BIOL1010/1011 > Genetics: Lecture 4 > Flashcards

Genetics: Lecture 4 Flashcards

History of DNA: Isolation, Structure and Replication (83 cards)

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1
Q

Who did the isolation of nuclein? When did this happen?

A

Friedrich Miescher is responsible for the isolation of nuclein in 1869.

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2
Q

What was Miescher’s question?

A

How to purify nuclein (nucleic acids) and extract them. Also wanted to determine their chemical composition.

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3
Q

Where did he get the nuclein from?

A

He got nuclein from white blood cells from the bandages of patients at a hospital (pus)

  • Later on he also used salmon sperm for a nuclein source as it was rich with nuclein.
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4
Q

How did he purify the nuclein?

A

He used detergent and alcohol to purify the nuclein.

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5
Q

Findings on chemical composition?

A

Miescher established information on the chemical composition. It is made up of:

  • hydrogen
  • nitrogen
  • oxygen
  • phosphorous
  • carbon

He also found that there is a unique ratio of phosphorous to nitrogen.

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6
Q

Main accomplishment or observation?

A

He isolated a new molecule from the nucleus – nuclein. This is what we now call DNA.

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7
Q

Who and when was the transformation of bacteria demonstrated?

A

Fred Griffith demonstrated the transformation of bacteria in 1928.

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8
Q

Question/Goal?

A

Griffith’s goal was to make a vaccine against Diplococcus pneumoniae, the causative agent of pneumonia.

  • He wished to kill the deadly bacterium by heating to make a safe vaccine.
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9
Q

What did he use in his experiment?

A

He used a harmless R-strain of D. pneumoniae which produced rough shaped colonies on a petri plate.

He also used a virulent S-strain of the bacterium produced smooth shaped colonies on a petri plate (they are sugar coated and give protection).

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10
Q

What was his purpose?

A

He wanted to kill the pathogen by heat and inject it into humans top build their immune systems by making antibodies.

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11
Q

How did he do this?

A

Griffith injects live and heat-killed Diplococcus pneumonidae into mice. He performed 4 different experiments.

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12
Q

Griffith Experiment #1

A

He injected living S cells into the mouse (pathogenic control group). As a result, the mouse died.

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13
Q

Griffith Experiment #2

A

He injected living R cells into the mouse (nonpathogenic control). As a result, the mouse survived.

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13
Q

Griffith Experiment #3

A

He injected heat-killed S cells into the mouse (nonpathogenic control). The mouse lived as a result.

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14
Q

Griffith Experiment #4

A

He injected a mixture heat-killed S cells and living R cells into the mouse. As a result the mouse died. When it was looked at closer, there were living S cells found in the mouse.

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15
Q

Interpretation of Griffith’s experiment?

A

Q. Perhaps, the heat-killed S-cells in the mixture in Expt. #4 were not really dead?
A. Not true, because in Expt. #3, the mice injected with heat killed S-cells did not die.

Q. Perhaps, the R-cells in the mixture in Expt. #4 had mutated into the killer form?
A. Not true; Expt. #2 serves as a “control”.

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16
Q

Griffith’s conclusion?

A
  • The ability to cause infection has been transferred from the dead S-cells to the harmless R-cells to make them virulent.
  • The R-cells had been “Transformed”, i.e., a permanent change in the hereditary system of the R-strain of the bacterium coming from the S cells.
  • But what is the transforming principle?
    ➢Protein or DNA?
  • the use of the word principle means that we have no idea what type of thing it is.
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17
Q

Who purified the transforming principle (DNA) and when did they do that?

A

In 1944, Oswald Avery purifies the transforming principle, DNA.

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18
Q

What did Avery do?

A

Avery repeated Griffith’s experiment, focusing on what the principle is, or further questioning the fourth experiment done by Griffith.

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19
Q

What was Avery’s question?

A

Avery’s questions was what is the principle that is transferred from dead S cells to R cells that makes them transform into S cells?

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20
Q

What was the main difference between Avery and Griffith’s experiments?

A

Avery did his experiment in-vitro (in class). This isolated variables and made them easier to control.

Avery first replicated/repeated Griffith’s experiment but added an extra step to separate out what was the principle?

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21
Q

What did Avery use in his experiments?

A

He used detergent to lyse the dead S cells.

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22
Q

What did he do in his experiment?

A
  • He took lysate (liquid left after centrifuging) and mixed it with R cells and they became S cells. He knew the principle was there.
  • He treated the lysate with enzymes to remove the sugar coat. When R cells were added, S cells still formed.
  • He next added enzymes trypsin and chymotrypsin to remove proteins. When R cells were added, S cells were still formed.
  • Next he employed Miescher’s technique to purify DNA by adding alcohol.
  • Next, he got rid of RNA by adding enzyme RNAse. S cells were still formed altering adding R cells
  • Finally, to prove his theory, they used DNAse to chew up the DNA. Finally, when R cells were added. No S cells were formed this time.
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23
Q

What was found in the lysate?

A

There are four components:

  • Sugar coat of S cells
  • Protein
  • RNA
  • DNA
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24
Avery's conclusion?
Avery concluded that the transforming principle was DNA.
25
What are viruses composed of?
Viruses are composed of only nucleic acid (DNA and RNA) and proteins.
26
What are phages? Bacteriophage?
Phages are viruses that attack vertebrates and mammals. Bacteriophage are viruses that attack bacteria. They land on bacteria and inject their DNA. The injected DNA creates more phage and a bunch more is freed when the bacteria bursts open. Then they infect others.
27
Hershey and Chase's Questions?
They wanted to determine if it was the DNA or the proteins that was infecting one another. - Differentiate proteins from nucleic acids.
28
When was the Hershey-Chase Experiment?
1952
29
Theoretical Basis of Hershey-Chase Experiment?
- Protein contains sulphur, but lacks phosphorous. Protein can be specifically labelled with radioactive sulphur (S35) - DNA contains phosphorous, but lacks sulphur. DNA can be specifically labelled with radioactive phosphorous (P32). - By this method, either DNA or protein can be detected in cells.
30
Hershey-Chase Experiment? Batch 1
Batch 1: Phages were grown with radioactive sulphur (s35), which was incorporated into phage protein. 1. Mixed radioactively labelled phages with bacteria. The phages infected the bacterial cells. 2. Agitated the mixture in a blender to free phage parts outside the bacteria from the cells. 3. Centrifuged the mixture so that bacteria formed a pellet at the bottom of the test tube; free phages and phage parts, which are lighter, remained suspended in the liquid. 4. Measured the radioactivity in the pellet at the bottom and the liquid at the top. Conclusion: Whatever makes more phage is not protein.
31
Hershey-Chase Experiment? Batch 2
Batch 2: Phages were grown with radioactive phosphorous (P32), which was incorporated into phage DNA. - The same exact method was done. 1. Mixed radioactively labelled phages with bacteria. The phages infected the bacterial cells. 2. Agitated the mixture in a blender to free phage parts outside the bacteria from the cells. 3. Centrifuged the mixture so that bacteria formed a pellet at the bottom of the test tube; free phages and phage parts, which are lighter, remained suspended in the liquid. 4. Measured the radioactivity in the pellet at the bottom and the liquid at the top. The capsids are not radioactive. Conclusion: Only bacterial DNA injected by the phage is radioactive. This determined that DNA = genes and were the infectious material.
32
Chemical Structure of DNA ?
DNA is composed of a double helix, arranged in an antiparallel arrangement.
33
What is the nucleotide?
The nucleotide is the building block of DNA.
34
What is the nucleotide composed of?
- A five carbon ribose sugar group (pentose). - A phosphate group on the 5' carbon of the ribose. - A base on the 1' carbon of the deoxyribose. * the directionality of the pentose sugar is important. 1' to 5'.
35
Nucleotides joined together form?
Nucleotides are joined together to form a polymer of a DNA strand.
36
Antiparallel Arrangement?
Strands of the DNA molecule are anti-parallel: 5' - 3' 3' - 5'
37
Nitrogenous Bases?
Nitrogenous bases are called nitrogenous because they contain a lot of N atoms (in the % of the atom). - Purines vs. Pyrimidines
38
Purines?
Purines are composed of Adenine and Guanine.
39
Pyrimidines?
Pyrimidines are composed of Cytosine, Thymine, and Uracil (in RNA).
40
What are the two types of Nucleic Acids?
Deoxyribonucleic Acid (DNA) Ribonucleic Acid (RNA)
41
Differences between Nucleic Acids?
- Deoxyribonucleic Acid is missing an oxygen. - DNA is double stranded and RNA is single stranded. - Deoxyribose (sugar) is missing an oxygen whilst ribose in RNA has the O in the OH group. - DNA has nitrogenous bases A-T and C-G - RNA has nitrogenous bases A-U and C-G
42
Bonds in DNA?
- Nucleotides are joined together by covalent phosphodiester bonds. - Nitrogenous bases are joined together by Hydrogen bonds.
43
How is DNA read?
-Each DNA strand has a 5' and a 3' end. - Order of bases in a strand of DNA molecule can be read directionally Example: 5' - TACG - 3'
44
Chargaff's Rule for DNA base composition?
He got experimental evidence that we haver similar (roughly exact) amounts of A&T alongside C&G.
45
A&T vs. C&G
A-T has two H bonds C-G has three H bonds
46
DNA measures?
- One full helix spiral is 3.4 nm long (10 nucleotides) - The radius of DNA is 1 nm - The distance between nucleotides is 0.34 nm.
47
Franklin?
Rosalind Franklin made an ultra pure crystal of the form of DNA (A form).
48
What was her question?
Determine the helical structure of DNA.
49
What did she do for her experiment?
She modified her X-ray diffraction machine to be able to create an image of DNA by shining an X-ray beam at the right place, amplitude, and strength through that crystal.
50
What did she determine?
She established the structure of DNA from this photograph. - She used math and the distance between the blotches to determine this structure.
51
Franklin vs. Watson & Crick?
At the time of Franklin's discovery, Watson & Crick were also working to discover the structure of DNA. - They were originally working together, Franklin even giving her photo to the men to collaborate. - When it was officially determined, both Watson & Crick alongside Franklin published their findings, Franklin's published below. - The media made it out that Franklin confirmed their idea, putting the entirety of the discovery on Watson and Crick despite the work being done by Franklin.
52
What did Watson & Crick do?
Watson & Crick had to “fit” the base pairings within the diameter specified by Franklin’s X-ray.
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How did they do this?
- They measured the space between the blotches on both sides. This determined the spacing of base pairs. - They measured the space between the small dots which shows the helix turn.
54
What did Watson & Crick find?
- Purine and purine was too wide - Pyrimidine and pyrimidine was too narrow - Purine and pyrimidine was a width consistent with X-ray data.
55
What did Watson & Crick find?
- Determined the purine and pyrimidine combination - the helix shape - the presence of a double helix
56
W&C + Franklin Structure of DNA?
- Double-stranded molecule made of polymers of nucleotides. - DNA strands are complementary (A = T, G = C). - Nucleotides in one strand of the DNA determines the nucleotides in the complementary strand. - Strands of the DNA molecular are anti-parallel: 5’ - 3’ 3’ - 5’
57
Structure Details?
- Distance between two rungs is 0.34 nm - 10 rungs of bp complete 1 full turn of DNA helix - Full turn is a 3.4nm distance
58
Meselson & Stahl Question?
How is DNA replicated?
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When did Meselson & Stahl do their experiment?
1958
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What models did they propose?
They proposed 3 models. - Conservative model (old stays together and so does new) - Semiconservative model (mix of equally integrated old and new DNA) - Dispersive model (chunks of new and old DNA mix together)
61
When and where does DNA replication occur?
DNA replication occurs in the nucleus and occurs in S phase in prep for mitosis and meiosis.
62
What was the basis of their experiment?
DNA is largely composed of nitrogen. - There are different isotopes of nitrogen that they used, allowing them to determine/find which DNA is new and which is old. - They have different masses that help distinguish new/old.
63
Meselson & Stahl Experiment?
1. Bacteria cultured in medium with N15 (heavy isotope). All DNA replicated in here will be incorporated with heavy nitrogen. 2. Bacteria was transferred to medium with N14 (lighter isotope) meaning that new DNA in this medium will be lighter. 3. DNA sample is centrifuged after first replication 4. DNA sample centrifuged after second replication.
64
Centrifuging technique?
- They used a cesium chloride (salt) density gradient by centrifuging. - Less dense cesium would be at the top of the tube and more dense cesium would be at the bottom after centrifuging. - Mixed DNA and CsCl, turned on centrifuge, then observe the gradient.
65
Results observed from Meselson & Stahl?
- the first generation was purely heavy - the second generation was purely intermediate - the third generation was a mix of light and intermediate. *** These results supported the second model, the semi-conservative model.
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Issues with other models?
Conservative: wrong after both first and second replication Semiconservative: correct Dispersive model: right after first replication, wrong after second replication
67
How are nucleotides added?
New nucleotides are ALWAYS added at the 3' end of the strand during replication. This is called the template strand.
68
When did Kornberg do his experiment?
In the 1960s
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What was his goal?
Discovery of DNA polymerase: how DNA replication works.
70
What were Kornberg's two requirements for DNA synthesis?
- DNA replication by any DNA polymerase needs to be initiated (started) from a double-stranded segment of nucleic acid, either a DNA:DNA, or DNA:RNA hybrid. -Synthesis of newly replicated DNA can only proceed in the 5ʹ to 3ʹ direction.
71
Helicase?
Unwinds parental double helix at replication forks.
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Single stranded binding protein?
Binds to and stabilizes single-stranded DNA until it is used as a template. - makes sure it does not rehybridize/join together.
73
Primase?
Synthesizes an RNA primer at 5' end of the leading strand and at 5' end of each Okazaki fragment of lagging strand.
74
DNA Polymerase III?
Using parental DNA as a template, it synthesizes new DNA stands by adding nucleotides to an RNA primer or a pre-existing DNA strand. - adding new nucleotides at 3' end of leading strand. Follows the helicase
75
DNA Ligase?
Joins Okazaki fragments of lagging strand; on leading strand, joins 3' end of DNA that replaces primer to the rest of the leading strand DNA.
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DNA Polymerase I with Exonuclease?
Removes the primer from the lagging strand, replacing RNA with DNA to fill in the gaps.
77
Okazaki Fragments?
Okazaki fragments are made up of DNA nucleotides and are small fragments that are synthesized/replicated in the direction away from the replication fork.
78
Leading strand?
Synthesized continuously by DNA Polymerase III.
79
Lagging strand?
Synthesized in fragments by primate/primers and polymerase III completing the synthesis. DNA polymerase I removes primer whilst Ligase puts everything back together.
80
Direction of Replication?
Look at slide/do a drawing
81
How is the Replication Fork formed?
The replication fork is formed by the action of the enzyme DNA helicase at the site of origin of replication. DNA helicase separates the strands of DNA, creating a replication bubble.
82
How do Okazaki fragments form?
Okazaki fragments are formed on the lagging strand, as DNA polymerase synthesizes a section and then must wait for helicase to open up more of the DNA helix upstream. As helicase opens the DNA, primase comes in and lays down a new complementary RNA primer.
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