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Berkeley MCELLBI 140 - Genetic dissection of biochemical pathways; Complementation

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LECTURE 8: Genetic dissection of biochemical pathways; ComplementationReading: Ch. 3, p. 59, Fig. 3.15 and 3.18; Ch. 7, p. 206-7, 213-215Problems: Ch. 7, solved prob II, III; 7-12, 7-16, 7-19, 7-21, 7-24 – 7-28; Ch. 3, 3-18; Ch.5, 5-31Today, our lecture will basically be a discussion of the Definition of a Gene. We willconcentrate on the definition of a gene as a unit of function; you will discuss the definition of agene 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 onespecific phenotypic trait. So Mendel’s definition of a gene might well have been “one gene, onephenotypic trait”.At about the same time that Mendel’s work was rediscovered, Dr. Archibald Garrod wasstudying several congenital metabolic diseases. In 1902, he published his work onalkaptonuria, a harmless condition in which the urine of affected individuals turns black uponexposure to air. He performed biochemical analyses of affected individuals and showed that asubstance called homogentisic acid, which blackens upon exposure to oxygen, accumulates intheir urine. Unaffected individuals do not excrete this substance, even if they ingest it. Heproposed that affected individuals were incapable of metabolizing homogentisic acid to itsnormal breakdown products. He noted in his paper that “the abnormality is apt to make itsappearance in two or more brothers or sisters whose parents are normal and among whoseforefathers there is no record of its having occurred” and that “of alkaptonuric individuals a verylarge proportion are children of first cousins”. The horizontal pattern of inheritance shouldquickly lead you to the conclusion that the condition is caused by a recessive allele. Garrod, whohad 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 washypothesized at the time that homogentisic acid was a breakdown product of tyrosine, the actualbiochemical pathway required to induce the change from tyrosine to homogentisic acid to finalbreakdown products was unknown. He proposed that affected individuals had an “alternativecourse of metabolism” and excreted homogentisic acid instead of the normal byproducts. Garrodstudied other inborn errors of metabolism and proposed that each arose from a mutation in a generequired 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 + H20Other mutations causing human disease in this metabolic pathway include:Phenylketonuria (PKU): mutation in phenylalanine hydoxylase. Phenyalanine accumulates andis converted to phenylpyruvic acid which is toxic to the developing nervous system.Tyrosinosis (very rare): mutation in tyrosine transaminase. Tyrosine levels are elevated in bloodand urine. Various congenital abnormalities.Tyrosinemia: mutation in p-hydroxyphenylpyruvic acid oxidase. Tyrosine and p-hydroxyphenylpyruvate are elevated in blood and urine. Liver failure, usually within 6months of birth.Albinism: mutation in tyrosinase, the first enzyme in the pathway that converts tyrosine tomelanin.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 werereadily 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 noobvious pecularities and which could only be revealed by chemical analysis? If such exist andare equally rare with the above they may well have wholly eluded notice up till now. Adeliberate search for such, without some guiding indications, appears as hopeless an undertakingas 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 inhaystacks! In the 1940’s, George Beadle and Edward Tatum carried out a series of experimentsto show a clear relationship between genes and enzymes that catalyze the steps of biochemicalreactions. 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 andundergo meiosis to form haploid ascospores).(2) Can induce mutations!(3) Requires very little to grow [grows on “minimal medium” containing only inorganic salts, asimple sugar, and one vitamin (biotin)]Beadle and Tatum reasoned Neurospora must make everything else it needs to grow (aminoacids, other vitamins, nucleic acids, etc) and that the biosynthesis of these substances was undergenetic control. Instead of having to rely upon existing diseases (mutations) to work outbiochemical pathways (like Garrod did), they could instead select mutants in which chemicalreactions 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 orminimal media supplemented with vitamins (and of course the controls: minimal media versuscomplete media). Amino acid auxotrophs were then tested for growth on minimal mediasupplemented with individual amino acids to identify the amino acid that the mutant Neurosporacould no longer synthesize.They isolated four auxotrophs that could only grow on minimal media if it was supplementedwith 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 geneswere required for the biosynthesis of arginine. They named these genes ARG-E, ARG-F, ARG-G, and ARG-H.


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Berkeley MCELLBI 140 - Genetic dissection of biochemical pathways; Complementation

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