Bacterial transposons

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Bacterial transposons

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Transposons and transposable elements : 

Transposons and transposable elements Dr. M.K.SATEESH Bangalore University Bangalore-56

Jumping genes : 

Jumping genes Transposable elements were first discovered by Barbara McClintock in the 1950's and recognized as genetic elements which cause unstable mutations, produce chromosomal aberrations (including breaks) and which change location in the genome.

Slide 3: 

Barbara McClintock (1902-1992), 1983 Nobel Prize in Medicine or Physiology for discovering transposons, or mobile genetic elements

Insertion sequences : 

Insertion sequences They were "re-discovered" in bacteria in the 1970's as "insertion sequences", genetic elements which move from one genetic location to another and that cause premature transcriptional termination in bacteria operons.

Transposable elements : 

Transposable elements “mobile genetic elements” comprise 45% of human chromosomal DNA “middle repetitive DNA” contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material aid speciation and genomic change (in bacteria transposons are often associated with antibiotic resistance genes) cells must depress transposition to insure genetic stability

Types of transposable elements : 

Types of transposable elements DNA vs. RNA viral vs. nonviral replicative mechanism vs. excision mechanism

Slide 7: 

transposon

Slide 8: 

transposon insertion mutation

Discovery of transposons : 

Discovery of transposons Barbara McClintock 1950’s Ac -Ds system in maize influencing kernel colorunstable elementschanging map positionpromote chromosomal breaks Rediscovery of bacterial insertion sequencessource of polar mutationsdiscrete change in physical length of DNAinverted repeat ends: form “lollipops” in EM after denaturation/reannealing

Transposable Elements in Prokaryotes : 

Transposable Elements in Prokaryotes Prokaryotic examples include: a. Insertion sequence (IS) elements. b. Transposons (Tn). c. Bacteriophage Mu (replicated by transposition) 1. IS elements are the simplest transposable elements found in prokaryotes, encoding only genes for mobilization and insertion of its DNA. IS elements are commonly found in bacterial chromosomes and plasmids. 2. IS elements were first identified in E. coli’s galactose operon, wheresome mutations’ were shown to result from insertion of a DNA sequence now called IS1

The insertion sequence (IS) transposable element, IS1 : 

The insertion sequence (IS) transposable element, IS1 inverted terminal repeats (IR) inverted terminal repeats (IRs)

Slide 12: 

Bacterial IS elements

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Prokaryotic IS elements range in size from 768 bp to over 5 kb. Known E. coli IS elements include: a. IS1 is 768 bp long, and present in 4–19 copies on the E. coli chromosome. IS2 has 0–12 copies on the chromosome, and 1 copy on the F plasmid. IS10 is found in R plasmids. 4. The ends of all sequenced IS elements show inverted terminal repeats (IRs) of 9–41 bp (e.g., IS1 has 23 bp of nearly identical sequence).

Integration of IS elements may: : 

Integration of IS elements may: a. Disrupt coding sequences or regulatory regions. b. Alter expression of nearby genes by the action of IS element promoters. c. Cause deletions and inversions in adjacent DNA. d. Serve as a site for crossing-over between duplicated IS elements.

When an IS element transposes: : 

When an IS element transposes: a. The original copy stays in place, and a new copy inserts randomly into the chromosome. b. The IS element uses the host cell replication enzymes for precise replication. c. Transposition requires transposase, an enzyme encoded by the IS element. d. Transposase recognizes the IR sequences to initiate transposition. e. IS elements insert into the chromosome without sequence homology (illegitimate recombination) at target sites (Figure 2). i. A staggered cut is made in the target site, and the IS element inserted. ii. DNA polymerase and ligase fill the gaps, producing small direct repeats of the target site flanking the IS element (target site duplications). f. Mutational analysis shows that IR sequences are the key

Fig. 2 Schematic of the integration of an IS element into chromosomal DNA : 

Fig. 2 Schematic of the integration of an IS element into chromosomal DNA

Transposons : 

Transposons Transposons are similar to IS elements, but carry additional genes, and have a more complex structure. There are two types of prokaryotic transposons: a. Composite transposons carry genes (e.g., antibiotic resistance) flanked on both sides by IS elements (IS modules). i. The IS elements are of the same type, and called ISL (left) and ISR (right). ii. ISL and ISR may be in direct or inverted orientation to each other. iii. Tn10 is an example of a composite transposon (Figure 3). It is 9.3 kb, and contains: (1) 6.5 kb of central DNA with genes that include tetracycline resistance (a selectable marker). (2) 1.4 kb IS elements (IS10L and IS10R) at each end, in an inverted orientation. iv. Transposition of composite transposons results from the IS elements, which supply transposase and its recognition signals, the IRs. (1) Tn10’s transposition is rare, because transpose is produced at a rate of ,1 molecule/generation. (2) Transposons, like IS elements, produce target site duplications (e.g., a 9-bp duplication for Tn10).

Slide 19: 

Bacterial transposons ISL (left) ISR (right)

Composite bacterial transposons : 

Composite bacterial transposons repeated ends, usually inverted, sometimes direct repeated ends themselves are IS elements and can independently transpose ends mobilize all intervening DNA often antibiotic resistance genes (examples Tn3 (ampicillin), Tn5 (kanamycin), Tn10 (tetracycline) often reside on plasmids

Fig. 3 Structure of the composite transposon Tn10 : 

Fig. 3 Structure of the composite transposon Tn10

Structure of Tn3 : 

Structure of Tn3 4957 bp 3 trans-acting genes: 2 cis-acting sites:

Slide 23: 

b. Noncomposite transposons also carry genes (e.g., drug resistance) but do not terminate with IS elements. i. Transposition proteins are encoded in the central region. ii. The ends are repeated sequences (but not IS elements). iii. Noncomposite transposons cause target site duplications (like composite transposons). iv. An example is Tn3. (1) Tn3’s length is about 5 kb, with 38-bp inverted terminal repeats. (2) It has three genes in its central region: (a) bla encodes β-lactamase, which breaks down ampiciliin. (b) tnpA encodes transposase, needed for insertion into a new site. (c) tnpB encodes resolvase, involved in recombinational events needed for transposition (not found in all transposons). (3) Tn3 produces a 5-bp duplication upon insertion (Figure 5).

Fig..4 Structure of the noncomposite transposon Tn3 : 

Fig..4 Structure of the noncomposite transposon Tn3

Fig. 5 DNA sequence of a target site of Tn3 : 

Fig. 5 DNA sequence of a target site of Tn3

Slide 26: 

Physical structure of transposons The insertion of a Tn into a plasmid RTF: resistance-transfer functions

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Prokaryotic transposons Movement of transposons Each transposon can be transferred independently

Fig. 7 Organizational maps of bacterial plasmids with transposable elements : 

Fig. 7 Organizational maps of bacterial plasmids with transposable elements

2 types of DNA tranposons : 

2 types of DNA tranposons Excisive mechanismexamples: Tn5, Tn10, P elements Replicative mechanismexamples: Tn3, bacteriophage Mu

Replicative transposons : 

Replicative transposons copy-and-paste mechanism orignal cut of transposon is only nick and only one strand is initially ligated element replicates through itself produces as intermediate a “co-integrate” structure co-integrate is resolved by resolvase (as TnpR of Tn3) and at specific site (as res of Tn3)

Slide 31: 

Mechanism of transposition in prokaryotes Replicative transposition * A new copy of the transposable element is generated in the transposition event * The structure of transposon 3 * Transposition of Tn3 takes place through a cointegrate intermediate

Excisive transposons : 

Excisive transposons cut-and-paste mechanism cut themselves out of original site, producing double strand break cut target site and ligate to element ends, thereby inserting at new site original site break repaired usually with sister chromosome, restoring transposon at original site sometimes end healed without transposon, can also be associated with deletion at excision site

Slide 33: 

Mechanism of transposition in prokaryotes Conservative transposition Some transposons excise from the chromosome and integrate Into the target DNA Generation of heteroduplex and homoduplex Tn10 elements

Slide 35: 

Phage mu can mediate the transposition of a bacterial gene into a plasmid Phage mu can cause deletion or inversion of adjacent bacterial segment Phage mu

Slide 36: 

Thank you

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