Transpozoni

TranspozoniElemente genetice mobile,care dovedesc PLASTICITATEA genomuluiTRANSPOZITIASCHIMBAREA LOCALIZARII UNEI SECVENTE DE ADN dublu catenar IN GENOM

ARE LOC o recombinarea genetica DESI NU SE PRODUCE UN SCHIMB DE MATERIAL GENETIC

1948 BARBARA McCLINTOCK descoperea genele saritoare investigand porumbul1983 este rasplatita cu Premiul Nobel

http://www.google.ro/imgres?imgurl=&imgrefurl=http%3A%2F%2Fwaynesword.palomar.edu%2Ftranspos.htm&h=0&w=0&sz=1&tbnid=fy_YUhFc3lhpeM&tbnh=148&tbnw=341&zoom=1&docid=pY8hv84RzIWHpM&hl=en&ei=UpF2UpK2M4SVtQbg34C4Cw&ved=0CAUQsCUhttps://www.google.ro/search?q=transposons+cartoons&rlz=1C1CHMO_roRO504RO504&espv=210&es_sm=93&source=lnms&tbm=isch&sa=X&ei=JdR-UoedO8SUtAaVhYHwCQ&ved=0CAcQ_AUoAQ&biw=1366&bih=667#facrc=_&imgdii=_&imgrc=j5kMuq0g0PH_8M%3A%3BvuQ_9cpgl1PdIM%3Bhttp%253A%252F%252Fdev.epubbud.com%252Fuploads%252F1%252F3%252F3%252F6%252F13360196%252Fimages%252FClinical_Microbiology_Made_Ridiculously_Simple_pdf%252Findex-29_1.jpg%3Bhttp%253A%252F%252Fwww.epubbud.com%252Fread.php%253Fg%253D2RBLFKRP%2526p%253D1%3B460%3B2112

Exista transpozoni ADN, dar si ARNhttps://www.google.ro/search?hl=en&site=imghp&tbm=isch&source=hp&biw=1366&bih=624&q=TRANSPOSONS&oq=TRANSPOSONS&gs_l=img.3..0l8j0i5l2.3538.6764.0.7508.11.10.0.1.1.0.158.1173.2j8.10.0....0...1ac.1.30.img..3.8.724.ik0AbnNBQPE#facrc=_&imgdii=_&imgrc=iewQTwD5aRjxQM%3A%3BUErl5w_agTQ6QM%3Bhttp%253A%252F%252Fwww.thenakedscientists.com%252FHTML%252Fuploads%252Ftx_naksciimages%252Fjamil1_Figure_3.gif%3Bhttp%253A%252F%252Fwww.thenakedscientists.com%252FHTML%252Farticles%252Farticle%252Fjamilcolumn1.htm%252F%3B450%3B2663

Ce urmeaza:http://www.nature.com/nrg/journal/v12/n9/fig_tab/nrg3030_ft.htmlDNA transposons. Many DNA transposons are flanked by terminal inverted repeats (TIRs; black arrows), encode a transposase (purple circles), and mobilize by a 'cut and paste' mechanism (represented by the scissors). The transposase binds at or near the TIRs, excises the transposon from its existing genomic location (light grey bar) and pastes it into a new genomic location (dark grey bar). The cleavages of the two strands at the target site are staggered, resulting in a target-site duplication (TSD) typically of 48bp (short horizontal black lines flanking the transposable element (TE)) as specified by the transposase. Retrotransposons (bandc) mobilize by replicative mechanisms that require the reverse transcription of an RNA intermediate.b| LTR retrotransposons contain two long terminal repeats (LTRs; black arrows) and encode Gag, protease, reverse transcriptase and integrase activities, all of which are crucial for retrotransposition. The 5 LTR contains a promoter that is recognized by the host RNA polymerase II and produces the mRNA of the TE (the start-site of transcription is indicated by the right-angled arrow). In the first step of the reaction, Gag proteins (small pink circles) assemble into virus-like particles that contain TE mRNA (light blue), reverse transcriptase (orange shape) and integrase. The reverse transcriptase copies the TE mRNA into a full-length dsDNA. In the second step, integrase (purple circles) inserts the cDNA (shown by the wide, dark blue arc) into the new target site. Similarly to the transposases of DNA transposons, retrotransposon integrases create staggered cuts at the target sites, resulting in TSDs.c| Non-LTR retrotransposons lack LTRs and encode either one or two ORFs. As for LTR retrotransposons, the transcription of non-LTR retrotransposons generates a full-length mRNA (wavy, light blue line). However, these elements mobilize by target-site-primed reverse transcription (TPRT). In this mechanism, an element-encoded endonuclease generates a single-stranded 'nick' in the genomic DNA, liberating a 3-OH that is used to prime reverse transcription of the RNA. The proteins that are encoded by autonomous non-LTR retrotransposons can also mobilize non-autonomous retrotransposon RNAs, as well as other cellular RNAs (see the main text). The TPRT mechanism of a long interspersed element 1 (L1) is depicted in the figure; the new element (dark blue rectangle) is 5 truncated and is retrotransposition-defective. Some non-LTR retrotransposons lack poly(A) tails at their 3 ends. The integration of non-LTR retrotransposons can lead to TSDs or small deletions at the target site in genomic DNA. For example, L1s are generally flanked by 720bp TSDs

4Initial Barbara Mc Clintock observa rupturi frecvente la nivelul unui cz. (respectiv 9), rupturi care se asociaza cu modificarea culorii bobului la porumb (Zea mays). Ea numeste gena implicata in modificarea culorii disociator (Ds). Dsproduce mutatii instabile sarind in diverse pozitii pe cz.Prin insertia transpozonului Ds gena de la nivelul locusului C este inactivata. Gena C fiind implicata in producerea culorii bobului acesta devine alb. Dar mutatia este reversibila, caci transpozonul va sari iarasi, astfel ca gena pentru pigmentul bobului va fi din nou activa.

INTRODUCERE

De asemenea B. Mc Clintock a descoperit gena Ac (Activator), care controlea-za transpozitia geneiDs, ceea ce are ca urmare ruperea cromozomului.https://www.google.ro/search?rlz=1C1CHMO_roRO504RO504&hl=en&site=imghp&tbm=isch&source=hp&biw=1366&bih=667&q=ac+ds+transposable+elements&oq=AC+Ds+&gs_l=img.1.3.0i24l6.9105.20593.0.25639.8.7.1.0.0.0.213.1075.0j6j1.7.0....0...1ac.1.30.img..0.8.1077.erQAVqDTIzs#facrc=_&imgdii=_&imgrc=iDhQc5pbPeZ-9M%3A%3BUZFf_9XHchmosM%3Bhttp%253A%252F%252Fbio3400.nicerweb.com%252FLocked%252Fmedia%252Fch15%252F15_22b-transposable_elements.jpg%3Bhttp%253A%252F%252Fbio3400.nicerweb.com%252FLocked%252Fmedia%252Fch15%252Ftransposable_elementsb.html%3B800%3B4186

Gena Ac poate sari datorita prezentei unei enzime (transpozaza). https://www.google.ro/search?hl=en&site=imghp&tbm=isch&source=hp&biw=1366&bih=667&q=transposase+mechanism&oq=transposase&gs_l=img.1.1.0l2j0i24l8.9606.15563.0.20582.15.12.1.2.2.0.119.1098.3j9.12.0....0...1ac.1.30.img..3.12.785.Y1Ec0aixEPw#facrc=_&imgdii=_&imgrc=oMocqBzyUFTxxM%3A%3B3hSwOWlcmIye_M%3Bhttp%253A%252F%252Fwww.chrisdellavedova.com%252Fwp-content%252Fuploads%252F2008%252F01%252Ftransposons1.jpg%3Bhttp%253A%252F%252Fwww.chrisdellavedova.com%252F2008%252F01%252F29%252Fscience-tuesday-one-cells-junk-is-another-cells-treasure%252F%3B600%3B7357

Gena Ds poate sari doar in prezenta genei Ac, caci Ds nu are transpozaza.

https://www.google.ro/search?rlz=1C1CHMO_roRO504RO504&hl=en&site=imghp&tbm=isch&source=hp&biw=1366&bih=667&q=ac+ds+transposable+elements&oq=AC+Ds+&gs_l=img.1.3.0i24l6.9105.20593.0.25639.8.7.1.0.0.0.213.1075.0j6j1.7.0....0...1ac.1.30.img..0.8.1077.erQAVqDTIzs#facrc=_&imgdii=_&imgrc=Q9xpDArVr7OOtM%3A%3Bv5ieGqdJ-kZ9jM%3Bhttp%253A%252F%252Fbarleyworld.org%252Fsites%252Fdefault%252Ffiles%252Ffigure-13-21.jpg%3Bhttp%253A%252F%252Fdejeka.wz.cz%252Fac-ds%3B703%3B10008

Marimea petelor de culoare este determinata de momentul transpozitiei in decursul dezvoltarii plantei.

C este gena normalac-m este gena mutantaTranspozitia geneiDsin diferite celule este intamplatoare, de unde aparitia mozaicului coloristic.

https://www.google.ro/search?rlz=1C1CHMO_roRO504RO504&hl=en&site=imghp&tbm=isch&source=hp&biw=1366&bih=667&q=ac+ds+transposable+elements&oq=AC+Ds+&gs_l=img.1.3.0i24l6.9105.20593.0.25639.8.7.1.0.0.0.213.1075.0j6j1.7.0....0...1ac.1.30.img..0.8.1077.erQAVqDTIzs#facrc=_&imgdii=_&imgrc=VOqlKZDi3eS9xM%3A%3BOAeRhuObS5EAiM%3Bhttp%253A%252F%252Fwww.bio.miami.edu%252Fdana%252Fpix%252FDs_Ac_variants.jpg%3Bhttp%253A%252F%252Fwww.bio.miami.edu%252Fdana%252F250%252F250S11_16print.html%3B504%3B5179

Prima dovada a controlului unei gene asupra alteia (fiind baza conceptului de reglare genica).Prima dovada si definire a elementelor genetice mobile. Dovada teoriei cz. demonstrand ruperea cromozomilor si fuziunea fragmentelor.Dezvoltarea celor mai bune tehnici de studiere a cz. la plante.

Barbara Mc Clintock a fost premiata la varsta de 81 ani, datorita contributiei deosebite si anume:http://de.wikipedia.org/wiki/Barbara_McClintockhttp://www.cas.muohio.edu/~wilsonkg/old/gene2005/manipulation/geneengneering/code/MobilE.htm10Un citat din spusele laureatei NobelOver the years I have found that it is difficult if not impossible to bring to consciousness of another person the nature of his tacit assumptions when, by some special experiences, I have been made aware of them. This became painfully evident to me in my attempts during the 1950s to convince geneticists that the action of genes had to be and was controlled. It is now equally painful to recognize the fixity of assumptions that many persons hold on the nature of controlling elements in maize and the manners of their operation. One must await the right time for conceptual change. (1973)Transpozon (Tn) - sinonimeELEMENTUL GENETIC MOBIL produce o ruptura cromozomiala la nivelul unui situs de insertie, ceea ce determina mutarea locusului genei saritoare (jumping gene) = TRANSPOZITIE

ELEMENTUL TRANSPOZABIL nu apare niciodata in forma libera, individualizat, in genom sau in celula in general.

Genom dinamicAdica un genom sigur nu este:nici fixnici imuabil

http://www.nature.com/nmeth/journal/v2/n10/fig_tab/nmeth1005-735_F1.htmlExoni albastru

IR gri

Promotor rosu

Gena reglatoare mov

SD, SA donor si acceptor de splicing

InsertieDeletie si insertieExpresie genica anormala

Figure1.Transposon-based vectors.The type of spliced transcript resulting from a transposition event depends on the transposon-based trapping construct. The three major classes of trapping vectors (delimited by the transposon inverted terminal repeats indicated by gray arrows) and the transcripts resulting from their insertion are depicted in the context of a hypothetical transcriptional unit composed of an upstream regulatory element (purple box), a promoter (red arrow), three exons (dark blue boxes) and a polyadenylation signal (pA). SD and SA represent splice donor and splice acceptor sites, respectively.13

IR = repetitii terminale inversate (in oglinda)Transpozonii pot fi o sursa majora de modificari evolutive in genomRearanjamentele creeaza secvente noi si schimba functia secventei initale/tintaUneori pot aparea maladii ca urmare a insertiei intr-o gena functionala, efectul fiind asemanator mutatiilor

La om elementele transpozabile sunt de 2 tipuri: transpozoni si retrotranspozoni (predomina in genomul uman)Al treilea mecanism/tip il reprezinta secventele de inserat, care se intalnesc numai la procariote.

Integrarea segmentelor IS (inserted sequence) in ADNul cromozomialI . SECVENTE DE INSERATSecventa de inserat (IS) este flancata de 9 13 pb = repetitii terminale inversate

Repetitiile directe si inverse/inversate (IR) indica prezenta elementelor genetice mobile la nivelul genomului.

TranspozazaSecventa de inserat contine:o regiune unica codanta pt. enzima transpozaza si/sauo zona centrala ce poate fi utilizata ca marker genetic, de ex. gena pt. rezistenta la antibiotice

II. TRANSPOZONII (Tn) comporta 2 tipuri de transpozitii: replicativa (1) si non replicativa (2)1. Tn original ramane la locul sau, se copiaza, iar copia este inserata la alt nivel creste nr.de copii ale Tn in genom;Mecanism:copy - paste

1. Transpozitie replicativa (~Copy-and-paste)Elementul transpozabil este copiat; copia se insera in segmentul cromozomial tinta.Elementtrans-pozabilSegm.tintaRecipientDonorIntegrareReplicare2. Transpozitia conservativa (non-replicativa)Mecanism: cut and pasteTn insusi ca entitate fizica se muta direct pe alt situsTranspozaza (se leaga de capetele transpozonului original, le sectioneaza lasand in urma capete coezive, sticky ends - si alipeste Tn in alt loc al genomului)ADN polimeraza umple golurile dintre capetele in zig-zag (staggered) ale ADN-ului tinta, iarADN ligaza inchide scheletul glucidofosforic (dupa unii autori, iar mai nou s-au evidentiat enzime din grupa recombinazelor de tip rezolvaza/invertaza si integraza)Nu se cunosc foarte multe lucruri cu privire la ADNul donor.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC179374/22

Tn5 si Tn10 (ale E.coli) se excizeaza din cromozom, se deplaseaza, apoi se re-integreaza. Totul cu ajutorul actiunii transpozazei.Complexul transpozon + transpozaza necesita cationi de Mg pentru a se re-insera . Daca Mg+ lipseste in mediu (de cultura), ia nastere o structura stabila numita transpozom; acesta se va insera aleator in genomul gazda daca este introdus in celule prin electroporare.

TRANSPOZOMhttp://utminers.utep.edu/rwebb/html/non-replicative_transposition.htmlhttp://www.epibio.com/item.asp?id=28423

In ADN-ul tinta au loc rupturi in zig-zag. Polimeraza umple golurile, astfel luand nastere segmentele repetitive ce flancheaza transpozonul. http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/transposons/non-repl-tpn.html24 Transpozaza Se leaga:Specific de un situsOriunde in ADNSecventele repetitive scurte directe si inverse sunt importante in excizia transpozonilor de catre transpozaza, dar si pt ADN polimeraza

2. Transpozitia conservativa (~Cut-and-paste)Elementul se muta prin excizie si integrare. Element transpozabilSegment tintaRecipientDonorExcisie & IntegrareIII. Retrotranspozonii

http://www.biotecharticles.com/DNA-Article/Types-of-Pseudogenes-Decoding-Pseudo-Notions-823.html27Retrotranspozitia apare cel mai frecvent la om1. ADN-ul elementului mobil este transcris in RNA.2. ARN-ul este revers-transcris in ADNc (complementar), care este apoi inserat in segmentul cromozomial acceptor.TargetDonorIntegrareTranscriereRNAADNcRevers transcriereExista 3 clase importante de retrotranspozoni la mamifere:Retrovirusurile endogene au secvente asemanatoare retrovirusurilor, dar nu pot infecta celule noi avand actiune limitata la un singur genom; au revers-transcriptaza si LTR (long terminal repeats) Retrotranspozonii nevirali (sau Tn ADN fosili), fara 3LTR au revers-transcriptaza fiind capabili de transpozitie independentaRetro-pseudogenele = pseudeogene procesate; sunt de 2 tipuri:cu nr.redus de copii (LINEs:L1, L2, L3 ) respectiv aprox. 850.000 copiicu nr. inalt de copii (aprox. 1,7 milioane copii): SINEs; Ex. fam. Alu (1,5 mil. copii)

Genetica Medicala, Covic, ultima editie31

L-1 LINEs (long-interspersed sequences): elemente lungi de spatiere sau elemente nucleare dispersate lungiElemente de 6,5 kb, repetate de 50.000-100.000 de ori (~21% din genom).SINEs (short-interspersed sequences) elemente nucleare dispersate scurte < 500 pb, repetate de peste 1.500.000 de oridispersate in intregul genom, in medie o copie la 3000pb; reprezinta 10% -13% din genomul uman

Alu = 300pb; reprezinta 5% din genomul uman

Enzima de restrictie al carei nume il poarta provine de la Arthrobacter luteus

Retrotranspozonii sau retropozonii: elemente transpozabile a caror mobilitate este ARN-mediata

Little Alberts 6-34 Garland Reverse transcriptaseencoded by the LINE-1 element Activitatea L1 presupune:- ARN polimeraza transcrie ADN in ARN Endonucleaza taie ADNul tinta Revers-transcriptaza copiaza L1 ARN in L1 ADN, care este inserat

Astfel creste numarul elementelor L1 in genom; acestea sunt uneori mai scurte decat L1 initiale (au deletii)Datorita diversitatii lor la nivelul genomului uman LINEs sunt folosite in analize moleculare (DNA fingerprint) ca markeri.http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Transposons.html33L-1 LINEs Contin gene cu valoare functionala cum sunt cele ce codifica polimeraza (pol) si transcriptaza; Migreaza in genom prin intermediul ARN Nu contin structuri de control (promotori) si de aceea nu se exprima (sunt pseudogene).

Unele cazuri de hemofilie A au aparut prin insertia unui element L1 in gena pentru factorul de coagulare VIII.

Cromozomi hibridizati cu probe pt secvente Alu (verzui); oarece corespondenta cu benzile R

Elemetele Alu constau dintr-o secventa de 260 perechi de baze (in medie) si contin o secventa pe care o recunoaste enzima de restrictie AluI respectiv:SINEs

Nu codifica transcriptaza si depind de prezenta unui element activ (L1).Wikipedia (Andreas Bolzer, Gregor Kreth, Irina Solovei, Daniela Koehler, Kaan Saracoglu, Christine Fauth, Stefan Mller, Roland Eils, Christoph Cremer, Michael R. Speicher, Thomas Crmer).

35Densitatea elementelor repetitive variaza in diferite regiuni genomice

Elementele LINEs se acumuleaza in regiuni bogate in ATElementele Alu se acumuleaza in regiuni bogate in GC; de asemenea in intronii genelor putand fi recunoscute de spliceozom si pastrate in ARNm matur. Astfel apare matisarea alternativaRetroTn nu sunt activi la om. S-au identificat mai multe mecanisme de inhibare a retrotranspozitiei printre care si unele epigenetice precum compactarea cromatinei si metilarea ADNului.

36http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Transposons.htmlSunt componente normale si ubicuitare ale genomului procariot si eucariot.

Procariote-transpozitie de la/catre cromozomul celulei, plasmidic, fagic.

Eucariote-transpozitie in cadrul aceluiasi cromozom sau pe un altul.

Produc modificari genomice; prin amplificarea si dispersia lor, prin rearanjamentele induse de excizie/insertie au o contributie importanta in evolutia genomului speciilor.

Determina rearanjamente cromozomiale (deletii, insertii, duplicatii, translocatii). Cresc dimensiunea genomului (in special la plante), Deseori creste nr. de copii ale elementului mobil in genom. Genele sau secventele genice se muta in alte regiuni ale genomului avand urmari cu efecte fenotipice; Se creeaza pseudogene. TRANSPOZONII = elemente genetice mobile ale cromozomilor, deoarece au capacitatea de a-si modifica pozitia in genom.Concluzii:

+/- efecte in urma transpozitieihttp://www.personal.psu.edu/rch8/workmg/TranspositionCh9.pdf38Alterari la nivel genicInactivarea genelor prin insertii;Crearea unor secvente noi in cadrul genelor care pot determina modificarea functiei .Alterarea secventelor reglatoare modificari de expresie genicaCrearea de noi introni;Asamblarea de noi gene prin componentele purtate de un element mobil.

Agenti mutageni biologici: modifica structura/ expresia genelorEfectele elementelor mobile asupra evolutiei genomului

In decursul evolutiei elementele mobile (initial doar cele in galben) s-au replicat, pentru ca in final sa intre si in secventele informationale.Lecture 2C Useful Genetics, Coursera40

http://www.nature.com/nrg/journal/v12/n9/fig_tab/nrg3030_ft.htmlTranspozitia poate avea loc oricand in timpul evolutiei unui organismGermline transposable element (TE) integration events can result from TE mobility in cells that give rise to gametes or from TE mobility post-fertilization during early development. Embryonic TE mobility in cells that do not contribute to the germ line or mobility at later developmental stages can, in principle, lead to somatic TE integration events. The overlapping brackets signify that some TE insertions in early development can contribute to the germ line, whereas others may not. Endogenous long interspersed element 1 (L1) retrotransposition events can occur in certain tumours, and experiments using engineered human L1s suggest that L1 retrotransposition may also occur during mammalian neurogenesis. Examples of the developmental timing of TE integration events are described in the main text.41Concluzii actualeDatorita prevalentei lor in orice tip de genom, transpozonii sunt un subiect mult discutat, totusi inca nu in totalitate elucidat.Inca nici macar nu este clar, daca ar trebui considerati o parte integranta a unui genom specific unei specii sau paraziti de succes!Precum un virus se deplaseaza de la un individ la altul, poate si transpozonii se pot muta de la un anumit genom la altul (de la un individ la altul) si nu numai in interiorul genomului unui individ dintr-o anumita specie? Datorita importantelor efecte la nivel genic si fenotipic, secventele transpozabile sunt intens cercetate fiind o posibila cale de aplicare a terapiei genice, exemplu :the sleeping beauty

http://www.personal.psu.edu/rch8/workmg/TranspositionCh9.pdfhttps://www.google.ro/search?q=transposons+cartoons&rlz=1C1CHMO_roRO504RO504&espv=210&es_sm=93&source=lnms&tbm=isch&sa=X&ei=JdR-UoedO8SUtAaVhYHwCQ&ved=0CAcQ_AUoAQ&biw=1366&bih=667#facrc=_&imgdii=_&imgrc=sexnyQZ5gqJT_M%3A%3B6QztGQMdcJ92lM%3Bhttp%253A%252F%252Fwww.jamesandthegiantcorn.com%252Fwp-content%252Fuploads%252F2010%252F11%252Ffig2.png%3Bhttp%253A%252F%252Fwww.jamesandthegiantcorn.com%252F2010%252F11%252F27%252Fhybrid-vigor-and-missing-genes%252F%3B1009%3B23142

http://en.wikipedia.org/wiki/Transposase43Sistemul transpozonSleeping Beautyeste untranspozonADN sintetic (artificial), construit pentru a introduce secvente de ADN precis definite in cromozomii vertebratelor, cu scopul de a determina noi caractere si a descoperi noi gene (oncogene) si functiile lor. este compus din transpozazaSleeping Beauty(SB) si transpozonul ADN construit in 1997 pentru a se insera dupa modelul cut-and-pastea fost desemnat ca Molecula anului 2009 de catre International Society for Molecular and Cell Biology and Biotechnology Protocols and Researches comparativ cu vectorii virali folositi in terapia genica, costurile de productie si utilizare sunt mai reduse. Primele studii clinice sunt in curs si vizeaza tumori maligne (limfoame cu celule B)Hum Gene Ther.2011 Sep;22(9):1043-51.Nonviral gene delivery with the sleeping beauty transposon system.Ivics Z,Izsvk Z.SourceMax Delbrck Center for Molecular Medicine, Berlin 13125, Germany. zivics@mdc-berlin.de

44

Lectura recomandata: Sleeping Beautytransposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cellshttp://nar.oxfordjournals.org/content/early/2012/12/25/nar.gks1305.full