during interphase

• is DNA + proteins

• equal mass ratio of protein and DNA

  • can be isolated with an isotonic buffer ( preserves natural structure because has the same salt concentration as the cell’s interior) to be studied

  • when we remove the salt we see the “beads on a string” structure of the nucleosomes

    • this is when the chromatin is extended

    • => even in its loose state the genetic material is still a highly organized DNA protein

• when it is condensed is is about 30nm and fiber)like

nucleosomes

• composed of

  • histones (proteins)

    • this is the protein core 

  • DNA

    • winds around the surface 

      • almost two full turns

• separated by linker DNA (10-90 bp in length)

histones

positively charged proteins that interact with the negatively charged DNA 

• have a core part that wraps itslef with DNA

• and tails that stick out of nucleosome and are accesible to other proteins

what does the protein core of a nucleosome consist of

• an octamer containing two copies of 4 types of histones

  • H2A

  • H2B

  • H3

  • H4

what are the two models for the structure of the condensed 30nm chromatin fiber?

1. Solenoid Model

• Consecutive nucleosomes are stacked along a single helical path connected by linker DNA

2. Two-start Helix Model

• aka the zigzag model, nucleosomes are arranged in alternating rows with straight linker DNA connecting non-consecutive nucleosomes in a zigzag pattern

what does the fifth histone H1 do?

it stabilizes the 30nm chromatin fiber

what regulates chromatin condensation?

• the modification of histone tails

• this is a form of post translational modificaiton

how are histone tails modified?

chemical modificaiton of histone proteins regulate the compaction of chromarin which in turn controls gene expression …by making the DNA more or less accessibel to the transcription machinery

• Acetylation of lysine

  • neutralizes the positive charge of the AA group 

• other modifications can change the charge of the side chains

  • e.g.: methylation, phosphorylation and ubiquination (not a mark for degradation in this scenario since just one Ub)

example of a chemical modification: Lysine to Acetyl Lysine

• Lysine is a positively charged residue

• acetylation removes that charge

• leaves us with Acetyl Lysine

In what way is transcription correlated to chromatin condensation

• decondensed chromatin

  • more active

• condensed chromatin

  • not active or less active

What is special about Polytene Chromosomes and what have we studied with them?

• There are many parallel identical chromosomes

• we often study their giant interphase chromosomes 

  • like that of the Drosophila‘s salivary glands

  • we amplify it to then observe condensed chromatin that presents as dark bands (aka topological domains)

How has polytene chromosome studying shown us that interphase chromatin organization is dynamic?

• puffs

  • show chromatin decondensation

    • with transcriptional activation — are associated with active form of RNA polymerase II (active transcription)

Heterochromatin

• compacted regions

• tend to be rich in repetitive DNA

• poor in genes

Euchromatin

• decondensed regions

• gene rich 

• poor in repetitive DNA

Loops

• organized by non-histone proteins (which play other structural roles)

• tend to be gene-rich because their primary function is to organize the genome in a way to facilitate gene expression

• 1-4Mb in length

Structural Maintenance Chromosomes (SMC)

• an evolutionary conserved family of proteins

  • we find them in bacteria

• they mediate DNA looping

• each monomer contains a coiled coil

  • has a dimer of SMC2 and SMC4

what is the model of loop formation

• SMC proteins help hug/encircle DNA to form loops

  • they slide along DNA/chromatin to make the loops larger or shorter

when do chromosomes occupy most of the nucleus?

during interphase

• each one has a particular territory

why does DNA need to be compacted?

• for segregation

  • DNA in our genome has a contour length of 2m so we need to condense them to overcome entangling

how are mitiotic chromosomes organized

as a series of loops around a central core

• condensin II forms a central scaffold with loops arounf it

• condensin I further compacts these loops into clusters of small nested loops

=> achieves a 10,000 fold compaction of chromatin into linearly organized chromosomes

condensins

set of specialized SMCs that condense the chromosomes during metaphase

• Condensin I: forms a central scaffold with loops around it

• Condensin II: further compacts loops into clusters of smaller nested loops

what are the 3 functional elements required for replication and stable inheritance of chromosomes?

1. origin of replication

2. centromere

3. 2 telomeres (ends)

how many origins or replication are there?

• multiple 

• up to 100,000 in the genome

how have experiments done in budding yeasts helped us understand chromosme elements? — yeast origin of replication

• In these experiments, we use a circular DNA molecule that carries a gene coding for the enzyme that synthesizes Leucine. Then the cells are grown in environment where there is no leucine so they have t produce their own —> those that can’t die and so we can tell where the gene has been transcribed

• ARS (Autonomously Replicating Sequence) is required for plasmid replication

  • it is the yeast origin of replication

how have experiments done in budding yeasts helped us understand chromosme elements? — yeast centromere

• The genomic fragment CEN is the DNA sequence from a yeast chromosome centromere

• CEN is required for good segregation

• centromere is where genes are linked to microtubules

spindle microtubules

the cytoskeletal structures that pull DNA apart

Centromere

• constricted region of the chromosome that holds the two sister chromatids together

• link to the spindle microtubules

Chromatids

During metaphase, each chromosome consists of two identical DNA molecules called sister chromatids, which were produced during DNA replication.

They are joined together at the centromere and are the most visibly prominent feature of a metaphase chromosome

what are two structures that interact with the metaphase chromsome

• kinetochore

  • a multi-protein complex that assembles on the centromere of each sister chromatid

• spindle microtubule

  • fibers that extend from the spindle poles and attach to the kinetochores. The microtubules are responsible for pulling the sister chromatids apart during anaphase, after the metaphase is complete

how does the centromere link to the spindle microtubules?

• there are sequences common to the various yeast centromeres

• contains a nucleosome that includes a centromere-specific histone variant called CENP-A (centromeric protein A)

• CENP-A linked to a specialized protein complex called the kinetochore which then connects the centromeres to the microtubules

how have experiments done in budding yeasts helped us understand chromosme elements? — yeast telomere

• Here, we use a restriction enzyme to produce a linear plasmid to mimic eukaryotic chromosomes

• linear chromosomes lacking TEL sequence for telomere are unstable

  • linear plasmids containing ARS and CEN behave like normal chromosomes if TEL is added to both ends

Telomere

• Protect from exonuclease

• Prevent end-to-end fusion

• Solve the replication pb faced by linear DNA

  • by acting as non-coding buffer zones that are progressively shortened with each division, thus protecting the cell's essential genetic information

What is the Telomere Pb? 

• lagging strand cannot be completed: chromosomes should shorten at the ends in each replication (removal of primers)

  • chromosome shortening is unsustainable

    • because at some point will loose an essential gene

  • telomerase is the solution because it extends the templat to giev primase more template DNA to prime on (preventing the shortening of the ends)

telomerase

• a DNA polymerase

• can extend telomeres 

• hence restores chromosome length to overcome lagging strand end-shortening 

• it is a reverse transcriptase that carries its own template RNA complementary to the DNA repeat

what are telomeres at the DNA level

simple DNA repeat sequences

reverse transcriptase

a DNA polymerase that uses RNA as a template

where is telomerase active

• germ cells

• stem cells

(not needed in somatic cells because only divide a few tomes so exoisting telomeric repeats are enough)

what is the link between cancer and telomerase

• The enzyme is often reactivated in cancer cells

  • hence could be a target for cancer therapy