The connection between genes, proteins and metabolism CAMPBELL BIOLOGY
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1 Lecture 6 The connection between genes, proteins and metabolism CAMPBELL BIOLOGY 1
2 What do genes do? What cellular processes do they affect? Garrod described: inherited disorders of metabolism also called: Inborn Errors of Metabolism He was studying the disorder called alkaptonuria - affected individuals produce urine that turns black - the disorder runs in families inherited as a recessive trait - affected individuals have a metabolic block preventing: homogentistic acid maleyl aceto acetic acid - homogentistic acid accumulates and is excreted in urine - is not a life-threatening condition Phenylketonuria (PKU) - a more serious inherited defect - a defect in the metabolism of the amino acid: phenylalanine phenylalanine tyrosine - leads to accumulation of a toxic metabolite = phenylpyruvic acid - if untreated, can cause severe mental retardation - all newborns are routinely tested for PKU and other inherited metabolic disorders = the Guthrie heel prick test a single drop of blood is assayed - PKU is a recessive genetic disorder - frequency of occurrence = 1/ 11,000 births 2
3 The discovery of Inborn Errors of Metabolism indicated that genes control metabolism How? Beadle and Tatum (1940 s) proposed: - genes control the production of (or code for) enzymes that function in metabolic pathways But what is the nature of the relationship between genes and enzymes? Does one gene code for one enzyme? Does one gene code for several enzymes? Do several genes code for one enzyme? (Nobel Prize 1958 for their work) Beadle and Tatum proposed: that ONE GENE codes for ONE ENZYME Neurospora crassa This is known as the ONE GENE : ONE ENZYME hypothesis - they studied nutritional mutants = auxotrophs of the red bread mould Neurospora crassa - the wild-type fungus can grow on minimal medium - auxotrophs can t grow on minimal medium they require extra nutritional supplements 3
4 The WILD-TYPE fungus can make all the complex biochemicals necessary for life (amino acids, vitamins, etc) from Minimal Medium (MM): (a) inorganic salts: nitrate, phosphate, potassium, etc (b) Carbon source: sugar AUXOTROPHS are unable to make certain biochemicals and therefore require MM + supplements: (c) amino acids (20) (d) vitamins (e) purines (f) pyrimidines required for protein synthesis required as enzyme cofactors precursors for DNA/RNA precursors for DNA/RNA Beadle and Tatum s experiments: 1. Create mutants by exposing fungal spores to radiation X-rays 2. Pick up individual spores and grow them in test-tubes containing complete medium Result: Both mutant and wild-type spores grow well 3. Transfer fungus to minimal medium Result: The wild-type grows well but the mutant can t grow 4
5 Why can t the mutant grow on minimal medium? - because it can t make an essential biochemical Which essential biochemical can t it make? i.e. What nutritional supplement when added to minimal medium will allow it to grow? Experiment 1 Complete MM MM MM MM Medium + vitamins + purines + amino acids NO GROWTH GROWTH Conclusion: the mutant can t make one or more amino acids Beadle & Tatum 5
6 Experiment 2 Which one of 20 amino acids can the mutant not make? Inoculate mutant onto MM + each amino acid MM MM MM MM MM + glycine + alanine + serine + tyrosine + arginine NO GROWTH GROWTH Conclusion: supplying arginine allows the mutant to grow therefore the mutant can t make arginine itself Beadle & Tatum 6
7 Can these studies tell us anything about the metabolic pathway responsible for biosynthesis of arginine? YES Beadle and Tatum went on to identify 3 different classes of mutants that could not synthesize arginine Each mutant class had a metabolic block at a different step in the metabolic pathway that produces arginine Because they isolated 3 classes of mutants that couldn t synthesize arginine, this suggested that 3 genes were involved in arginine biosynthesis i.e. that the metabolic pathway contained 3 enzymatic steps Metabolic pathways usually involve multiple enzymatic steps Each unique enzyme is encoded by a unique gene??? arginine 7
8 Class C mutants - can t grow if supplied with ornithine nor citrulline - can only grow if they are supplied with arginine - therefore the metabolic block must be in the last enzymatic step that converts citrulline arginine MM Orn Cit Arg Class B mutants - can t grow if supplied with the ornithine - but can grow if they are supplied with citrulline or arginine - therefore the enzymatic block must be in the enzymatic step that converts ornithine citrulline MM Orn Cit Arg 8
9 Class A mutants - Will grow if supplied with either ornithine or citrulline or arginine - Therefore the metabolic block must lie upstream of ornithine MM Orn Cit Arg 9
10 Beadle and Tatum s experiments showed: 1. It is possible to work out the order in which the enzymatic steps occur in a metabolic pathway using a genetic approach 2. That one gene codes for one enzyme 3. This definition was modified when it was discovered that many genes code for proteins that are not enzymes e.g. hemoglobin one gene codes for one protein 4. It was modified again when it was discovered that some proteins contain more than one polypeptide chain each of which is encoded by a separate gene e.g. hemoglobin one gene codes for one polypeptide Beadle and Tatum won the Nobel Prize in
11 geneticsofmd.webnode.com/ dwe00212g01 Duchenne muscular dystrophy.gif 11
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13 Quick lesson on Introns & Exons Example DMD gene: a typical eukaryotic gene is made up of two types of DNA sequence (a) coding sequences called exons and (b) non-coding sequences called introns. After transcription, introns are removed from the mrna (this is called RNA splicing). (We ll be learning about promoters early next week) Transcription (DNA transcribed into RNA) Promoter Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 RNA processing intron removal Mature mrna + addition of a polya tail AAAAAAA Diffential splicing of RNA can lead to generation of multiple proteins 13
14 Immunocytochemisty of muscle biopsies from untreated patient (DMD), healthy control (HC) and the 4 treated patients. New Eng J Med 357:2679 Dec 2007 Review - Foster et al Hum Gene Therapy July
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16 Stamer and Stuber
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