Microbial Genetics (Micr340)

1. Microbial Genetics (Micr340) Lecture 12 Transposition

2. Nonhomologous recombination • No requirement for two DNAs being of the same or similar nucleotide sequences • Needs enzymes that recognize specific regions in DNA • Mechanisms include: • transposition • phage integration and excision • resolution of cointegrates

3. Transposition • Transposons – DNA elements that can hop (transpose) from one place in DNA to another • Movement by a transposon is called transposition, catalyzed by enzymes called transposases • Transposons usually encode their own transposases • Transposons are known to exist in all organisms on earth

4. History of Transposons • Transposons (jumping genes) were first discovered in Maize (corn) by Barbara McClintock. • Transposons were later found in bacteria, flies and humans. • This discovery led to the awarding of Nobel Prize to McClintock in 1983.

5. Transposition • Many transposons are essentially cut out of one DNA and inserted into another • Other transposons are copied and then inserted elsewhere • Donor DNA and target DNA

6. http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_5.htmlhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_5.html

8. Structure of Bacterial Transposons • All contain repeats at their ends, usually inverted repeats (IR) • Presence of short direct repeats in the target DNA that bracket the transposon • The sites of insertion are different among target DNAs

9. Types of Bacterial Transposons • Smallest bacterial transposons are called insertion sequence elements (IS elements); they only encode transposase enzymes • Composite transposons – formed by two IS elements of the same type, bracketing other genes • Noncomposite transposons are bracketed by two short inverted repeats, with multiple resistance markers.

10. Structure of a Bacterial transposon

11. IS Elements and Composite Transposons

12. Composite transposons

13. Noncomposite transposons

14. Assay of transposition

15. Transposon Mutagenesis • A gene that has been marked with a transposon is relatively easy to map • Genes marked by transposons are very easy to be cloned • Transposon mutagenesis to identify essential genes (related to research of my lab).

16. Properties of transposons for mutagenesis • Transpose at a fairly high frequency • Not very selective in target sequence • Carry selectable marker (antibiotic resistance genes) • Broad host range for transposition

17. Tn5 mutagenesis

18. Cloning genes mutated by transposition

19. Isolating random gene fusions