Problem Results: Question: 1. You screen two libraries- cDNA; genomic 2. Clones are isolated having homology to PSY- 10 clones from each library 3. These are subcloned into pBluescript. 4. Protein expression is induced with IPTG and proteins separated by SDS-PAGE. Results: Genomic clones: 0/10 gave expression cDNA clones: 2/10 gave expression Question: Why zero genomic clones Why only 2 cDNA clones

Transgenic Organisms Lecture 6 Reading: Chapter 9 Molecular Biology syllabus web site

Genetic Markers RFLP/ RAPDS and other newer PCR-based methods -to create maps -to study evolutionary relationships Mapping markers -in situ hybridization, fluorescent tags -Southern analysis (linked markers co-segregate) -chromosome walking to generate physical maps -comparison of physical and genetic maps

DNA polymorphisms can be used to map human mutations Analysis of restriction fragment length polymorhpisms (RFLPs)

Isolation of a contiguous stretch of DNA and construction of a physical map in that region Chromosome walking

Physical maps of entire chromosomes can be constructed by screening YAC clones for sequence-tagged sites Ordering of contiguous overlapping YAC clones

Gene replacement and transgenic organisms Some genes are identified through means other than mutant analysis To determine the function of these genes, it is possible to replace an organism’s wild type gene with an inactive gene to create a “gene knockout” It is also possible to introduce additional genes (transgenes) to create a transgenic organism

In vitro mutagenesis of a cloned gene Gene knockout and transgenic techniques usually involve mutagenesis of cloned genes prior to transfer into the organism

Transgenic Approaches Methods spheroplasts-yeast, plants chemical methods; microinjection- animal cells electroporation particle gun bombardment bacterial-plants Stable or transient selection with markers Knockouts (homologous recombination) “gene replacement” Transgenic Organisms

Purposes of transgenic research Basic- understanding gene function Applied- gene therapy to introduce functional genes improvement (foods; create novel sources of drugs; increasing plant production to provide more food)

Creation of mice ES cells carrying a knockout mutation

Production of transgenic Drosophila Eye color, a screenable phenotype encoded by w+ gene. Drosophila, red-eyed wild type (left) & white-eyed mutant (right).

Transgenic Plants Plants cells are totipotent and can regenerate from undifferentiated tissue to produce viable, seed-bearing plants. Methods: electroporation, microinjection, bombardment, use of Agrobacterium tumefaciens

Production of transgenic plants with Ti plasmids

Reporter Genes as Transgenes Examples Advantage: Easy to assay compared to native gene GUS- b-glucuronidase GFP- green fluorescent protein LACZ- b-galactosidase LUC- luciferase

Gene X is an enzyme,GGPPS How do we determine where in the plant this gene is expressed? Fuse the promoter of Gene X to the coding region encoding GUS (a bacterial enzyme, betaglucuronidase). Assay enzyme activity of GUS using a chromogenic substrate. Active enzyme catalyzes formation of a blue product.

Reporter Genes as Transgenes Example: assaying the promoter of Gene X Coding Region ORF Promoter REPORTER ORF

Reporter Genes as Transgenes GUS b–glucuronidase is a bacterial enzyme that acts on a chromogenic substrate to produce a blue product. Arabidopsis promoter-GUS fusions expressed in Arabidopsis. (Okada et al., 2000, Plant Physiology 122:1045-56.)

Artificial Promoters Promoter Coding Region Promoter Coding Region ORF To alter natural expression with respect to time, place, or level of expression Promoter Coding Region ORF Promoter Coding Region ORF

Combining artificial promoters and reporter genes Promoter for constitutive expression (35S) GFP coding region 35 S Promoter REPORTER (GFP) ORF + 35 S Promoter REPORTER (GFP) ORF

Constitutive expression of GFP GFP, Green Fluorescent Protein- is a bacterial protein that will normally localize to the cytoplasm. Transient expression of GFP in tobacco (Zhu, Li, Wurtzel, unpub.)

Gene X is a chloroplast protein How do we determine which part of the protein is needed to direct it to a chloroplast Fuse DNA encoding the putative transit sequence to the coding sequence of GFP (jellyfish green fluorescent protein) which is driven by a constitutive promoter (35S). Use a fluorescence microscope to detect the fluorescence of GFP.

Promoter Coding Region 35 S Promoter REPORTER (GFP) Combining reporters & constitutive promoters to assay gene elements Example: assaying transit sequence of Gene X Gene X Promoter Coding Region ORF 35 S Promoter REPORTER (GFP) ORF

Fusion of maize PSY transit sequence to GFP directs GFP to tobacco chloroplasts. Untransformed PSY-GFP Green Red Merged Zhu, Li, & Wurtzel unpublished

Reporter Genes as Transgenes GUS- b-glucuronidase GFP- green fluorescent protein LACZ- b-galactosidase LUC- luciferase Arabidopsis promoter-GUS fusions expressed in Arabidopsis. (Okada et al., 2000, Plant Physiology 122:1045-56.) Transient expression of GFP in tobacco (Zhu, Li, Wurtzel, unpub.)

Turning off genes Antisense Promoter Coding Region Promoter ORF Promoter Coding Region ORF

Turning off genes RNAi