Download Gene - Oregon State University

What all good citizens should know
about genes and plant
biotechnology
Steve Strauss
[email protected]
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
Poplar
Rice
Maize
Barley
Hexaploid wheat
Fritillaria (lilly family)
120 Mbp (120,000,000 bp)
460 Mbp
450 Mbp
2,500 Mbp
5,000 Mbp
16,000 Mbp
>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
Basic structure and processing of
genes
5’
3’
Introns
GENE
Exon 1
Exon 2
Exon 3
Exon 4
Transcription (inside nucleus)
mRNA
Exon 1 Exon 2Exon 3Exon 4
Translation (outside nucleus)
Protein
Regulatory elements are mostly
upstream (5’) and called “promoters”
5’
3’
Introns
Exon 1
Regulatory
Elements
Start site
Exon 2
Exon 3
Exon 4
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)
Examples of promoter - gene
combinations in GE plants
Promoter
(controls expression)
Gene
(encodes protein)
Tomato Fruit 5X
Lycopene (tomato)
35S-CAMV
(plant virus)
Round-up Ready© (bacteria)
How are genes cut and spliced?
Restriction enzymes
allow DNA to be cut at
specific sequences
Cut
fragments
are
separated
on gels
Ligases allow DNA to
be spliced together
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 singlestarnded 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
Samples
Control
Treatment
mRNA purification
PCR
Analyze
Labeling (Probes)
Spotting
Cy3
Slide (microarray, chip)
Cy5
Hybridization
Scanning
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
Arrays are useful
for physiological
diagnosis
“Good prognosis
profile”
“poor prognosis
profile”
Gene expression profiling predicts clinical outcome of breast cancer. Nature (2002):
415:530-535
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.