Advanced Genetics. Why Study Genetics?
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1 Advanced Genetics
2 Advanced Genetics Why Study Genetics?
3 Why Study Genetics? 1. Historical and aesthetic appreciation
4 Why Study Genetics? 1. Historical and aesthetic appreciation 2. Practical applications - Recognize genetic experiments
5 Why Study Genetics? 1. Historical and Aesthetic appreciation 2. Practical applications - Recognize genetic experiments - Interpret genetic experiments
6 Why Study Genetics? 1. Historical and Aesthetic appreciation 2. Practical applications - Recognize genetic experiments - Interpret genetic experiments - Conduct genetic experiments
7 What is a genetic experiment?
8 Classification of Experiments 1. Observational 2. Interventional
9 Classification of Experiments 1. Observational - Visual (microscopy) - Physiology (blood pressure) - Chemical analysis (serum glucose) - Biochemical analysis (sequence genome, proteome, metabalome, etc.)
10 Classification of Experiments Interventional 1. Environmental - pharmacology - nutrition - temperature
11 Classification of Experiments Interventional 1. Environmental - pharmacology - nutrition - temperature 2. Mechanical - biochemistry - physiology - surgical (transplantation)
12 Classification of Experiments Interventional 1. Environmental 2. Mechanical 3. Genetic - manipulate genes Interventional experiments are powerful because cause/effect relationships can be inferred
13 Classification of Experiments Interventional Genetic Experiments 1. Manipulate genes 2. Observe phenotype 3. Conclusion Interventional experiments are powerful because cause/effect relationships can be inferred
14 Cause How does an experiment establish cause? Why is it important to establish cause?
15 Cause Observation 1 Observation 2
16 Cause Observation 1 Observation 2 Correlation
17 Cause Observation 1 Observation 2 Correlation
18 Cause Experiment Intervention 1 Observation 1 Control No Intervention Observation 1 Positive Conclusion: Intervention causes change Negative Conclusion: Intervention does not cause change
19 Cause Experiment Intervention 1 Observation 1 Observation 2 Control No Intervention Observation 1 Observation 2 Positive Conclusion: Intervention causes change of 1 and 2 What conclusion about relationship of 1 and 2?
20 Cause Experiment Intervention 1 Observation 1 Observation 2 Control No Intervention Observation 1 Observation 2 Positive Conclusion: Intervention causes change of 1 and 2 What conclusion about relationship of 1 and 2? CORRELATION
21 Cause Intervention 1 JUSTIFIED Observation 1 Observation 2 Intervention 1 Observation 1 NOT JUSTIFIED Observation 2
22 Categories of genetic experiments 1. Method - Classical (mutagens and breeding) - Molecular (Molecular biology) 2. Logic - Forward (phenotype to gene) - Reverse (gene to phenotype)
23 Example: Mendel s peas Found mutant pea strains wrinkled and smooth. Bred to generate heterozygotes. Observed 3:1 segregation ratio. Conclusion regarding dominant and recessive alleles.
24 Example: Transcription factor binding Perform ChIP-seq with histone mark. Perform ChIP-seq with transcription factor. Identify transcription factor binding sites that have the histone mark. Draw conclusion regarding how histone mark affect transcription factor binding.
25 Example: Protein crystallography Express wild-type protein from plasmid in bacteria, purify, crystallize and solve 3-D structure. Perform site directed mutagenesis on plasmid. Express mutant protein from plasmid in bacteria, purify, crystallize and solve 3-D structure. Draw conclusion regarding residue and structure.
26 Example: Dominant negative protein Design mutant ERK kinase protein that interferes with function of wild-type protein. Express in cells from plasmid, measure signal transduction. Draw conclusion regarding role of ERK in signaling.
27 Example: Dominant negative protein Design mutant ERK kinase protein that interferes with function of wild-type protein. Synthesize protein with a machine. Inject protein into cells, measure signal transduction. Draw conclusion regarding role of ERK in signaling.
28 Proposition: The essence of genetics is the analysis of the relationship between GENOTYPE and PHENOTYPE Geneticists primarily: 1. Manipulate genotype (Intervention) 2. Analyze phenotype (Observation) 3. Draw conclusions
29 Genotype 1. Definition: All the genetic information contained in an organism; the genetic constitution of an organism with respect to one or a few genes under consideration. 2. Distinctions: A. One gene vs. Entire genome B. Genetic vs. Molecular Alleles known by phenotype they cause Alleles known by DNA sequence Allele Phenotype DNA unc-1(+) wild type wild type unc-1(e1) mild uncoordinated missense codon unc-1(e2) severe uncoordinated deletion C. Wild type is by definition D. Complete description - genetic: wild type or mutant at every locus - molecular: sequence of the genome
30 Phenotype 1. Definition: The observable characteristics of an individual, which are a result of genotype and environment. 2. Common ways to determine phenotype: - visual inspection, including microscopy, staining, etc. - biochemical assay - chemical assay - behavioral assay
31 Caenorhabditis elegans Brenner, Sidney (1974). The gene5cs of Caenorhabdi+s elegans. Gene5cs 77: (9,310 cita5ons, 2002 Nobel prize)
32 What makes an organism suitable for genetic analysis? 1. Ease of culture. 2. Reproduc5ve system (self fer5le hermaphrodites and males). 3. Small genome and limited number of chromosomes.
33 Introduction/Rationale 1. How genes might specify the complex structures found in higher organisms is a major unsolved problem in biology 2. Necessary to find mutants (genotype) and analyze the structure of the nervous system (phenotype). 3. Some eight years ago, when I embarked on this problem, I decided that what was needed was an experimental organism which was suitable for gene5cal study and in which one could determine the complete structure of the nervous system. Drosophila, with about 10 5 neurons, is much too large, and, looking for a simpler organism, my choice eventually sewled on the small nematode, Caenorhabdi+s elegans.
34 Properties of C. elegans - Self- fer5lizing hermaphrodite: Male: XX XO - 1 mm length day life cycle - Small, possibly fixed number of cells
35 Results 1. Isolate muta5ons - Mutagenize with chemical mutagen - Screen for mutant animals
36 Results 2. Posi5on in the genome (mapping) A. Autosomal vs. sex linked B. Two- factor mapping (Distance) Measure recombina5on frequency between two muta5ons a b + +
37 Results 2. Posi5on in the genome (mapping) A. Autosomal vs. sex linked B. Two- factor mapping (Distance) Measure recombina5on frequency between two muta5ons a b + + C. Three- factor mapping (Order) + a b a + b c c +
38 Results 3. Complementa5on - Purpose, determine if two muta5ons affect the same gene m 1 m 2
39 Results Table 4 1. Linkage to par5cular autosome or X chromosome 2. Complementa5on
40 Results Figure 3 and maps factor mapping factor mapping
41 Discussion/Conclusions Brenner developed methods for manipula5ng the genotype of C. elegans, making gene5c analysis possible. 1. Isola5on of muta5ons 2. Mapping - Linkage to chromosome - Posi5on on a chromosome - 2- factor - 3- factor 3. Complementa5on tests These techniques made it possible to address biological ques5ons.
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