Lecture 1

Lecture11 Bacterial and Eukaryotic Transposons •Molecular Genetics I: MLGN 301 2016 15/12/2016 By Prof.Dr /Ahmed Mansour Alzohairy

Genetics Department, Zagazig University, Zagazig, Egypt

Recommended book for further information

Lecture PowerPoint to accompany

Molecular Biology Fourth Edition

Robert F. Weaver

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Learning objectives Learning about:

• • • • •

Bacterial Transposons Eukaryotic Transposons Rearrangement of Immunoglobulin Genes Retrotransposons Group II Introns

Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

Molecular Genetics I: MLGN 301 2016 Trainer name: Prof. Ahmed Mansour Alzohairy

Learning outcomes By the end of this session and practical, students are expected to be able to understand what are: •Insertion Sequences •Complex Transposons •Replicative Transposition of Tn3 •Examples of Transposable Elements •Transposable Elements in Maize •Antibody Structure •Immune System Diversity •Antibody Heavy Chain Coding Regions •Model for Synthesis of Provirus DNA •Ty Transcription •Non-LTR Retrotransposons •Nonautonomous Retrotransposons Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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23.1 Bacterial Transposons • A transposable element moves from one DNA address to another • Originally discovered in maize, transposons have been found in all kinds of organisms – Bacteria – Plants – Humans Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Discovery of Bacterial Transposons

• • • • •

Phage coat is made of protein Always has the same volume DNA is much denser than protein More DNA in phage, denser phage Extra DNAs that can inactivate a gene by inserting into it were the first transposons discovered in bacteria • These transposons are called insertion sequences (ISs) Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Insertion Sequences • Insertion sequences are the simplest type of bacterial transposon • They contain only the elements necessary for their own transposition – Short inverted repeats at their ends – At least 2 genes coding for an enzyme, transposase that carries out transposition

• Transposition involves: – Duplication of a short sequence in the target DNA – One copy of this sequence flanks the insertion sequence on each side after transposition Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Generating Host DNA Direct Repeats


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Complex Transposons • The term “selfish DNA” implies that insertion sequences and other transposons replicate at the expense of their hosts, providing no value in return • Some transposons do carry genes that are valuable to their hosts, antibiotic resistance is among most familiar Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Antibiotic Resistance and Transposons • Donor plasmid has Kanr, harboring transposon Tn3 with Ampr • Target plasmid has Tetr • After transposition, Tn3 has replicated and there is a copy in target plasmid • Target plasmid now confers both Ampr, Tetr Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Transposition Mechanisms

• Transposons are sometimes called “jumping genes”, DNA doesn’t always leave one place for another • When it does, nonreplicative transposition – “Cut and paste” – Both strands of original DNA move together from 1 place to another without replicating

• Transposition frequently involves DNA replication – – – –

1 copy remains at original site New copy inserts at the new site Replicative transposition “Copy and paste”


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Replicative Transposition of Tn3

• In first step, 2 plasmids fuse, phage replication, forms a cointegrate – coupled through pair of Tn3 copies • Next is resolution of cointegrate, breaks down into 2 independent plasmids, catalyzed by resolvase gene product Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Detailed Tn3 Transposition


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Nonreplicative Transposition • Starts with same 2 first steps as in replicative transposition • New nicks occur at arrow marks • Nicks liberate donor plasmid minus the transposon • Filling gaps and sealing nicks completes target plasmid and its new transposon Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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23.2 Eukaryotic Transposons • Transposons have powerful selective forces on their side • Transposons carry genes that are an advantage to their hosts – Their host can multiply at the expense of completing organisms – Can multiply the transposons along with rest of their DNA

• If transposons do not have host advantage, can replicate themselves within their hosts


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Examples of Transposable Elements

• Variegation in the color of maize kernels is caused by multiple reversions of an unstable mutation in the C locus, responsible for kernel color • Mutation and its reversion result from Ds (dissociation) element – Transposes into the C gene – Mutates it Transposes to wild type Robert F. – Weaver. Molecular Biology out again, Copyright revert © The McGraw-Hill Companies, Inc. Fourth Edition.

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Ds and Ac of Maize • Ds cannot transpose on its own • Must have help from an autonomous transposon, Ac (for activator) – Ac supplies transposase – Ds is an Ac element with most of its middle removed – Ds needs • A pair of inverted terminal repeats • Adjacent short sequences that Ac transposase can recognize


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Transposable Elements in Maize


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Structures of Ac and Ds


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P Elements • The P-M system of hybrid dysgenesis in Drosophila is caused by conjunction of 2 factors: – Transposable element (P) contributed by the male – M cytoplasm contributed by the female allows transposition of the P element

• Hybrid offspring of P males and M females suffer multiple transpositions of P element • Damaging chromosomal mutations are caused that render the hybrids sterile • P elements have practical value as mutagenic and transforming agents in genetic experiments with Drosophila Robert F. Weaver. Molecular Biology Copyright © The McGraw-Hill Companies, Inc. Fourth Edition.

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23.3 Rearrangement of Immunoglobulin Genes • Mammalian genes use a process that closely resembles transposition for: – B cell antibodies – T cell receptors

• Recombinases involved in these processes have similar structures Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Antibody Structure • Antibody is composed of 4 polypeptides – 2 heavy chains – 2 light chains

• Sites called variable regions – Vary from 1 antibody to another – Gives proteins their specificity

• Rest of protein is Robert F. Weaver. Molecular Biology constant region

Fourth Edition. Copyright

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© The McGraw-Hill Companies, Inc.


Immune System Diversity • Enormous diversity of immune system is generated by 3 basic mechanisms: – Assembling genes for antibody light chains and heavy chains from 2 or 3 component parts – Joining the gene parts by an imprecise mechanism that can delete bases or add extra bases – Causing a high rate of somatic mutations, probably during proliferation of a clone if immune cells Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Rearrangement of Antibody Light Chain Gene


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Antibody Heavy Chain Coding Regions Human heavy chain is encoded in – – – –

48 variable segments 23 diversity segments 6 joining segments 1 constant segment


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Recombination Signals • The recombination signal sequences (RSSs) in V(D)J recombination consist of: – Heptamer – Nonamer – Separated by 12-bp or 23-bp spacers

• Recombination occurs only between a 12 signal and a 23 signal • Guarantees that only 1 of each coding region is incorporated into the rearranged gene Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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The Recombinase • Recombination-activating gene (RAG-1) stimulated V(D)J joining activity in vivo • Another gene tightly liked to RAG-1 also works in V(D)J joining, RAG-2 • These genes, RAG-1 and RAG-2, are expressed only in pre-B and pre-T cells


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Mechanism of V(D)J Recombination • RAG1 and RAG2 introduce single-strand nicks into DNA adjacent to either a 12 signal or 23 signal • Results in transesterification where newly created 3’OH group: – Attacks the opposite strand – Breaks it – Forms hairpin at the end of the coding segment

• Hairpins then break in an imprecise way that allows joining of coding regions with loss of bases or gain of extra bases Robert F. Weaver. Molecular Biology Copyright © The McGraw-Hill Companies, Inc. Fourth Edition.

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23.4 Retrotransposons • Retrotransposons replicate through an RNA intermediate • Retrotransposons resemble retroviruses – Retroviruses can cause tumors in vertebrates – Some retroviruses cause diseases such as AIDS

• Before studying retrotransposons, look at replication of the retroviruses Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Retroviruses • Class of virus is named for its ability to make a DNA copy of its RNA genome • This reaction is the reverse of the transcription reaction – reverse transcription • Virus particles contain an enzyme that catalyzes reverse transcription reaction


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Retrovirus Replication • Viral genome is RNA, with long terminal repeats at each end • Reverse transcriptase makes linear, ds-DNA copy of RNA • ds-DNA copy integrates back into host DNA = provirus • Host RNA polymerase II transcribes the provirus to genomic RNA • Viral RNA packaged into a virus Robert F. Weaver.particle Molecular Biology Copyright © The McGraw-Hill Companies, Inc. Fourth Edition.

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Model for Synthesis of Provirus DNA • RNase H degrades the RNA parts of RNA-DNA hybrids created during the replication process • Host tRNA serves as primer for reverse transcriptase • Finished ds-DNA copy of viral RNA is then inserted into the host genome • It can be transcribed by host polymerase II Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Retrotransposons • Several eukaryotic transposons transpose in a way similar to retroviruses – Ty of yeast – copia of Drosophila

• Start with DNA in the host genome – Make an RNA copy – Reverse transcribe it within a virus-like particle into DNA that can insert into new location

• HERVs likely transposed in the same way until ability to transpose lost – HERV = human endogenous retroviruses


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Ty Transcription


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Non-LTR Retrotransposons • LTR are lacking in most retrotransposons • Most abundant type lacking LTR are LINEs and LINE-like elements – Long interspersed elements – Encode an endonuclease that nicks target DNA – Takes advantage of new DNA 3’-end to prime reverse transcriptase of element RNA – After 2nd strand synthesis, element has been replicated at target site

• New round of transposition begins when the LINE is transcribed • LINE polyadenylation signal is weak, so transcription of a LINE often includes exons of downstream host DNA Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Nonautonomous Retrotransposons • Nonautonomous retrotransposons include very abundant human Alu elements and similar elements in other vertebrates • Cannot transpose by themselves as they do not encode any proteins • Take advantage of retrotransposition machinery of other elements such as LINE • Processed pseudogenes likely arose in same manner Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Group II Introns • Group II introns – Retrohome to intronless copies same gene by: • Insertion of an RNA intron into the gene • Followed by reverse transcription • Then second-strand synthesis

– Retrotranspose by: • Insertion of an RNA intron into an unrelated gene • Target-primed reverse transcription • Lagging-strand DNA fragments as primers

• Group II retrotransposition: – Forerunner of eukaryotic spliceosomal introns – Accounted for appearance in higher eukaryotes Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Practical (to try in your own time)

In few sentences describe the differences between

•Bacterial Transposons •Eukaryotic Transposons •Rearrangement of Immunoglobulin Genes •Retrotransposons •Group II Introns






Department of Genetics, Zagazig University, Zagazig, Egypt

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