3.01 Bio 1107 Midterm #3

What did Frederick Griffith's mouse experiment show?

Bacteria can take up DNA from the environment (transformation): the S (smooth, virulent) strain kills mice, the R (rough, nonvirulent) strain does not, and heat-killed S strain alone is harmless, but killed S strain + live R strain kills the mouse (p.5)

Which strain is virulent: S or R?

S strain (smooth-looking colonies) is virulent; R strain (rough-looking colonies) is nonvirulent (p.5)

Avery, McCarty & MacLeod: what is the transforming substance?

DNA (p.6)

In the follow-up to Griffith's work, which molecule transformed R strain into S strain?

Only DNA did (RNA, protein, lipid, and carbohydrate did not) (p.6)

What did Hershey & Chase show?

DNA infects cells, not protein; after centrifugation there was no sulfur (protein label) in cells, but phosphorus (DNA label) was in cells (p.7)

Key features of the Watson-Crick DNA structure?

Double helix of 2 antiparallel strands; phosphate backbone outside; hydrogen bonding between complementary bases inside; Chargaff's rule (A=T, G=C) (p.8)

What is Chargaff's rule?

In each base pair the amount of purine equals the amount of pyrimidine: A = T and G = C (p.8)

Which bases pair in DNA?

A with T and G with C (complementary base pairing by hydrogen bonds) (p.8)

What are the three proposed modes of DNA replication?

Conservative (DNA is copied, original stays intact), dispersive (DNA is cut, each piece copied, randomly reattached), semi-conservative (each strand serves as a template for a new strand) (p.9)

Describe semi-conservative replication

DNA strands separate and each serves as a template to synthesize a new strand (p.9)

Where does replication begin?

At the origin of replication, usually made up of an A-T rich region (p.10)

In which direction does replication proceed?

Bidirectionally, from the origin (p.10)

What does helicase do?

Unzips and unwinds DNA (p.11)

What does primase do?

Synthesizes the RNA primer (p.11)

What can DNA polymerase do?

Add nucleotides only to an existing 3' OH end; DNA is synthesized 5' to 3' (p.11)

Describe the leading strand

Synthesized continuously in the 5' to 3' direction, same direction as the unwinding/unzipping (into the replication fork) (p.12)

Describe the lagging strand

Synthesized as Okazaki fragments in the 5' to 3' direction, opposite the direction of unzipping (away from the fork) (p.12)

What seals Okazaki fragments together?

Ligase (p.12)

[Practice Q] Addition of each new nucleotide in DNA replication requires:

An existing 3'-OH end to add to (choice C) (p.26)

[Fig p.5 Griffith] In the four-panel mouse experiment, which two injections kill the mouse?

The live S (smooth, virulent) strain, and the R strain + heat-killed S strain combination; the R strain alone and the heat-killed S strain alone both leave the mouse alive (p.5)

[Fig p.7 Hershey-Chase] Which radioactive labels were used, and what entered the cells?

Sulfur-35 labeled the protein capsids and phosphorus-32 labeled the DNA; after blending and centrifugation there was no sulfur in the cells, but phosphorus was found in the cells, so DNA (not protein) entered the bacteria (p.7)

[Fig p.9 Meselson-Stahl] After ONE round of replication, which replication models give a single density band?

Semi-conservative and dispersive both give one band (hybrid density); the conservative model gives two separate bands (original heavy DNA plus new light DNA) (p.9)

[Fig p.9 Meselson-Stahl] After a SECOND round, how do semi-conservative and dispersive differ on the gradient?

Semi-conservative shows two bands (hybrid middle band plus light upper band); dispersive still shows a single band that keeps shifting upward (lighter) with each generation (p.9)

[Fig p.10 Replication bubble] In the replication bubble diagram, which way does helicase move and where is it located?

Helicase (yellow proteins) sits at both replication forks and moves outward from the bubble in opposite directions (bidirectional replication) (p.10)

[Fig p.13-14 Fork diagram] In the numbered fork-diagram exercise, what is the spot called where the two strands separate?

The replication fork (it's the Y-shaped point where the double helix is being unwound; the fork moves in one direction) (pp.13-14)

[Fig p.13-14 Fork diagram] In that diagram, the blue highlighted strand near DNA polymerase is which strand, and the grey highlighted one?

The blue strand paired with DNA polymerase running into the fork is the leading strand; the grey strand with the RNA primer and Okazaki fragment is the lagging strand (pp.13-14)

[Fig p.14 Fork diagram] Which enzyme sits just ahead of helicase on the unopened double-stranded DNA?

Topoisomerase (p.14)

What organism was Griffith working with in 1928, and what was he trying to do?

Streptococcus pneumoniae (the bacterium that causes pneumonia); he was trying to develop a vaccine. A 'strain' = a genetic variant or subtype of the same species (Lecture p.9)

What is the collective conclusion of Griffith + Avery/McLeod/McCarty + Hershey-Chase?

Griffith: bacteria can be 'transformed' by something from other bacteria; Avery et al: the transforming substance must be DNA; Hershey-Chase: phage DNA infects cells (not protein). Together: DNA is the genetic material (Lecture p.17)

[IKE] Griffith (1928) + Avery, McLeod, McCarty (1944) experiments demonstrated that…

C) DNA is the hereditary material (combined conclusion of both experiments) and D) bacteria can acquire hereditary information from their environment (Griffith only) (Lecture p.18)

What was Rosalind Franklin's contribution to the DNA structure?

X-ray crystallography of DNA provided key clues to its structure; Watson and Crick solved the structure by integrating Chargaff's and Franklin's data (Lecture pp.20-21)

[IKE] A newly discovered bacterial species has 35% G in its DNA. What is the % A?

15%. G = C so C is also 35%; G + C = 70%, so A + T = 30%, and A = T = 15% (35% is the most popular WRONG answer) (Lecture p.23)

[IKE] Write the complementary strand of 5'-CCCTGGGCTCT-3'

3'-GGGACCCGAGA-5'; re-written 5' to 3' by convention this is the same sequence as 5'-AGAGCCCAGGG-3' (label the 5' and 3' ends first, then pair C-G and T-A) (Lecture pp.26-31)

Memory hook: how much of the original molecule does each replication model conserve?

Semi-conservative 'conserves' HALF the original molecule; conservative 'conserves' the ENTIRE original molecule; dispersive breaks up the original molecule into little pieces (Lecture p.33)

Addition of each new nucleotide requires TWO things. What are they?

1) A complementary base to pair with, 2) a 3'-OH on an existing nucleotide strand to add to. DNA synthesis must be templated (base paired) and must start from an existing primer; the energy comes from the phosphates on the incoming nucleotide (Lecture p.41)

What do single-stranded binding (SSB) proteins do in replication?

They coat the separated single strands and stop them from re-annealing (re-pairing with each other) (Lecture p.46 fig)

In the replication enzyme diagram: what do DNA Pol I and DNA Pol III each do?

DNA Pol I removes the primer; DNA Pol III extends the chain (the main polymerase adding new nucleotides) (Lecture p.48 fig)

What does DNA gyrase do in replication?

Helps unwind DNA (relieves the twisting ahead of the helicase at the replication fork) (Lecture p.48 fig)

[TICA] If protein (not DNA) were the hereditary material, what would Hershey and Chase have observed?

B) 35S in the pellet (with the bacteria) and 32P in the supernatant: the labeled protein would enter the cells and the DNA would stay outside, the opposite of the real result (Lecture p.51)

Meselson-Stahl: exact band predictions after 2 generations for each model

Semi-conservative: 1/2 low-density (14N) DNA + 1/2 intermediate-density (hybrid). Conservative: 1/4 high-density (15N) + 3/4 low-density (14N). Dispersive: all intermediate-density (hybrid) (Lecture p.52 fig)

Why are the Hershey-Chase and Meselson-Stahl experiments considered good experiments?

They gave a clear answer by supporting only one hypothesis and ruling out all the others (Lecture p.53)

[IKE] A double-stranded DNA sample contains 20% adenine. What is the % guanine?

30%. A = T = 20% each, so A + T = 40%; the remaining 60% is G + C, split equally, so G = C = 30% (Lecture p.54)

What is a phosphodiester bond, and which bonds hold the two strands together?

Phosphodiester bonds (covalent) link nucleotides along each sugar-phosphate backbone; hydrogen bonds between the nitrogenous bases hold the two antiparallel strands together (Lecture pp.55, 58)

[IKE] The two strands in a DNA double helix are held together by hydrogen bonds between which parts?

The nitrogenous bases on one strand and the nitrogenous bases on the other strand (NOT sugars or phosphates; those form the backbones) (Lecture p.58)

[IKE] How would DNA synthesis be affected if RNA primase were not functional?

D) Synthesis of a complementary DNA strand would NOT initiate (without a primer there is no 3'-OH for DNA polymerase to add to) (Lecture p.62)

[IKE] How would DNA replication be affected if ligase were absent?

B) Replication would result in multiple small segments of DNA instead of a complete polymer (Okazaki fragments would never be sealed together) (Lecture p.63)

In the Hershey-Chase centrifugation, where did each label end up?

Sulfur (35S, protein capsids) stayed in the supernatant; phosphorus (32P, DNA) was in the pellet, where the cells were (Lecture p.15)