CHAPTER 20 Transposable Elements

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台大農藝系遺傳學 601 20000 Chapter 20 slide 1

CHAPTER 20

Transposable Elements

Peter J. Russell

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General Features of Transposable Elements

1. Transposable elements are divided into two classes on the basis of their

mechanism for movement:

a. Some encode proteins that move the DNA directly to a new position or replicate the DNA to produce a new element that integrates elsewhere. This type is found in both prokaryotes and eukaryotes.

b. Others are related to retroviruses, and encode reverse transcriptase for making DNA copies of their RNA transcripts, which then integrate at new sites. This type is

found only in eukaryotes.

2. Transposition is nonhomologous recombination, with insertion into DNA that

has no sequence homology with the transposon.

a. In prokaryotes, transposition can be into the cell’s chromosome, a plasmid or a phage chromosome.

b. In eukaryotes, insertion can be into the same or a different chromosome.

3. Transposable elements can cause genetic changes, and have been involved in

the evolution of both prokaryotic and eukaryotic genomes. Transposons may:

a. Insert into genes.

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Transposable Elements in Prokaryotes

1. Prokaryotic examples include:

a. Insertion sequence (IS) elements.

b. Transposons (Tn).

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Insertion Sequences

Animation: Insertion Sequences in Prokaryotes

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 (Figure 20.1)

3. 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.

b.

IS2 has 0–12 copies on the chromosome, and 1 copy on the F plasmid.

c.

IS10 is found in R plasmids.

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5. 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.

6. 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 20.2).

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).

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Transposons

1. 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 20.3). 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.

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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).

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2. Models have been generated for transposition:

a. Cointegration is an example of the replicative transposition that

occurs with Tn3 and its relatives (Figure 20.6).

i. Donor DNA containing the Tn fuses with recipient DNA.

ii. The Tn is duplicated, with one copy at each donor-recipient DNA

junction, producing a cointegrate.

iii. The cointegrate is resolved into two products, each with one copy

of the Tn.

b. Conservative (nonreplicative) transposition is used by Tn10, for

example. The Tn is lost from its original position when it transposes.

3. Transposons cause the same sorts of mutations caused by IS elements:

a. Insertion into a gene disrupts it.

b. Gene expression is changed by adjacent Tn promoters.

c. Deletions and insertions occur.

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Fig. 20.6 Cointegration model for transposition of a transposable element by

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IS Elements and Transposons in Plasmids

1. Bacterial plasmids are extrachromosomal DNA capable of self-replication.

Some are episomes, able to integrate into the bacterial chromosome. The

E. coli F

plasmid is an example (Figure 20.7):

a. Important genetic elements of the F plasmid are:

i. tra genes for conjugal transfer of DNA from donor to recipient. ii. Genes for plasmid replication.

iii. 4 IS elements: 2 copies of IS3, 1 of IS2, and 1 of γδ (gammadelta). All have homology with IS elements itt the E. coli chromosome.

b. The F factor integrates by homologous recombination between IS elements, mediated by the tra genes.

2. R

plasmids have medical significance, because they carry genes for resistance to

antibiotics, and transfer them between bacteria (Figure 20.7).

a. Genetic features of R plasmids include:

i. The resistance transfer factor region (RTF), needed for conjugal transfer. It includes a DNA region homologous to an F plasmid region, and genes for plasmid-specific DNA replication.

ii. Differing sets of genes, such as those for resistance to antibiotics or heavy metals. The resistance genes are transposons, flanked by IS module-like sequences, and can replicate and insert into the bacterial chromosome.

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Bacteriophage Mu

1. Temperate bacteriophage Mu (mutator) can cause mutations when it transposes.

Its structure includes:

a. A 37 kb linear DNA in the phage particle that has central phage DNA and unequal lengths of host DNA at the ends (Figure 20.8).

b. The DNA’s G segment can invert, and is found in both orientations in viral DNA.

2. Following infection, Mu integrates into the host chromosome by conservative

(non-replicative) transposition.

a. Integration produces prophage DNA flanked by 5 bp target site direct repeats.

b. Flanking DNA from the previous host is lost during integration.

c. The Mu prophage now replicates only when the E. coli chromosome replicates, due to a phage-encocled repressor that prevents most Mu gene expression.

3. Mu prophage stays integrated during the lytic cycle, and replication of Mu’s

genome is by replicative transposition.

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Fig. 20.8 Temperate bacteriophage Mu genome shown in (a) as in phage particles and

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Fig. 20.9 Production of deletion or inversion by homologous recombination between

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Transposable Elements in Eukaryotes

1. Rhoades (1930s) working with sweet corn, observed interactions between two

genes:

a. A gene for purple seed color, the Al locus. Homozygous mutants (a/a) have colorless seeds.

b. A gene on a different chromosome, Dt (dotted) that causes seeds with genotype a/a

Dt/-- to have purple dots.

i. Dt appears to mutate the a allele back to the Al wild-type in regions of the seed, producing a dotted phenotype.

ii. The effect of the Dt allele is dose dependent.

(1) One dose gave an average of 7.2 dots per seed. (2) Two doses gave an average of 22.2 dots/seed. (3) Three doses gave an average of 121.9 dots/seed. c. Rhoades interpreted Dt as a mutator gene.

2. McClintock (1940s-50s), working with corn

(Zea mays)

proposed the existence

of “controlling elements” that regulate other genes and are mobile in the

genome.

3. The genes studied by both Rhoades and McClintock have turned out to be

transposable elements, and many others have been identified in various

eukaryotes.

a. Most studied are transposons of yeast, Drosophila, corn and humans.

b. Their structure is very similar to that of prokaryotic transposable elements.

c. Eukaryotic transposable elements have genes for transposition and integration at a number of sites, as well as a variety of other genes.

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Transposons in Plants

Animation: Transposable Elements in Plants

1. Plant transposons also have IR sequences, and generate short direct target site repeats. 2. The result of transposon insertion into a plant chromosome will depend on the properties

of the transposon, with possible effects including:

a. Activation or repression of adjacent genes by disrupting a cellular promoter, or by action of transposon promoters.

b. Chromosome mutations such as duplications, deletions, inversions, translocations or breakage. c. Disruption of genes to produce a null mutation (gene is nonfunctional).

3. Several families of transposons have been identified in corn, each with characteristic numbers, types and locations.

a. Each family has two forms of transposon. Either can insert into a gene and produce a mutant allele.

i. Autonomous elements, which can transpose by themselves. Alleles produced by an autonomous element are mutable alleles, creating mutations that revert when the transposon is excised from the gene.

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4. Multiple genes control corn color, and classical genetics indicates that a

mutation in any of these genes leads to a colorless kernel. McClintock

studied the unstable mutation that produces spots of purple pigment on

white kernels (Figure 20.10).

a. She concluded that spots do not result from a conventional mutation,

but from a controlling element (now Tn).

b. A corn plant with genotype

c/c

will have white kernels, while

C/--

will

result in purple ones.

i. If a reversion of

c

to

C

occurs in a cell, that cell will produce purple

pigment, and hence a spot.

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iii. McClintock concluded that the

c

allele resulted from

insertion of a “mobile controlling element” into the

C

allele.

(1) The element is

Ds

(dissociation), now known to be a

nonautonomous transposon.

(2) Its transposition is controlled by

Ac

(activator), an

autonomous transposon (Figure 20.11).

c. McClintock’s evidence of transposable elements did not fit the

prevailing model of a static genome. More recent studies have

confirmed and characterized the elements involved.

i. The

Ac-Ds

system involves an autonomous element

(Ac)

whose insertions are unstable, and a nonautonomous element

(Ds)

whose insertions are stable if only

Ds

is present.

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iii.

Ac

is 4,563 bp, with 1 1-bp imperfect terminal IRs and 1 transcription

unit producing a 3.5 kb mRNA encoding an 807 amino acid transposase.

Insertion generates an 8-bp target site duplication (Figure 20.12).

iv.

Ac

activates

Ds

to transpose or break the chromosome where it is

inserted.

v.

Ds

elements vary in length and sequence, but all have the same terminal

IRs as

Ac,

and many are deleted or rearranged versions of

Ac.

vi. Unique to corn transposons, timing and frequency of transposition and

gene rearrangements are developmentally regulated.

vii.

Ac

transposes only during chromosome replication, and does not leave a

copy behind. There are two possible results of

Ac

transposition,

depending on whether the target DNA has replicated or not (Figure

20.13).

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(1) If

Ac

transposes during replication into a replicated target site, its

chromatid’s donor site will be empty since that copy of

Ac

has

inserted elsewhere. In the homologous donor site on the other

chromatid, a copy will remain. There is no net increase in copies of

Ac.

(2) Transposition to an unreplicated chromosome site also leaves one

donor site empty (and the other with a copy of

Ac).

The DNA into

which

Ac

inserts will then be replicated, resulting in a net gain of one

copy of

Ac.

viii. Replication of Ds is the same, except that the transposition protein is

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Fig. 20.12 The structure of the Ac autonomous transposable element of corn and of

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5. In Mendel’s wild-type (SS) peas the starch grains are

large and simple, while in wrinlded peas

(ss)

they are

small and fissured.

a. SS seeds contain more starch and less sucrose than

ss

seeds.

b. The sucrose difference makes

ss

seeds larger, with higher water

content, so that when dried they are wrinided.

c. One type of starch-branching enzyme (SBEI) is missing in

ss

plants, reducing their starch content.

d. The SBEI gene corresponding to the

s

allele has a 0.8 kb

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Ty

Elements in Yeast

1. Ty

elements share characteristics with bacterial transposons:

a. Terminal repeated sequences.

b. Integration at non-homologous sites.

c. Generation of a target site duplication (5 bp).

2. Ty

element is diagrammed in Figure 20.14:

a. It is 5.9 kb including 2 terminal direct repeats of 334 bp, the long terminal repeats (LTR) or deltas (δ).

b. Each delta contains a promoter and transposase recognition sequences.

c. Ty elements encode one 5.7 kb mRNA beginning at the delta 5’ promoter (Figure 20.14). d. There are two ORFs in the mRNA, designated TyA and TyB, encoding two different

proteins.

e. Ty copy number varies between yeast strains, with an average of about 35.

3. Ty

elements also share similarities with retroviruses, ssRNA viruses that replicate

via dsDNA intermediates.

a. Ty elements transpose by making an RNA copy of the integrated DNA sequence, them making DNA using reverse transcriptase. This DNA can integrate at a new

chromosomal site. Evidence for this includes:

i. An experimentally introduced intron in the Ty element (which normally lacks

introns) was monitored through transposition. The intron was removed, indicating an RNA intermediate.

ii. Ty elements encode a reverse transcriptase.

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Drosophila

transposons

1. It is estimated that 15% of the

Drosophila

genome is mobile! These

transposons fall into different classes:

a. The

copia

retrotransposons include several families, each highly

conserved and present in 5-100 widely scattered copies per genome

(Figure 20.15).

i. All

copia

elements in

Drosophila

can transpose, and there are

differences in number and distribution between fly strains.

ii. Structurally,

copia

elements are similar to yeast

Ty

elements:

(1) Direct LTRs of 276 bp flank a 5 kb DNA segment.

(2) The end of each LTR has 17 bp inverted repeats.

(3) An

RNA

intermediate and reverse transcriptase are used for

transposition.

(4) Virus-like particles (VLPs) occur with

copia.

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Fig. 20.15 Structure of the transposable element copia, a retrotransposon found in

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b. P elements cause hybrid dysgenesis, a series of defects

(mutations, chromosomal aberrations and sterility) that

result from crossing certain

Drosophila

strains (Figure

20.16).

i. A mutant lab strain female (M) crossed with a

wild-type male (P) will result in hybrid dysgenesis.

ii. A mutant lab strain male (M) crossed with a

wild-type (P) female (reciprocal cross) will have normal

offspring.

iii. Thus, hybrid dysgenesis results when

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iv. The model is based on the observation that the M strain has no

P

elements, while the haploid genome of the P male has about 40

copies.

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P

elements vary from full-length autonomous elements

through shorter versions resulting from a variety of internal

deletions.

(2)

P

element transposition is activated only in the germ line.

(3) The F1 of an M female crossed with a P male have

P

elements inserted at new sites, flanked by target site repeats.

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P

elements are thought to encode a repressor protein that

prevents transposase gene expression, preventing

transposition.

(5) Cytoplasm in an M oocyte lacks the repressor, and so when

fertilized with P-bearing chromosomes, transposition occurs

into the maternal chromosomes, leading to hybrid dysgenesis.

v.

P

elements are used experimentally to transfer genes into the germ

line of

Drosophila

embryos. For example (Figure 20.18):

(1) The wild-type

rosy (ry)

gene was inserted into a P element,

cloned in a plasmid and microinjected into a mutant

ry/ry

strain.

(2) Insertion of the recombinant P element into the recipient

chromosome introduced the

ry

allele, and produced wild-type

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Fig. 20.17 Structure of the autonomous P transposable element found in

Drosophila

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Fig. 20.18 Illustration of the use of P elements to introduce genes into the

Drosophila

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