080818.PMCB.lecture1

080818.PMCB.lecture1 - Plant Molecular and Cellular Biology...

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Unformatted text preview: Plant Molecular and Cellular Biology FOR5530 F. Altpeter, Agronomy J. Davis, Forest Resources A. Hanson, Horticultural Sciences G. Peter, Forest Resources 8/18/2008 1 Plant Molecular and Cellular Biology Lecture 1: Course Overview & Intro to Recombinant DNA Methodology Gary Peter 8/18/2008 2 Learning Objectives 1. 2. 3. 4. 5. Course Objectives Module 1 learning objectives, grading and expectations Use the fundamental & powerful concepts/framework of molecular & cellular biology Apply biological reasoning & evidence Explain methods and approaches that are used/needed to elucidate molecular mechanisms 8/18/2008 PMCB Lecture 1: G. Peter 3 Course Objectives Understand current knowledge of plant genomics and fundamental molecular mechanisms that mediate plant growth, development, function, and adaptation Understand experimental methods and strategies used to elucidate molecular mechanisms Promote students’ ability to interpret and design experiments to elucidate molecular and cellular mechanisms controlling plant growth, development, function, and adaptation Enable students’ ability to read and analyze primary literature in molecular biology and genomics 8/18/2008 PMCB Lecture 1: G. Peter 4 Course Introduction Syllabus Expectations Universal Intellectual Standards Clear Accurate Precise Relevant Depth Breadth 8/18/2008 PMCB Lecture 1: G. Peter 5 Learning Objectives for Module 1 List and explain the concepts, molecular mechanisms, and proteins and their functions that mediate DNA replication and repair List and explain the mechanisms that regulate DNA replication in prokaryotes and eukaryotes Apply the principles of recombinant DNA strategies and methods to investigate the function of genes involved with plant growth, development and adaptation 8/18/2008 PMCB Lecture 1: G. Peter 6 Grading & Expectations for Module 1 Expectations Actively engaged in learning material Check for extra information posted on course website Use of correct biological terminology, reasoning and sufficient level of detail Grading 1 Exam worth 50 points 4 Homework assignments a total of 50 points Assignments will be posted on the course website Problem Set 1 Assigned Due Date 8/18/2008 Problem Set 2 9/1 9/8 Problem Set 3 9/8 9/15 Problem Set 4 9/15 9/22 Exam Week 5 8/25 9/1 PMCB Lecture 1: G. Peter 7 Scales of Analysis 0.001 1 10,000 x107 1x109 1 Time (seconds) Primary Biologists' Focus Whole Organ Cell Molecule 1 x 10-9 1 x 10-5 0.1 10 Stand/ Field Plant Landscape / Region Primary Industry Focus 100 10,000 Space (meters) 8/18/2008 PMCB Lecture 1: G. Peter 8 Fundamental & Powerful Concepts of Molecular and Cellular Biology Replication DNA>RNA>Protein Regulation Structure/Function 8/18/2008 PMCB Lecture 1: G. Peter 9 What Do We Need To Elucidate Molecular Mechanisms? Understand the structure, function, interaction, regulation, and organization of molecules that mediate a process Way to identify individual and groups of genes, RNAs and proteins critical to a process Way to manipulate individual and groups of genes, RNAs, and proteins to affect the process to dissect their roles/functions in organisms 8/18/2008 PMCB Lecture 1: G. Peter 10 What Constitutes Evidence for a Particular Biological Mechanism? Proposition Genetic and biochemical data are the only kinds of biological evidence Genetic evidence permits identification of genes involved in particular processes and provides in vivo functional evidence in the context of the organism Biochemical evidence permits identification of genes based on in vitro function and provides detailed understanding a protein’s reaction mechanism(s) and mode of action 8/18/2008 PMCB Lecture 1: G. Peter 11 Example of Genetic Evidence for the Function of a Specific Gene An organism with an altered phenotype is identified The mutation which causes the altered phenotype is heritable and segregates in crosses between mutant and normal (wild type, nonmutants) 8/18/2008 PMCB Lecture 1: G. Peter 12 Example of Biochemical Evidence for the Function of a Specific Protein The association of specific proteins during purification to homogeneity with a measurable activity 8/18/2008 PMCB Lecture 1: G. Peter 13 Combined Genetic and Biochemical Evidence: Genes & Proteins Responsible for DNA Replication in E. coli Forward & Reverse Genetic Screening Temperature sensitive mutants impaired in DNA replication Quick stop Slow stop Biochemical Reactions In vitro reactions competent for DNA replication Complementation Purification Subunit structures 8/18/2008 PMCB Lecture 1: G. Peter 14 Biochemical Approaches Specific assay for activity of interestDevelop with crude extracts Test for stability of activity Conduct single or multistage purification of proteins or protein complexes Isolate and characterize protein structure and function 8/18/2008 PMCB Lecture 1: G. Peter 15 Genetic Approaches Forward genetics: Looking for mutations in natural or mutagenized populations that cause changes in phenotype Selection Screening Reverse genetics: Creating mutations in selected genes to determine their function in a process e.g., Shuman & Silhavy Nature Reviews Genetics (2003) 4: 419-432 8/18/2008 PMCB Lecture 1: G. Peter 16 Development of Molecular Biology Recombinant DNA methods evolved from advances in bacterial genetics and biochemical studies of the enzymology of DNA 8/18/2008 PMCB Lecture 1: G. Peter 17 Manipulating Molecules: Creating Novel Sequences Isolation of unique sequences Synthesis Chemical synthesis Cloning Plasmids, Phages, Polymerase chain reaction Transformation, Transduction, Conjugation Restriction enzymes, Ligation, Recombinases… Amplification of unique sequences Plasmids, Phages, Polymerase chain reaction Selection DNA sequencing 8/18/2008 PMCB Lecture 1: G. Peter 18 Cloning & Amplification: In Vivo DNA replication Plasmids Low copy High copy Origin of replication Phages Double stranded Single stranded 8/18/2008 PMCB Lecture 1: G. Peter 19 Putting Novel Genes into Cells Transformation Transduction Conjugation Selection medlib.med.utah.edu/.../Figures/ Lecture3/Conjgtn.JPG 8/18/2008 PMCB Lecture 1: G. Peter 20 Amplification: In Vitro In vitro reactions Purified DNA polymerases Polymerase chain reaction 8/18/2008 PMCB Lecture 1: G. Peter 21 Cutting Apart & Putting Back Together Restriction enzymes Methylases Ligases DNA – double strand RNA – single strand Eco RI enzyme – DNA complex Rosenberg,J.M. (1991) Curr. Opin. Struct. Biol., 1, 104-110. - Review of EcoRI Studies 8/18/2008 PMCB Lecture 1: G. Peter 22 Example: Cloning a Gene Need pure plasmid with selectable marker Restriction enzyme Ligase Transformation method Methods to analyze inserted DNA 8/18/2008 PMCB Lecture 1: G. Peter 23 History of Molecular Biology History of Genetics Timeline.htm Http://www.accessexcellence.org/AE/AEPC/WWC /1994/geneticstln.html Molecular-Biologist_com A Concise History of Molecular Biology & Genetics.htm Http://molecular-biologist.com/ 8/18/2008 PMCB Lecture 1: G. Peter 24 Summary Fundamental & powerful concepts of molecular and cellular biology Replication DNA>RNA>Protein (Central dogma) Regulation Structure/Function Genetic and biochemical evidence Recombinant DNA methods exploit natural processes for the manipulation of genes/proteins 8/18/2008 PMCB Lecture 1: G. Peter 25 Important Resources 1. 1. 2. 3. Databases available on web: NCBI TAIR JGI…. 2. Methods/Protocol Manuals – My Favorite Oldies 1. 2. Experiments in Molecular Genetics by JH Miller CSHL, 1972 Guide to Molecular Cloning Techniques, ed. SL Berger, AR Kimmel, Methods in enzymology v. 152, 1987 PMCB Lecture 1: G. Peter 26 8/18/2008 ...
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This note was uploaded on 05/31/2011 for the course PCB 5530 taught by Professor Peter,g during the Spring '08 term at University of Florida.

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080818.PMCB.lecture1 - Plant Molecular and Cellular Biology...

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