week 1 (DNA experiments)

Question 1

Who lead to the discovery that genes are made of DNA? (1928)

Answer 1

Fred Griffith

Question 2

What type of bacteria did Fred Griffith study in his experiments in 1928?

Answer 2

Streptococcus pneumoniae

Question 3

What was the virulence of the bacterium (streptococcus pneumoniae) know to depend on?

Answer 3

A surrounding polysaccharide capsule that protects the bacterium from the body’s defence system.

Question 4

What property does the polysaccharide capsule give bacterium?

Answer 4

Allowed them to produce smooth edged colonies on an agar surface

Question 5

What was different about the rough edged colony of bacterium?

Answer 5

They were non encapsulated and non-lethal/avirulent

Question 6

Why did Griffith conclude that live (R) bacteria was replaced or transformed by (S) bacteria.

Answer 6

• Mouse + (S) = Death
• Mouse + (R) = Survival
• Mouse + heat killed (S) = Survival
• Mouse + (S) + (R) = Death

Question 7

Why was it concluded that the cell extract contained the transforming principle?

Answer 7

Living (S) cells appeared even when the heat-killed (S) culture in the mixture was replaced by a cell extract prepared from broken (S) cells, which had been freed from both intact cells and the capsular polysaccharide by centrifugation.

Question 8

How did Oswald Avery determine the chemical nature of the transforming principle?

Answer 8

1. Isolated DNA from (S) cells 2. Added the DNA to live (R) bacterial cultures 3. Allowed the mixture to incubate for a period of time then placed the samples on an agar surface and incubated them until colonies appeared

Question 9

What did Avery discover about the colonies that appeared?

Answer 9

Some were (S) type

Question 10

As some of the new colonies were (S) type, what did Avery do to show that this was permanent genetic change?

Answer 10

He dispersed many of the newly formed colonies and placed them on a second agar surface.

Question 11

If the R colonies arising from the original mixture was dispersed, what happened? (S) colonies?

Answer 11

Only R bacteria grew in subsequent generations (S) colonies bred as true (S)

Question 12

How did Avery provide evidence that transformation was caused by DNA not an impurity?

Answer 12

1. Chemical analysis of samples containing the transforming principle showed that the major component was a deoxyribose containing nucleic acid
2. Physical evidence showed that the sample contained a highly viscous substance having the properties of DNA
3. Incubation with trypsin, chymotrypsin (enzymes that catalyse protein hydrolysis) or with ribonuclease (RNase) (a protein that catalyses RNA hydrolysis. This did not affect transforming activity thus is neither protein nor RNA
4. Incubation with DNase (an enzyme that catalyses DNA hydrolysis), inactivated the transforming principle

Question 13

The gene-as-protein theory posed 2 alternative explanations for the gene as protein results: Why could the first point be proved/ eliminated

Answer 13

1. The transforming principle might not be DNA but instead one of the proteins contaminating the sample.
2. DNA somehow affected capsule formation directly by acting in the metabolic pathway for biosynthesis of the polysaccharide and permanently altering this pathway. proved: DNase is not a pure enzyme eliminated: Original work showed insensitivity to proteolytic enzymes

Question 14

What did Hotchkiss’ experiment prove?

Answer 14

Extensive purification ( a sample with only 0.02%) did not reduce transforming activity.

Question 15

How did Hotchkiss prove that the transforming ability of DNA was not limited to capsule synthesis?

Answer 15

He transformed penicillin sensitive bacterial strain to penicillin resistance which is completely distinct from the rough/smooth character of the bacterial capsule

Question 16

What did Erwin Chargaff (1950) discover?

Answer 16

A wide variety of chemical structures in DNA were possible- thus allowing biological specificity

Question 17

What was the hypothesis about the tetranucleotide structure of DNA based on? How did this incorrect conclusion arise?

Answer 17

DNA contains equimolar quantities of A, G , C and T

1. Poor quantitative chemical analysis
2. The DNA isolated usually came from animals, plants and yeast which the 4 base pairs are present in nearly equimolar base concentrations

Question 18

What dareChargaffs rules?

Answer 18

1. DNA composition varies from sepcies to species
2. DNA from any cell of any organisms should have a 1:1 ratio (base Pair Rule) of pyrimidine and purine bases

[A] = [T] and [C] = [G]

Question 19

What are bacterial viruses called?

Answer 19

Bacteriophage or phage for short

Question 20

What experiment was performed my Hershey and Chase? (1952)

Answer 20

The blender experiment: -Viral replication was studied in the bacterium E.coli DNA injected by a bacteriophage T2 particle into a bacterium contains all the information required to synthesise progeny phage particles.

• A single phage particle consists of DNA encased in a protein shell and a long protein tail by which it attaches to sensitive bacteria
• An attached phage can be torn from a bacterial cell wall by violent agitation of the infected cells in a kitchen blender (liquidization) - Those components that could not be shaken free by agitation presumably had been injected into the bacterium

Question 21

How did Hershey and chase use radioactive elements in their experiment?

Answer 21

• DNA is the only phosphorus containing substance in the phage particle
• The proteins of the shell contain only sulphur atoms
  1. Bacteria was infected by T2 phage grown in a medium containing radioactive phosphate (32P)
  2. If the growth medium contained radioactive sulphur (35 S), phage containing radioactive proteins were obtained
  3. These phages are used to infect a bacterial host so the phage DNA and protein molecules can be located by their radioactivity
  4. The mixture was centrifuged and the mixture and collected the pellet containing phage-bacterium complexes
  5. The complexes were resuspended in aqueous solutions and blended

Question 22

What were the findings of Hershey and chase’ use radioactive elements in their experiment?

Answer 22

80% radioactive Sulphur was in the supernatant, 20% in the pellet 30% radioactive Phosphorous in the supernatant, 70% in the pellet When the pellet material was suspended in growth material and reincubated, it was found to be capable of phage production. Thus the ability of the bacterium to synthesise progeny phage was associated with the transfer of Phosphorous hence of DNA from the parental phage to the bacteria

Question 23

How did Franklin and Wilkins obtain a 3d image of DNA fibres? (1950)

Answer 23

X-ray diffraction patterns

Question 24

Define ‘Model’ in scientific terms

Answer 24

Hypothesis or tentative explanation of the way a system works, usually including components, interactions and sequence of events. A successful model suggests additional experiments and allows investigators to make predictions that can be tested in a laboratory If it makes many correct predictions it is more likely to be correct

Question 25

What are the key features of the Watson and Crick model for B-DNA?

Answer 25

1. Two polyribonucleotide strands twist about each other to form a double helix
2. Phosphate and deoxyribose groups form a backbone on the outside of the helix
3. Purine and Pyrimidine base pairs stack inside the helix and form planes perpendicular to the helix axis and the deoxyribose base groups
4. Helix diameter is 2nm
5. Adjacent base pairs are separated by an average distance of 0.24 nm along the helix axis, the structure repeats itself after about 10 base pairs
6. 3 hydrogen bonds stabilising A-T base pairs 2 hydrogen bonds stabilising C-G
7. Two strands are antiparallel: One stand runs 3’-5’ the other runs 5’-3’