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BIOC2003 > Molecular Biology > Flashcards

Molecular Biology Flashcards

1
Q

What are nucleotides composed of?

A
  • Cyclic nitrogenous bases
  • Pentose sugars
  • Phosphate groups
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2
Q

State which nucleotides are pyrimidines and purines, and how many rings each involves

A

PYRIMIDINES (ONE RING):
Uracil, Thymine, Cytosine

PURINES (TWO RINGS)
Adenine, Guanine

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3
Q

How are successive (deoxy)ribose residues joined?

A

They are joined by a phosphate group between the 3’ position of one sugar and the 5’ position of the next sugar.

A chain usually has a free 5’ end, and the other has a free 3’ end.

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4
Q

What is the ratio of concentrations of nucleotides found in the body?

A

[A=T] and [C=G]

[purines] = [pyrimidines]

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5
Q

Describe the polarity of polynucleotide chains.

A

DNA is polar as a result of the phosphodiester bond created between the 5C of one nucleotide and 3C of the other.

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6
Q

Describe the two ways that nucleases cleave phosphodiester bonds.

A
  1. Cleavage of phosphodiester bond leaves product with 5’-phosphate and 3’-hydroxyl ends
  2. Cleavage of phosphodiester bond leaves product with 3’-phosphate and 5’-hydroxyl ends
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7
Q

Describe the function of type II restriction endonucleases.

A

They cleave both strands of dsDNA at specific recognition sites that are palindromic.

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8
Q

Describe the function of DNA ligases.

A

DNA ligase catalyses the formation of phosphodiester bonds between the 5’-phosphate and 3’-hydroxyl ends of adjoining nucleotides in dsDNA

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9
Q

State Chargaff’s Rule.

A

[Purines] : [Pyrimidines] is always equal to 1.

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10
Q

How many nucleotides in the entire human genome?

A

Roughly 3 billion.

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11
Q

What is the packing ratio of DNA?

A 
   actual length
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12
Q

What are the two forms of chromatin present in the body?

A

Euchromatin and Heterochromatin

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13
Q

What are the packing ratios for interphase and mitotic chromosomes?

A

Interphase chromosomes: 1000

Mitotic chromosomes: 10,000

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14
Q

Outline the process by which DNA is organised and condensed into chromosomes.

A
  1. At the simples level, chromatin is a double-stranded helical structure of DNA
  2. DNA is complexed with histones to form nucleosomes
  3. Each nucleosome consists of eight histone proteins around which the DNA wraps 1.65 times
  4. A chromatosome consists of a nucleosome plus the H1 histone
  5. The nucleosomes fold up to produce a 30nm fiber that forms loops averaging 300nm in length
  6. The 300nm fibers are compressed and folded to produce a 250nm - wide fiber.
  7. Tight coiling of the 250-nm fiber produces the chromatid of a chromosome.
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15
Q

Elaborate on what ‘histone’ proteins are, and how they are structured.

A

Histones are the key protein related to DNA packaging.
It is comprised of two copies of 4 core histones, making up the histone octamer.

Structure:
- Histone 3 and 4 make up the H3-H4 dimer
- Two copies of this come together to make the
H3-H4 tetramer
- H2A and H2B make up the H2A-H2B dimer
- Two copies of this come together to make the
H2A-H2B tetramer
- These two tetramers come together to make
the histone octamer

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16
Q

What is a nucleosome?

A

The basic repeating unit of the 11 nm chromatin fibre:

the DNA double helix wraps TWICE around a central core of an histone octamer

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17
Q

Which protein fastens DNA to the nucleosome core?

A

Histone H1

18
Q

What is the result of histone tail modification?

A

Alteration of the nucleosome

19
Q

What factors account for DNA stability?

A

HYDROGEN BONDING - provides specifity, as well as overall stability

STACKING - induced dipole interactions b/w aromatic bases

PRESENCE OF CATIONS - counteracts electrostatic repulsion of anionic phosphate backbones

HYDROPHOBIC EFFECTS - burying the hydrophobic purine and pyrimidine rings in the interior of the double helix increases its stability

20
Q

List the main features of DNA.

A
  • DNA can be denatured by heat or chemicals
  • It is capable of re-annealing to deoxyribonucleotides (DNA), as well as ribonucleotides (RNA)
  • It is susceptible to enzymes
  • Carries on overall negative charges
21
Q

What is the melting temperature of DNA consisting of:

  • entirely of A/T base pairs
  • entirely of C/G base pairs
  • both (naturally occurring DNA)
A

≈ 70 celsius
≈ 110 celsius
≈ 85-90 celsius

22
Q

What is meant by ‘reannealing’?

A

After the double strand is separated by denaturation (heating), cooling the two strands will pair back and renature the double strand.

23
Q

What are the different types of enzymes used for recombinant DNA engineering?

A

Polymerases (DNA polymerases, reverse transcriptase)

Nucleases (e.g. restriction endonucleases, RNAse H)

DNA ligase

Alkaline phosphatase

24
Q

What is meant by FISH?

A

F - Flourescent
I - In
S - Situ
H - Hybridisation

FISH can show transcriptional activity in the alpha- and beta-globing loci which are located on separate chromosomes

25
Q

What are the two main features of DNA replication?

A
  • It is semiconservative

- It is bidirectional

26
Q

How are Okazaki fragments processed?

A

DNA polymerase I extends the Okazaki fragment while its 5’-3’ exonuclease activity removes the RNA primer.

This process, called ‘nick translation’, results in movement of the nick along the lagging strand.

DNA polymerase I dissociates after extending the Okazaki fragment 10-12 nucleotides. DNA ligase binds to the nick.

27
Q

What is a DNA Polymerase?

A

An enzyme that catalyses the polymerisation of deoxyribonucleotides into the DNA strand

28
Q

How many DNA polymerases are found in prokaryotes (E. coli)? State the function of each of them.

A

There are FIVE.

Pol I - RNA primer removal, DNA repair
Pol II - DNA repair
Pol III - Chromosome replication
Pol IV - DNA repair, translesion synthesis (TLS)
Pol V - TLS

29
Q

How many DNA polymerases are found in eukaryotes?

A

There are typically OVER 15.

30
Q

Which prokaryotic DNA has the highest processivity?

A

DNA Pol III (holoenzyme)

31
Q

List some features of DNA polymerases.

A

=> Multisubunit enzymes
=> Processive
=> Template required
=> Proofreading activity (3’-5’ exonuclease)
=> Replicative polymerases remain bound to the replication fork
=> Coordinate synthesis of leading and lagging strands

32
Q

Discuss the trombone model of DNA Replication.

A

The lagging-strand template loops back through the replisome so that the leading and lagging strands are synthesised in the same direction; the SSB tetramer binds to the single-stranded DNA

33
Q

Discuss the synthesis of the lagging strand template DNA.

A

Lagging strand synthesis requires multiple priming events.

As helicase unwinds the DNA template, primase synthesizes an RNA primer. The lagging-strand polymerase completes an Okazaki fragment.

When the lagging-strand polymerase encounters the preceding Okazaki fragment, it releases the lagging strand.

The lagging-strand polymerase binds to a newly synthesized primer and begins synthesizing another Okazaki fragment.

34
Q

What is the structure of E. coli DNA polymerase?

A

Made up of TEN SUBUNITS:

alpha, epsilon, theta, beta, tau, gamma, delta and (delta)’, chi, psi

35
Q

Define ‘genetic engineering’

A

The direct manipulation of the genome of an organism using biotechnology

36
Q

Outline the Polymerase Chain Reaction (PCR).

A

PCR is a technique used to amplify DNA using DNA polymerase, and allows for the exponential growth of DNA. There are (?) main steps involved:

  1. DENATURATION
    the bound template strands are denatured by heat (breaking H-bonds);
    ≈ 95 celsius for 20-30 seconds
  2. ANNEALING
    the sample is cooled just enough to allow the primers to bind to the ends of each of the two template strands;
    50-65 celsius for 20-40 seconds
  3. EXTENSION/ELONGATION
    DNA polymerase attaches to the primers and makes a copy of each template strand;
    ≈ 72 celsius for Taq polymerase

The total yield of DNA strands synthesized after ‘n’ cycles is 2^n.

37
Q

(add after 20)

Draw the DNA melting curve.

A

(insert drawings)

38
Q

(add after 23)

Describe the fractionation of nucleic acids in a gel electrophoresis.

A
  1. DNA restriction fragments in mixture are placed in the well of an agarose gel, and an electric field is applied
  2. Molecules move through the gel pores at a rate inversely proportional to their chain length
  3. Molecules are then subject to autoradiography or are incubated with fluorescent dye, resulting in the signal produced corresponding to the DNA band.

note that a marker is typically used to measure the size of the bands.

39
Q

(add after the one above)

What is Southern Blot and what is it used for?

A

Southern Blot involves transferring DNA molecules from an agarose gel to a membrane in order to locate a particular sequence of DNA within a complex mixture (e.g. locate a gene in a genome).

40
Q

(add after the one above)

Describe the process of Southern Blot.

A
  1. Digest the DNA with a restriction enzyme
  2. Run the digest on an agarose gel (electrophoresis)
  3. Denature the DNA and transfer to the membrane, then treat the membrane with UV light.
  4. Probe the membrane with labeled ssDNA
  5. Visualise the membrane either through autoradiography, colorimetry or luminescence.

BIOC2003 (8 decks)

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