8/18/2008 1

Plant Molecular and Cellular Biology

FOR5530

F. Altpeter, AgronomyJ. Davis, Forest Resources

A. Hanson, Horticultural SciencesG. Peter, Forest Resources

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Plant Molecular and Cellular BiologyLecture 1: Course Overview & Intro to Recombinant DNA Methodology

Gary Peter

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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

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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

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Course Introduction

SyllabusExpectations

Universal Intellectual StandardsClearAccuratePreciseRelevantDepthBreadth

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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

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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

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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

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Fundamental & Powerful Concepts of Molecular and Cellular Biology

ReplicationDNA>RNA>ProteinRegulationStructure/Function

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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

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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

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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)

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Example of Biochemical Evidence for the Function of a Specific Protein

The association of specific proteins during purification to homogeneity with a measurable activity

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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

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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

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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

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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

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Cloning & Amplification: In Vivo

DNA replicationPlasmids

Low copyHigh copyOrigin of replication

PhagesDouble strandedSingle stranded

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Putting Novel Genes into Cells

TransformationTransductionConjugationSelection

medlib.med.utah.edu/.../Figures/ Lecture3/Conjgtn.JPG

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Amplification: In VitroIn vitro reactions

Purified DNA polymerasesPolymerase chain reaction

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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

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Example: Cloning a Gene

Need pure plasmid with selectable markerRestriction enzymeLigaseTransformation methodMethods to analyze inserted DNA

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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/

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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

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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