Lecture 2 - DNA, Chromosomes, & Genomes Flashcards
Proof that genetic information is stored in DNA?
- Most DNA is located in chromosomes, whereas RNA/Proteins are distributed throughout the cell
- Precise correlation b/t amount of DNA and # of chromosomes
- In diploid organisms, somatic cells (diploid) have 2x DNA as germ cells (haploid)
- Molecular composition of DNA unchanged throughout cells of organism, while composition of RNA/protein are variable in different cell types
- DNA is chemically more stable than RNA or Proteins
Griffith experiment: what did it demonstrate, importance to understanding DNA function?
- Griffith develops an assay, which sets the stage for future experimenters to determine that DNA is genetic material
- Worked w/ Streptococcus pneumoniae => pathogenic in mammals
- Griffith wondered what genetic factors imparted pathogenic/nonpathogenic characteristics on this bacteria
- Type R (no polysaccharide capsule protection) -> avirulent
- Type S (polysaccharide capsule protection) -> virulent
Experiment: 4 variables and results
1. living S -> dead mouse
2. heat killed S -> live mouse
3. living R -> live mouse
4. heat killed S + living R ->dead mouse
Conclusion:
- Streptococcus pneumoniae transformation of avirulent R to virulent S strain
- Transformation is host independent
- Some S cell component is transforming principle
Avery, Macleod, & McCarty’s experiments: what did they demonstrate, importance to understanding DNA function?
Experiment: 4 variables and results, using R cells and DNA from heat killed S cells
1. Control, no enzyme -> S colonies
2. Protease -> S colonies
3. RNase -> S colonies
4. DNase -> no colonies
Conclusion: DNA is hereditary material
Watson & Crick Model of DNA
- DNA is a right-handed, double helix
- Bases are flat, perpendicular to sugar/phosphate backbone (stack together)
- ~ 10 bases per turn of helix, 1 turn = 3.4 nm in length
- Has major and minor grooves
- Base hydrogen bonds to complementary base in 2nd strand (Purines A/G H-bond to Pyrimidines T/C due to 3D alignment/fulfillment of H bonding)
- 2 chains are antiparallel
Chargaff’s Principle
[Thymine] = [Adenine]
[Cytosine] = [Guanine]
[T] + [C] = [A] + [G]
How many hydrogen bonds between A/T? C/G?
A/T: 2 hydrogen bonds
C/G: 3 hydrogen bonds
Why does twisting of DNA occur to form helix?
Occurs due to negative charge repulsion of phosphate groups, increasing the distance between negative charges to increase thermodynamic stability
How is DNA so stable?
- H-bond is weak, but thousands together are very strong
- Nitrogenous base-stacking hydrophobic effect => water fearing core (bases) and hydrophilic backbone
What does it mean to say that DNA exhibits polarity?
DNA has directionality, it is synthesized from the 5’ - 3’ end, and the complimentary strand is antiparallel
What are nucleotides? Difference b/t RNA and DNA? Describe all of them, know their structures
What are the nitrogenous bases? For DNA? For RNA
Nucleic acid (DNA/RNA) is polymer, nucleotide is monomer
Nucleotides have deoxyribose/ribose sugar, phosphate group, and nitrogenous base
DNA sugar is deoxyribose, no 2’ OH
RNA sugar is ribose, 2’ OH
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BASES
Purines: adenine, guanine (PURe As Gold)
Pyrimidines: cytosine, uracil, thymine (CUT the PY)
DNA & RNA:
- Adenine
- Guanine
- Cytosine
DNA:
- Thymine
RNA:
- Uracil
Nomenclature (DNA vs RNA nucleotides, nucleotide vs nucleoside, M/D/TP, dNTP vs NTP)
DNA nucleotides: deoxybase-5’-phosphate
RNA nucleotides: base-5’-phosphate
Nucleoside: no phosphate group
Nucleotide: phosphate group
dNMP vs dNDP vs dNTP: mono-, di-, triphosphate
dNTP vs NTP: deoxyribonucleotide triphosphate vs ribonucleotide triphosphate
How to remember structures of nucleotides?
PUrines: doUble ring
- Guanine has oxygen, adenine doesn’t (GO)
Pyrimidines: single ring
- Cytosine has one oxygen, Thymidine and Uracil have 2 oxygen (U TOO)
- ThyMidine has Methyl, uracil doesn’t
What is the bond b/t nucleotides?
Phosphodiester bond b/t 5’ phosphate and 3’ hydroxyl
Difference between DNA and RNA
- RNA has 2’ OH
- Uracil replaces thymine (no methyl)
- RNA is single stranded and folds back on itself (hairpin slide)
- RNA functions in expression of genetic information
Eukaryotic DNA is packaged in ______
Chromosomes
How much of the genome contains coding information?
1.5%
In which phase of the cell cycle does DNA synthesis occur? Chromosomal condensation?
DNA synthesis: interphase (S phase)
Chromosomal condensation: M phase
How many chromosomes do human somatic cells have?
23 x 2 (diploid) = 46 chromosomes
How many chromosomes do yeast cells have?
16 chromosomes
How many chromosomes do e. coli cells have?
1 chromosome (circular)
Where is genetic material contained in eukaryotes vs prokaryotes? What is the eukaryotic nucleolus?
Eukaryotes: nucleus
Nucleolus of euks: a region in the nucleus that functions in specialized RNA synthesis, partial biosynthesis of ribosomes, and synthesis of tRNAs + rRNAs
Prokaryotes: nucleoid
Breakdown of eukaryotic chromosome length
- Human haploid genome is 1 m, but most cells are diploid => 2 m worth of DNA in each cell.
- 2 m is divided in to the 23 different chromosomes => individual chromosomes are between 15-85 mm
- During metaphase, the 85 mm chromosome is 10 um long and 0.5 um wide (HOW IS THIS POSSIBLE, answered in other flashcard)
Unineme vs Multineme - which is correct, evidence?
Unineme: a model for DNA condensation that suggests there is only one strand of DNA per chromosome (CORRECT)
Multineme: a model for DNA condensation that suggests multiple double helices running through the chromosome
Evidence for unineme:
- Method - Pulsed field electrophoreses separates very large DNA
- Results - In lower eukaryotes like yeast, # of different sized DNA molecules = # of nonhomologous chromosomes
- Metabolically labeling drosophilia DNA
What is pulsed field electrophoresis?
An experimental method to separate very large DNA molecules (chromosome-size) ex: yeast (s. cerevisae)
How does DNA condense? What is chromatin? What are chromatin components?
- DNA is not naked in chromosomes => chromosomes contain a lot of protein and even small amounts of RNA
- Chromatin: complex of DNA with protein (histones and nonhistones) and RNA
- Basic component, + at neutral pH: histones (lots of lysines and arginines)
- Acidic component, - at neutral pH: group of proteins called nonhistone chromosomal proteins
What are the 4 levels of condensation to go form DNA double helix to condensed M-phase chromosome?
- DNA double helix (2 nm)
- DNA + histones “beads on a string” (10 nm)
- Solenoid or zigzag (30 nm)
- 30 nm fiber organized into loops via chromatin “scaffold”
What is a nucleosome?
The basic structural unit of eukaryotic chromosomes
Describe DNA bending in a nucleosome
Minor groove facing histone core: AA, TT, and TA dinucleotides preferred
Minor groove facing outside: GC dinucleotide preferred
What are the five classes of histone proteins? What are the molar ratios? What is their amino acid composition?
H1, H2a, H2b, H3, and H4
1 H1: 2 H2a: 2H2b: 2H3: 2 H4 (1:2:2:2:2)
AA comp: 20-30% lysine and arginine => + charge at neutral pH
Describe nucleosome/histone core structure. How are the histone core proteins arranged? How many bp of DNA wrap around nucleosome?
2 molecules each of H2a, H2b, H3, and H4, form nucleosome/histone core octamer, which form ellipsoid beads structure
Arrangement: N-terminal tails exposed, as they are required for formation of 30 nm fiber (higher order structure)
147 bp of DNA wrap around nucleosome => 1.75 turns
Describe nucleosome assembly
- Form 2H3*2H4 tetramer
- Tetramer binds to DNA -> DNA-2H3*2H4 complex
- Complex recruits two copies of H2a*H2b dimer to complete nucleosome core
The nucleosome has a twofold ________
Axis of symmetry
What does the H1 subunit do?
Facilitates nucleosome binding to outside edge of DNA (linker DNA) via H-bond, tightening nucleosome+linker DNA reactions and completing nucleosome => ~166-200 bp and 2 full turns
Histone-DNA interactions: How many points of contact? How many H-bonds? What drives the bending of DNA around nucleosome core? What role do basic amino acids of the core play?
- 14 points of contact, one for each time minor groove of DNA faces histone octamer
- ~ 40 H-bonds involved (majority occur b/t proteins of core and oxygens of phosphodiester bonds w/in minor groove)
- Bending of DNA around nucleosome core driven by force of so mnay H-bonds
- Basic amino acids (+ charge) of the core neutralize negatively charged phosphates of DNA backbone => bending of DNA /o repulsive forces b/t phosphates on inside of bend
What are the two types of chromatin?
Heterochromatin: highly condensed, normally associated w/ low gene expression activity (gene silencing)
Euchromatin: less condensed, associated w/ active gene expression
Chromatin remodeling/modifications (covalent): Consequences and examples (acetylation, methylation, phosphorylation)
- Chromatin can undergo covalent changes/modifications, some of which are reversible
- Consequences include changes in gene expression, which can thus be controlled/regulated => huge implications for organismal development and activation/deactivation of cell functions
- Can affect nucleosomal core (affects gene expression) or N-terminal tails (regulates ability to form 30 nm fiber)
Examples:
Acetylation - neutralizes positive charge of basic AA (like lysine/arginine) => reduces degree of e-static interactions b/t DNA and AA => formation of euchromatin (HATs acetylate, HDACs deacetylate)
Methylation - opposite of acetylation; addition of methyl groups stabilizes positive charge => formation of heterochromatin
Phosphorylation: confers a negative charge to the AA => repulsion b/t DNA and AA => favor formation of euchromatin
Describe nonhistone proteins
- Large number of different proteins (heterogenous)
- Composition of fraction varies from cell type to cell type in the same organism
- Function: regulating expression of different sets of genes
What two models were proposed to explain why chromatin is 30 nm when nucleosome fiber is 11 nm in diameter?
Solenoid Model and Zigzag model
What is the solenoid model? (what supports this, how many nucleosomes per turn, what does the resulting 30 nm fiber look like, where is linker DNA)
- Model supported by electron microscopy & X-ray diffraction studies
- ~ 6 nucleosomes per turn
- 30 nm fiber made up of nucleosome discs stacked on edge in shape of helix
- Linker DNA buried in center of helix but never passes through axis of fiber
What is the zigzag model? (what supports this, what does the resulting 30 nm fiber look like, where is linker DNA, what linker DNA favors this)
- Model supported by biophysical studies showing a spring like property of isolated 30 nm fibers
- 30 nm fiber made up of zigzag pattern of nucleosomes formed through binding of histone H1
- Linker DNA passes through central axis of fiber
- Favored by longer linker DNA
Which level 3 model of chromatin condensation is correct?
Since length of linker DNA varies from species to species, both models may be correct
Describe the final level of condensation - 30 nm scaffold
- A scaffold of nonhistone proteins condenses the 30 nm chromatin fiber (solenoid/zigzag) into loops or supercoiled domains
- Keep loops from tangling
- Nature of scaffold still poorly understood
Specialized structures of chromosomes (Beginning/end vs middle)
- Beginning/end: Telomeres
- Middle: Centromere
Describe centromere structure
- Center of two sister chromatids
- During anaphase, spindle fiber attaches to centromere and separate sisters to opposite ends of cell to become daughter chromosomes
- Constricted region of metaphase chromosome
Centromeres in eukaryotes (unicellular vs multicellular)
Fully understood in unicellular eukaryotes (yeast)
- 110-120 bp in length, interchangeable b/t chromosomes
- 3 regions (I and III are short conserved boundary sequences, II is 88 bp AT rich region for spindle fiber attachment)
Not yet fully understood in multicellular eukaryotes (humans)
- Much bigger and complex than yeast, structure uncertain
- Large amounts of repetitive DNA
- Alpha satellite sequence - 5k-15k copies of 171 bp sequence)
- 450,000 bp segment of x-chromosome sufficient (Contains repetitive DNA and centromere protein binding sites)
Describe telomere structure (end of telomere, eukaryotic telomeres)
- Ends of chromosomes
- 3 functions
End of telomere
- Single stranded overhang of 3’ strand
- Overhang length is organism dependent (125-127 bases in humans)
- G-rich => allows for non WC base pairing, hoogstein bping, G-quartet => like tying loose knot in the end for protection of overhang
Eukaryotic telomeres
- Unique sequence 5’ TAG 3’
- 500-3300x repeat sequence TTAGGG (conserved among vertebrates)
- Shorten w/ age in normal somatic cells
- Don’t shorten w/ age in cancerous and germ cells
What are the 3 functions of telomeres?
- Prevent degradation
- Prevent fusion of ends w/ other DNA molecules
- Facilitate faithful replication of linear end of DNA
What are the two structures of telomeres? Explain
- G-quartet (H-bond b/t 4 individual guanine nucleotides => stabilize 3’ overhang)
- T-loop (3’ overhang does intrastrand bping to protect it, and it is further stabilized by TRF protein complex)
What is in situ (specific site) hybridization?
An experimental tool that takes advantage of the COMPLEMENTARITY of nucleotides to elucidate physical structures of chromosomes
Uses short pieces of radioactively/fluorescently labeled DNA (sometimes RNA) called probes for detecting
