Question What is the chemical nature of the repressor 1 MCB 140 11 8 2006 If you think about it The repressor has to directly bind to a specific DNA sequence the operator in the E coli genome From a reverse engineering perspective the simplest way to design something that interacts with DNA sequencespecifically is to use a nucleic acid that is complementary to that DNA 2 MCB 140 11 8 2006 In 1959 a biochemical experiment was done proving that the lac repressor is not a protein and is most likely an RNA molecule In 1965 a genetic experiment was done proving that biochemical experiment entirely wrong 3 MCB 140 11 8 2006 1959 When the z gene and the i gene arrive in an i cytoplasm no repressor the z gene becomes active and stays active for about 2 hours At that point the repressor is made and shuts the z gene off This offers an elegant opportunity to determine the biochemical nature of the repressor add an inhibitor of protein synthesis and let the cells spend their first two hours post mating in that good stuff A B Pardee and L S Prestidge BBA 36 545 If the repressor is a protein then inhibiting its synthesis following transfer of z into an i cytoplasm should allow for constitutive synthesis of galactosidase 4 MCB 140 11 8 2006 The result Inhibiting protein synthesis does not destroy the plateau effect gal still goes off Conclusion the repressor probably is not a protein since it was made when protein synthesis was inhibited Ribonucleic acid would seem a likely candidate for the role of the repressor A B Pardee and L S Prestidge BBA 36 545 5 MCB 140 11 8 2006 A distinction between genetics and biochemistry The validity of Pardee s conclusion is unequivocally dependent on a biochemical phenomenon total inhibition of protein synthesis by 5 methyltryptophan It turns out that not all protein synthesis in E coli is inhibited by 5 me T A genetic experiment crosses between strains of different genotype was performed to prove Pardee wrong 6 MCB 140 11 8 2006 8 28 7 MCB 140 11 8 2006 Let s mate 1 Make an i strain of E coli constitutive 2 Make several such strains different kinds of mutations 3 Mate those i E coli to other E coli carrying nonsense suppressor tRNA genes 4 See what happens 8 MCB 140 11 8 2006 Suppression of and complementation among mutants of the regulatory gene of the lactose operon of Escherichia coli Bourgeois S Cohn M Orgel LE JMB 14 300 1965 1 2 Cross an i strain with a tRNAsup strain Measure activity of gal or lactose in the medium Suppressor lactose Strain i i none wt no yes 8 4400 su1 no 6 yes 4400 su3 no 14 yes 3900 7600 7000 1200 1550 3400 7100 9 MCB 140 11 8 2006 In their own words We know that the suppressor strains used here act at the level of translation by allowing the chain terminating codon to be read as an amino acid We have shown suppression of i mutations by these suppressors and conclude therefore that the i gene of the lactose operon codes for a protein 10 MCB 140 11 8 2006 stimulus Regulation of genes occurs via the interaction of transacting factors proteins with cis acting sequences near the genes themselves 11 MCB 140 11 8 2006 Fran ois Jacob If it s true for E coli it must be true for E lephant 12 MCB 140 11 8 2006 Budding brewer s and baker s yeast Saccharomyces cerevisiae 13 MCB 140 11 8 2006 Yeast ferment all available sugar even in the presence of oxygen Mammals sugar glycolysis pyruvate respiration TCA O P CO2 H2O S c sugar glycolysis pyruvate fermentation C2H5OH CO2 14 MCB 140 11 8 2006 A major evolutionary conservation of cellular response to sugar in the medium In Saccharomyces cerevisiae budding yeast a fungus a eukaryote 1 Enzymes for utilization of the sugar galactose are induced 1000 fold by galactose 2 These enzymes are also severely repressed by glucose in the medium 3 Thus for these genes to be induced fully the medium must contain galactose and no glucose Just like E coli 15 MCB 140 11 8 2006 Analogy and homology as tools in genetic investigation Animal Mandibular Arch ventral Mandibular Arch dorsal Hyoid Arch dorsal Shark Meckel s cartilage Palatoquadrate cartilage Hyomandibular cartiliage Amphibian Articular bone Quadrate bone Stapes Mammal Malleus Incus Stapes 16 MCB 140 11 8 2006 17 MCB 140 11 8 2006 First experiment Howard Douglas 1963 Goal make i yeast that is yeast that synthesize galactose metabolizing enzymes constitutively 1 Take mutant strain gal3 that has a markedly delayed response to galactose and does not grow on it very well at all 2 Grow on galactose see what grows Douglas HC Penroy G 1963 A gene controlling inducibility of the galactose pathway enzymes in Saccharomyces Biochim Biophys Acta 68 155 18 MCB 140 11 8 2006 What grew 1 One would expect revertants of the gal3 mutation Those didn t show up 2 What did show up was true i cells yeast that synthesized the GAL enzymes constitutively that s why they grew 3 They made i i cells they were inducible 19 MCB 140 11 8 2006 Conclusion ta daaa The inducibility in gene in yeast fits the description proposed by Monod and Jacob for regulator genes By analogy with the lactose system in E coli the galactoseinducible state in yeast corresponds to the production of a repressor due to i while the constitutive state due to i represents a failure to repression 20 MCB 140 11 8 2006 Nothin but net Indeed the i gene is now called GAL80 Its product Gal80p is a repressor of GAL genes 21 MCB 140 11 8 2006 But We might have expected by further analogy with the systems in E coli to find mutations linked to the structural galactose genes and to be expressed as cis dominant constitutives but these have not yet been detected in our material In other words they wanted to find operator mutations and didn t find them 22 MCB 140 11 8 2006 Screen for gal cells H Douglas D Hawthorne 1964 1 Take wt haploid yeast 2 Zap them with UV light 3 Replica plate to find those that are gal 4 1 1000 are mutant Douglas HC Hawthorne D 1964 Enzymatic expression and genetic linkage of genes controlling lactose utilization in Saccharomyces Genetics 49 837 23 MCB 140 11 8 2006 7 5a 24 MCB 140 11 8 2006 A 8 25 MCB 140 11 8 2006 Initial analysis of mutants 1 2 3 4 Cross mutant to wt Sporulate Dissect the tetrad ascus Confirm that 2 spores are GAL and 2 are gal 26 MCB 140 11 8 2006 What came out Large number of mutants in different genes GAL1 six mutants GAL2 five mutants GAL3 six mutants GAL4 two mutants GAL5 nine mutants GAL7 four mutants GAL10 one mutant 27 MCB 140 11 8 2006 When genes are linked PDs exceed …
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