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Molecular Genetics > Transposons I > Flashcards

Transposons I Flashcards

1
Q

WHAT IS A TRANSPOSON?
- what is it? Other names? Found in?

A

1 * A segment of DNA or a genetic element THAT MOVES TO NEW CHROMOSOMAL LOCATIONS = A NEW LOCATION IN THE GENOME, BY A PROCESS NAMED TRANSPOSITION.

2 * other names - controlling elements, cassettes, jumping genes, mobile genes, mobile genetic elements & transposons

3 * transposons are found in BACTERIA, FUNGI, PLANTS, ANIMALS AND VIRUSES.

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2
Q

WHAT IS TRANSPOSITION?

A

1 * A SPECIFIC FORM OF GENETIC RECOMBINATION that MOVES certain genetic elements – transposons – from one DNA site to another.

2 * transposition CAN CAUSE MUTATION by insertion into other genes or causing chromosomal rearrangements

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3
Q

Discovery of transposable elements: 7

A

1 ▪ Barbara McClintock - American scientist

2 ▪ 1940s, McClintock studied coloured kernels of maize and found that colouration is linked to two genetic loci called ! Ac and Ds !

3 ▪ Predicted that Ds IS A MOBILE GENETIC ELEMENT THAT CAN TRANSPOSE

4 ▪ In non-coloured kernels, Ds is located within a colour gene.

5 ▪ When Ds moves out of the colour gene, the gene becomes active and we observe the colour.

6 ▪ This happens at random and not in all cells ➔ colour mosaicism.

7 ▪ Nobel prize in Physiology or Medicine in 1983

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4
Q

Acceptance of McClintock’s transposon theory came with the discovery of BACTERIAL TRANSPOSONS:

Detection of Transposable elements in E. coli = 3

A

1 ▪ Comparing transducing phages λdgal+ (wild-type) and λdgal –

2 ▪ λdgal - contains gal gene from E. coli gal - mutants, which have insertions of a genetic element called IS in the gal gene

3 ▪ λdgal - DNA carrying IS mutation is longer than wild- type λdgal+ DNA

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5
Q

Experimental demonstration of transposon presence in a bacterial genome:

A

*Electron micrograph of a λdgal+ / λdgal- DNA heteroduplex provides proof that IS mutation is due to insertion of a piece of DNA

1 * mix denatured DNA of wild- type and mutant phage

2 * hybrid DNA molecules renature

3 * single-stranded loop corresponds to inserted IS element in mutant

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6
Q

Classification of transposons
By Kingdom, Structural, Mechanistic, Outcome for the genome:

A

1 * By kingdom
– Prokaryotic, Eukaryotic

2 * Structural
– Simple = non-composite transposons, composite transposons

3 * Mechanistic
– Cut and paste, copy and paste, reverse transcription
– DNA transposon, class II transposons ➔ all bacterial transposons, also found in eukaryotes
– Retrotransposon, class I transposons: viral, non-viral➔eukaryotes

4 * Outcome for the genome
– Movement: without duplication = conservative, with duplication = replicative

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7
Q

The simplest transposon:
Insertion Elements or Insertion Sequences (IS)

(size)?

A

▪ simplest transposons are called Insertion Sequences or IS elements

▪ ~ 1kb

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8
Q

IS elements belong to the simple transposons =

A

“conservative transposons“

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9
Q

IS Element Structure (4):

A

1 ▪ Each IS element has a different sequence but all have the same overall structure.

2 ▪ Ends composed of SHORT INVERTED REPEATS - usually 15-25 bp long

3 ▪ Between ends is TRANSPOSON GENE ➔ IS ELEMENTS ENCODE FOR ONE PROTEIN ➔ NEEDED FOR TRANSPOSITION.

4 ▪ IS elements are surrounded by direct repeats which are NOT part of the IS element

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10
Q

IS ELEMENT

A

Direct Repeats….. Inverted Repeats… Transposase gene….Inverted Repeats….Direct repeats

IS ELEMENT = inverted repeats,m Transposase gene

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11
Q

Many DNA transposable elements have common characteristics: 2

A

1 * Most DNA transposons are FLANKED BY DIRECT SEQUENCE REPEATS.
- The direct repeats are NOT PART OF THE TRANSPOSON.
- These direct repeats have something to do with the mechanism of transposition.

2 * Many DNA transposable elements also possess TERMINAL INVERTED REPEATS.
- These inverted repeats are PART OF THE TRANSPOSON.

  1. Flanking direct repeat, Terminal inverted repeat, ………., Terminal inverted repeat, Flanking direct repeat.

Transposable element: (Terminal inverted repeat……terminal inverted repeat)

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12
Q

Understanding Simple or conservative transposons: 5

A

1 * A SIMPLE TRANSPOSON = “conservative transposon” , or non-composite transposon

2 * Minimum: TRANSPOSASE ENCODING GENE BETWEEN SHORT, INVERTED, REPEATED SEQUENCES 50 bp.
– Enzyme for transposition

3 * CAN ALSO ENCODE OTHER GENES, e.g. resolvase - responsible for resolution of the transfer (see later) or/and antibiotic resistance genes

4 * IS element can be considered the simplest simple transposon

  1. Example Tn3

STRUCTURE:
…IR..TRANSPOSASE…RESOLVASE… AM^R (Ampicillin resistance)..IR…
= Transposon Tn3.

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13
Q

Understanding Composite transposon: SIMILARITY TO SIMPLE TRANSPOSON AND IS ELEMENTS:

A

They ALLHAVE PROTEIN CODING GENES FLANKED BY INVERTED REPEAT SEQUENCES that can be RECOGNISED BY TRANSPOSASE ENZYMES.

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14
Q

Understanding Composite transposon:

Difference to simple transposons and IS elements: 3

A
  1. Composite transposons are FLANKED BY TWO SEPARATE IS ELEMENTS which may or may not be exact replicas.

– Instead of each IS element moving separately, the entire length of DNA spanning from one IS element to the other is transposed as one complete unit using the ‘external’ IR sequences.

2 * One of the IS elements provides the transposase gene / function

3 * Composite transposons will also often carry one or more genes conferring antibiotic resistance.

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15
Q

How does transposition work - Overview

A

1 * Movement of a transposon occurs through RECOMBINATION between DNA sequences at the very ends of transposons with a DNA sequence at a NEW GENOMIC LOCATION.

2 * WITH OR WITHOUT DUPLICATION of the transposon

Genomic DNA (old site) –> movement without duplication –> excised from old site, and inserted in new site.

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16
Q

Conservative versus replicative transposition

A
  • Conservative – excision and insertion, e.g. cut and paste transposition
  • Replicative - new copy generated

look at image slide (14)

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17
Q

Cut and paste or conservative transposition - overview

A

1 * They transpose by EXCISION (cutting) of the transposable sequence from one position in the genome and its INSERTION (pasting) to another position within the genome

2 * The cut-and-paste transposition involves TWO TRANSPOSASE SUBUNITS. Each transposase submit BINDS to the SPECIFIC SEQUENCES AT THE TWO ENDS of transposon. These subunits of transposase protein then COME TOGETHER and lead to the EXCISION of transposon.

3 * This excised ‘Transposon-Transposase Complex’ then gets INTEGRATED to the target recipient site. In this manner, the transposon is cut from one site and then pasted on other site by a mechanism mediated by transposase protein

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18
Q

Cut-and-Paste Transposons: Examples in prokaryotes are

A

IS-elements, Tn10

  • also in eukaryotes
19
Q

Cut-and-Paste Transposons

  • Consequence for the genome:
A

transposon number does NOT INCREASE.

A direct repeat duplication remains at the donor site.

20
Q

Cut and paste transposition mechanism: 3

A
  • Also called NON-REPLICATIVE transposition

➔transposes without making a new copy

➔transposon number does not change in the genome

21
Q

Cut and paste transposition mechanism STEPS =7

A

1) The transposase gene is transcribed. The resulting mRNA is translated to synthesise the transposase protein

2) Multimers of the transposase bind to the inverted repeats of the donor DNA

3) Transposase proteins interact forming a SYNAPTIC OR PAIRED END’ COMPLEX with the Donor DNA.

4) Transposases make DOUBLE-STRANDED CUT in the DONOR DNA at the ends of the transposon

4a) Transposon is EXCISED from donor DNA

5) Transposase makes a STAGGERED CUT in
the RECIPIENT / target DNA.

6) Each 3’-END of the DONOR DNA is then COVALENTLY JOINED to an overhanging 5’-END of the RECIPIENT DNA.

6a) This leaves a GAP between the transposon 5’-end and the recipient DNA’s 3’-end

7) DNA POLYMERASE FILLS IN in the short, overhanging sequences
➔ results in a SHORT, DIRECT REPEATS on the sides of the transposon insertion in the recipient DNA

22
Q

Cut and paste transposition mechanism.. SIMPLIFIED

A
  1. mRNA transposase IR Transposase gene IR
  2. binding to multimer of transposase
  3. synaptic complex
  4. DNA cleavage of both strands

4a. excised transposon

  1. Target DNA
    5’ to 3’ 3’OH
  2. DNA strand stransfer
    6a.
  3. DNA-repair synthesis to fill gaps.
    Ligation of nicks
    Element in new DNA location
23
Q

UNDERSTANDING:Cut and paste transposons are surrounded by short direct repeats = 5

A

1 ▪ At the site of insertion, IS element and many other transposons are flanked by very short direct repeats➔not part of transposon

2 ▪ During cut and past transposition: Insertion requires NICKING of the two recipient DNA strands at the insertion site

3 ▪ The nicks are SEPARATED by a FEW BASE PAIRS

4 ▪ This generates TWO SHORT COMPLEMENTARY SINGLE STRANDED DNAs stranded DNAs from the insertion site surrounding the transposon

5 ▪ Single stranded DNAs will be FILLED IN ➔ now a direct repeat flanking the transposon

SLIDE 19

24
Q

What are Copy and paste or Replicative Transposons?

A
  • Replicative transposons transpose by a mechanism which involves replication of transposable sequence, i.e. they make a TRANSPOSON COPY.
25
Q

Copy and paste or Replicative Transposons PROCEDURE:

A

1 * The copy of the transposon is INSERTED into the recipient / target site

2 * The original transposon REMAINS located in the donor site

3 * The consequence for the genome is a DUPLICATION of the transposon ➔ the number of transposons increases in the genome

26
Q

Copy and paste or Replicative Transposons:

Example in prokaryotes:

A

Tn3

27
Q

Copy and paste or Replicative Transposons:

Consequence for the genome

A
  1. Both, the donor and the recipient DNA molecule are
    having a transposable sequence each, after transposition.
  2. Both transposons can transposase again ➔ this could lead to a large INCREASE IN TRANSPOSON NUMBERS IN THE GENOME
28
Q

Copy and paste or Replicative transposition of Tn3:
Proposed by who?

4 steps? RESULTS?

A

Mechanism proposed by James A. Shapiro in 1979:

1) Transposase makes FOUR SINGLE-STRAND CLEAVAGES
– one on each strand of the DONOR DNA
– one on each strand of the TARGET DNA

2) LIGATION of the donor and target DNA leads to formation of a COINTEGRATE also called a Shapiro intermediate or a THETA FIGURE after the Greek letter:

2a) The cointegrate has two regions made up of SINGLE STRANDED DNA. These regions represent the two strands of the ORIGINAL TRANSPOSON.

3) Using the single stranded DNA as template, DNA REPLICATION synthesises the second strand.
* ➔ transposon is replicated = two copies (original and new)

4) The donor and target DNAs are separated by an enzyme called RESOLVASE

  • Resolvase recognises and binds to IRS or RES elements in the transposon and executesSITE SPECIFIC RECOMBINATION between the old and the new transposon copy
  • This results in TWO DNAs, ONE WITH THE OL AND ONE WITH THE NEW TRANSPOSON insertion site.
29
Q

Transposition can increase genome size: EXPLAIN (3)

A
  • Copy and paste or Replicative transposition creates a new transposon in addition to the original transposon
  • This increase the amount of DNA in a genome
  • If this occurs many times, the increase in DNA can be significant.
30
Q

Understanding The C-value Paradox:

SOLUTION?

A
  • The term C-values WERE CONSIDERED AS A MEASURE FOR GENOME SIZE.
    It was thought that C-values (genome size) reflect the complexity of genomes, i.e. how many genes the genomes contain.

– If this is correct, we would expect that more complex organisms have bigger genomes.

– This is not necessarily true, for example some salamander genomes may contain 40 x more
DNA than human genomes➔C value paradox

SOLUTION?
- the PRESENCE OF NON-CODING DNA in genomes: Most of eukaryotic DNA is non-coding and hence does not reflect genes. The human genome has less than 2% protein encoding regions

– A large proportion of this non-coding DNA can be made up of transposons

31
Q

UNDERSTANDING BEFORE/AFTER Transposon insertions can cause mutations:

A

Before transposition:
The target gene is active
promoter -> gene coding region – transposon inserted

After transposition:
1) The target gene may be inactive
promoter -> no transcription or transposon

2) The target gene may have altered transcription levels
- stronger transcription
- weak or no transcription
or transposon -> gene coding region

3) The transposition may have no effect
- promoter; 1. transcription or gene coding region -> transposon

32
Q

Transposition of an IS element can create a new composite transposon

A

IS element1…Gene A…Gene B……(transposition) IS element 2 inserteted

—> then : CREATES A NEW COMPOSITE TRANSPOSON

IS element 1…..Gene A… Gene B…IS element 2….

PAGE 26

33
Q

Bacterial transposons can be localised on plasmids:

Distribution of IS and Tn elements in E. coli …SLIDE 27

A

IS1 : 5 – 8 copies on chromosome

IS2 : 5 copies on chromosome, 1 copy on F PLASMID

IS3 : 5 copies on chromosome, 2 copies on F PLASMID

IS elements can jump from one location to another, eg chromosome to an F plasmid

Distribution of IS elements and Tns in one F plasmid

Because IS elements are regions of identical sequence, they are sites where RECOMBINATION occurs.

34
Q

Bacterial plasmids: self-replicating circular genetic elements that are separate from the bacterial chromosome

A

**Transposons play a major role in generating R plasmids

RESPONSIBLE FOR THE MULTI-DRUG RESISTANT BACTERIA – SERIOUS PROBLEM IN HOSPITALS

35
Q

Bacterial plasmids: self-replicating circular genetic elements that are separate from the bacterial chromosome

  • e.g. F plasmid, R plasmids (resistance or R factors)
A
  • e.g. F plasmid, R plasmids (resistance or R factors)

1 * resistance-transfer section mediates conjugation, contains Tn10

2 * resistance-determinant section has cluster of resistance genes on 3 Tns, can transpose as a whole or each Tn can transpose independently

3 * Tn3 (simple) is within Tn4 (composite)

36
Q

Examples of genetic determinants found on transposons - Tn3

A

ampicillin resistance

37
Q

Examples of genetic determinants found on transposons - Tn5

A

kanamycin resistance

38
Q

Examples of genetic determinants found on transposons - Tn9

A

chloramphenicol resistance

39
Q

Examples of genetic determinants found on transposons - Tn10

A

tetracycline resistance

40
Q

Examples of genetic determinants found on transposons - Tn501

A

mercury resistance

41
Q

Examples of genetic determinants found on transposons - Tn951

A

lactose utilization

42
Q

Examples of genetic determinants found on transposons - Tn1681

A

enterotoxin production

43
Q

Examples of genetic determinants found on transposons - Tn4451

A

chloramphenicol resistance
- C.perfringens

44
Q

Summary

A

1 * Bacterial IS elements - first transposable elements identified at the molecular level. They encode only transposase.

2 * IS element are very simple DNA transposons belonging to the simple or conservative transposons so do some Tn transposons (Tn3)

3 * Composite transposons are flanked by two IS elements, one of the IS elements encodes for the transposase. Example: Tn10

4 * Tn transposons are flanked by terminal repeat sequences and confer extra properties on their host cell such as antibiotic resistance

5 * Two modes of transposition – conservative = cut and paste and replicative = copy and paste

6* Tns insert into plasmids such as R plasmids and can be transferred by conjugation to recipient bacteria➔multiply-antibiotic-resistant pathogens

7 * Transposition can increase genome size or alter gene transcription or create new composite transposons.

Molecular Genetics (21 decks)

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