Problem

1. Problem 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

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

3. 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

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

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

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

7. 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

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

9. 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

10. 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)

11. Creation of mice ES cells carrying a knockout mutation

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

19. 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

20. Production of transgenic plants with Ti plasmids

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

22. 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.

23. Reporter Genes as TransgenesExample: assaying the promoter of Gene X Gene X Promoter Coding Region ORF Promoter REPORTER ORF

24. 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.)

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

26. 35 S Promoter 35 S Promoter REPORTER (GFP) REPORTER (GFP) ORF ORF Combining artificial promoters and reporter genes • Promoter for constitutive expression (35S) • GFP coding region +

27. 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.)

28. 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.

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

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

31. 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.)

32. Promoter Promoter Coding Region Coding Region ORF ORF Turning off genes • Antisense

33. Turning off genes • RNAi