LECTURE 8 Genetic dissection of biochemical pathways Complementation Reading Ch 3 p 59 Fig 3 15 and 3 18 Ch 7 p 206 7 213 215 Problems Ch 7 solved prob II III 7 12 7 16 7 19 7 21 7 24 7 28 Ch 3 3 18 Ch 5 5 31 Today our lecture will basically be a discussion of the Definition of a Gene We will concentrate on the definition of a gene as a unit of function you will discuss the definition of a gene as a unit of structure linear array of DNA base pairs in other courses Mendel was the first to describe the unit of heredity Although he didn t coin the term gene his characters or constant factors were basically defined as factors that controlled one specific phenotypic trait So Mendel s definition of a gene might well have been one gene one phenotypic trait At about the same time that Mendel s work was rediscovered Dr Archibald Garrod was studying several congenital metabolic diseases In 1902 he published his work on alkaptonuria a harmless condition in which the urine of affected individuals turns black upon exposure to air He performed biochemical analyses of affected individuals and showed that a substance called homogentisic acid which blackens upon exposure to oxygen accumulates in their urine Unaffected individuals do not excrete this substance even if they ingest it He proposed that affected individuals were incapable of metabolizing homogentisic acid to its normal breakdown products He noted in his paper that the abnormality is apt to make its appearance in two or more brothers or sisters whose parents are normal and among whose forefathers there is no record of its having occurred and that of alkaptonuric individuals a very large proportion are children of first cousins The horizontal pattern of inheritance should quickly lead you to the conclusion that the condition is caused by a recessive allele Garrod who had read Bateson s translation of Mendel s work came to the same conclusion Garrod hypothesized that alkaptonuria was an inborn error of metabolism Although it was hypothesized at the time that homogentisic acid was a breakdown product of tyrosine the actual biochemical pathway required to induce the change from tyrosine to homogentisic acid to final breakdown products was unknown He proposed that affected individuals had an alternative course of metabolism and excreted homogentisic acid instead of the normal byproducts Garrod studied other inborn errors of metabolism and proposed that each arose from a mutation in a gene required for a specific biochemical reaction Garrod s definition of a gene might well have been one mutant gene one metabolic block We now know that the biochemical pathway is as follows Phenylalanine Tyrosine p Hydroxyphenylpyruvate 2 5 Dihydroxyphenylpyruvate Homogentisic Acid Maleylacetoacetic Acid CO2 H20 Other mutations causing human disease in this metabolic pathway include Phenylketonuria PKU mutation in phenylalanine hydoxylase Phenyalanine accumulates and is converted to phenylpyruvic acid which is toxic to the developing nervous system Tyrosinosis very rare mutation in tyrosine transaminase Tyrosine levels are elevated in blood and urine Various congenital abnormalities Tyrosinemia mutation in p hydroxyphenylpyruvic acid oxidase Tyrosine and phydroxyphenylpyruvate are elevated in blood and urine Liver failure usually within 6 months of birth Albinism mutation in tyrosinase the first enzyme in the pathway that converts tyrosine to melanin In describing his work on alkaptonuria and and other inborn errors of metabolism like albinism Garrod notes that these pecularities are rare in the population as a whole but that they were readily identifiable because of their overt phenotypes Near the end of his 1902 paper he states May it not well be that there are other such chemical abnormalities which are attended by no obvious pecularities and which could only be revealed by chemical analysis If such exist and are equally rare with the above they may well have wholly eluded notice up till now A deliberate search for such without some guiding indications appears as hopeless an undertaking as the proverbial search for a needle in a haystack BUT that s where genetics comes in We can do genetic screens to find the needles in haystacks In the 1940 s George Beadle and Edward Tatum carried out a series of experiments to show a clear relationship between genes and enzymes that catalyze the steps of biochemical reactions They chose the bread mold Neurospora for their work Why 1 Life cycle was known can grow vegetatively as haploid or diploid cells can mate and undergo meiosis to form haploid ascospores 2 Can induce mutations 3 Requires very little to grow grows on minimal medium containing only inorganic salts a simple sugar and one vitamin biotin Beadle and Tatum reasoned Neurospora must make everything else it needs to grow amino acids other vitamins nucleic acids etc and that the biosynthesis of these substances was under genetic control Instead of having to rely upon existing diseases mutations to work out biochemical pathways like Garrod did they could instead select mutants in which chemical reactions were blocked Their experiment genetic screen Fig 7 20 p 214 Mutagenize asexual spores conidia of Neurospora with Xrays or UV light Cross the mutagenized spores with the opposite wild type mating type Collect individual ascospores and grow them on complete medium containing vitamins amino acids etc Conidia from each culture tested on minimal media for growth Those that fail to grow auxotrophs were tested again for growth on minimal media supplemented with amino acids or minimal media supplemented with vitamins and of course the controls minimal media versus complete media Amino acid auxotrophs were then tested for growth on minimal media supplemented with individual amino acids to identify the amino acid that the mutant Neurospora could no longer synthesize They isolated four auxotrophs that could only grow on minimal media if it was supplemented with arginine These arginine auxotrophs carried mutations that blocked arginine biosynthesis Each mutation mapped to a different linkage group so they concluded that at least four genes were required for the biosynthesis of arginine They named these genes ARG E ARG F ARGG and ARG H They then asked whether the arginine synthesis mutants could grow on minimal media supplemented with known intermediates in the biochemical pathway Supplements added to minimal media Strain Wildtype Arg E mutant Arg F mutant Arg G mutant Arg
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