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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.