TransposableGenetic Elements

MBIOS 520/420

September 22, 2005

MBios 420/520 Announcements

• PowerPoint lectures are now available for download at the web site http://mbios420.tripod.com/mbios420.htm

• No longer holding office hours; please e-mail me at marter@wsu.edu for an appointment

• Thoughts on a review session?

• Excellent source for questions and review:

Go to http://www.ncbi.nlm.govSearch in BOOKS.Enter keywords.Hit GO.

Transposon Introduction

• Transposable elements are stretches of DNA that can move to new locations in a genome

• These elements can contain genes or be non-coding

• Large portions of higher eukaryotes’ genomes are composed of either inert or active transposons (often as repetitive DNA)

• Transposons are thus important evolutionarily

• Transposons can also be used to isolate genes or introduce foreign genes into cells

Bacterial Transposon Discovery

• First transposons characterized, these are the simplest

• Detected because experimental lac- strains kept reverting back to wild type (ie, colonies kept turning blue)

• lac- mutants were due to transposons which then moved back out of the gene

Agar w/X-Gal

lac gene

Agar w/X-Gal

lac gene

lac gene

transposonTransposition

event

Bacterial Transposons

• Can occur in bacterial genome or in plasmid, and can move between these two

• Consist of two major types:

Insertion Sequences (IS) small, <2500 bp

Composite Transposons large, flanked by two IS elements

• Consists of a pair of inverted terminal repeats at each end(cannot be mutated without loss of transposition activity)

• Between this is a stretch of DNA, often containing the gene for transposase – the enzyme that catalyzes transposition

• Flanking the terminal repeats are a pair of direct repeats that result from the transposition process

Insertion Sequences

Insertion Sequence Transposition

Transposase moves the element by creating a

staggered cut at either end in a

random spot of the genome

The IS element then moves then inserts into

this region

DNA polymerase fills in the resulting single-

stranded areas

The result is termed a target site duplication

Composite Transposons

• Denoted Tn

• Created when two IS elements insert near each other

• The elements can be either in inverse or direct orientation to each other

• These two then move together and transpose the sequence between them (often carrying genes)

• Movement of these large elements is how bacteria become antibiotic resistance (often using viral intermediates)

Composition Transposon Transposition

• Involves both IS elements

• Two types:

Replicative Transposon transposon is copied & moved(ie, a copy remains in place)

Non-Replicative Tranposon the whole element is moved(aka “cut & paste”) (no copy remains)

• Similar to conserved & semi-conservative DNA replication

Tn3 Transposition• A combination of replication & recombination

Tn10 TranspositionHow can we determine if a transposon uses replicative or “cut and paste” transposition?

Gene Tagging with Transposons

• We can use transposons to tag and isolate genes

• Ex: let’s say we want to isolate a blue flower color gene

STEP 1

A. Transform plant with a vector containing a

transposon.

B. Grow progeny from that plant and pick out the

mutant phenotype (in this case, an uncolored flower).(Thousands of plants needed,

depending on genome size)

C. This plant should have the transposon inserted

somewhere in the gene.

transposon

transposon

Blue color gene

M2

Gene Tagging with Transposons

STEP 2

A. Isolate genomic DNA from the mutant plant.

B. Make genomic DNA library from this sample (ex: using

BAC vectors).

C. Pool clones of the library into 96-well plate.

THE MATH Ex: RiceGenome size = 400 million bases

BAC insert size = 200,000 bases

# clones needed for 1X = 2,000# clones needed for 6X = 12,000# of clones per well = 125

BAC clones

(many thousands of these)

Gene Tagging with Transposons

STEP 3

A. Find the well containing the BAC clone with the transposon (use PCR).

B. Grow the cells from this well on a plate.

C. Hybridize with transposon probe to locate exact colony

that has the clone.

D. Sequence this clone.You’ve found the gene!

P32-labeled transposon

probe

1 2 3 4 5

1 2 3 4 5

PCR w/ transposon primers

Gene Tagging with Transposons & Inverse PCR• There is a faster way of identifying the gene without having to build an entire library, by using a technique called inverse PCR

A. Follow STEP 1 like before. Isolate mutant & extract its

DNA.

B. Cut DNA with restriction enzymes, then re-ligate to

form circular segments.

C. Use the transposon-based primers & do PCR. This

amplifies the flanking gene regions. Sequence it.

STEP 2b

EcoRI

DNA ligase(high volume

reaction)

SEQUENCE

Gene Tagging with Transposons - Troubleshooting• What if we get many transposons inserting into our mutant genome? How do we tell which one is in our color gene?

OPTION 1

Do inverse PCR as in STEP 2B.

BLAST sequence & search for similarity with other known

pigment genes.

SEQUENCE

(GENBANK)

Gene Tagging with Transposons - Troubleshooting

OPTION 2

Cross mutant plant back to wild-type plant.

Produce an F2 (or more) generation.

Do Southern blot with tranposon probe. Find

marker that segregates with mutation. Many plants

needed.

Make a library out of a plant with only this marker (Plant

D in our example).

XP

F1

F2

6.5 kb

4.3 kb

9.4 kb

2.3 kb2.0 kb

0.5 kb

23.0 kb

λ HindIIIMarker

A B C D E F

Gene Tagging with Transposons - Troubleshooting

OPTION 3

Pick out all BAC clones with transposons via PCR (STEP

3).

Do RFLP ofF2 mutant plant using transposon as a probe.

Cut BACs with same restriction enzyme as in your RFLP. Hybe with tranposon

probe.

Transposon probe should bind at same MW in mutant

and BAC digest.

A B C D

6.5 kb

4.3 kb

9.4 kb

2.3 kb2.0 kb

0.5 kb

23.0 kb

λ HindIIIMarker

F2 MutantPlant A B C D

• Similar in structure to bacterial transposons

• Most are thought to be derived from viral genomes that have integrated into a host cell genome

• Some eukaryotic transposons move via an RNA intermediate

• Some transpositions are utilized for programmed genome rearrangements

• Movement of transposons in genomes can inactive or activate genes, and can cause cancer

• The movement and buildup of transposable sequences has had an effect on the evolution of eukaryotice genomes

Eukaryotic Transposons

• Two best characterized transposons are Ac (Activator) and Ds (Dissociation) elements (both are “cut & paste” types)

• Can occur in many copies of the cell, but must work together

Maize Transposons

• Ac is ~4.6 kb, with same basic structure as insertion elements

• Ds is similar (identical inverted terminal repeats) but has deletions of various sizes in it

• Because of the deletions, Ds does not have transposase & cannot transpose itself (Ac needed)

Transposase Gene

Transposase Missing

• Barbara McClintock, maize geneticist

• Observed mosaic corn kernels, despite presence of CI, a dominant colorless allele

• Concluded that chromosome was breaking, causes CI loss

• Only occurred when another segregating factor, Ac, was present

Discovery of Transposons

CI Present

CI Absent

• When we do transposon mutagenesis, how can we control when transposition stops & starts?

• Ac can be introduced via a vector

• But how do we know that Ac won’t transpose into our gene instead of Ds?

Using Ac & Ds Elements for Mutagenesis

Mutate the inverted terminal repeats of

Ac, then it can’t transpose!

XP

F1

F2

Ac+/+ Ds-/- Ac-/- Ds+/+

Ac+/- Ds+/-

XAc+/? Ds+ Ac-/-

BC1F1

BC1F2

Ac-/- Ds+

StableMutant

• Known as P elements, similar in structure to Ac & Ds

• P elements can be incomplete (no transposase) or complete (functional transposase) – analogous to Ac/Ds

Drosophila Transposons

• P elements are only active in germ line cells, because a stop codon exists in transposase

• In germ cells, alternative splicing removes exon 2 to remove the codon

• Demonstrated by engineering a P element without intron 3

• These transpose via an RNA intermediate

• Transposon is transcribed into RNA, then reverse transcriptase creates “cDNA” that inserts into genome

• Two categories exist, based on their origin & structure:

Retro-Transpososons

Retroviruslike Elements Possess Long Terminal RepeatsHave viral genes gag & polDerived from retroviruses

Retroposons Non-LTR retrotransposonsHave poly A:T tractDerived from reverse transcribed mRNA

• Ty1 of yeast is best studied

• Long terminal repeats called δ regions (not inverted) flank a coding region

• Coding region has TyA & TyB; these genes are gag & pol derived

• ~35 copies per yeast cell; sometimes solo δ regions are found (formed by recombination between δ)

• Form target site duplications

Retroviruslike Elements

• RNA is synthesized by normal RNA pol II transcription

• δ elements act as strong promoters (can activate genes)

• TyB gene has reverse transcripase activity and produces dsDNA from the RNA

• DNA integrates into the genome (δ element is replicated)

• copia and gypsy are Drosophila retrotransposons very similar to Ty1 (gypsy even has viral env gene)

Ty1 Transposition

• How can we prove Ty1 transposes as an RNA molecule?

• Constructed Ty1 element with a galactose-inducible promoter and an intron

• Used galactose to stimulate transcription, then found that all the new copies transposed had the intron spliced out

Proof of Ty1 Transposition

• F, G and I elements in Drosophila; LINEs in humans

• Also called non-LTR retrotransposons because they lack inverted or direct repeats at their ends (do have target site repeats)

• Retroposons all have a poly-A region at the end, evidence that these are reverse transcribed mRNAs that re-inserted in the genome

• These function by reverse transcription, followed by insertion

• LINE-1 or L1 = only known active human transposon (make up 5% of human genome) & can cause mutations (ex: hemophilia)

Retroposons

• Can create duplications, deletions, inversions, translocations

• Means of creating pseudogenes – no selection pressure (can gain novel function)

Transposition & Chromosome Rearrangement

Deletions & Duplication Created byTransposition:

Transposition & Chromosome RearrangementDeletions & Inversions Created byTransposition:

• Antibody genes have powerful enhancers and use recombination to produce diverse sets of antibodies

• c-myc can recombine with this region & cause cancer

Transposition & Cancer

• Technique use transposons to identify tissue-specific enhancers

• Add lacZ gene between the inverted terminal repeats of a P element

• Transform Drosophila with P element

• Dissect Drosophila & grind tissue in X-Gal

• Blue color change shows that P element inserted near a tissue specific enhancer

Enhancer Trapping

Or do inverse PCR

• All active transposons have the potential to cause mutations

• These can be deleterious or potentially beneficial

• Duplication of genes via chromosome rearrangements can produce pseudogenes which can eventually gain new function

• Which came first Retroviruses or Retrotransposons?

Transposons & Genome Evolution