Lecture 6: How we use DNA [G] Flashcards
What did Sir Archibald Garrod say in 1902?
- That defects in your metabolism can alter your phenotype i.e diseases like Albinism and Alkaptonuria
- Found that there was a connection between defective enzymes and altered phenotypes.
Albinism
Failure to produce melanin due to a tyrosinase enzyme defect.
When was the physical mapping of genes to chromosomes?
1910
What is Alkaptonuria?
When individuals secrete homogentisic acid into their urine, which goes black following exposure to air. This is due to a metabolic block.
What did Sir Archibald Garrod state?
That in each case (of both of these diseases) an inheritable factor for a metabolic step (production of pigment; conversion of homogentisic acid to tyrosine) was DEFECTIVE due to a defective gene.
The Neurospora findings revealed that most genes correspond to a region of the genome that directs the synthesis of a single enzyme, leading to the view that each gene encodes one polypeptide chain.
The Neurospora findings revealed that most genes correspond to a region of the genome that directs the synthesis of a single enzyme, leading to the view that each gene encodes one polypeptide chain.
What did Beadle and Tatum do in 1941?
- 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). [UNLESS MUTATED]
- They generated mutants of Neurospora crassa with different missing or altered steps in a biochemical pathway to test Sir Archibald’s idea… that a defective metabolic step could alter the phenotype… altered genes
What is the benefit of Neurospora crassa of being a haploid?
Its haploid lifecycle ensures that mutations are expressed without a second allele masking the effects.
Describe the life cycle of Neurospora crassa
- 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.
Are the nuclei of Neurospora crassa multinucleate?
Yes, but all are identical
If a mutant can’t grow in minimal media, what does this mean?
That the mutant can’t make arginine
What does it mean if a mutant can grow only with arginine?
The mutation blocks the final step in the pathway (citrulline → arginine)
What does it mean if the mutant grows with citrulline or arginine, but not ornithine?
The mutation blocks the first step in the pathway (precursor → ornithine).
Does the pathway of precursor -> ornithine -> citruline -> arginine have one gene controlling the whole conversion, or is there a gene for each step.
To answer this, they made arginine auxotrophs of Neurospora crassa
Define Auxotroph
Auxotrophs are mutants that cannot synthesize certain compounds necessary for growth, and so requires an additional particular nutrient.
Define Prototroph
the normal strain which does not require that nutritional supplement. Contains all the enzymes needed to synthesise molecules, like amino acids.
Describe Beadle and Tatum’s experiment
Mutagenesis
1) Grow mating type little a on rich medium and then illuminate with x- rays. The x rays will induce random mutations in the DNA of Neurospora spores, altering specific genes, some of which disrupt metabolic pathways.
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 the little a mating type 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
Some spores became mutants with non-functional genes.
These mutations affected the mold’s ability to grow on minimal media.
1) Transfer each survivor to minimal medium. Wild- type grows on minimal medium. Failure of these mutants to grow on minimal medium identified a potential nutritional requirement. Hence, these were identified as auxotrophs because they could not synthesize an essential compound, amino acids.
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
Summarise the experiment
Mutate spores with X-rays → Create mutants.
Grow mutants on complete media → All grow.
Transfer mutants to minimal media → Only prototrophs grow.
Supplement minimal media with individual nutrients → Identify auxotrophs.
Use growth patterns to determine where the mutation blocked the metabolic pathway.
Cross mutants to test complementation → Confirm which genes are involved.
What does complementation mean?
To make whole or to make complete
What do the complementation tests by Beadle and Tatum suggest?
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
Describe Beadle and Tatum’s results
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.
What was minimal media supplemented with?
Intermediates (e.g., ornithine, citrulline, arginine).
