Chapter 17 Transposable Genetic Elements

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Chapter Outline
Transposable Elements: An Overview Transposable Elements in Bacteria Cut-and-Paste Transposons in Eukaryotes Retroviruses and Retrotransposons Transposable Elements in Humans The Genetic and Evolutionary Significance of Transposable Elements
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Transposable Elements: An Overview

Transposable elements transposonsare found in the genomes of many kinds of organisms; they are structurally and functionally diverse.
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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 cells 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.
b. Increase or decrease gene expression by insertion into regulatory sequences. c. Produce chromosomal mutations through the mechanics of transposition.
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Types of Transposition
In cut-and-paste transposition, an element is cut out of one site in a chromosome and pasted into a new site. In replicative transposition, an element is replicated, and one copy is inserted at a new site; one copy also remains at the original site. In retrotransposition, an elements RNA is used as a template to synthesize DNA molecules, which are inserted into new chromosomal sites.
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Three different mechanisms for transposition

Conservative transposition
Replicative transposition Retrotransposition

Three different mechanisms for transposition

Conservative transposition: The element itself moves from the donor site into the target site Replicative transposition: The element moves a copy of itself to a new site via a DNA intermediate Retrotransposition: The element makes an RNA copy of itself which is reversed-transcribed into a DNA copy which is then inserted (cDNA)
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Conservative transposition

Replicative transposition

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Retrotransposition

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Key Points
A cut-and-paste transposon is excised from one genomic position and inserted into another by an enzyme, the transposase, which is usually encoded by the transposon itself.
A replicative transposon is copied during the process of transposition.

A retrotransposon produces RNA molecules that are reverse-transcribed into DNA molecules; these DNA molecules are subsequently inserted into new genomic positions.
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Transposable Elements in Bacteria

Bacterial transposons move within and between chromosomes and plasmids.
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Bacterial Transposons
Insertion Sequences (IS Elements) Composite Transposons Tn3 Elements

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IS Elements
Insertion Sequences (IS elements) are the simplest bacterial transposons. IS elements were first detected in certain lacmutations of E. coli. IS elements are compactly organized and contain only genes whose products are involved in transposition. Inverted terminal repeats are found at the ends. Some IS elements encode transposase.
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Insertion sequence (IS) elements

Simplest type of transposable element found in bacterial chromosomes and plasmids Encode only genes for mobilization and insertion Range in size from 768 bp to 5 kb IS1 first identified in E. colis glactose operon is 768 bp long and is present with 4-19 copies in the E. coli chromosome Ends of all known IS elements show inverted terminal repeats (ITRs)
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Insertion Sequences
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. colis 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 419 copies on the E. coli chromosome. b. IS2 has 012 copies on the chromosome, and 1 copy on the F plasmid. c. IS10 is found in R plasmids. 4. The ends of all sequenced IS elements show inverted terminal repeats (IRs) of 941 bp (e.g., IS1 has 23 bp of nearly identical sequence).
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Characteristics of Transposable Elements

Carry gene for enzyme that catalyzes transposition
transposase for elements that use a DNA intermediate reverse transcriptase for elements that use an RNA intermediate

May contain other genes

The IS50 Element

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5. Integration of IS elements may:

a. Disrupt coding sequences or regulatory regions.

b. Alter expression promoters.

of

nearby

genes

by

the

action

of

IS

element

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). f. Mutational analysis shows that IR sequences are the key

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Insertion of an IS Element Causes Target Site Duplication

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Multiple IS Elements
The bacterial chromosome may contain several copies of an IS element. Plasmids may also contain IS elements.
When a particular IS element is found on both a plasmid and a chromosome, homologous recombination may occur.
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Conjugative R Plasmids
Conjugative R plasmids have spread multiple drug resistance in bacterial populations. These plasmids have two components.
The resistance transfer factor (RTF) contains genes required for conjugative transfer between cells. The R-determinant contains the genes for antibiotic resistance.
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Formation of Conjugative R Plasmid by Recombination of IS Elements

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Composite Transposons
Composite transposons are bacterial cut-and-paste transposons and are denoted by the symbol Tn. Composite transposons are created when two IS elements insert near each other.

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Composite Transposons
Tetracycline resistance is carried by a transposable element The transposon is a composite transposon, composed of IS-elements flanking an included sequence, in this case containing an antibiotic resistance gene IS10R is an autonomous element while IS10L is non-autonomous Composite transposons probably evolved from IS elements by the chance location of a pair in close proximity to one another. Inactivation of one element by mutation would not harm ability to transpose and would assure continued 31 transposition of the entire transposon

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) Tn10s transposition is rare, because transpose is produced at a rate of ,1 molecule/generation. (2) Transposons, like IS elements, produce target site duplications (e.g., a 9bp duplication for Tn10). (Table 20.1)
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Genetic Organization of Composite Transposons

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Tn3 Elements
Tn3 elements are larger than the IS elements. Tn3 elements (like composite transposons) contain genes that are not required for transposition. Tn3 elements have simple inverted repeats at each end (not IS elements). Tn3 elements produce target site duplication when they transpose.
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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) Tn3s length is about 5 kb, with 38-bp inverted terminal repeats. (2) It has three genes in its central region:
(a) bla encodes -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).

(3) Tn3 produces a 5-bp duplication upon insertion (Figure 20.5).

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Noncomposite transposons (Tn)

Carry genes (e.g., a gene for antibiotic resistance)
Ends are non-IS element repeated sequences Tn3 is 5 kb with 38-bp ITRs and includes 3 genes; bla (-lactamase), tnpA (transposase), and tnpB (resolvase, which functions in recombination)
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Genetic Organization of Tn3

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Transposition of Tn3

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Key Points
Insertion sequences (IS elements) are cutand-paste transposons that reside in bacterial chromosomes and plasmids. IS elements can mediate recombination between different DNA molecules. Conjugative plasmids can move transposons that contain genes for antibiotic resistance from one bacterial cell to another.
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Key Points
Composite transposons consist of two IS elements flanking a region that contains one or more genes for antibiotic resistance. Tn3 is a replicative transposon that transposes by temporarily fusing DNA molecules into a cointegrate; when the cointegrate is resolved, each of the constituent DNA molecules emerges with a copy of Tn3.

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Key Points
Bacterial transposons are demarcated by inverted terminal repeats; when they insert into a DNA molecule, they create a duplication of sequences at the insertion site (a target site duplication).

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Transposable Elements in Bacteria

Insertion Sequences contain only the elements needed for transposition

Composite Transposons contain DNA that has insertion sequences on both sides

Antibiotic resistance genes are often included

Cut-and-Paste Transposons in Eukaryotes

Transposable elements were discovered by analyzing genetic instabilities in maize; genetic analyses have also revealed transposable elements in Drosophila.
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Ac and Ds Elements in Maize

Discovered by Barbara McClintock Aleurone color is affected by the Cl allele, which encodes dominant inhibitor of aleurone coloration. Mosaics with pigmented patches were caused by loss of the Cl allele.
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The Ac/Ds System

The Dissociation Factor (Ds) is located at a site on chromosome 9 in mosaic kernels where chromosome breakage occurs. Ds cannot induce chromosome breakage by itself. The Activator Factor (Ac) stimulates chromosome breakage at the site of Ds.
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Activities of the Ac/Ds Elements

The Ac element encodes a transposase that is responsible for excision, transposition, mutation, and chromosome breakage.

The Ac transposase interacts with sequences at the ends of Ac and Ds elements and catalyzes their movement.
Deletions or mutations in the Ac gene abolish its catalytic function.
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Hybrid Dysgenesis in Drosophila

Crosses between M and P strains produce dysgenic hybrids if the male in the cross is from the P strain. The chromosomes of P strains carry genetic factors that are activated in the eggs of M females; these factors cause mutations and chromosome breakage.
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P Elements
The chromosomal element in P strains is called a P element. P elements are transposons that are present in multiple copies and at different locations in the genomes of P strains but are absent from the genomes of M strains.
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The Structure of P Elements

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Key Points
The maize transposable element Ds, discovered because of its ability to break chromosomes, is activated by another transposable element, Ac, which encodes a transposase. Transposable P elements are responsible for hybrid dysgenesis, a syndrome of germ-line abnormalities that occurs in the offspring of crosses between P and M strains of Drosophila.
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Key Points
Within the germ line, P element activity is regulated by small RNAs (piRNAs) derived from the P elements themselves.

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Retroviruses and Retrotransposons

Retroviruses and related transposable elements utilize the enzyme reverse transcriptase to copy RNA into DNA. The DNA copies of these entities are subsequently inserted at different positions in genomic DNA.
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Retroviruses
Retroviruses possess an RNA-dependent DNA polymerase (reverse transcriptase), which allows them to synthesize DNA from an RNA transcript.
The human immunodeficiency virus (HIV), which causes acquired immune deficiency syndrome (AIDS), is a retrovirus.
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The Life Cycle of HIV

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Replication of the HIV Genome

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Integration of the HIV Double-Stranded DNA into Chromosomal DNA

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Retrotransposons
Retroviruslike elements (LTR retrotransposons) resemble integrated retroviruses.
Retroposons are DNA copies of polyadenylated RNA.

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Retroviruslike Elements
Found in yeast, plants, and animals.
Structure: central coding region flanked by long terminal repeats (LTRs) oriented in the same direction.

The coding region contains homologues of the gag and pol genes of retroviruses.
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Transposition of the Yeast Ty1 Element

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Retroposons (non-LTR Retrotransposons)

Retrotransposons are a large and widely distributed class of retrotransposons.
Retroposons move through an RNA molecule that is reverse transcribed into DNA. Retroposons have a homologous sequence of A:T base pairs at one end that is derived from the poly(A) tail of retroposon RNA.
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Retroposons in Drosophila
In Drosophila, the retroposons HeT-A and TART are found at the telomeres of chromosomes and replenish DNA that is lost by incomplete chromosome replication.

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Key Points
Retrovirus genomes are composed of singlestranded RNA comprising at least three genes: gag (coding for structural proteins of the viral particle), pol (coding for a reverse transcriptase/integrase protein), and env (coding for a protein imbedded in the virus lipid envelope).
The human retrovirus HIV infects cells of the immune system and causes the life-threatening disease AIDS.

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Key Points
Retroviruslike elements possess genes homologous to gag and pol, but not to env. Retroviruslike elements and the DNA forms of retroviruses inserted in cellular chromosomes are demarcated by long terminal repeat (LTR) sequences.

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Key Points
Retroposons lack LTRs; however, at one end they have a sequence of A:T base pairs derived from the reverse transcription of a poly(A) tail attached to the retroposons RNA.
The retroposons HeT-A and TART are components of the ends of Drosophila chromosomes.
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Transposable Elements in Humans

The human genome is populated by a diverse array of transposable elements that collectively account for 44 percent of all human DNA.
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The L1 Element
The L1 element is a retroposon belonging to a class of sequences known as the long interspersed nuclear elements (LINEs). The human genome contains 3000-5000 complete L1 elements and more than 500,000 truncated L1 elements. Complete L1 elements are about 6 kb long, have an internal promoter, and have two open reading frames that encode a nucleic-acid binding protein and a protein with endonuclease and reverse transcriptase activities.
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Short Interspersed Nuclear Elements (SINEs)

SINEs retroposons are the second most abundant class of transposable elements in the human genome. SINE familes are the Alu, MIR, and Ther2/MIR3 elements.
SINEs are usually less than 400 base pairs long and do not encode proteins. The reverse transcriptase required for SINE transposition is provided by a LINE-type John Wiley & Sons, Inc. element.

Key Points
The human genome contains four basic types of transposable elements: LINEs, SINEs, retroviruslike elements, and cut-and-paste transposons.
The L1 LINE and the Alu SINE are transpositionally active; other human transposons appear to be inactive.
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The Genetic and Evolutionary Significance of Transposable Elements

Transposable elements are used as tools by geneticists. In nature, they play a role in genome evolution.
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The Evolutionary Status of Transposable Elements

Do transposable elements perform any useful function, or are they merely genetic parasites?
Where did transposable elements come from?

What mechanisms have evolved to control and limit their movement?

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Transposons and Chromosome Structure

Transposable elements have been implicated in the formation of chromosome rearrangements. Crossing over may occur between homologous transposons located at different positions on the same chromosome or on different chromosomes. These events are referred to as ectopic intrachromosomal exchanges or ectopic interchromosomal exchanges, respectively.
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Intrachromosomal Recombination Between Transposons in the Same Orientation Produces a Deletion

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Unequal Crossing Over Between Transposons on Sister Chromatids Produces a Gene Duplication

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Evolutionary Issues Concerning Transposable Elements

Why have transposable elements spread? Do they confer a selective advantage, or are they genetic parasites?
How did transposable elements evolve?

How are retroviruslike elements related to retroviruses?

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Key Points
Transposons are used in genetic research to induce mutations.
Transposons are used as vectors to move DNA within and between genomes. Crossing over between paired transposons can create chromosome rearrangements.

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Effects of Transposition
Transposable elements can:
Cause mutations in adjacent genes Cause chromosomal rearrangements Relocate genes

Possible Advantages of Transposable Elements Transposable elements may: Create genetic diversity Act as promoters Allow recombination between plasmid and genomic DNA when multiple copies of the element are present Carry antibiotic resistance genes, conferring an advantage on bacterial cells Increase the number of copies of an exon or gene