1. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Lectures Introduction. The basis of diversity –Mutation, homologous recombination and repair (3 lectures) Non-homologous recombination –Mechanisms of transposition (2 lectures) Genomics and genome mapping –Pre-genomic techniques –Prokaryote genome sequencing –Revision tutorial

2. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Non-homologous recombination Transposable elements A. Discovery B. Classification C. Examples and distribution –Antibiotic resistance spread –Cassette model D. Mechanisms E. Regulation F. Methods of study and uses LECTURE 4 5

3. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. A. Discovery First noted in 1967 in E.coli as cause of polar mutations in; gal operon (Saedler) / lac operon (Shapiro) High frequency of spontaneous reversion to gal or lac + Hedges and Jacob (1974) demonstrated 1st Transposon Tn1 (Tn3 related): Amp r in plasmid RP4 P O ETK MUTATION gal operon on defective lambda phage ; dgal TRANSCRIPTION BLOCKED. NO ENZYME EXPRESSION

4. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. IS1 mediated excisions in the gal operon gal::IS1 in this sector reverts as red WT (gal+) colonies on McConkey Agar. Note the high frequency  gal (deletion). No revertants possible gal point (amber) mutation. Supression revertants at low frequency

5. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Deletion of adjacent genes due to activity of IS1 inserted in the gal operon gal IS1 chlD locus mutations confer resistance to chlorate Chlorate resistant colonies on chlorate/McConkey agar. A. IS1 has transposed to chlD locus but still reverts as papillae to gal+ B. IS1 has transposed to chlD locus then gal has been deleted; hence no papillae

6. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Deletion of adjacent genes due to activity of IS1 inserted in the gal operon Chlorate resistant colonies on chlorate/McConkey agar. A. IS1 has transposed to chlD locus but still reverts as papillae to gal+ B. IS1 has transposed to chlD locus then gal has been deleted; hence no papillae Close-up of colonies with gal+ papillae

7. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. B. Classification There are four basic types TYPE I :The Insertion sequences and their composite elements TYPE II: The Tn3 family of elements TYPE II: The transposing bacteriophages (e.g. mu - not covered here) The conjugative transposons (e.g. Tn916 carrying tet resistance around a range of host cells in Enterococcus and other bacteria). Large family found in these Gram positive bacteria with broad host range. Carry Integration / excision determinants and plasmid transfer genes. INTEGRATE - EXCISE -TRANSFER ON PLASMID (not covered in detail here).

8. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. B. Classification Cont... Many features in common but with exceptions. All transpose as DISCRETE sequences ALL have transposase which serves to recognise ENDS MUST have precise end recognition EITHER use terminal inverted repeat sequences OR in some cases integrate at specific sequences to produce a consensus sequence for end recognition

9. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. C. Examples and distribution TYPE I: IS1, IS2, IS5, IS10 General structure Inverted repeat COMPOSITE TRANSPOSONS E.g. Tn10 IS10 Tet r Target DNA Target duplication- direct repeat Transposase genes

10. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. C. Examples and distribution cont... TYPE II The Tn3 like elements. Much BIGGER! Many ANTIBIOTIC RESISTANCE DETERMINANTS Type Kbps Marker Inverted repeats Target dup’ Tn 1 5.0 amp r 38 5 Tn 3 5.0 amp r 38 5  5.0 NONE 38 5 Tn 1721 5.0 tet r and INTEGRON system 38 5

11. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. C. Examples and distribution cont... Antibiotic resistance spread largely due to transposable elements R100 shows cassette model of evolution IS2 tra IS10 Tn10 Tn2571 IS1 Tn4 Tn3 on R1 Tn903 on R6 Resistance Determinants mer amp sul str kan cm

12. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. D. Transposition Mechanisms CONSERVATIVE VS REPLICATIVE Independent of RecA + Donor Target sequence REPLICATIVE TRANSPOSITION RESOLUTION CONSERVATIVE TRANSPOSITION + + TRANSPOSON Donor may be degraded

13. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. E. Regulation All transposons are under negative regulation Recombinational frequencies down to around 10 -3 to 10 -6 In E. coli the growth temperature greatly affects many transposition events. Higher frequencies at lower temperatures (below 37 o C) Especially IS1 and Tn3. Basis not known. EXAMPLES: a. Repressor molecule Tn3 b. Antisense RNA (Tn10) c. Methylation (Tn10 and many IS elements) d. Transcriptional frameshift IS1. fusion of two reading frames

14. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. E. Regulation cont…... a. Tn3 Repressor  -lactamase Repressor Transposase

15. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. E. Regulation cont…... b/c. Antisense RNA and methylation. IS10 best studied GA(me)TC Transposase pOUT antisense pIN

16. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. F. Methods of study and uses Various methods used to demonstrate transposition. 1. Deletion formation (as for IS1 before) 2. Cointegrate formation (as for practical 3) 3. Non-replicating plasmids as delivery vectors 4. Defective phage such as lambda as a vector Can be used a a means to TAG genes for mapping (see practical) Can be used for insertional mutagenesis (esp’ Tn5 in Gm negatives)  Dale Chapter 7.

17. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Plasmid cointegration mediated by IS1. See practical exercise Crosses of: A. E.coli DP990 (pOXKm, pKPG16 (pBR322::IS1)) with C600 nal R B. E.coli DP990 (pOXKm, pBR322 (Control)) with C600 nal R A. 100µl undiluted and plated on Km,Tet and Nal agar plates. Cointegrates grow B. 100µl undiluted and plated on Km,Tet and Nal agar plates. No cointegrates detected 100µl of 10-3 dilution plated on Km, Nal plates. Indicates the pOXKm transconjugants