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Lectures

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bessie
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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.

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  1. 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. Non-homologous recombinationTransposable elements LECTURE 4 • A. Discovery • B. Classification • C. Examples and distribution • Antibiotic resistance spread • Cassette model • D. Mechanisms • E. Regulation • F. Methods of study and uses 5

  3. P O E T K MUTATION TRANSCRIPTION BLOCKED. NO ENZYME EXPRESSION 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): Ampr in plasmid RP4 gal operon on defective lambda phage ; dgal

  4. 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 point (amber) mutation. Supression revertants at low frequency gal (deletion). No revertants possible

  5. chlD locus mutations confer resistance to chlorate gal IS1 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

  6. Deletion of adjacent genes due to activity of IS1 inserted in the gal operon Close-up of colonies with gal+ papillae 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

  7. 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. 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. Target DNA Target duplication- direct repeat Transposase genes IS10 IS10 Tetr E.g. Tn10 C. Examples and distribution Inverted repeat General structure • TYPE I: IS1, IS2, IS5, IS10 COMPOSITE TRANSPOSONS

  10. Type Kbps Marker Inverted repeats Target dup’ Tn 1 5.0 ampr 38 5 Tn 3 5.0 ampr 38 5  5.0 NONE38 5 Tn 1721 5.0 tetr and INTEGRON system38 5 C. Examples and distribution cont... • TYPE II The Tn3 like elements. Much BIGGER! • Many ANTIBIOTIC RESISTANCE DETERMINANTS

  11. Resistance Determinants IS1 Tn3 on R1 mer amp sul str kan cm Tn4 tra Tn2571 IS2 Tn10 Tn903 on R6 IS1 IS10 IS10 C. Examples and distribution cont... • Antibiotic resistance spread • largely due to transposable elements • R100 shows cassette model of evolution

  12. TRANSPOSON Target sequence + RESOLUTION + + Donor may be degraded D. Transposition Mechanisms CONSERVATIVE VS REPLICATIVE Independent of RecA Donor CONSERVATIVE TRANSPOSITION REPLICATIVE TRANSPOSITION

  13. 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 37oC) 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. E. Regulation cont…... a. Tn3 Repressor -lactamase Transposase Repressor

  15. E. Regulation cont…... b/c. Antisense RNA and methylation. IS10 best studied pOUT antisense pIN GA(me)TC Transposase

  16. 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. Plasmid cointegration mediated by IS1. See practical exercise Crosses of: A. E.coli DP990 (pOXKm, pKPG16 (pBR322::IS1)) with C600 nalR B. E.coli DP990 (pOXKm, pBR322 (Control)) with C600 nalR A. 100µl undiluted and plated on Km,Tet and Nal agar plates. Cointegrates grow 100µl of 10-3 dilution plated on Km, Nal plates. Indicates the pOXKm transconjugants B. 100µl undiluted and plated on Km,Tet and Nal agar plates. No cointegrates detected