©m j larkin biology & biochemistry. the queen’s university of belfast. lectures introduction....

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

LecturesLectures• 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

• 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

http://www.qub.ac.uk/


Non-homologous recombinationTransposable elements

Non-homologous recombinationTransposable elements

• A. Discovery• B. Classification• C. Examples and distribution

– Antibiotic resistance spread

– Cassette model

• D. Mechanisms• E. Regulation• F. Methods of study and uses

• 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



A. Discovery 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

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

P O E T K

MUTATION

gal operon on defective lambda phage ; dgal

TRANSCRIPTION BLOCKED. NO ENZYME EXPRESSION



IS1 mediated excisions in the gal operonIS1 mediated excisions in the gal operon

gal::IS1in this sectorreverts as redWT (gal+) colonies onMcConkeyAgar. Note thehigh frequency

gal (deletion). No revertants possible

gal point(amber)mutation.Supressionrevertantsat lowfrequency



Deletion of adjacent genes due to activity of IS1 inserted in the gal operon


gal

IS1

chlD locusmutations conferresistance to chlorate

Chlorate resistant colonies on chlorate/McConkey agar.A. IS1 has transposed to chlD locusbut still reverts as papillae to gal+B. IS1 has transposed to chlD locusthen gal has been deleted; hence no papillae





Chlorate resistant colonies on chlorate/McConkey agar.A. IS1 has transposed to chlD locusbut still reverts as papillae to gal+B. IS1 has transposed to chlD locusthen gal has been deleted; hence no papillae

Close-up of colonies withgal+ papillae



B. ClassificationB. Classification

There are four basic typesTYPE I :The Insertion sequences and their composite elements TYPE II: The Tn3 family of elementsTYPE 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).

There are four basic typesTYPE I :The Insertion sequences and their composite elements TYPE II: The Tn3 family of elementsTYPE 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).



B. Classification Cont...B. Classification Cont...

Many features in common but with exceptions.

All transpose as DISCRETE sequences

ALL have transposase which serves to recognise ENDSMUST 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

Many features in common but with exceptions.

All transpose as DISCRETE sequences

ALL have transposase which serves to recognise ENDSMUST 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



C. Examples and distributionC. Examples and distributionTYPE I: IS1, IS2, IS5, IS10TYPE I: IS1, IS2, IS5, IS10

General structure Inverted repeat

COMPOSITE TRANSPOSONS

E.g. Tn10 IS10 IS10Tetr

Target DNA

Target duplication-direct repeat

Transposase genes



C. Examples and distribution cont...C. Examples and distribution cont...

TYPE II The Tn3 like elements. Much BIGGER!Many ANTIBIOTIC RESISTANCE DETERMINANTS

TYPE II The Tn3 like elements. Much BIGGER!Many ANTIBIOTIC RESISTANCE DETERMINANTS

Type Kbps Marker Inverted repeats Target dup’

Tn 1 5.0 ampr 38 5

Tn 3 5.0 ampr 38 5

5.0 NONE 38 5

Tn 1721 5.0 tetr and INTEGRON system 38 5



C. Examples and distribution cont...C. Examples and distribution cont...

Antibiotic resistance spreadlargely due to transposable elements

R100 shows cassette model of evolution

Antibiotic resistance spreadlargely due to transposable elements

R100 shows cassette model of evolution

IS2tra

IS10 IS10

Tn10

Tn2571

IS1

IS1

Tn4

Tn3 on R1

Tn903 on R6

Resistance Determinants

merampsulstrkancm



D. Transposition MechanismsD. Transposition Mechanisms

CONSERVATIVE VS REPLICATIVEIndependent of RecA

+Donor

Target sequence

REPLICATIVE TRANSPOSITION

RESOLUTION

CONSERVATIVETRANSPOSITION

+ +

TRANSPOSON

Donor may be degraded



E. RegulationE. Regulation

All transposons are under negative regulationRecombinational frequenciesdown to around 10-3 to 10-6

In E. coli the growth temperature greatly affects manytransposition 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



E. Regulation cont…...E. Regulation cont…...

a. Tn3 Repressor

-lactamase

RepressorTransposase



E. Regulation cont…...E. Regulation cont…...

b/c. Antisense RNA and methylation.

IS10 best studied

GA(me)TC

Transposase

pOUT antisense

pIN



F. Methods of study and usesF. 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 vectors4. Defective phage such as lambda as a vector

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 vectors4. 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)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.



Plasmid cointegration mediated by IS1. See practical exercise

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,Tetand Nal agar plates.Cointegrates grow

B. 100µl undiluted and plated on Km,Tetand Nal agar plates.No cointegrates detected

100µl of 10-3dilution plated onKm, Nal plates.Indicates thepOXKmtransconjugants


©m j larkin biology & biochemistry. the queen’s university of belfast. lectures introduction....

Documents

gal papillae

j larkin biology biochemistry

queens university of

revertants possible

high frequency gal deletion

red wt gal colonies

activity of is1

low frequency slide