Topic C: Biochemical Pathways and Complementation Flashcards
What was known about genes at the time of Beadle and Tatum (1940’s)?
- A gene is the fundamental unit of heredity inherited according to Mendel’s two laws
- Genes are located on chromosomes (Morgan) (but we didnt know that the hereditary material was DNA)
- Enzymes catalyze chemical reactions
- Biochemical pathways (they knew that enzymes could work together in a pathway to transform a precursor to an end product)
- Inborn errors of metabolism (deficiencies in biochemical pathways can be hereditary)
Beadle and Tatu provided evidence that a single gene encoded a single enzyme. Why isn’t this super accurate? What did we change to this statement to make it more accurate?
-We know that some enzymes are made of multiple polypeptides, and so for some enzymes they are made of multiple genes.
- So this is more accurately stated as “one gene one polypeptide”, meaning a single gene encodes a single polypeptide
What did Beadle and Tatum’s seek to find out with their experiment?
- all previously known information suggested there was a link between the hereditary unit – a gene – and enzymes. We did not know what this link was.
- Were interested in the relationship between genes and enzymes
What were the material Beadle and Tatum use for their experiment?
- Neurospora (n), fungus
- Minimal media
- Complete media
- fungus Mutated using x-rays to introduce single point mutations to generate auxotrophic mutants
*Prototroph: Wildtype neurospora (no mutations)
*Auxotroph: Mutated neurospora
What is the difference between minimal media plates and complete media plates (in terms of Beadle and Tatums experiment)? Why is it important to have both plates, and both auxotrophs and prototrophs?
- Minimal media: contains the minimum ingredients for neurospora to grow. This includes simple precursors that Neurospora can convert to things like vitamins and amino acids that are necessary to its survival, using biochemical pathways. Only Prototrophs (wildtype Neurospora) can grow on mm.
Auxotrophs cannot survive on mm without additional vitamins or amino acids. - Complete media: contains all end products. Complete media has all of the end products (like vitamins and amino acids) so that even if a biochemical pathway is deficient (not working) the neurospora can grow. Both prototrophs and auxotrophs can survive on complete media
it is important to have both as Prototroph’s are used as a control to ensure growth is possible on all the plates
The complete media control ensures that spores from the mutant strain are still able to grow (still viable) when they have everything they need to survive.
The minimal media ensures that the spores are still auxotrophic so you did not mix up your strains, for example.
How did Beadle and Tatum determine what the fungus was deficient in?
- using their mutated strains, they added them each to minimal media (control), minimal media with amino acids, minimal media with vitamins, and a complete media plate.
- the mutated strains only grew in the compete media, and the minimal media with amino acids.
- Here they confirmed that this strain was an amino acid auxotroph – it had lost the ability to synthesize an essential amino acid from the simple precursors in the minimal media.
How did Beadle and Tatum determine what Amino acid the fungus was deficient in?
- To explore which amino acid the auxotroph could not produce, they separated the auxotrophs into 20 vials and then added an amino acid to each one.
- After testing all of the amino acids by adding them one by one to the minimal media, only the addition of MET (methionine) allowed this strain to grow.
- This meant that this strain was unable to synthesize methionine from the simple precursor in the minimal media.
How do we use mutant strains to determine the order of biochemical pathways? (ex. Methionine)
- each ‘strain’ is an auxotroph that has a different mutation. All cannot produce Methionine, but each strain is ‘blocked’ at a different step/ enzyme
- all of these strains were place on minimal media plates, with the addition of a single amino acid (each amino acid was a different intermediate AA, that was necessary in the production of methionine.)
- For example, Mutant strain 2 could not grow on minimal media that contains homoserine, or on mimimal media with O-acetylhomoserine. It could grow, however, on all of the subsequent intermediates. What this demonstrated was that mutant strain 2 is unable to synthesize Enzyme 2. It can synthesize all of the other enzymes in this pathway. However, because it is blocked at the second step of this pathway it is a met- auxotroph. - The more mutant strains that can grow, the closer the chemical intermediate is to the end product.
- We can use this understanding to construct biosynthetic pathways.
- To find the order of steps to produce Methionine.
- Over and over again they showed that a mutation in a single gene resulted in function of a single enzyme.
What is a heterodimer? a Homodimer?
Heterodimer: A protein/enzyme made of two different polypeptides
Homodimer: Protein/enzyme made of 2 of the same polypeptides
If an enzyme contains multiple subunit polypeptides, that means it has _______
multiple genes
If an enzyme contains multiple subunit polypeptides, that means it has multiple genes. Mutations in any one of these genes could disrupt _________…
disrupt the function of the enzyme entirely
Alleles are
sequence variants of a gene
Diploid individuals will have _____ alleles of a gene (in an individual)
2
How do we determine if a gene is dominant or recessive?
- We determine if a gene is dominant by how the gene is expressed in a heterozygous individual. Whichever phenotype is shown in the heterozygous individual is the dominant gene. (an “Aa” individual will show “A” phenotype)
- Recessive phenotypes are then only observed in individuals that are homozygous for the recessive allele (two ‘a’ genes, will show up as ‘a’ phenotype”
What is homozygous? Heterozygous?
A genotype is heterozygous if the two alleles of a single gene are different in a diploid individual (Aa)
A genotype is described as homozygous if the two alleles of a single gene are the same (AA) in a diploid individual
