TRANSPOSITION

1. INTRODUCTION. 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. They were "re-discovered" in bacteria in the 70's as "insertion sequences", genetic elements which move from one genetic location to another and that cause premature transcriptional termination in bacteria operons (i. e. they cause “polar” mutations).. Transposons can be classified into 3 types based on the mechanism which is used for transposition: 1. replicative or co-integrate-forming transposons 2. conservative (non-replicative) transposons 3. retro-transposons which transpose via an RNA intermediate. The first lecture will deal with replicative and conservative transposons. Next we'll cover retro-transposons. Transpositional recombination is site-specific in one sense that recombination occurs precisely at the ends of transposon but is in another sense is non-specific because the target recombination sequence is (somewhat) random.

2. Transposons in bacteria. Bacterial transposons are common and varied. Normal E. coli strains carry more than one copy of transposons IS1, IS2, IS3, and IS4. Many more are found on naturally occurring plasmids including the antibiotic resistance transposons Tn3 and Tn4 on R factor plasmids and Tn1000 on F (fertility factor). The smaller transposons such as the IS elements do not encode additional functions to transposition. Larger transposons often carry genes that confer selective advantage to the transposon-carrying cell: resistance to antibiotics is most common, resistance to Hg, toxin production and fermentation genes are also found. Larger transposons are often composite transposons. That is, they are made up of IS elements flanking a drug-resistance gene. The IS elements are capable of independent transposition without carrying drug-resistance. Bacteriophage Mu is essentially a 40kb transposon carrying capsid genes which replicates via transposition and is then packaged into virions. Transposons play an important role in the evolution of bacterial genomes by providing a mechanism for inheritance of genes via nonhomologous recombination. In this way transposible elements also facilitate horizontal gene transfer between unrelated organisms.

3. Transposons in eukaryotes. Approximately 15% of the human genome is believed to be composed of transposable elements. Most eukaryotic transposons are of the retrotransposon type but some DNA elements are also found. Examples include P elements from Drosophila which have been used extensively for gene transfer and have been well-studied; also the Ac/Ds elements originally discovered by McClintock in maize. Both elements are of the conservative transposition type and create a double-strand break in DNA during excisive tranposition. Both types of elements can be found in “degenerate” form. Degenerate transposible elements no longer encode transposase (the enzyme that catalyzes the breaking and joining of DNA during tranposition). They nonetheless may transpose when transposase is provided in trans since they retain the ends of the transposon, sites upon which the transposase can act. The “Ds” element in maize is a degenerate element that transposes when “Ac” provides transposase in trans. P elements in Drosophila melanogaster are a recent (ca. 1950) acquisition which are believed to have been acquired from an unrelated Drosophila species, D. willistoni, by a parasitic mite. Mating of male flies from natural populations (P element+) to females from lab stocks (P element-) produces a phenomenon known as “hybrid dysgenesis”: sterility, high mutation rate, high freq of chromosomal aberrations and nondisjunction. (The converse cross female P+ x male P- does not produce hybrid dysgenesis. Why?) These results can understood by the notion that the P+ strains carry transposable elements that are repressed for transposition. Zygotic induction of P elements occurs because the female oocyte, lacking P elements, cannot repress tranposition of the P elements introduced by the sperm.

4. Evolution of transposons. The fact that some transposible elements such as IS elements of bacteria carry no other genes other than those involved in transposition has raised the question of how they evolved:

a. No selective advantage. Selfish gene hypothesis. IS elements are like viruses, parasitic on host.

b. Cell tolerates IS because they facilitate quick genetic change. The evolution of an enormous variety of antibiotic resistance transposons and their spread among bacterial species since the introduction of these drugs 40 years ago certainly is an example of genetic adaptation via natural selection.

5. Genetic properties of transposons.

a. simple transposition

b. replicon fusion

c. adjacent deletion

d. adjacent inversion

6. Structure of transposons:

a. inverted repeat at ends

b. direct repeat generated in target 5-13 bp.

c. transposase gene tnp

7. 4 classes of bacterial transposons:
a. Class I. e.g.. IS10 (ends of compositeTn10), IS50 (ends of Tn5), IS4. CONSERVATIVE.

Transposase related in sequence.

b. Class II. e.g. IS1, IS2. MAINLY CONSERVATIVE. Transposase different from class I.

c. Class III. e.g.. Tn3 gd. REPLICATIVE. Obligate co-integrate intermediate. Transposase gene, resolvase gene (for site-specific resolution of co-integrate intermediate) and res (resolution site).

d. Class IV. e.g.. Tn554, Tn7. The odd ones. Tn554 and Tn7 transpose site-specifically into only one site on the bacterial chromosome.

8. Replicative mechanism of transposition.

a. 3 genes: tnpA, tnpR, res.

1. tnpA: Transposition-negative.

2. tnpR: Elevated levels of transposition (acts genetically like repressor). Co-integrate intermediate accumulate.

3. res. ICs-acting. Generally like tnpR phenotype.

b. Modified Shapiro model of transposition explains:

1. Direct repeat in target.

2. Obligate co-integrate intermediate.

3. Dependence on replication. Side reaction without replication (as seen at low freq. in tnpR mutants) give conservative type.

9. Conservative mechanism.

a. Suicide-donor model. Repair can occurs by homologous recombination with homolog or sister.

1. Explains why no tnpR-type gene needed.

2. Predicts low level of co-integrant in general but could be produced.

3. Kleckner experiment with heteroduplex transposon.

10. What keeps transposition frequencies down.

a. Excessive transposition would kill cell. Only high frequency transposon is Mu, which does kill cell.

b. Low levels of transposition are probably because transposase is poorly expressed:

1. Poor promoters. Dam methylation inhibits transcription initiation. Terminator for outside promoters. Anti-sense pOUT RNA inhibits translation of pIN mRNA. Translation inhibited by hairpin over ribosome binding site if transcription initiated from outside.

2. Transposase almost cis-acting : Transposase walks over or diffuses out, binding first to closest transposon ends but then binding to non-specific DNA?

3. Transposition immunity: e.g. Tn3. One and only one transposon per replicon allowed.

c. Transposition definitely higher in "virgin" cells.

d. Is transposition induced by environmental conditions? Stress: probably not. However, in some cases, transposition is much higher at low temperatures.

11. How geneticists use transposons

a. Random mutagenesis. Under certain circumstance, tranposons can be induced to transpose to the genomes of organisms. Geneticists can screen through populations of transposon-mutageneized organisms for their mutation of interest. There are several advantages to this approach

1) Insertion mutations are most likely null if they are within a coding sequence. Note however that insertion into regulatory elements can reduce or increase expression of a gene, not necessarily a null phenotype.

2) Insertion mutations can provide a molecular probe for isolation of the mutant gene. If the transposon carries sequences that are not present in the genome otherwise the junction DNA fragments between the element and the genome can be cloned by hybridization to specific transposon sequence probe or PCRed by primers unique to the transposon.

3) The insertion mutation can provide a convenient genetic marker for a gene with a difficult to assay phenotype. Strain constructions can be facilitated by monitoring the exogeneous marker (e. g. drug-resistance and prototrophy in microorganisms, color markers etc.)

4) The insertion mutation can include other genes to monitor expression of the locus. For instance, lacZ can be inserted within an element and used to monitor expression of the insertion site. DNA damage inducible genes of E. coli were isolated in such a manner in the late 1970s. Tissue specificity of expression can also be monitored in this way in higher organisms.

b. Insertion of engineered sequences at random positions back into the genome. This techique is used quite often in Drosophila by cloning the gene-of-interest into a P element.

c. Transposon excision (conservative transposition) can create double-strand breaks. Triggered by P-element excision, this has been used in Drosophila to promote homologous gene replacement.