What all good citizens should know about genes and plant biotechnology Steve Strauss...
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Transcript of What all good citizens should know about genes and plant biotechnology Steve Strauss...
What all good citizens should know about genes and plant
biotechnology
Steve Strauss
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.