Lecture 6 - DNA: The Genetic Material PDF

Lecture 6 – DNA is the genetic material Part 1 – what do genes do? Sir Archibald Garrod – concept of the inborn error of metabolism E.g. Albinism – lack of pigment and alkaptonuria – individuals secrete homogentisic acid into their urine (urine goes black due to exposure to air). Inheritable fact...

Lecture 6 – DNA is the genetic material Part 1 – what do genes do? Sir Archibald Garrod – concept of the inborn error of metabolism E.g. Albinism – lack of pigment and alkaptonuria – individuals secrete homogentisic acid into their urine (urine goes black due to exposure to air). Inheritable factor for a metabolic step was defective Beadle and Tatum – one gene enzyme Used Neurospora crassa as a model organism ( can grow rapidly on a very simple medium containing only salts, carbon and nitrogen sources, biotin (vitamin H) They have haploid nuclei A/a fuse together through meiosis followed by mitosis which makes haploid ascospores Generated mutants with different missing or altered steps in a biochemical pathway. Question Does one gene control the whole conversion or is there a separate gene for each step? To answer this, they made arginine auxotroph's of Neurospora crassa Arginine = ornithine + citrulline Auxotroph: a mutant that requires a particular additional nutrient Prototroph: the normal strain which does not require that nutritional supplements Experiment Step 1: Gene explanation There is only one copy of a gene in each nucleus of a haploid colony. If a gene is mutated, then there is no other copy of the gene to mask that mutation. Step 2: Grow all survivors  1. Dissect individuals microscopic ascospores 2. Transfer each one to a culture tube containing complete medium 3. Do this with hundreds of asci and grow colonies If there is no growth, then discard. Step 3: identify mutants Every survivor was transferred to minimal medium. Wild type grows on minimal medium. Failure to grow identified a potential nutritional requirement. Step 4: identify nutritional requirements Growth on minimal medium containing amino acids identifies a requirement for an amino acid Step 5: identify arginine auxotroph's  Growth on MM supplemented with arginine identifies a requirement for arginine (R) Testing the arginine auxotroph's If the auxotroph's came from one ascus, they have the same mutation. If two of these were mated then every nucleus in the heterokaryon would have the same mutation, and so the heterokaryon would be unable to grow without arginine. 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 expected for every arg- cross, since every nucleus would be defective in the same gene. If the auxotroph's came from different asci, they would probably have different mutations. If these were defective in different parts of the arginine synthesis pathway and they were mated. Then the arg-1 mutant might be complemented by the agr-2 mutant: and the heterokaryon would grow in the absence of arginine. How does complementation work? Step 1: precursor 1 is converted into precursor 2 Step 2: precursor 2 is converted into product Nucleus One (Red nucleus): Defect in Step 1: This nucleus has a mutation that prevents the conversion of Precursor 1 into Precursor 2. As a result, it cannot produce Precursor 2 or the final product, and the pathway is blocked at Step 1. Nucleus Two (Green nucleus): Defect in Step 2: This nucleus has a mutation that prevents the conversion of Precursor 2 into the final product. It can successfully carry out Step 1, so Precursor  2 is produced but cannot be further converted into the final product, blocking the pathway at Step 2. Because the heterokaryons contain both nuclei, each cell can perform step 1 and step 2 resulting in COMPLEMENTATION of the phenotypes: each defect complementing the other. Complementation tests suggested that Beadle and Tatum had isolated 3 classes of mutants defective in arginine biosynthesis Gene control every step of metabolism Part 2 – A key proposal and some key experiments: DNA is the genetic material 1869 – the discovery of nucleic acid Investigated the nuclei of leukocytes (white blood cells) During his experiments, he noticed a substance he called nuclein with unexpected properties which phosphorus was rich in it, but it did contain carbon, nitrogen and hydrogen like known proteins. 1928 – the discovery of transformation He demonstrated bacterial transformation where a bacterium changes its form and function through the action of a transforming principle or transforming factor. He can convert rough cell to smooth cell Streptococcus pneumoniae (Pneumococcus in 1928) R (rough colonies): non-pathogenic S (smooth colonies): pathogenic, secrete a gelatinous polysaccharide capsule The Griffith Experiments 1928 – R cells do not cause disease The Griffith Experiments 1928 – R cells do not cause disease The Griffith Experiments 1928 – only living S cells cause disease The Griffith Experiments 1928 – biological transformation Dead S cells converted to living R cells due to transformation of some R cells to liviing S cell 1944: the Avery – MacLeod – McCarty experiments Method: Heat-killed S strain (virulent bacteria) was used to extract genetic material, which was treated with different enzymes: Protease (destroys proteins) RNase (destroys RNA) DNase (destroys DNA) He chose these enzymes because there are most common micromolecules in bacteria Test: The treated S extract was mixed with R strain (non-virulent bacteria). Results: Protease & RNase treatments: R strain transformed into S strain (indicating transformation occurred). DNase treatment: No transformation, only R strain remained (indicating DNA was the transforming material). Conclusion: DNA is the genetic material responsible for transformation and inheritance. This experiment provided crucial evidence that DNA carries genetic information. 1952 – protein is not the hereditary material Alfred D Hershey and Martha Chase examined the function of protein and DNA in bacteriophage (phage) T2 The structure of phage T2 DNA is tightly packed Inside an icosahedral head That is surrounded by a sheath That is terminated with a base plate from which tail fibers emerge The state of phage research 1948-1952 Differential labelling Labelling phage protein The fate of 35S labeled protein Labelling phage DNA The fate of 32P-labelled DNA