Biotechnology: Principles, Applications, and Social Implications

From Protein to Product

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

Phil McClean Department of Plant Science North Dakota State University

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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 the biological function of an organism by adding genes from another organism
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These definitions imply biotechnology is needed because:

Nature has a rich source of variation Here we see bean has many seedcoat colors and patterns in nature

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

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But nature does not contain all the genetic 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
Definitions are needed for this statement: Gene - a piece of DNA that controls the expression of a trait

Allele - the alternate forms of a gene

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What is the difference between genes and alleles for Mendels Traits?
Mendels Genes Plant height Seed shape

Smooth Wrinkled Allele

Tall

Short Allele

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This Implies a Genetic 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 Mendels Genes Plant Height Gene

Gene: gibberellin 3--hydroxylase Function: adds hydoxyl group to GA20 to make GA1 Role of GA1: regulates cell division and elongation Mutation in short allele: a single nucleotide converts an alanine to threonine in final protein Effect of mutation: mutant protein is 1/20 as active
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Allelic Differences for Mendels Seed Shape Gene

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

(The guiding principle that controls trait expression)

Protein
Translation

Trait (or phenotype)

Seed shape

DNA (gene)

Transcription

RNA
Plant height

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In General, Plant Biotechnology Techniques Fall Into Two Classes

Gene Manipulation Identify a gene from another species which controls a trait of interest Or modify an existing gene (create a new allele) Gene Introduction Introduces that gene into an organism Technique called transformation Forms transgenic organisms
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Gene Manipulation Starts At the DNA Level

The nucleus

contains DNA

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Source: Access Excellence

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DNA Is Packaged
Double-stranded DNA

is condensed into

Chromosomes
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Source: Access Excellence

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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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Homology Cloning
Clones transferred to filter

Human clone library

Mouse probe added to filter

Hot-spots are human homologs to mouse gene

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Complementary Genetics
1. Protein sequence is related to gene sequence
NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COOATG 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

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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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Complementary Genetics (cont.)

3. Use PCR to amplify gene fragment
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

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

3 5 5 3

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PCR Animation

Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated

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PCR Again

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Complementary Genetics (cont.)

4. Gene fragment used to screen library
Clones transferred to filter

Human clone library

PCR fragment probe added to filter

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Hot-spots are human gene of interest

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Map-based Cloning
1. Use genetic techniques to find marker near gene 2. Find cosegregating marker 3. Discover overlapping clones (or contig) that contains the marker
Gene Marker

Gene/Marker

Gene/Marker

4. Find ORFs on contig

Gene/Marker

5. Prove one ORF is the gene by Mutant + ORF = Wild type? transformation or mutant analysis 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 enzyme Increased 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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RoundUp Sensitive Plants

Shikimic acid + Phosphoenol pyruvate

+ Glyphosate
Plant EPSP synthase

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

Without amino acids, plant dies

X
X

Aromatic amino acids

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RoundUp Resistant Plants

Shikimic acid + Phosphoenol pyruvate

+ Glyphosate
Bacterial EPSP synthase
RoundUp has no effect; enzyme is resistant to herbicide

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

With amino acids, plant lives

Aromatic amino acids

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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 doesnt exist to deliver vitamin pills
Improved vitamin A content in widely consumed crops an attractive alternative
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-Carotene Pathway in Plants

IPP Geranylgeranyl diphosphate
Phytoene synthase

Phytoene

Problem: Rice lacks these enzymes

Phytoene desaturase

-carotene desaturase

Lycopene
Lycopene-beta-cyclase
Normal Vitamin A Deficient Rice

-carotene (vitamin A precursor)

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The Golden Rice Solution

-Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate
Daffodil gene Phytoene synthase

Phytoene

Vitamin A Pathway is complete and functional

Phytoene desaturase Single bacterial gene; performs both functions

-carotene desaturase

Lycopene
Daffodil gene Golden Rice

Lycopene-beta-cyclase

-carotene (vitamin A precursor)

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Metabolic Pathways are Complex and Interrelated

Understanding pathways is critical to developing new products

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Modifying Pathway Components Can Produce New Products

Turn On Vitamin Genes = Relieve Deficiency Modified Lipids = New Industrial Oils

Increase amino acids = Improved Nutrition

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Trait/Gene Examples
Trait RoundUp Ready Gene Bacterial EPSP

Golden Rice
Plant Virus Resistance Male Sterility Plant Bacterial Resistance Salt tolerance

Complete Pathway
Viral Coat Protein Barnase p35 AtNHX1
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Introducing the Gene or

Developing 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
Promoter TP Coding Region

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)

Transit Peptide Targets protein to correct organelle

ex.: RbCS (RUBISCO small subunit; choloroplast target

Coding Region Encodes protein product

ex.: EPSP -carotene genes

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Selectable Marker
Promoter Coding Region

Promoter Region Normally constitutive

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

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]

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Effect of Selectable Marker

Non-transgenic = Lacks Kan or Bar Gene
Plant dies in presence of selective compound

X
Transgenic = Has Kan or Bar Gene
Plant grows in presence of selective compound

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Insertion Sequences
TL TR

Required for proper gene insertions Used for Agrobacterium-transformation

ex.: Right and Left borders of T-DNA

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Lets Build A Complex Cassette

pB19hpc (Golden Rice Cassette)

TL

aphIV

35S Gt1

psy

35S rbcS

crtl

TR

T-DNA Border

Hygromycin Resistance

Phytoene Synthase

Phytoene Desaturase

T-DNA Border

Insertion Sequence

Selectable Marker

Gene of Interest

Gene of Interest

Insertion Sequence

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Delivering the Gene to the Plant

Transformation cassettes are developed in the lab
They are then introduced into a plant

Two major delivery methods

Agrobacterium Gene Gun
Tissue culture required to generate transgenic plants

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Plant Tissue Culture

A Requirement for Transgenic Development

Callus grows A plant part Is cultured Shoots develop Shoots are rooted; plant grows to maturity

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Agrobacterium
A natural DNA delivery system A plant pathogen found in nature Infects many plant species Delivers DNA that encodes for plant hormones DNA incorporates into plant chromosome Hormone genes expressed and galls form at infection site
Gall on stem

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Gall on leaf

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The Galls Can Be Huge

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Natural Infection Process Is Complex

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But Natures Agrobacterium Has Problems

Infected tissues cannot be regenerated (via tissue culture) into new plants
Why? Phytohormone balance incorrect regeneration Solution? Transferred DNA (T-DNA) modified by
Removing phytohormone genes Retaining essential transfer sequences

Adding cloning site for gene of interest

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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
Tissue must be capable of developing into normal plants Leaf, germinating seed, immature embryos

Introduce DNA
Agrobacterium or gene gun

Culture plant tissue

Develop shoots Root the shoots

Field test the plants

Multiple sites, multiple years

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The Lab Steps

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Lab Testing The Transgenics

Insect Resistance Cold Tolerance

Transgene= Bt-toxin protein

Transgene= CBF transcription factors

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More Modern Examples

Salt Tolerant Mercury Resistance

Transgene= Glyoxylase I

Transgene= Mercuric ion reductase

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The Next Test Is The Field

Herbicide Resistance

Non-transgenics

Transgenics

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Final Test
Consumer Acceptance RoundUp Ready Corn

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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?

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