Lecture 6: How we use DNA

Question 1

What did Sir Archibald Garrod say in 1902?

Answer 1

That defects in your metabolism can alter your phenotype i.e Albinism and Alkaptonuria

Question 2

What is Alkaptonuria?

Answer 2

When individuals secrete homogentisic acid into their urine, which goes black following exposure to air.

Question 3

What did Sir Archibald Garrod state?

Answer 3

that in each case an inheritable factor for a metabolic step (production of pigment; conversion of homogentisic acid to tyrosine) was DEFECTIVE

Question 4

What did Beadle and Tatum do in 1941?

Answer 4

• They used Neurospora crassa (the red bread mould) as a model organism, as It can grow rapidly on a very simple medium containing only salts, carbon and nitrogen sources, and biotin (vitamin H).
• They generated mutants of Neurospora crassa with different missing or altered steps in a biochemical pathway to test Sir Archibald’s idea.

Question 5

Describe the life cycle of Neurospora crassa

Answer 5

• There are 8 haploid spores in a spore case called an ascus. 4 belong to one mating type, 4 to another.
• The ascus spores germinate and once they germinate, they will form haploid vegetative mycelia, which are fungal haploid masses.
• These haploid masses are maintained asexually as they make conidiospores, which regenerate more haploid mycelium.
• The 2 vegetative mycelia grow into each other. The cells of the opposite mating type will then fuse to form a binucleate heterokaryon.
• The A and a nuceli will then fuse in pairs to form a transient diploid phase.
• Meiosis, then mitosis occurs.
• 8 asca spores are then formed, They will be wrapped in a fruiting body.
• Then the life cycle repeats itself.

Question 6

Are the nuclei of Neurospora crassa multinucleate?

Answer 6

Yes, but all are identical

Question 7

Does the pathway of ornithine -> citruline -> arginine have one gene controlling the whole conversion, or is there a gene for each step.

Answer 7

To answer this, they made arginine auxotrophs of Neurospora crassa

Question 8

Define Auxotroph

Answer 8

a mutant that requires a particular additional nutrient.

Question 9

Define Prototroph

Answer 9

the normal strain which does not require that nutritional supplement.

Question 10

To answer this, they made arginine auxotrophs of Neurospora crassa

Question 11

Describe Beadle and Tatum’s experiment

Answer 11

Mutagenesis
1) Grow mating type little a on rich medium and then illuminate with x- rays.

2) As there is only one copy of each gene in each nucleus, of a haploid colony , if a gene is mutated there is no other copy of that gene to mask that mutation. So, breed with big A mating type to find the mutated spore.

2) This will produce the fruiting bodies, with a very rare ascus with 4 auxotrophs and 4 prototrophic spores in that ascus.

3) Then, you screen for growth in order to find the mutants.

Grow all surviving spores
1) Dissect individual microscopic ascospores

2) Pick up each spore individually and transfer each one to a culture tube containing complete medium.

3) Do this with hundreds of asci and grow colonies.

Identify mutants
1) Transfer each survivor to minimal medium. Wild- type grows on minimal medium. Failure to grow identified a potential nutritional requirement.

Identify nutritional requirement
1) Grown on minimal medium containing amino acids.

Identify arginine auxotrophs
1) Make 20 different tubes, each suplemented with minimal medium and a different amino acid. Look for what only grows when Arginine is added.

• If there were ONLY ONE GENE for the whole of arginine biosynthesis, the same result would be expected for every arg- cross, since every nucleus would be defective in the same gene.
• Growth of a heterokaryon on minimal medium would provide evidence of multiple steps

Question 12

What does complementation mean?

Answer 12

To make whole or to make complete

Question 13

What do the complementation tests by Beadle and Tatum suggest?

Answer 13

Because the heterokaryons contain both nuclei, each cell can perform step 1 and step 2 resulting in COMPLEMENTATION of the phenotypes: each defect complements the other. Complementation tests suggested that Beadle and Tatum had isolated 3 classes of mutants defective in arginine biosynthesis

Question 14

Describe Beadle and Tatum’s results

Answer 14

They isolated 3 classes of arginine auxotrophs: a gene controlled each step and so Archibald Garrod was correct. Hence proving that genes control every single step in our metabolism.

Question 15

What did Swiss physician Friedrich Miescher suggest in 1869?

Answer 15

That nuclein may be the basis of hereditary.

Question 16

Who discovered transformation in 1928?

Answer 16

Frederick Griffith. He demonstrated bacterial transformation, where a bacterium changes its form and function through the action of a transforming principle or transforming factor.

Question 17

What are the 2 forms of Streptococcus pneumoniae ?

Answer 17

R and S

Question 18

Describe R colonies

Answer 18

They are rough and non-pathogenic

Question 19

Describe S colonies

Answer 19

They are smooth, pathogenic, and they secrete a gelatinous polysaccharide capsule

Question 20

What animal did Frederick Griffith work on?

Answer 20

Mice

Question 21

Describe the Griffith experiments

Answer 21

• He injected mice with living R cells and the mice remained healthy.
• He then killed them and cut their hearts out. He spread the heart tissue on to an agar plate and recovered living R cells.
• He then came to the conclusion that R cells survive in mice, but do not cause pneumonia, so R cells are non-pathogenic.
• He then injected the mice with living S cells and the mice got pneumonia.
• Living S cells could then be recovered from the mouse heart tissue.
• He then came to the conclusion that S cells survive in mice, and cause pneumonia. S cells are pathogenic.
• Mice were then injected with heat-killed S cells. The mice remained healthy. So the dead cells were defective and did not cause disease. So living streptococci was needed to cause pneumonia.
• He then came to the conclusion that the living cell was pathogenic, not something that it makes.
• He then mixed heat-killed S cells with living R cells, and injected this mixture into the mice.
• The mice got pneumonia.
• He found a mixture of both R cells and S cells in the mouse heart tissue.
• He concluded that an S cell transforming principle that survives heat treatment has altered (transformed) some of the R cells.

Question 22

Describe the Avery-MacLeod-McCarty experiments

Answer 22

• They grew the living S cells in a flask and then boiled them to kill them.
• They then spun out all of the insoluble material and a soluble extract of S cells was left behind.
• The soluble S cell extract was then divided into 3 portions .
• In the first test tube, a protease was added. In the second test tube, RNase was added. In the third test tube, DNase was added. (The 3 most common macromolocules that you can extract from bacteria).
• They then tipped what was left after the enzymes did their work into living R cells. They then grew this, and looked to see what they had.
• In the protease, there was a mixture of R cells and S cells. So protease did not destroy the transforming principle, and so the transforming princilple isn’t a protein.
• With the RNase, there was also a mixture of R cells and S cells, so RNA cannot be the transforming principle.
• With the DNase, there was only the living R cells. So the trasnforming principle is DNA.

-The conclusion was that DNAse destroys the transforming principle

• They then purified DNA from S cells and they added that to cultures of R cells, and saw transformation of S cells into R cells.
• We would now consider these Avery – MacLeod – McCarty experiments as providing definitive evidence that DNA is the hereditary material.
• However, these results were not received well at the time because they were medics and scientific opinion favoured protein as the hereditary material.

Question 23

what is protease?

Answer 23

An enzyme that degraded protein

Question 24

what is RNase?

Answer 24

An enzyme that degrades RNA

Question 25

What is DNase?

Answer 25

An enzyme that degrades DNA.

Question 26

Describe the structure of phage 2

Answer 26

• DNA is tightly packed…
• …inside an icosahedral head…
• …attached to a core…
• …that is surrounded by a sheath…
• …that is terminated with a base plate from which tail fibres emerge

Question 27

Dsescribe the state of phage research between 1948 and 1952

Answer 27

• T2 phage infects E coli, with attachment mediated by the base plate and fibres.
• After infection, the phage particles remain attached to the bacterium, but the heads appear empty, forming ‘ghosts’.
• The E coli undergoes lysis and releaseas new virions.

Question 28

How did Beadle and Tatum summarise their findings?

Answer 28

As one gene, one enzyme/ One gene, one function

Question 29

What did Alfred D Hershey and Martha Chase (his research assistant) do?

Answer 29

examined the functions of protein and DNA in bacteriophage (phage) T2

Question 30

What did Martha Chase do to trace the fate of phage protein and DNA?

Answer 30

She labelled them radioactively

Question 31

Is it true that Avery et al even added purified DNA from S cells to cultures of R cells and saw transformation of some R cells to S cells?

Answer 31

Yes

• We would now consider these Avery – MacLeod – McCarty experiments as providing definitive evidence that DNA is the hereditary material.
• However, these results were not well received at the time, as scientific opinion favoured protein as the hereditary material … because of an experimental approach from c.1910.

Question 32

In order to work out the role that both protein and DNA played in T2 phage, what did Martha Chase do?

Answer 32

She worked out how to differentially lable the protein or the DNA.

Question 33

What did Martha Chase do if she wanted to label the protein in T2 phage?

Answer 33

• She grew E coli in the presence of radioactive 35 S. (radioactive methionine)
• She then infected the E coli cells with T2 phage.
• Collect the cells when they’re bursting and centrifuge them. The living and lysed bacteria will go to the bottom of the test tube and be discarded. Whilst the phage suspension will be collected.
• What’s left will be a suspension of radio-labelled phage, where the proteins are radioactively labelled.

Question 34

What did Martha Chase do if she wanted to label the DNA in T2 phage?

Answer 34

• She infected E coli in the presence of radioactive 32P
• The new viriants that were generated then had their DNA radioactively labelled.

Question 35

Is sulfur present in proteins or in DNA?

Answer 35

Sulfur is present in proteins (in methionine and cysteine) but not in DNA.

Question 36

Is phosphorous present in proteins or DNA.

Answer 36

Phosphorous is present in large amounts in nucleic acids (in the sugar-phosphate backbone), but generally not in proteins (although some can be phosphorylated).

Question 37

What is the fate of 35 S labelled protein?

Answer 37

• They added the radio-labelled phage to fresh E coli.
• After the attachment of the phage and the formation of ghosts, they then knocked off the ghosts using a blender.
• They then centrifuged. At the top, they had the ghosts and the unadsorbed phage, which they discarded of.
• They grew the pellets and they could not detect the S 35 in this pellet.
• This hence supported the conclusion that protein is not the genetic material, as there was no inheritance of S 35.

Question 38

What is the fate of 32 P labelled DNA?

Answer 38

• Add the radio-labelled phage to E-coli, allow infection, knock off the ghosts, and centrifuge.
• The cells in the pellet were radioactive and labelled by 32 P.
• Radioactive DNA passed from one generation to the next, and so DNA was clearly the genetic material.

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Question 39

Why was Hershey scared to say that the DNA was the genetic material?

Answer 39

Because if he did, then he could lose his job.

Instead he said: ‘the DNA has some function. Further chemical inferences should not be drawn from the experiments presented.”