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8/18/2008 1
Plant Molecular and Cellular Biology
FOR5530
F. Altpeter, AgronomyJ. Davis, Forest Resources
A. Hanson, Horticultural SciencesG. Peter, Forest Resources
8/18/2008 2
Plant Molecular and Cellular BiologyLecture 1: Course Overview & Intro to Recombinant DNA Methodology
Gary Peter
8/18/2008 PMCB Lecture 1: G. Peter 3
Learning Objectives
1. Course Objectives2. Module 1 learning objectives, grading and
expectations3. Use the fundamental & powerful
concepts/framework of molecular & cellular biology 4. Apply biological reasoning & evidence5. Explain methods and approaches that are
used/needed to elucidate molecular mechanisms
8/18/2008 PMCB Lecture 1: G. Peter 4
Course ObjectivesUnderstand current knowledge of plant genomics and fundamental molecular mechanisms that mediate plant growth, development, function, and adaptationUnderstand experimental methods and strategies used to elucidate molecular mechanismsPromote 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 5
Course Introduction
SyllabusExpectations
Universal Intellectual StandardsClearAccuratePreciseRelevantDepthBreadth
8/18/2008 PMCB Lecture 1: G. Peter 6
Learning Objectives for Module 1
List and explain the concepts, molecular mechanisms, and proteins and their functions that mediate DNA replication and repairList and explain the mechanisms that regulate DNA replication in prokaryotes and eukaryotesApply 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 7
Grading & Expectations for Module 1
ExpectationsActively engaged in learning material Check for extra information posted on course websiteUse of correct biological terminology, reasoning and sufficient level of detail
Grading1 Exam worth 50 points4 Homework assignments a total of 50 points
Assignments will be posted on the course website
Problem Set 1
Problem Set 2
Problem Set 3
Problem Set 4 Exam
Assigned 8/25 9/1 9/8 9/15 Week 5
Due Date 9/1 9/8 9/15 9/22
8/18/2008 PMCB Lecture 1: G. Peter 8
Scales of AnalysisTi
me
(sec
onds
)
Landscape /RegionStand/
FieldWhole Plant
OrganCell
Molecule
Space (meters)1 x 10-5 0.1 10 100 10,0001 x 10-9
0.00
11
10,0
00 1x1
071x
109
Primary Industry Focus
Primary Biologists' Focus
8/18/2008 PMCB Lecture 1: G. Peter 9
Fundamental & Powerful Concepts of Molecular and Cellular Biology
ReplicationDNA>RNA>ProteinRegulationStructure/Function
8/18/2008 PMCB Lecture 1: G. Peter 10
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 processWay 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 11
What Constitutes Evidence for a Particular Biological Mechanism?
PropositionGenetic and biochemical data are the only kinds of biological evidence
Genetic evidence permits identification of genes involved in particular processes and provides in vivofunctional 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 12
Example of Genetic Evidence for the Function of a Specific Gene
An organism with an altered phenotype is identifiedThe 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 13
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 14
Combined Genetic and Biochemical Evidence: Genes & Proteins Responsible for DNA Replication in E. coli
Forward & Reverse Genetic ScreeningTemperature sensitive mutants impaired in DNA replication
Quick stopSlow stop
Biochemical ReactionsIn vitro reactions competent for DNA replication
Complementation PurificationSubunit structures
8/18/2008 PMCB Lecture 1: G. Peter 15
Biochemical ApproachesSpecific assay for activity of interest-
Develop with crude extractsTest for stability of activityConduct single or multistage purification of proteins or protein complexesIsolate and characterize protein structure and function
8/18/2008 PMCB Lecture 1: G. Peter 16
Genetic ApproachesForward genetics: Looking for mutations in natural or mutagenized populations that cause changes in phenotype
SelectionScreening
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
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
8/18/2008 PMCB Lecture 1: G. Peter 18
Manipulating Molecules: Creating Novel Sequences
Isolation of unique sequencesSynthesis
Chemical synthesisCloning
Plasmids, Phages, Polymerase chain reactionTransformation, Transduction, ConjugationRestriction enzymes, Ligation, Recombinases…
Amplification of unique sequencesPlasmids, Phages, Polymerase chain reactionSelection
DNA sequencing
8/18/2008 PMCB Lecture 1: G. Peter 19
Cloning & Amplification: In Vivo
DNA replicationPlasmids
Low copyHigh copyOrigin of replication
PhagesDouble strandedSingle stranded
8/18/2008 PMCB Lecture 1: G. Peter 20
Putting Novel Genes into Cells
TransformationTransductionConjugationSelection
medlib.med.utah.edu/.../Figures/ Lecture3/Conjgtn.JPG
8/18/2008 PMCB Lecture 1: G. Peter 21
Amplification: In VitroIn vitro reactions
Purified DNA polymerasesPolymerase chain reaction
8/18/2008 PMCB Lecture 1: G. Peter 22
Cutting Apart & Putting Back Together
Restriction enzymesMethylasesLigases
DNA – double strandRNA – 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 23
Example: Cloning a Gene
Need pure plasmid with selectable markerRestriction enzymeLigaseTransformation methodMethods to analyze inserted DNA
8/18/2008 PMCB Lecture 1: G. Peter 24
History of Molecular Biology
History of Genetics Timeline.htmHttp://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 25
SummaryFundamental & powerful concepts of molecular and cellular biology
ReplicationDNA>RNA>Protein (Central dogma)RegulationStructure/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 26
Important Resources1. Databases available on web:
1. NCBI2. TAIR3. JGI….
2. Methods/Protocol Manuals – My Favorite Oldies1. Experiments in Molecular Genetics by JH Miller
CSHL, 19722. Guide to Molecular Cloning Techniques, ed. SL
Berger, AR Kimmel, Methods in enzymology v. 152, 1987