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Biotechnology:Principles, Applications,and Social Implications

From Protein to Product

Phil McCleanDepartment of Plant Science

North Dakota State University

The techniques used by the biotechnology industryto modify genes and introduce them into transgenic organisms

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What is Biotechnology?

How about some definitions

General Definition

The application of technology to improve a biological organism

Detailed Definition

The application of the technology to modify thebiological function of an organism by adding genesfrom another organism

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But we know nature does not have all of the traits we need

• Here we see bean has many seedcoat colors and patterns in nature

•Nature has a rich source of variation

These definitions imply biotechnologyis needed because:

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But nature does not contain all thegenetic variation man desires

•Fruits with vaccines

•Grains with improved nutrition

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What controls this natural variation?

Allelic differences at genes control a specific trait

Gene - a piece of DNA that controls the expression of a trait

Allele - the alternate forms of a gene

Definitions are needed for this statement:

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What is the difference betweengenes and alleles for Mendel’s Traits?

Mendel’s GenesPlant height Seed shape

Tall ShortAllele

Smooth WrinkledAllele

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This Implies aGenetic Continuum

A direct relationship exists between the gene, its alleles,and the phenotypes (different forms ) of the trait

Alleles must be:• similar enough to control the same trait• but different enough to create different phenotypes

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Allelic Differences for Mendel’s GenesPlant Height Gene

Gene: gibberellin 3--hydroxylaseFunction: adds hydoxyl group to GA20 to make GA1

Role of GA1: regulates cell division and elongationMutation in short allele: a single nucleotide converts an alanine to threonine in final proteinEffect of mutation: mutant protein is 1/20 as active

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Gene: strach branching enzyme (SBE) isoform 1Function: adds branch chains to starchMutation in short allele: transposon insertionEffect of mutation: no SBE activity; less starch, more sucrose, more water; during maturation seed looses more water and wrinkles

Allelic Differences for Mendel’s Seed Shape Gene

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Central Dogma of Molecular Genetics

(The guiding principle that controls trait expression)

DNA(gene)

RNA

Protein Trait(or phenotype)

Transcription

Translation

Plant height

Seed shape

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In General, Plant Biotechnology TechniquesFall Into Two Classes

• Identify a gene from another species which controls a trait of interest• Or modify an existing gene (create a new allele)

Gene Manipulation

• Introduces that gene into an organism• Technique called transformation• Forms transgenic organisms

Gene Introduction

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Gene Manipulation StartsAt the DNA Level

The nucleus

contains DNA

Source: Access Excellence

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DNA Is Packaged

Source: Access Excellence

Double-strandedDNA

Chromosomes

is condensedinto

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Chromosomes Contain Genes

Chromosome

Gene

Source: Access Excellence

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Genes Are Cloned Based On:

Similarity to known genes

Homology cloning (mouse clone used to obtain human gene)

Protein sequence

Complementary genetics (predicting gene sequence from protein)

Chromosomal location

Map-based cloning (using genetic approach)

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Human clonelibrary

Clones transferredto filter

Mouse probeadded to filter

Hot-spots are humanhomologs to mouse gene

Homology Cloning

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Complementary Genetics

1. Protein sequence is related to gene sequence

NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COO-

ATG GAT-GCT TGG-AGT-AAA C C C G A TCT G C A G

2. The genetic code information is used to design PCR primers

Forward primer: 5’-ATGGAT/CGCN-3’Reverse primer: 5’-T/CTTNC/GT/ACCA-3’

Notes: T/C = a mixture of T and C at this position; N = a mixture of all four nucleotides Reverse primer is the reverse complement of the gene sequence

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3. Use PCR to amplify gene fragment

Complementary Genetics(cont.)

a. template DNA is melted (94C)3’ 5’5’ 3’

3’ 5’

5’ 3’

b. primers anneal to complementary site in melted DNA (55C)

3’ 5’

5’ 3’

3’ 5’

5’ 3’

c. two copies of the template DNA made (72C)

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Denaturation: DNA meltsAnnealing: Primers bindExtension: DNA is replicated

PCR Animation

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PCR Again

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Human clonelibrary

Clones transferredto filter

PCR fragmentprobe added to filter

Hot-spots are human geneof interest

Complementary Genetics(cont.)

4. Gene fragment used to screen library

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Map-based Cloning

1. Use genetic techniques to find marker near gene

Gene Marker

2. Find cosegregating markerGene/Marker

3. Discover overlapping clones (or contig) that contains the marker Gene/Marker

4. Find ORFs on contigGene/Marker

5. Prove one ORF is the gene by transformation or mutant analysis

Mutant + ORF = Wild type?Yes? ORF = Gene

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Gene Manipulation

• It is now routine to isolate genes

• But the target gene must be carefully chosen

• Target gene is chosen based on desired phenotype

Function:Glyphosate (RoundUp) resistance EPSP synthase enzymeIncreased Vitamin A content Vitamin A biosynthetic pathway enzymes

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The RoundUp Ready Story

• Glyphosate is a broad-spectrum herbicide• Active ingredient in RoundUp herbicide • Kills all plants it come in contact with• Inhibits a key enzyme (EPSP synthase) in an amino acid pathway

• Plants die because they lack the key amino acids

• A resistant EPSP synthase gene allows crops to survive spraying

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+ Glyphosate

X

RoundUp Sensitive Plants

X

X

Shikimic acid + Phosphoenol pyruvate

3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)

Plant EPSP synthase

Aromaticamino acids

Without amino acids, plant dies

X

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BacterialEPSP synthase

Shikimic acid + Phosphoenol pyruvate

3-enolpyruvyl shikimic acid-5-phosphate(EPSP)

Aromaticamino acids

RoundUp Resistant Plants

+ Glyphosate

With amino acids, plant lives

RoundUp has no effect;enzyme is resistant to herbicide

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The Golden Rice Story

• Vitamin A deficiency is a major health problem

• Causes blindness• Influences severity of diarrhea, measles

• >100 million children suffer from the problem

• For many countries, the infrastructure doesn’t existto deliver vitamin pills

• Improved vitamin A content in widely consumed cropsan attractive alternative

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-Carotene Pathway in Plants

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Problem:Rice lacks

these enzymes

NormalVitamin A

“Deficient”Rice

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The Golden Rice Solution

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene

Single bacterial gene;performs both functions

Daffodil gene

-Carotene Pathway Genes Added

Vitamin APathway

is completeand functional

GoldenRice

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Metabolic Pathways are Complexand Interrelated

Understanding pathways is critical to developing

new products

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Modifying Pathway ComponentsCan Produce New Products

Modified Lipids =New Industrial Oils

Turn On Vitamin Genes = Relieve Deficiency

Increase amino acids = Improved Nutrition

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Trait/Gene Examples

RoundUp Ready Bacterial EPSP

Golden Rice Complete Pathway

Plant Virus Resistance Viral Coat Protein

Male Sterility Barnase

Plant Bacterial Resistance p35

Salt tolerance AtNHX1

Trait Gene

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Introducing the Gene orDeveloping Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

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Transformation Cassettes

Contains

1. Gene of interest

• The coding region and its controlling elements

2. Selectable marker

• Distinguishes transformed/untransformed plants

3. Insertion sequences• Aids Agrobacterium insertion

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Gene of Interest

Coding Region• Encodes protein product

ex.: EPSP -carotene genes

Promoter Region• Controls when, where and how much the gene is expressed

ex.: CaMV35S (constitutive; on always) Glutelin 1 (only in rice endosperm during seed development)

Promoter Coding RegionTP

Transit Peptide• Targets protein to correct organelle

ex.: RbCS (RUBISCO small subunit; choloroplast target

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Selectable Marker

Coding Region• Gene that breaks down a toxic compound;non-transgenic plants die

ex.: nptII [kanamycin (bacterial antibiotic) resistance] aphIV [hygromycin (bacterial antibiotic) resistance] Bar [glufosinate (herbicide) resistance]

Promoter Region• Normally constitutive

ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter

Promoter Coding Region

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Effect of Selectable Marker

Transgenic = Has Kan or Bar Gene

Plant grows in presenceof selective compound

Plant dies in presenceof selective compound

Non-transgenic = Lacks Kan or Bar Gene

X

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Insertion Sequences

• Used for Agrobacterium-transformationex.: Right and Left borders of T-DNA

Required for proper gene insertions

TL TR

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Let’s Build A Complex Cassette

pB19hpc (Golden Rice Cassette)

TL TRaphIV 35S Gt1 psy 35S rbcS crtl

HygromycinResistance

PhytoeneSynthase

PhytoeneDesaturase

T-DNABorder

T-DNABorder

SelectableMarker

Gene ofInterest

Gene ofInterest

InsertionSequence

InsertionSequence

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• Transformation cassettes are developed in the lab

• They are then introduced into a plant

• Two major delivery methods

Delivering the Geneto the Plant

• Agrobacterium

• Gene GunTissue culturerequired to generatetransgenic plants

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Plant Tissue CultureA Requirement for Transgenic Development

A plant part Is cultured

Callusgrows

Shootsdevelop Shoots are rooted;

plant grows to maturity

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AgrobacteriumA natural DNA delivery system

• A plant pathogen found in nature

• Hormone genes expressed and galls form at infection site

• Delivers DNA that encodes for plant hormones

• Infects many plant species

Gall onstem

Gall onleaf

• DNA incorporates into plant chromosome

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The Galls Can Be Huge

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Natural Infection Process Is Complex

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But Nature’s AgrobacteriumHas Problems

Infected tissues cannot be regenerated (via tissue culture)into new plants

Transferred DNA (T-DNA) modified by

• Removing phytohormone genes

• Retaining essential transfer sequences

• Adding cloning site for gene of interest

• Phytohormone balance incorrect regeneration

Solution?

Why?

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The Gene Gun

• DNA vector is coated onto gold or tungsten particles

• Particles are accelerated at high speeds by the gun

• Particles enter plant tissue

• DNA enters the nucleus and incorporates into chromosome

• Integration process unknown

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Transformation Steps

Prepare tissue for transformation

Introduce DNA

Culture plant tissue• Develop shoots• Root the shoots

Field test the plants

• Leaf, germinating seed, immature embryos

• Tissue must be capable of developing into normal plants

• Agrobacterium or gene gun

• Multiple sites, multiple years

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The Lab Steps

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Lab Testing The Transgenics

Insect Resistance

Transgene=Bt-toxin protein

Cold Tolerance

Transgene=CBF transcription factors

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Salt Tolerant Mercury Resistance

More Modern Examples

Transgene=Glyoxylase I

Transgene=Mercuric ion reductase

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The Next Test Is The Field

Non-transgenics

Transgenics

Herbicide Resistance

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Final TestConsumer Acceptance

RoundUp Ready Corn

Before After

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The Public Controversy

• Should we develop transgenics?

• Should we release transgenics?

• Are transgenics safe?

• Are transgenics a threat to non-transgenic production systems?

• Are transgenics a threat to natural eco-systems?