Lect.10 Transposition + Transposon mutagenesis

Transposons

• “jumping genes”

• DNA elements that can hop or transpose from one place in the DNA to another

Transposition

Movement of transposons (hopping) (requires transposases)

Transposases

• Enzymes that promote transposition and are encoded within the transposon

• Cuts the donor DNA at ends of transposon and inserts the transposon into target DNA

Transposition offers a way of introducing __

Genes from one bacterium into the chromosome of another to which it has little DNA sequence homology

Homologous recombination

• account for the majority of recombination in a cell

• Results from the breaking and rejoining of 2 DNA molecules that have similar or identical sequence

• Benefit of using “homology arms” in cloning

Non-homologous recombination

Does NOT require 2 DNAs to have the same/similar sequence and depends on the enzymes that recognize specific regions in the DNA that may/may not have similar sequences

Result of transposition

Transposon appears in a different site in the genome from where it was originally

• DNA is cut out of one DNA strand —> may/may not be copied —> inserted into another location in the genome

Donor DNA

The DNA strand that the transposon originated from

Recipient DNA

The DNA strand that the transposon hops into

Why does transposition need to be tightly regulated and occur rarely?

• don’t want it to jump into important genes and disable them

• Worry about frameshift mutations

Smaller transposons (~1000bp) encode only the genes for the ___

Transposase that will promote movement

Larger transposons often encode additional genes for ___

• regulation of movement

• Factors beneficial for the host (like antibiotic resistance)

2 common features of transposons:

1. Inverted repeats

2. Direct repeats

Inverted repeats

• found at the ends of bacterial transposons

• Recognized by transposases that bind to form synapse for excision

Direct repeats

• formed in the target DNA that bracket the transposon (AFTER integration of the transposon into the new area of DNA)

• Not a part of the transposon; it sits right outside it

Insertion sequence elements (IS elements)

• smallest bacterial transposons

• Carry no selectable genes

• Encode transposase

IS elements generally inactivate genes they hop into, resembling __ BUT they can _

• deletion mutations

• Revert

When an IS element inactivates a gene, why is it possible for it to be reverted

The IS element can simply “hop out” of that gene and into a new area of DNA

IS elements can cause __ effects

Polar

Composite transposons

2 copies of the same IS elements can combine to form a large transposon that will transfer everything in between

• often contain selectable genes

IS element vs Composite transposon

• IS element (only has the transposase gene and inverted repeats)

• Composite transposon: made up of 2 IS elements and will carry that genes that are in between the IS elements

Reverse genetics

Creating a targeted mutation and then studying the resulting phenotype (gene locus isolated —> introduce mutation —> test for phenotype/function)

Forward genetics

Discover the gene responsible for phenotypes (phenotype/function —> locus) (take phenotype, see what genotype caused it)

4 qualities of effective mutagenic transposons

1. Transpose at high frequency

2. Does not have a very selective target sequence

3. Carry an easily selectable marker (ex: AbxR)

4. Has a broad host range for transposition

(Transposon mutagenesis): Once selected, these transposons can be cloned into a vector that ___ inside the recipient. Then the transposon can move from the plasmid into the genome and __ a gene(s).

• can NOT replicate (aka suicide system)

• Interrupt