Download The Molecular Biology of Gene Function

The Molecular Biology of Gene
Function
Mark Jordan
Cereal Research Centre
AAFC
Typical Eukaryotic Gene
From Wikipedia.org
http://www.ucl.ac.uk/~sjjgsca/promoter1.gif
5’ UTR Impacts Translation
Start Codon-Kozak Sequence
Biota
Phylum
Consensus sequences
gccRccATGG
Vertebrate
Fruit fly (Drosophila spp.)
Arthropoda
cAAaATG
Budding yeast
(Saccharomyces cerevisiae)
Ascomycota
aAaAaAATGTCt
Slime mold (Dictyostelium
discoideum)
Amoebozoa
aaaAAAATGRna
Ciliate
Ciliophora
nTaAAAATGRct
Malarial protozoa
(Plasmodium spp.)
Apicomplexa
taaAAAATGAan
Toxoplasma (Toxoplasma
gondii)
Apicomplexa
gncAaaATGg
Trypanosomatidae
Euglenozoa
nnnAnnATGnC
Terrestrial plants
AACAATGGC
http://en.wikipedia.org/wiki/Kozak_consensus_sequence
http://cnx.org/content/m11416/latest/
Stop Codon
• TAA, TAG, TGA
• Mutations cancel the stop signal – incorrect or
no protein.
• Mutations introduce a stop codon- truncated
mRNA and incorrect or no protein
3’ UTR
http://cnx.org/content/m44539/latest/
miRNA and 3’ UTR
http://www.laskerfoundation.org/awards/2008_b_description.htm
miRNA and 3’ UTR
Li and Zhang (2013) Trends in Genetics 29:116-124
• What is necessary for controlled expression is a
complete package of all elements. Failure to take into
account (or lack of knowledge of) all the elements can
lead to unexpected results due to improper expression.
Eg. If alternative splicing occurs will get a different
protein with different effects. Eg. Different
backgrounds have different miRNA and could target
the new gene.
• Possible to test early on for most of these changes to
core gene regulatory regions. Eg. Can test to see if
mRNA properly spliced or if degraded or if improper
start/stop or low expression levels from promoter
effects.
Pleiotropy
One gene affects multiple traits
Direct Pleiotropy
• The secondary traits are derived from the
primary trait.
Direct Pleiotropy
Mat QTL
Hd QTL
FN QTL
Cabral et al. 2103
Indirect Pleiotropy
• One gene affects multiple traits
independently.
• Eg. a regulatory gene such as a TF which binds
to many different promoters. Mutations that
affect binding could cause many traits to
change.
Mechanisms of Pleiotropy
• Alternative splicing means one gene can code
for multiple proteins with different effects.
• Gene can affect multiple pathways –
regulatory proteins like TF or kinases
• Gene can affect a basic cellular function or
pathway needed for many traits
• Antagonistic pleiotropy- gene has positive and
negative effects – sickle cell
Natural Pleioptropy and Breeding
• The case of Lr34
• Using classical genetics genes were identified
for the traits:
– Leaf rust resistance (Lr34)
– Stripe rust (Yr10)
– Powdery mildew (Pm19)
– Leaf tip necrosis (Ltn1)
– Also virus resistance and stem rust
A. Lr34 carrying genotype on left and NIL without Lr34
on right. B and C. Lr34 carrying genotype on left
Keller et al. 2012
http://www.globalrust.org/traction/permalink/bgriworkshop13
• Using genetic mapping a genomic
region on chrom 7D was found to
be responsible
• Gene was cloned and found to be
an ABC transporter
• The largest and arguably the most
important family of membrane
transport proteins. They are
ubiquitous in biology and power
the translocation of substrates
across the membrane, often
against a concentration gradient,
by hydrolyzing ATP. (http://wwwssrl.slac.stanford.edu/research/highlights_a
rchive/rees_abc.html)
• The substrate(s) for Lr34 is
unknown
Vit B12 transporter
Lr34 is antagonistic pleiotropy
• Resistant form has 2 base changes from the wild
type suscep form.
• Resistance is at adult stage and plants not
completely resistant. Small amount of disease
reduces selection pressure so is very durable.
• Used for over 100 years.
• Amount of disease below economic levels.
• “Most important gene in wheat”.
• LTN is a small price to pay for resistance to
disease.
Lr34 inserted
into barley using
genetic
transformation
causes
premature LTN
and stunted
mostly sterile
plants.
Resistance is
observed to
multiple
pathogens at
seedling stage.
Risk et al. Plant Biotech. J.
(2013) 847-854
• Lr34 is toxic- if expressed at certain levels or
under certain conditions it causes cell death
• Wheat has regulatory mechanisms to control
the gene to maximize benefit and reduce
detriment (selected by breeding). Other
species (even related ones) do not have those
controls.
• Extent of LTN varies among wheat genotypes.
Regulatory mechanisms may vary.
Can We Predict Pleiotropic Effects
• Knowledge of mechanism of action – regulatory molecules (TF, miRNA)
and basic cellular function proteins (eg. ABC transporters) more likely to
exhibit pleiotropy.
• In many cases all changes may be positive. Could change a TF to affect a
suite of genes and get multiple benefits.
• Origin of gene- could be more pleiotropy if gene from a distant source due
to lack of regulatory controls (eg Lr34) OR less pleiotropy due to lack of
pathway/function in new species (eg in native species affects 10 pathways
but in new species only one is present). In general a gene that has multiple
effects also has multiple controls – more closely related more likely to
have the controls.
• Not only at species level – different varieties of same species can have
different effects (what plant breeding is about).
• Prediction needs knowledge of function and also how that gene is
regulated. Whole genome sequence will help. Can find all genes with
binding sites for a TF and predict phenotypes based on function of those
genes. Same with miRNA. Not there yet.