Lecture 1

Question 1

Miescher isolated nuclei from white blood cells in pus in 1869. Through this, what did he discover?

Answer 1

Found a novel phosphorus-bearing substance = “nuclein”.

Question 2

What is nuclein?

Answer 2

Nuclein is mostly chromatin, a complex of DNA and chromosomal proteins

Question 3

What was the key experiment performed by Frederick Griffith in 1928 that demonstrated transformation in bacteria?

Answer 3

• Observed mutation resulting in a change in Streptococcus pneumoniae — from virulent (S) smooth colonies where bacteria had capsules, to avirulent (R) non pathogenic rough colonies of bacteria without capsules
• Heat-killed virulent colonies could transform avirulent cells into virulent ones. The strain that causes disease can mutate avirulent Streptococcus pneumoniae such that they can cause disease

Question 4

Avery, MacLeod and McCarty did similar experiment that indicated that it was DNA being transferred in the transformation of bacteria. What was this experiment?

Answer 4

Instead of just mixing a heated and lysed virulent cell (S) extract with live non-virulent cells they treated the virulent cell extracts with different hydrolytic enzymes:
1) Deoxyribonuclease (DNase) → Digested DNA such that avirulent was no longer transformed to virulent
2) Ribonuclease (RNase) → Still transformed into virulent
3) Mixture of proteases → Protease treated extract: still converted to virulent

Question 5

What were the 2 results of Avery, MacLeod and McCarty’s experiment that looked at virulent cell extracts and different hydrolytic enzymes?

Answer 5

1) They found that only DNase treatment prevented transformation.
2) They also showed that highly purified DNA from virulent cells could be used to transform non-virulent cells. Experiments excluded both protein and RNA as the chemical agent of transformation

Question 6

What was the procedure for Hershey-Chase transformation experiments (1952) with bacteriophage (aka phage) viruses that infect bacteria in 5 steps?

Answer 6

1) T2 phage (T2 infects E. coli) made up of only protein and DNA
2) Protein can be radioisotopically labeled with 35S → found in protein, but not DNA
3) DNA can be radioisotopically labeled with 32P → abundant in DNA, but not protein
4) Phage coat can be physically removed from cell surface after infection (injection of genetic material)
5) 32P detected in pellet and not in supernatant where 35S was detected in supernant

Question 7

Nucleotides made up of three main components:

Answer 7

1) Nitrogenous base
2) Pentose sugar
3) Phosphate group(s)

Question 8

What is a nucleoside?

Answer 8

base (A, G, C, T, U) + sugar (pentose sugar = deoxyribose)

Question 9

What bases are purines and which are pyrimidines?

Answer 9

• Purine: Adenine and guanine are related structurally to the parent molecule purine
• Pyrimidine: Cytosine, thymine and uracil resemble the parent molecule pyrimidine

Question 10

What forms part of the backbone of nucleic acids?

Answer 10

Pentose sugars (5-carbon sugars)

Question 11

What are the pentoses found in DNA and RNA?

Answer 11

DNA = 2-deoxyribose
RNA = Ribose

Question 12

What’s different about deoxyribose compared to ribose?

Answer 12

Missing the OH group at the 2 position

Question 13

What forms ribonucleosides?

Answer 13

A, U, C, or G plus ribose

Question 14

What forms deoxyribonucleosides?

Answer 14

A, T, C, or G plus 2´-deoxyribose

Question 15

What are Nucleotides?

Answer 15

Nucleoside phosphates (nucleosides plus one or more phosphate groups). Composed of base, sugar, and phosphate group(s)

Question 16

What are the phosphates labelled as in nucleotides?

Answer 16

α, β, and γ

Question 17

What do nucleotides store?

Answer 17

Nucleotides store chemical energy (e.g. ATP is main energy carrier in cells). Energy of nucleoside triphosphate (NTP) > nucleoside diphosphate (NDP) > nucleoside monophosphate (NMP)

Question 18

What are the bonds within nucleic acids (DNA and RNA)?

Answer 18

phosphodiester bonds

Question 19

What are phosphodiester bonds?

Answer 19

3’ carbon of one sugar to the 5’ phosphate group of the next in DNA and RNA

Question 20

What is the process of DNA polymerization?

Answer 20

• 3’ OH attacks the alpha phosphate to form phosphodiester bind with added nucleotide releasing diphosphate in the process → releases energy stored in the nucleoside trisphosphate
• Reaction catalyzed by DNA polymerase using energy stored in incoming dNTP

Question 21

In what direction does DNA polymerization occur?

Answer 21

From 5’ to 3’ end

Question 22

What experiment performed by James Watson, Francis Crick, Rosalind Franklin, Maurice Wilkins – 1953 determined the structure of DNA (Nobel Prize 1962 to Wilkins, Watson, and Crick)?

Answer 22

X-ray diffraction indicated that DNA has simple repeating structure, proposed double helix model of DNA

Question 23

What are the 3 “Chargaff’s rules”?

Answer 23

1) Content of A’s equal to T’s
2) Content of C’s equal to G’s
3) Content of purines (A’s and G’s) equal to that of pyrimidines (C’s and T’s)

Question 24

What did chargaff’s findings hint to?

Answer 24

First hint of DNA base pairing

Question 25

What is specific base pairing (Clue to Structure of Double-stranded DNA)?

Answer 25

H-bonding in DNA: - A-T form 2 H-bonds - G-C form 3 H-bonds

Question 26

Depending on conditions, DNA helix can twist in multiple ways. What are 3 of these ways?

Answer 26

A-, B-, and Z-form DNA

Question 27

What form of DNA is found in physiologically relevant conditions?

Answer 27

B-form DNA

Question 28

What are 5 characteristics of B-form DNA?

Answer 28

1) Right-handed helix 2) ~10 (10.5) residues per turn 3) Major groove is 22 Å (2.2 nm) wide 4) Minor groove is 12 Å (1.2 nm) wide 5) Either side of each base pair faces out from either a major groove or a minor groove and the width of each groove is the distance between backbones of opposing strands

Question 29

True or false: Complimentary DNA strands are antiparallel

Answer 29

TRUE

Question 30

What enables DNA replication?

Answer 30

Base pairing mechanism enables DNA replication

Question 31

What is semi-conservative replication of DNA?

Answer 31

When double stranded DNA is unwound, each single stranded DNA is able to serve as a template for the synthesis of a new strand with a complementary sequence

Question 32

What is the variation in DNA between Organisms?

Answer 32

Ratios of G + C to A + T differ between organisms → still must follow chargaff’s rules: A = T and G = C! --> The total percentage of G + C varies over a range of 22% to 73%

Question 33

What is Tm?

Answer 33

The temperature at which the DNA strands are ½ denatured is the melting temperature or Tm

Question 34

What has a significant effect on Tm?

Answer 34

Higher G/C content has higher melting temp due to the fact that G/C base pairs form stronger interactions since they form 3 H-bonds between them thus requiring more energy to break them.

Question 35

What is denaturation via heat?

Answer 35

With heating, noncovalent forces holding DNA strands together weaken and the bonds break. When the bonds break, the two strands come apart in denaturation or melting

Question 36

In addition to heat, DNA can be denatured by what 2 additional methods?

Answer 36

1) organic solvents 2) high pH (DNA denatures more readily in low salt concentration)

Question 37

When does DNA denature more readily?

Answer 37

DNA denatures more readily in low salt concentration

Question 38

What is DNA renaturation?

Answer 38

After 2 DNA strands separate, under proper conditions the strands can come back together → Process is called annealing or renaturation

Question 39

What are the 3 most important factors involved in DNA renaturation?

Answer 39

1) Temperature: best at about 25C below Tm 2) DNA Concentration: within limits, higher concentration better likelihood that 2 complementary will find each other 3) Renaturation Time: as increase time, more annealing will occur

Question 40

What is Polynucleotide Chain Hybridization?

Answer 40

Process of putting together a combination of 2 different nucleic acids: Strands could be 1 DNA and 1 RNA, could also be 2 DNA with complementary or nearly complementary sequences

Question 41

DNA size is expressed in 3 different ways, what are they?

Answer 41

1) Number of base pairs 2) Molecular weight: 660 is molecular weight of 1 base pair 3) Length: 34 Å (3.4 nm) per helical turn of 10.5 base pairs

Question 42

How can DNA be measured (2 ways)?

Answer 42

DNA size can be measured by electron micrograph or gel electrophoresis

Question 43

What is supercoiled DNA?

Answer 43

Supercoiled DNA coils or wraps around itself like a twisted rubber band