Lecture 9 One Gene One enzyme

1. Lecture 9One Gene One enzyme

2. Genes DNA sequences that code for RNA- prm, RBS, ORF, Term Is there a ribosome binding site upstream of the ATG Is there a promoter upstream of the ribosome binding site Prokaryotic Genes PROMOTER 3’ 5’ antisense ---TTGACAT------TATAAT-------AT-/-AGGAGGT-/-ATGCCC CTT TTG TGA ---AACTGTA------ATATTA-------TA-/-TCCTCCA-/-TAC GGG GAA AAC ATT sense (-35) 3’ (-10) 5’ RIBOSOME BINDING SITE 3’ 5’ U-/-AGGAGGU-/-AUGCCC CUU UUG UGA Met Pro leu leu stp When ALL OF THESE RULES ARE SATISFIED THEN AND ONLY THEN WILL A PIECE OF DNA GENERATE A PROTEIN. EUKARYOTES ARE EVEN MORE COMPLICATED.

3. In the next few lectures, the following questions will be Addressed: What is the structure of a gene? How does a gene function? How is information stored on the gene? What is the relationship between genotype and phenotype? One gene One enzyme hypothesis

4. DMD Duchene muscular dystrophy is a human disease caused by an X-linked recessive mutation. DMD affects one in 3500 males. Age of onset is between one and six years. Affected individuals are often initially identified because they rise from the prone position in an unusual manner.

5. Pathways Biologists and clinicians want to address the question of how altering a particular set of base pairs that make up the 3 billion base pairs in the human genome led to this phenotype. Formal genetics provides little information about these intermediary steps. Since the 1970’s, a set of techniques have been developed that enable us to elucidate each step in a pathway These techniques are generally placed under the rubric of Molecular Biology or Molecular Genetics

6. DMD Through these techniques, it was found that DMD patients have a mutation in a single gene. The normal function of the gene is to enable muscle fibers to make a protein called dystrophin. Dystrophin localizes to the plasma membrane in muscle cells. The normal dystrophin protein stabilizes the muscles during muscle contractions. Muscle fibers in people affected with DMD are extremely deficient in dystrophin. Without this protein, the plasma membrane ruptures during muscle contraction and degeneration of the muscle tissue occurs.

7. Huntington's Disease Huntington's disease (HD) results from degeneration of neurons, in certain areas of the brain. This degeneration causes uncontrolled movements, loss of intellectual faculties, emotional disturbance and early death This disease is caused by a single dominant mutation on the forth chromosome. Each child of an HD parent has a 50-50 chance of inheriting the mutation. A person who inherits the mutation will sooner or later develop the disease! What is the normal function of the Huntington gene? What happens in the mutant? Can it be blocked? To understand this disease we need an interdisciplinary approach. DNA Geneticist Bioinformatics RNA Molecular biologist Protein Biochemist Protein complex Biochemist Cellular phenotype Cell biologists Organism phenotype Physicians

8. Alkaptonuria Degenerative disease. Darkening of connective tissue, arthritis Darkening of urine Garrod characterized the disorder- using Mendels rules- Autosomal recessive. Affected individuals had normal parents and normal offspring. 1909 Garrod termed the defect- inborn error (genetic) of metabolism. Homogentisic acid is secreted in urine of these patients. This is an aromatic compound and so Garrod suggested that it was an intermediate that was accumulating in mutant individuals and was caused by lack of enzyme that splits aromatic rings of amino acids. Garrods results and his explanation were ignored 1958 La Du showed that accumulation of homogentistic acid is due to absence of enzyme in liver extracts 1994 Seidman mapped gene to chromosome 3 in human 1996 Gene cloned and mutant identified P230S &V300G 2000 Enzyme principally expressed in liver and kidneys

9. Ornithine Citruline Arginine Enzyme1 Enzyme2 The work of biochemists showed that chemical compounds in the cell are synthesized through a series of intermediates-a biochemical pathway Glutamic acid- How do you link genes to enzymes

10. How does a gene generate a phenotype? The experiments of Beadle and Tatum in the 1940’s provided the first insight into gene function. They developed the one gene/one enzyme hypothesis This hypothesis has three tenets: Products are synthesized as a series of steps Each step is catalyzed by an unique enzyme Each enzyme is specified by a unique gene The logic: Precursor Int1 Int2 Product EnzA EnzB EnzC GeneA GeneB GeneC

11. Precursor Int1 Int2 Product EnzA EnzB EnzC GeneA GeneB GeneC Consequences of mutations Lets say we know the biochemical pathway. With this pathway, what are the consequences of a mutation in geneB? Would the final product be produced? Would intermediate2 be produced? Would intermediate1 be produced? What happens if we add intermediate1 to the media? What happens if we add intermediate2 to the media?

12. Neurospora Beadle and Tatum analyzed biosynthetic mutations in the haploid fungus Neurospora (Red bread mold) It had the advantage in that it could be grown on a defined growth medium. Given salts like Na3 citrate, KH2PO4, NH4NO3, MgSO4, CaCl2 and sugars like sucrose Neurospora can synthesize the amino acids, vitamins etc required and grow to form colonies on agar plates.

13. Prototroph: a strain that utilizes sugar, salt and water to grow. Auxotroph: Mutant strain that needs a specific amino acid or vitamin along with sugar, salt and water to grow.

14. Arginine biosynthetic mutants Beadle and Tatum set out to identify genes involved in the biosynthetic pathway that led to the production of the amino acid arginine. Neurospora has approximately 15,000 genes and only 4-5 of these genes are involved in synthesizing arginine. How do you identify five genes from 15,000? The POWER OF GENETICS!!!!!! Typically the organism is exposed to a strong mutagen. This randomly mutagenizes genes. Then you look for a mutant in the pathway of interest

15. Logic of experiment ARGININE BIOSYNTHESIS PATHWAY Irradiate (mutagenize) spores. Grow on medium containing arginine Transfer to medium lacking arginine DO THEY GROW OR NOT? If the cells cannot grow on medium lacking arg, then they must have a mutation in a gene required for making ARGININE Mutant needed arginine to grow. Conclusion: Enzyme for making arginine was missing

16. The method Irradiate spores. Take mutant spores. Plate individual spores on complete media (sugar, salts and water, AND vitamins AND all 20 amino acids). Complete All mutants grow To identify mutants Transfer mutants to minimal media (water, sugar, salts) 1 2 3 4 5 6 7 8 9 10 minimal Strain1 and 7 can grow on complete media but not minimal media. They have a mutation in a gene required for growth on minimal media!!!

17. Analogy In the class: There are two kinds of students: Students who can climb trees Students that cannot climb trees. Under normal growth conditions when supermarkets are open, both kinds of students live happily When supermarkets are closed Students who can climb trees grow happily because they can climb trees and eat fruit Students who cannot climb trees do not grow. They cannot climb trees, and go hungry.

18. Conclusion- strain1 Strain1 and 7 are defective in either amino acid production or Vitamin production Take Strain 1 Complete media (salt+sugar+ Vitamin + amino acids) Minimal media (salt+sugar) + 20 amino acids Minimal media (salt+sugar) Minimal media (salt+sugar) + vitamins Conclusion: strain1 is defective in the production of Vitamins and the mutant is rescued by adding back vitamins

19. Conclusion- strain7 Strain1 and 7 are defective in either amino acid production or Vitamin production Take strain 7 Complete media (salt+sugar+ Vitamin + amino acids) Minimal media (salt+sugar) + 20 amino acids Minimal media (salt+sugar) + vitamins Minimal media (salt+sugar) complete media (salt+sugar) Vitamin + amino acids Conclusion: strain7 is defective in the production of Amino acids and the mutant is rescued by adding back amino acids Which of the 20 amino acids does strain7 fail to produce

20. Which amino acid Minimal media + vitamin + all 20 amino acid Growth Minimal media + vitamin + lysine No growth Minimal media + vitamin + glutamine No growth Minimal media + vitamin + arginine Growth Mutant7 is in a gene required for the production of Arginine. Beadle and Tatum found that three mutants could not produce arginine Arg1 Arg2 Arg3 The biochemical pathway for arginine synthesis was kind of known. Ornithine and citrulline are closely related to arginine and were thought to be precursors The pathway for arginine biosynthesis is : Precursor -----> ornithine -----> citrulline -----> arginine enz1 enz2 enz3

21. Add back Precursor -----> ornithine -----> citrulline -----> arginine enz1 enz2 enz3 There are three different enzymes required for arginine synthesis Enz1, enz2 and enz3 Beadle and Tatum isolated three different mutations in genes (three genes) Arg1 Arg2 Arg3 ?????Which mutant gene codes for which enzyme???? Instead of arginine, if they added ornithine or citrulline to the media, some mutants were rescued and others were not Ornithine Citrulline Arginine Mutant1 Mutant2 Mutant3

22. Add back Precursor -----> ornithine -----> citrulline -----> arginine enz1 enz2 enz3 Arg1 Arg2 Arg3 Instead of arginine, if they added ornithine or citrulline to the media, some mutants were rescued and others were not Ornithine Citrulline Arginine Mutant1 + + + Mutant2 - + + Mutant3 - - +

23. Mutant in Arg1- only precursor made Add ornithine or citrulline to media, downstream enzymes are functional and pathway continues---> arginine synthesized Mutant in Arg2- You need to supplement media with citrulline for the pathway to continue. Adding the precursor or ornithine does not help. Mutant in Arg3- You need to supplement media with arginine. Adding the precursor, ornithine or citrulline does not help. These experiments demonstrated that a single gene (mutation) coded for a single enzyme. In addition, the combination of appropriate mutations and intermediates enabled Beadle and Tatum to define the biochemical pathway leading to Arginine synthesis. The Results Also show that THREE different Genes/enzymes are necessary for ONE phenotype- synthesis of ARG! This would affect phenotype ratios in a cross

24. Analogy Walnut Laurel Bay UCSC

25. Another example I get three mutants for a particular pathway I add back various intermediates in this pathway and determine the results Compound E B N A Mut1 - - + + Mut2 - - + - Mut3 + - + + What is the order of the compounds and mutations in the pathway?

26. Another example Compound E B N A Mut1 - - + + Mut2 - - + - Mut3 + - + + Rearrange the mutants Compound E B N A Mut3 + - + + Mut1 - - + + Mut2 - - + - Rearrange the compounds Compound B E A N Mut3 - + + + Mut1 - - + + Mut2 - - - + B----> E----> A----> N mut3 mut1 mut2

27. The steps in a biochemical pathway identified by this procedure are dependent on the available intermediates and mutations. This procedure does not identify every step in the pathway This process does not identify every step in the pathway! B----> E----> A----> N B----> E----> S-----> A----> N This process might also identify multiple mutants for the steps in the pathway! B----> E----> A----> N Mut3 mut5 Mut1 mut4 Mut2

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29. This rationale currently is being used in many laboratories to elucidate more complex pathways in multicellular organisms • ｷDevelopment- formation of the body axis • ｷBehavior- courtship and mating • ｷBiological clocks • ｷAging • ｷCell cycle and Cancer • Review • ｷBiochemical processes occur as a series of discrete stepwise reactions • ｷEach reaction is catalyzed by a single enzyme • ｷEach enzyme is specified by a unique gene • Solving biochemical pathways: • The more mutations that a compound rescues, the later in the pathway the compound is located • Conversely, the later a mutation is in a pathway, the fewer compounds will rescue it:

30. Temperature-sensitive mutations The one gene/one enzyme concept explains a number of genetic phenomena Temperature-sensitive mutations Some mutations exhibit a phenotype at one temperatures (the restrictive temperature) but function normally at another temperature (permissive temperature). Reasons: Slight destabilization/alteration of the 3D conformation of the enzyme or its ability to interact with other proteins Low temp- structure of enzyme- normal- activity normal High temp- structure of enzyme-altered- No activity These kinds of conditional mutants allow you to turn on and off a function of a protein.

31. Heat sensitive mutants Cold sensitive Protein is functional at high temp and inactive at low temp Active at 30C but inactive at 15C Temperature sensitive Protein is functional at low temperature but inactive at high temperature Active at 23C but inactive at 32C PCNA Interacts very stably with RFC Mis folding Cs Ts K253E C752T

32. An example of a Ts mutation: Dogs and cats that are white with black feet or vice versa The genes for coat color are normal at one temperatures but are inactive at another temperatures One of the genes for coat color is Albino - in cats This gene affects melanin production. The normal or dominant form, C, is 'full color'. Various mutant alleles. These mutants are temperature sensitive -

33. In order of decreasing dominance we have C, Cb, Cs and c. C is wild-type or full color. It is dominant to all other alleles. Cb- 'Burmese' factor- it causes a slight lightening of color and is slightly temperature sensitive. Cs- 'Siamese' factor; it has a much greater lightening effect and is temperature sensitive. c is the most recessive form, also known as albino. In the homozygote cc this causes complete absence of any pigment and white fur. Cb is incompletely dominant over Cs; the heterozygote (Cb/Cs) gives a phenotype intermediate between Burmese and Siamese, known as Tonkinese.

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35. Biosynthetic pathways at the grocery store Most of the red and blue colors found in higher plants are a result of pigments synthesized from one of two metabolic pathways, the carotenoid or the anthocyanin pathway. The biosynthetic pathway for corn kernel color is as follows: Precursor-----> Chalcone ----> Flavanone ----> Anthocyanins (white) (yellow) (white) (blue) Grocery store corn is usually yellow. Which step in the pathway must be mutated to produce yellow corn? Beadle/Tatum Results Also show that THREE different Genes/enzymes are necessary for ONE phenotype- synthesis of ARG! Similarly for blue corn multiple genes/enzymes are required. Let’s see how this would affect phenotype ratios in a cross

36. Mutants and Genetic pathways Altered PHENOTYPE RATIOS! The one gene/one enzyme helps explain altered phenotype ratios observed in a standard dihybrid cross: (2 genes segregating independently) If the Two genes being analyzed affect the same genetic pathway Precursor----> intermediate----> product yellow white blue EnzA EnzB Parental cross white x yellow

37. Multiple genes affecting a single phenotype Precursor----> intermediate----> product yellow white blue EnzA EnzB ｷA, B = normal alleles ｷa, b= nonfunctional mutant alleles Parental cross: AAbb x aaBB white yellow F1 AaBb (blue) x AaBb (blue) F2

38. Multiple genes affecting a single phenotype Precursor----> intermediate----> product yellow white blue EnzA EnzB Parental cross: AAbb x aaBB white yellow F1 AaBb (blue) x AaBb (blue) F2 aB Ab ab AB AaBb AB AABB AABb AaBB 4:3:9 Y:W:B AAbb AAbB Aabb AabB Ab aaBb aABb aABB aaBB aB aabB ab aAbb aAbB aabb 9 A-B- blue 3A-bb white 3aaB- yellow 1aabb yellow

39. Labradors recessive Epistasis give 9:4:3 ratio Parental Cross: black x yellow BBEE bbee BbEe (black) x BbEe (black) Yellow-------> brown--------> black E B Given the pathway show above, what phenotypic ratios would be produced in progeny from the dihybrid cross: BbEe x BbEe eB Eb eb EB EeBb EB EEBB EEBb EeBB 4:3:9 Y:Br:Bl EEBb EEbb EeBb Eb Eebb Recessive epistasis Homozygous ee gene alleles mask effect of B gene alleles e is epistatic to B EeBb EeBB eeBB eeBb eB eb Eebb eebb EeBb eeBb Epistasis= When the Alleles of One Gene Mask the Expression of Alleles of a Second Gene

40. Gene interactions give 9:7 Precursor----> intermediate----> product white white blue EnzA EnzB AaBb x AaBb aB Ab ab AB AB Ab AaBb AABB AABb AaBB aB AAbb AAbB Aabb AabB ab aaBb aABb aABB aaBB aabB aAbb aAbB aabb 9 A-B- blue 3A-bb white 3aaB- white 1aabb white

41. WT -- Brown WT -- Vermilion WT -- White Enz V+ Precursor -----Brown pigment \ (white) \ transporter W+ --------- Red / Precursor ----- Vermilion pigment / (white) Enz B+

42. Gene Interaction: A range of Phenotypes Arise From Combined Action of Alleles of Two Genes The 9:3:3:1 ratio in the F2 suggests two genes control coat color.

43. Dominant Epistasis give 13:3 ratio or 12:3:1 ratio B/b is the epistatic gene. Any chicken with a dominant B in their genome will have white feathers. Being homozygous recessive bb at this locus enables the expression of genes coded for at the hypostatic locus (A). At the hypostatic locus A/a the dominant allele A codes for colored feathers while the recessive a codes for no color. Hence, a chicken that is homozygous recessive aa will also be white giving you a 13:3 ratio In white leghorn and white wyandotte chickens, a dominant B allele masks color production associated with the dominant A allele of a second gene. Altered 9:3:3:1 ratios are a hallmark of epistasis involving two genes. Dominant allele of one gene hides effects of both alleles of second gene= 12:3:1 ratio Dominant allele of one gene hides effect of dominant allele of second gene = 13:3 ratio

44. Summer squash color GeneW determines pigment production W No pigment w Pigment GeneY determines color of pigment Y yellow y green 9 W-Y- white 3 W-yy white 3 wwY- yellow 1 wwyy green

45. Multiple genes regulate a single phenotype • Pepper Color • Gene 1: • R=red • r=yellow • Gene 2: • Y=absence of chlorophyll (no green) • y=presence of chlorophyll (green) • Possible genotypes: • R-/Y- : red (red/white no chlorophyll) • R-/yy : brown/orange (red/green chlorophyll) • rr/Y- : yellow (yellow/white no chlorophyll) • rr/yy : green (yellow/green chlorophyll)

46. Two genes affect Chicken Combs • 4 different chicken comb phenotypes result: • Rose Combs (R-pp) • Walnut Combs (R-P-) • Pea Combs (rrP-) • Single Combs (rrpp)

47. Multi-gene traits Many human traits, such as height, skin color etc are determined by multiple genes. Multigenic ("many gene") traits exhibit a mode of inheritance that would have surprised Gregor Mendel. Complex interactions between genes. Number of traits result from mutations in single genes- MONOgenic trait. Multigenic diseases result from less severe mutations in more than one gene. Any of these mutations alone might not affect a trait, but together, they can lead to significant phenotypic differences. Interaction among the phenotypic effects of different genes, called epistasis, adds a layer of complexity to the study of genetic disease. Genes don't function alone; rather, they constantly interact with one another. These gene-gene interactions result in an output phenotype. Certain genes are known to modify the phenotype of other genes. This implies that multiple genes may interact to increase or decrease disease susceptibility. If the effect of the disease-bearing gene is masked or altered by the effects of a second gene (by say altering expression level of the disease bearing gene), then identifying the first gene can be complicated. In addition, if more than one genetic interaction occurs to cause a disease, then identifying the multiple genes involved and defining their relationships becomes even more difficult.

48. Alzheimers Alzheimer's disease, is a progressive neurodegenerative disorder that causes memory loss and dementia. A gene called apolipoprotein E4 was associated with a higher risk of developing Alzheimer's. While having one or two copies of mutant apolipoprotein E4 increase one's risk of Alzheimer's, not all carriers of apolipoprotein E4 develop the disease. This suggested that gene-gene interactions were involved. They confirmed 27 different genetic interactions in 4 different biochemical pathways: cholesterol metabolism, beta-amyloid production, inflammation, oxidative stress. Some interactions were synergistic, while others were antagonistic. The synergistic interactions indicate that the pair of involved genes together increase the risk of Alzheimer’s. The strongest interactions involved the pairing of apolipoprotein E4 mutation with mutations in three different genes: alpha(1)-antichymotrypsin, β-secretase, and butyrylcholinesterase K. These genes are not acting alone, but in a pathway that affect one another. The APP gene produces a transmembrane protein that is modified, cleaved by secretase and then inserted into membranes. Function of APP is not fully known- could be membrane or Ca+ trafficking in neurons. APP interacts with chymotrypisn and Apolipoprotein. Alterations in these interactions lead to formation of APP plaques (amyloid fibers) leading to Alzheimers. ApoE4 mutation leads to alterations in cholesterol in membranes. The ApoE4 allele causes greater aggregation of APP and greater amyloid deposition.

49. Four genes for hair color • Hair Color • Hair color is controlled by multiple genes on chromosomes 3, 6, 10, and 18. • The more dominant alleles that appear in the genotype, the darker the hair!

50. Multiple genes affect a single phenotype- additive effects