What all good citizens should know about genes and plant

biotechnology

Steve Strauss

Steve.Strauss@oregonstate.edu

Richardson Hall 338

541 737 6578

FS/BI 430/530(H), Fall 2005

Science and GMO-relevant technology

• Genes and genomes - Today– Genomes and their inheritance and variation– Genes and their structure– Important methods: Gene cloning, PCR and

microarrays

• Biotechnology – Next week– Basic concepts of cloning/regeneration– Transformation methods– Transgene structure/expression

Cells are highly complex in structure and heredity

A genome refers to a complete set of genetic material in a cell or part of a cell – such as all nuclear chromosomes, all chloroplast DNA molecules

Plant genomes• Nuclear – diploid or higher

– Inherited from both parents– Diploid – two copies– Polyploid – many copies; aneuploid – partial

copies– Most plant genomes result from polyploid

events! • Many copies of most genes, which diverge or lose

function over evolution = gene families! Many genes with similar/overlapping functions

• Related concept of redundancy in genomes and development – gene knock-outs often without effects

Plant genomes• Nuclear – diploid or higher

– Lots of repetitive, non-genic, non-translated DNA (“junk DNA”)

– Lots of “jumping genes” (transposable elements)• Mostly stable, but can move on occasion, in response to

stress or other factors – takes other genes with them, changes expression

– Lots of variation in genome size between and within species

• C-value paradox – genome size weakly correlated with complexity

– Readily transformed with foreign DNA

Nuclear genome size varies widely

Gene density varies widely

Examples of nuclear genome size variation among plants

• Arabidopsis thaliana 120 Mbp (120,000,000 bp)

• Poplar 460 Mbp

• Rice 450 Mbp

• Maize 2,500 Mbp

• Barley 5,000 Mbp

• Hexaploid wheat 16,000 Mbp

• Fritillaria (lilly family) >87,000 Mbp

Transposable elements comprise most of the maize genome (and many other plants)

Organelle genomes• Chloroplast

– Only in plants: partial set of genes for photosynthesis• Endosymbiotic origin: Bacterial structure and function

– About 100 genes – Inherited from one parent (usually female) – partial containment

method– Tens to hundreds of copies per cell

• Enables super high gene expression levels for making and storing proteins, super high resistance gene toxin levels

– Hard to transform

• Mitochondrion: Similar story, fewer genes, highly complex and variable structure– Cell respiration– Very hard to transform

• Cytoplasmic male-sterility genes known or created in both organelle genomes

Structure and function of genes

• Basic double-helix biology and what it means for genetic science

• Basic gene function/expression and analysis

About 50 years ago……“ This structure has novel features which are of considerable biological

interest…”

Chromosomes are made of

wound-up DNA and proteins

called chromatin

The tight wrapping, and chemistry of associated

proteins, helps to control gene

expression

DNA extraction methods use

strong detergents like CTAB to lyse

cell membranes

and separate cellular

biochemicals from one

another, then DNA is

chemically partitioned from rest of

cellular material

• What is a gene? • Segment of a

chromosome that allow a cell to produce a specific function or molecule

• DNA which exists as 2 complementary strands containing adenine (A), thymine (T), cytosine (C), or guanine (G)

Base pairing among

nucleotides due to hydrogen

bonding is basis of replication, gene function, and much DNA methodology

The longer the perfect match,

the stronger the bond – depends

on chemistry, temperature,

length of DNA molecule, and %

identity during annealing or

hybridization of DNA strands

Gene function: The central dogma of molecular biology

transcription

translation

RNA undergoes complex

processing before and after movement from

nucleus to cytoplasm that affects gene expression – Proteins also modified and degraded in

complex ways

Exon 1 Exon 2 Exon 3 Exon 4

5’ 3’Introns

Exon 1Exon 2Exon 3Exon 4 mRNA

Protein

Transcription (inside nucleus)

Translation (outside nucleus)

GENE

Basic structure and processing of genes

Regulatory elements are mostly upstream (5’) and called

“promoters”

Exon 1 Exon 2 Exon 3 Exon 4

5’ 3’Introns

RegulatoryElements

Start site Stop site

Promoters

• They determine under what environmental conditions, in what cells, and to what level a gene is expressed

• They can be excised from coding region and transferred to other genes

• Many function over wide phylogenetic distances (e.g., all dicot plants)

Tomato Fruit 5X Lycopene (tomato)

Promoter (controls expression)

Gene (encodes protein)

Examples of promoter - gene combinations in GE plants

Round-up Ready© (bacteria)35S-CAMV (plant virus)

Restriction enzymes allow DNA to be cut at

specific sequences Ligases allow DNA to be spliced together

How are genes cut and spliced?Cut

fragments are

separated on gels

Gene cloning allows lots of

DNA for specific

genes to be made,

required for study and

manipulation

The polymerase chain reaction allows gene cloning without bacteria

• Extremely sensitive– Amplifies target DNA in repetitive cycles, thus called

“chain reaction”– Capable of detecting DNA from a single cell– Basis of most DNA forensic procedures and other

kinds of DNA fingerprinting and mapping• Basis of most systems for detecting

“contaminating” transgenic DNA– Can be highly quantitative

The polymerase chain reaction allows gene cloning without bacteria

• Requires synthesis of small DNA molecules called primers identical to target gene (oligonucleotides, or oligos)– Thus must know target sequence

• Requires heat-stable DNA polymerase that continues to work after heat to near boiling temperature– Hot springs bacterial sources, re-engineering

Genomics: High throughput sequencing or analysis of lots of genes

Key genomics concepts• Many genomes have been sequenced in

mammals, insects, plants, microbes– Most of genome does not express genes– But gets at promoters/regulatory regions near genes– Gets all, including those genes expressed very rarely

or at low levels• Annotation critical: Many genes not recognized

or wrongly recognized (genome bioinformatics = computational analysis of genomic data)– Comparative methods compare well-known models to

crop species greatly speed biotechnology

Plants exhibit extensive conservation of gene content

Loblolly pine and Arabidopsis thaliana differ greatly in form, ecological niche, evolutionary history, and genome size. Yet most genes of substantial length have an Arabidopsis gene homolog. Kirst et. al. 2003.

Key genomics concepts• The expressed sequences can be identified by

isolating the messenger RNA, converting to DNA, and sequencing all or part of them– Called ESTs or expressed sequence tags – Many very large databases of them exist – Can compare tissues/cells/environmental treatments to

learn about general function• Comparison of genomic and EST sequences

shows how genes are spliced (intron/exons)• Using either genomic or EST information, the

expression or genetic state of tens of thousands of genes can be studied at once using “gene chips” (microarrays)

Microarrays = lots of single-starnded DNA in a very small area

Arrays are typically the size of a microscope slide or less, and can contain tens of thousands of DNAs – with either full or partial genes

Affymetrix array with thousands of 25 bp gene signals

Overview of a microarray experiment

Target DNA

PCR

Spotting

Slide (microarray, chip)

SamplesControl Treatment

mRNA purification

Labeling (Probes)

Cy3 Cy5

Hybridization Scanning

Analyze

Uses of arrays• Genotyping/sequencing tens of thousands of genes

to speed conventional breeding or genetic analysis• Profiling gene expression in response to

environment or in specialized tissues to identify new genes with important physiological roles– Can see coordinated changes of gene expression in

specific biological pathways (e.g., oils, nutrients)• Understanding physiological state of cells/organisms

under stress • Diagnosing or predicting response to

disease/environmental stress• Monitoring physiological changes due to GE or other

forms of breeding

“Good prognosis profile”

“poor prognosis profile”

Gene expression profiling predicts clinical outcome of breast cancer. Nature (2002): 415:530-535

Arrays are useful for physiological

diagnosis

Summary• We know a lot about how to control expression

of genes via changes to promoters and other proximal sequences– Properties usually transfer after gene splicing,

regardless of the gene they control • Promoters and genes are highly conserved and

usually work across broad phylogenetic distances– Model/microbe to crop transfers common in first

generation of GMOs – Bacterial and viral genes and promoters common

Summary• Information can be rapidly transferred among species

for any gene due to genomics databases and analytical methods– Sequence comparisons followed by immediate cloning via

PCR– Provides new options for GE and non-GE gene-based

breeding• PCR is a highly sensitive way to monitor transgenes

– Makes historical imprecision of agricultural practices a large legal risk in GMO era

• Microarrays are a powerful research method– New ways to genotype and monitor plant physiology and gene

expression– Obstacle to broad, commercial use: cost & complexity – Likely diagnostic uses in near-term

Discussion questions• What aspects of gene function or genomics are

most unclear?– What are most important to understand for

interpreting biotechnologies? • What is PCR and why is it important for

intellectual property and legal enforcement of GMO regulations? What do you think was done before PCR?

• Give an example of how genomics, combined with GMO methods, could be used to produce a new kind of GMO crop.