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MOLECULAR GENETICS 681:502 Spring 2007 Tuesdays and Thursday, 10-11:30 AM

MOLECULAR GENETICS 681:502 Spring 2007 Tuesdays and Thursday, 10-11:30 AM Waksman Institute Auditorium Dr. Andrew Vershon, Waksman Institute, Rm 234 Phone: 445-2905 E-mail: vershon@waksman.rutgers.edu Course Web Site: http://mmg.rutgers.edu/502.html Username: 502 Password: 502.

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MOLECULAR GENETICS 681:502 Spring 2007 Tuesdays and Thursday, 10-11:30 AM

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  1. MOLECULAR GENETICS 681:502 Spring 2007 Tuesdays and Thursday, 10-11:30 AM Waksman Institute Auditorium Dr. Andrew Vershon, Waksman Institute, Rm 234 Phone: 445-2905 E-mail: vershon@waksman.rutgers.edu Course Web Site: http://mmg.rutgers.edu/502.html Username: 502 Password: 502

  2. Course structure • Lectures - • Reading - General genetics/ Mol. Biol. texts for review Modern Genetic Analysis 7th Ed , Griffiths et al., Freeman & Co, 1999 (is available online) • Assigned Papers - to help with understanding and provide perspective • Problem sets - For practice, some will be collected/graded • Exams - Three closed book - UMDNJ East Lecture Hall

  3. Genetic approaches used in different model systems • Will discuss methods of classic and molecular genetics • Will learn genetic approaches in different systems • bacteria and phages, used to establish the paradigms • yeast - simple eukaryote • worms - model systems for studying development, • flies - complex development, short generation time • plants - compare differences with animals, crops • mice - mammalian systems • humans - ultimate goal • c. Goal is to be able to read papers in these systems • Have a background in the genetic approaches used

  4. E1 E2 E3 A B C D • 1. What is genetics? - Study and manipulation of heredity • a. Genetic manipulation - • b. Genetic analysis started with Mendel • c. Genetic analysis is not just used to study heredity • 2. The Genetic Approach - The Salvation of Doug • How to use genetics to dissect a biological process

  5. 3. Genotype vs. Phenotype a. Genotype - describes the complete set of genes inherited by an individual b. Phenotype - (derived from Greek - the form that is shown) describes the aspects of individuals morphology, physiology, behavior, etc. c. No two individuals have the same genotype- Always some slight differences, even in bacteria DNA Pol 1/107 mutation rate d. If referring to same genotype or phenotype then referring to a subset of traits that are of interest 2-3 diff

  6. 4. Mendel • a) Pick the right organism for the research - pea plants • Can cross strains- cross pollinate • Fast generation time • Relatively cheap to grow • Produce many offspring • Have markers (phenotypes) that can score, • Can follow traits • pea color (yellow/green), • flower color (white/purple) • pea characteristic (round/wrinkled)

  7. b. The Experiment: Bred Strains True Parent W P P F1 P P P W F2 P 3P:W W F3 P W

  8. c. Segregation of the markers P W AA aa Two alleles of the gene W P AA aa Parent P F1 Aa P P P W F2 aa AA Aa Aa P 3P:W W F3 AA aa AA:Aa:aa 1:2:1

  9. d. What would happen if Mendel did not breed his strains true? P W F1 1:1 No dominance of traits P W W P Aa aa Parent

  10. e. What would happen if the phenotype is caused by a dosage effect of the gene? P W AA aa W P AA aa Parent R F1 Aa P R R W F2 aa Aa AA Aa

  11. f. Test Cross - Used to determine the genotype of a strain Mate strains with a recessive tester strain W W P:W P:W aa Aa:aa 1:1 Aa:aa 1:1 W P AA aa Parent P F1 Aa P P P W F2 aa Aa AA Aa P F3 Aa

  12. 6. Segregation of two traits RryY RRyy (ROUND, green) rrYY (wrinkled, YELLOW) Homozygous RryY (ROUND, YELLOW) Punnett Square Phenotype Ratio RY:Ry:rY:ry 9:3:3:1 R:r - 12:4 - 3:1 Y:y - 12:4 - 3:1

  13. 7. Methods for calculating frequencies: • Product rule: probability of independent traits occurring (r,y) is the product of the individual events r or y • Ex: If probability of rr is 1/4 and yy is 1/4 • then ry phenotype (rryy) is 1/4 X 1/4 = 1/16 • Ry = 3/4 x 1/4 = 3/16 • RY= 3/4 x 3/4 = 9/16 • 9:3:3:1 ratio of the phenotypes

  14. c. Ex: Cross two strains: AabbCcDd X AaBbccDd Want abcd phenotype (aabbccdd) How many colonies would you have to screen? then Aa X Aa = 1/4 aa bb X Bb = 1/2 bb Cc X cc = 1/2 cc Dd x Dd = 1/4 dd 1/4 x 1/2 x 1/2 x 1/4 = 1/64 Want AbCd (A-bbC-dd) 3/4 x 1/2 x 1/2 x 1/4 = 3/64

  15. d) Can also be used to calculate probabilities of a disease (xx). XX and Xx are healthy If X and x alleles are present at the same frequency, then 1/4 of population will be xx However if carriers (Xx) are 1/25 of the population: Getting the disease allele has a frequency of 1/50, Probability to get the disease (xx) is therefore 1/2500 1/25 1/25 Xx Xx 1/2 1/2 xx (1/25 x 1/2)(1/25 x 1/2) = 1/2500

  16. 8. Lethality- If homozygous marker is lethal Aa X Aa AA Aa aa Genotype 1 2 1 Phenotype WT No Tail Gene is pleiotropic - has more than one distinct phenotype Mutant must be recessive

  17. 9. Relationship between phenotype and genotype • a) Characteristics of an organism is determined by the phenotype of its parts, • Ultimately decided by which proteins are expressed in which cell types • Frequently those that are studied have to have clear and distinct phenotypes, • A given phenotype indicates a particular genotype • b) One-to-one relationship dominates genetics examples but rare in real life • However not always the case: • curly mutant with curled wings at 25C is WT at 19 • purple has different color when young but WT when adult

  18. c. How can mutations affect a biological process? Enz1 A B If you screened for mutants that would not grow in the absence of B, what types of mutations would you get?

  19. G C A A T T T T Reverse Changes revert back to WT G A A C • Mutations - process where genes change from one allelic form to another • Forward changes away from WT Reverse mutation rate is less than a forward mutation Because you need a specific change

  20. 12. Types of mutations • a) Single base pair substitutions • transition A->G, G->A, C->T, T->C • transversion, A->C or T, G->C/T • WT UAU -> Tyr Wild type • Silent UAC -> Tyr Wild type • Misense UCU -> Ser Non-conserved • Neutral UUU -> Phe Conserved • Nonsense UAG -> Stop Truncated Mutant or WT? Mutant or WT? Mutant or WT?

  21. b) Small deletions or insertions: • Leu Lys Arg Leu • CUC AAG CGC UUA A • CUA AGC GCU UAA • Leu Ser Ala STOP • CUA AGC AGC UUA A • Leu Ser Ala Leu

  22. c) Large deletions or insertions:

  23. d) Reverse mutations: • WT UUA Leu UAU Tyr • Mutant GUA Val UAA Stop • True UUA Leu UAU Tyr • Equivalent CUA Leu UAC Tyr

  24. e) Intragenic Suppressors: Compensating mutation within protein

  25. How can you tell between a true revertant and a suppressor? Back cross vs WT. If revertant only get WT If mutant/suppressor will get mutants R E E R Phenotype + WT R E + Mut/Supp E R - -

  26. f) Extrageneic suppressors: i) Nonsense suppressors - mutations in tRNA

  27. ii) Suppressor mutations in associated proteins

  28. iii) Mutations that over expresses protein, or with higher activity

  29. g) Null vs leaky or conditional mutations

  30. i) Conditional mutations - Phenotype is only observable under specific conditions • i) Temperature Sensitive

  31. ii) Protein Dependent

  32. 13. Making Mutations: a) Spontaneous mutations - very rare naturally occurring due to errors in replication or repair b) Induced mutations - Use of base analogs, 5 bromouracil, 2-amino-purine Alkylating agents - EMS, nitrosoguanidine Hydoxylamine- GC->AT Nitrous Acid - deaminate C's Intercalating agents - slip between bases and mimic bases, Cause insertions/deletions Activating SOS repair - UV, aflatoxin B Mutator strains - MutS, MutY, MutT, Contain mutants in proteins involved in repair mutY - G>T mutT - A>C Transposon mediated - Tn etc

  33. A -> G Mutation by deamination of adenine

  34. c) Mutation frequency- number of mutants found in population d) Mutation rate- number of mutations that occur over time usually the organismal generation span number of mutations per cell division

  35. 14. Genetic interactions between genes: a) None - 4 distinct phenotypes - 1234 9 3 3 1

  36. 14. Genetic interactions between genes: b) Complementation - Need a WT copy of both genes - A B AAbb- +- X----> Y----> Z aaBB- 9 7

  37. 14. Genetic interactions between genes: c) Duplication- multiple genes, aabb to obtain a phenotype A or B + - X--------->Y 15: 1

  38. 14. Genetic interactions between genes: d) Suppressors - cancel the effects of a mutant phenotype, aa restore the WT phenotype on bb+- 13 3

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