Molecular biology - Chapter 23: Transposition

Molecular BiologyFourth EditionChapter 23TranspositionLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.123.1 Bacterial TransposonsA transposable element moves from one DNA address to anotherOriginally discovered in maize, transposons have been found in all kinds of organismsBacteriaPlantsHumans2Discovery of Bacterial TransposonsPhage coat is made of proteinAlways has the same volumeDNA is much denser than proteinMore DNA in phage, denser phageExtra DNAs that can inactivate a gene by inserting into it were the first transposons discovered in bacteriaThese transposons are called insertion sequences (ISs)3Insertion SequencesInsertion sequences are the simplest type of bacterial transposonThey contain only the elements necessary for their own transpositionShort inverted repeats at their endsAt least 2 genes coding for an enzyme, transposase that carries out transpositionTransposition involves:Duplication of a short sequence in the target DNAOne copy of this sequence flanks the insertion sequence on each side after transposition4Generating Host DNA Direct Repeats5Complex TransposonsThe term “selfish DNA” implies that insertion sequences and other transposons replicate at the expense of their hosts, providing no value in returnSome transposons do carry genes that are valuable to their hosts, antibiotic resistance is among most familiar6Antibiotic Resistance and TransposonsDonor plasmid has Kanr, harboring transposon Tn3 with AmprTarget plasmid has TetrAfter transposition, Tn3 has replicated and there is a copy in target plasmidTarget plasmid now confers both Ampr, Tetr7Transposition MechanismsTransposons 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 replicatingTransposition frequently involves DNA replication1 copy remains at original site New copy inserts at the new siteReplicative transposition“Copy and paste”8Replicative Transposition of Tn3In first step, 2 plasmids fuse, phage replication, forms a cointegrate – coupled through pair of Tn3 copiesNext is resolution of cointegrate, breaks down into 2 independent plasmids, catalyzed by resolvase gene product9Detailed Tn3 Transposition10Nonreplicative TranspositionStarts with same 2 first steps as in replicative transpositionNew nicks occur at arrow marksNicks liberate donor plasmid minus the transposonFilling gaps and sealing nicks completes target plasmid and its new transposon1123.2 Eukaryotic TransposonsTransposons have powerful selective forces on their sideTransposons carry genes that are an advantage to their hostsTheir host can multiply at the expense of completing organismsCan multiply the transposons along with rest of their DNAIf transposons do not have host advantage, can replicate themselves within their hosts12Examples of Transposable ElementsVariegation in the color of maize kernels is caused by multiple reversions of an unstable mutation in the C locus, responsible for kernel colorMutation and its reversion result from Ds (dissociation) elementTransposes into the C geneMutates itTransposes out again, revert to wild type13Ds and Ac of MaizeDs cannot transpose on its ownMust have help from an autonomous transposon, Ac (for activator)Ac supplies transposaseDs is an Ac element with most of its middle removedDs needs A pair of inverted terminal repeatsAdjacent short sequences that Ac transposase can recognize14Transposable Elements in Maize15Structures of Ac and Ds16P ElementsThe P-M system of hybrid dysgenesis in Drosophila is caused by conjunction of 2 factors:Transposable element (P) contributed by the maleM cytoplasm contributed by the female allows transposition of the P elementHybrid offspring of P males and M females suffer multiple transpositions of P elementDamaging chromosomal mutations are caused that render the hybrids sterileP elements have practical value as mutagenic and transforming agents in genetic experiments with Drosophila1723.3 Rearrangement of Immunoglobulin GenesMammalian genes use a process that closely resembles transposition for:B cell antibodiesT cell receptorsRecombinases involved in these processes have similar structures18Antibody StructureAntibody is composed of 4 polypeptides2 heavy chains2 light chainsSites called variable regions Vary from 1 antibody to anotherGives proteins their specificityRest of protein is constant region19Immune System DiversityEnormous diversity of immune system is generated by 3 basic mechanisms:Assembling genes for antibody light chains and heavy chains from 2 or 3 component partsJoining the gene parts by an imprecise mechanism that can delete bases or add extra basesCausing a high rate of somatic mutations, probably during proliferation of a clone if immune cells20Rearrangement of Antibody Light Chain Gene21Antibody Heavy Chain Coding RegionsHuman heavy chain is encoded in 48 variable segments23 diversity segments6 joining segments1 constant segment22Recombination SignalsThe recombination signal sequences (RSSs) in V(D)J recombination consist of:HeptamerNonamer Separated by 12-bp or 23-bp spacersRecombination occurs only between a 12 signal and a 23 signalGuarantees that only 1 of each coding region is incorporated into the rearranged gene23The RecombinaseRecombination-activating gene (RAG-1) stimulated V(D)J joining activity in vivoAnother gene tightly liked to RAG-1 also works in V(D)J joining, RAG-2These genes, RAG-1 and RAG-2, are expressed only in pre-B and pre-T cells24Mechanism of V(D)J RecombinationRAG1 and RAG2 introduce single-strand nicks into DNA adjacent to either a 12 signal or 23 signalResults in transesterification where newly created 3’-OH group: Attacks the opposite strandBreaks itForms hairpin at the end of the coding segmentHairpins then break in an imprecise way that allows joining of coding regions with loss of bases or gain of extra bases 2523.4 RetrotransposonsRetrotransposons replicate through an RNA intermediateRetrotransposons resemble retrovirusesRetroviruses can cause tumors in vertebratesSome retroviruses cause diseases such as AIDSBefore studying retrotransposons, look at replication of the retroviruses26RetrovirusesClass of virus is named for its ability to make a DNA copy of its RNA genomeThis reaction is the reverse of the transcription reaction – reverse transcriptionVirus particles contain an enzyme that catalyzes reverse transcription reaction27Retrovirus ReplicationViral genome is RNA, with long terminal repeats at each endReverse transcriptase makes linear, ds-DNA copy of RNAds-DNA copy integrates back into host DNA = provirusHost RNA polymerase II transcribes the provirus to genomic RNAViral RNA packaged into a virus particle28Model for Synthesis of Provirus DNARNase H degrades the RNA parts of RNA-DNA hybrids created during the replication processHost tRNA serves as primer for reverse transcriptaseFinished ds-DNA copy of viral RNA is then inserted into the host genomeIt can be transcribed by host polymerase II29RetrotransposonsSeveral eukaryotic transposons transpose in a way similar to retrovirusesTy of yeastcopia of DrosophilaStart with DNA in the host genomeMake an RNA copyReverse transcribe it within a virus-like particle into DNA that can insert into new locationHERVs likely transposed in the same way until ability to transpose lostHERV = human endogenous retroviruses30Ty Transcription31Non-LTR RetrotransposonsLTR are lacking in most retrotransposonsMost abundant type lacking LTR are LINEs and LINE-like elementsLong interspersed elementsEncode an endonuclease that nicks target DNATakes advantage of new DNA 3’-end to prime reverse transcriptase of element RNAAfter 2nd strand synthesis, element has been replicated at target siteNew round of transposition begins when the LINE is transcribedLINE polyadenylation signal is weak, so transcription of a LINE often includes exons of downstream host DNA 32Nonautonomous RetrotransposonsNonautonomous retrotransposons include very abundant human Alu elements and similar elements in other vertebratesCannot transpose by themselves as they do not encode any proteinsTake advantage of retrotransposition machinery of other elements such as LINEProcessed pseudogenes likely arose in same manner33Group II IntronsGroup II intronsRetrohome to intronless copies same gene by:Insertion of an RNA intron into the geneFollowed by reverse transcriptionThen second-strand synthesisRetrotranspose by: Insertion of an RNA intron into an unrelated gene Target-primed reverse transcription Lagging-strand DNA fragments as primersGroup II retrotransposition: Forerunner of eukaryotic spliceosomal intronsAccounted for appearance in higher eukaryotes34