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Unformatted text preview: MCB 121 Spring 2010 Molecular Biology of Eukaryotic Cells
Dr. Ted Powers Molecular and Cellular Biology College of Biological Sciences TA: Leyda Evers, CDB Graduate Group Prerequisite: Biological Sciences 101
1 Goal of class: I. Explore relationship between Genotype and Phenotype Genotype: an organisms genetic constitution or allelic makeup Phenotype: the physical manifestation that results from the expression of this genetic constitution II. Understand the molecular basis for the transition between Genotype and Phenotype: The Central Dogma: DNA --> RNA --> Protein III. Understand how genomes are maintained and propagated IV. Experimental basis for our understanding of these events
2 “You know, the proper method for inquiring after the properties of things is to deduce them from experiments.” Isaac Newton, 1672 3 Course Resources: Lectures: PowerPoint Presentations Reading assignments Text Book Additional literature posted as .pdf files Problem Sets Sample Exams Office Hours (Instructor & TA) Basis for Exams: Lectures, Problem Sets, Sample Exams Textbook: Molecular Biology of the Gene, Watson et al. 6th Edition Resources available on the course website (SmartSite)
4 Basic Modules for the quarter: I. II. III. IV. V. Lectures 1-4: Introduction to molecular genetics: Genetic analysis: Approaches & Tools Lectures 5-7: Protein Synthesis Lectures 8-10: DNA related issues Lectures 11-14: mRNA formation/regulation Lectures 15-18: RNA processing & beyond Testing/Grades: • 2 Midterms (100 points each) • Final Exam (100 points) • Final grade based on 300 point total • All quizzes scantron based (bring UCD 2000 form) 5 6 Lectures 1: The Spirit of Molecular Biology: Combining Classical Genetics and Biochemistry to solve an interesting biological problem
Reading: Watson: Primary: Optional: pp. 783-789 Wood & Edgar Scientific American, 1967 Jarvik & Botstein Proc. Natl. Acad. Sci. Edgar, Genetics, 2004 Problem Set 1
7 Genetics versus Biochemistry The Geneticist: Observe and Deduce
“The geneticist is inclined to let reproductive processes take their normal course and then, by analyzing progeny, to deduce the molecular events that must have occurred within them” The Biochemist: Grind and Reconstitute “The biochemist is eager to break the organism open and search among the remains for more direct clues to what is going on inside”
Wood & Edgar, 1967 Also check out: http://bio.research.ucsc.edu/people/sullivan/savedoug.html 8 The issue Late 1960’s: Bob Edgar and co-workers studied the Assembly of the bacteriophage T4 What is T4? •Virus that infects E. coli •Genome is single circular chromosome of 165 Kb that encodes ~100 genes ~ 50 genes required for viral assembly •Used extensively in the 1950’s - 1960’s for genetic analysis into nature of the genetic code [Seymour Benzer’s work on rII locus] •Distinct morphological features 9 Components of Bacteriophage T4 [Critical features simplified: Head, Tail, Tail Fibers]
10 T4 Genome: genes code for
1. 2. 3. 4. Structural components Viral Replication Interference with host cell function Host cell lysis Distinct Steps during infection 1. Binding of phage to Bacterium 2. Injection of chromosome 3. Repression of host cell protein functions and replication of viral DNA (~5 minutes post infection) 4. Synthesis of viral structural components (heads, tails, etc) (~8 minutes post infection) 5. Assembly and packaging of DNA (13 minutes) 6. Host cell lysis and liberation of new virus (25 minutes) 1 virus will yield ~200 new viruses per infected cell Edgar and co-workers interested in Step 5 above
11 Replicative cycle of T4 12 The Question: How to study assembly process using a genetic approach? 1. Identify genes involved 2. Study mutants to learn about mechanism The Problem: Individual genes involved are essential, so mutants will die and can not be recovered. How to propagate mutants? The Solution: Use “Conditional Lethal Alleles” 13 Q: What is a conditional allele? A: Mutation in a gene that is functional under one condition but not another Here conditions refer to temperature of growth: Heat Sensitive or Temperature Sensitive (ts) or Cold Sensitive (cs) ts mutants often result in a thermosensitive protein: 14 In vivo complementation analysis used to identify T4 genes Experiments used a “plaque assay”
Phage Cause Clear zone Bacterial Lawn
15 •Important: Co-infection assay measures 1 round of infection Therefore- complementation can be observed without Recombination (formation of wild type viral genomes) •However, recombination frequencies between alleles used To determine linkage between genes and establish a Genetic Map •Electron Microscope used to look at ts mutants at the Non-permissive temperatureInsights gained into nature of assembly defect 16 Primitive genetic Map for T4 Based in part on recombination Frequencies between known Genes: ~60 genes 17 The Genome of Bacteriophage T4: 1994
Sequenced genome: ~ 300 genes: 168 with known function A preview of things to come: genetics only gets you so far sometimes!
18 More about complementation vs recombination Recombination: creation of new combinations of genes through physical breaking and rejoining of chromosomes progeny that result from a cross in which recombination has occurred have new genotypes that are different from parental genotypes Complementation: no physical change in genotypes of individual chromosomes necessary represents mixing of gene products [Source: Griffiths, An Introduction To Genetic Analysis 2000] 19 Important aside for the use of conditional alleles: Extragenic versus Intragenic complementation 20 Important aside for the use of conditional alleles: Extragenic versus Intragenic complementation (cont.) This occurs because many proteins acts as oligomers 21 Q: Is there an alternative approach to generate conditional mutants that are less susceptible to displaying Intragenic complementation? A: Yes, Amber mutants 22 Amber mutants = UAG stop codon 23 Amber mutants less susceptible to intragenic complementation Question for later: how do you propagate an amber mutant?
24 Question: What role do individual genes play during viral assembly? Approach: In Vitro analysis of mutant extracts
Hypotheses: 1. Assembly of complete viral particles from specific substructures should be possible in vitro 2. If true, then studying these reactions should yield an assembly map of the virus 25 T4 in vitro Assembly Map Overall stages Heads + Tails Assemble separately Tail fibers assemble and then attach Question: How was this done?
26 Approach: 1. Infect different cultures of E. coli with a different mutant phage at the non-permissive temperature 2. Make a cell free extract from these infected cells and, in some cases, purify viral substructures 3. Incubate extracts (or purified components) in a test tube at the permissive temperature 4. Assay for productive viral assembly by EM and/or by infecting E. coli and using the plaque assay 27 Experiment I: Question: can tail fibers assemble in vitro? Conclusion: 1. Product of reaction infectious 2. EM analysis revealed fully assembled virus The answer is YES 28 Experiment II: Question: can a more complex reaction occur in vitro? Answer is yes! Multiple reactions can proceed simultaneously: 1. Attachment of heads and tails 2. Attachment of tail fibers to newly attached head-tail particles
29 In vitro functional complementation groups 30 •Conclusion: in vitro assembly experiments using ts mutants can provide insight into assembly process e.g. gene5 involved in tail formation gene20 involved in head formation •Food for thought: Why is there more than one gene in an in vitro complementation group? •Real value of in vitro approach is sorting out genes with no obvious function e.g. genes 13,14,15,18 31 in vitro complementation and viral assembly
•Possible functions for genes 13,14,15,18? EM analysis of extracts prepared at non-permissive temperature showed free heads, free tails, unattached tail fibers Possibilities •Head maturation •Tail maturation •Head-tail joining
How to figure it out?
32 What would you predict? Predictions? •Head maturation •Tail maturation •Head-tail joining 33 Genes 13 and 14 therefore involved in head maturation 34 T4 in vitro Assembly Map Overall stages Heads + Tails Assemble separately Tail fibers assemble and then attach 35 What about a purely genetic approach for ordering gene function? Jarvik and Botstein, 1973 Realization: If you have a ts mutant gene product and a cs mutant gene product that function in the same pathway, then you can order their time of action relative to one another. How? Reciprocal temperature shift experiments 36 Consider generic reaction pathway A --> B --> C step I step II A --> B --> C each step carried out by Gene1 or Gene2 Both steps required in the order listed and both are essential for growth of organism 37 Two possibilities:
Gene1 Gene2 A --> B --> C or Gene2 Gene1 A --> B --> C You can distinguish between these if you have a ts and cs mutant in each gene: example: ts Gene1 (no growth at 40°C, growth at 20°/30°C) cs Gene2 (no growth at 20°C, growth at 30°/40°C) How? Combine each mutation into the same genome (double mutant)
38 Control Experiments 1 20°C --> 20°C 40°C --> 40°C 2 20°C --> 30°C 40°C --> 30°C Gene1 Gene2 ts cs A --> B --> C or Gene2 Gene1 cs ts A --> B --> C No growth Yes growth No growth Yes growth 39 Now the real experiments 1 20°C --> 40°C 2 40°C --> 20°C Gene1 Gene2 ts cs A --> B --> C or Gene2 Gene1 cs ts A --> B --> C Yes growth No growth No growth Yes growth 40 Important assumptions for reciprocal shift experiments using double mutant (ts/cs) viruses 1. The ts and cs mutant phenotypes must be readily reversible upon a shift back from non-permissive to permissive conditions. 2. Both mutations must be in the same virus, if they are recessive mutants. Thus, double infections would not work. Why not? Note: exceptions to these assumptions and their consequences discussed at length by Jarvik and Botstein. 41 ...
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This note was uploaded on 09/23/2010 for the course NPB 8746546 taught by Professor Goldberg during the Spring '10 term at UC Davis.
- Spring '10