Recombination & Cloning of Dna

8/18/2019 Recombination & Cloning of Dna

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DNA RECOMBINATION &CLONING

By:Dedy Agus Setiawan (083 !"0 0" #

$% y Lai'a Rusda (083 !"0 #S%ni Sya i uddin (083 !"0 3 #

E i)a A* i'ia + (083 !"0 33 #


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• Meselson and Weigle’s experiment. Density-labeled, “heavy” phage,symbolized as B! phage, "as #sed to $oin%e$t ba$teria along "ith“light”phage, the ab$ phage. &he progeny %rom the in%e$tion "ere $olle$tedand s#b'e$ted to !s!l density gradient $entri%#gation . (arental-type B!

and ab$ phage "ere "ell separated in the gradient, b#t re$ombinant phage) B$, b$, aB$, aB!, and so on* "ere distrib#ted di%%#sely bet"een the t"oparental bands be$a#se they $ontained $hromosomes $onstit#ted %rom%ragments o% both “heavy” and “light”D+


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De initi%n % DNA Re,%-.inati%n

•

Geneti, e,%-.inati%n is t/e natu a' * %,ess .yw/i,/ geneti, in % -ati%n is ea anged to %ormne" asso$iations.

• &here are three types o% D+ re$ombination:

. omologo#s - o$$#rs bet"een se #en$es that arenearly identi$al )e.g., d#ring meiosis*/. +on-homologo#s - When very di%%erent n#$leotide

se #en$es re$ombine0. &ransposition 1 D+ element moves %rom one site

to another, #s#ally little se #en$e similarityinvolved.


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%-%'%g%us Re,%-.inati%n•

&he pro$ess #nderlying homologo#s re$ombinationis termed gene a' e,%-.inati%n be$a#se theenzymati$ ma$hinery that mediates the ex$hange$an #se essentially any pair o% homologo#s D+

se #en$es as s#bstrates.• omologo#s re$ombination o$$#rs in all organisms

and is parti$#larly prevalent d#ring the prod#$tion o%gametes ) meiosis * in diploid organisms.

•

omologo#s re$ombination $an also o$$#r inba$teria.

• 2ndeed, even viral $hromosomes #ndergore$ombination.


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Ai-s % DNA Re,%-.inati%n

&he 3eneral ims o% D+ 4e$ombination are:• 2solate and st#dy ea$h o% genes %or obtaining the

in%ormation abo#t %#n$tion and their $ontrol’sme$hanism

•

5btaining the gene’s prod#$t in short time andmore o% amo#nts than gene’s prod#$tion in$onventional method.


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In1%'1ed C%-*%nents•

&he 6nzymes o% 3eneral 4e$ombination 2n$l#de4e$ , 4e$B!D , 4#v , 4#vB , and 4#v!• &he prin$ipal players in the pro$ess are the Re,BCD

en2y-e ,%-*'e 4 "hi$h initiates recombination 7•

&he Re,A * %tein4 w/i,/ .inds sing'e5st andedD+ , %orming a n#$leoprotein %ilament $apable o%strand invasion and homologo#s pairing7 and

• &he Ru1A4 Ru1B4 and Ru1C * %teins4 w/i,/ d i1e. an,/ -ig ati%n and pro$ess the olliday '#n$tioninto re$ombinant prod#$ts.


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6 %,ess % DNA Re,%-.inati%n•

4e$ombination o$$#rs by the brea8age and re#niono% D+ strands so that a physi$al ex$hange o% partsta8es pla$e.

• 2n 9 ;, 4obin olliday proposed a model %orhomologo#s re$ombination that has proved to be$orre$t in its essential %eat#res


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6xamples o% 4e$ombination


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olliday Model

4. olliday ) 9 ;*

- olliday=#n$tions %ormd#ringre$ombination

- =s $an beresolved / "ays,only one

prod#$es tr#ere$ombinantmole$#les


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6M o% a olliday =#n$tion ">a %e" meltedbase pairs aro#nd '#n$tion


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recBCD (ath"ay o% omologo#s4e$ombination

(art 2: +i$8ing and 6x$hanging. ni$8 is $reated in one strand by recBCD at a Chi

se #en$e )3!&33&33*, %o#nd every ?@@@ bp./. An"inding o% D+ $ontaining Chi se #en$e by

recBCD allo"s binding o% B and recA. 3. recA promotes strand invasion into homologo#s

D+ , displa$ing one strand.;. &he displa$ed strand base-pairs "ith the single

strand le%t behind on the other $hromosome.?. &he displa$ed and no" paired strand is ni$8ed )by

recBCD? * to $omplete strand ex$hange.


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recBCD (ath"ay o%

omologo#s4e$ombination

(art 22:

Bran$hMigrationand4esol#tion


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recBCD (ath"ay o% omologo#s 4e$.(art 22: Bran$h Migration and 4esol#tion

. +i$8s are sealed olliday =#n$tion/. Bran$h migration ) ruvA C ruvB *0. 4esol#tion o% olliday =#n$tion ) ruvC *


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RecBCD : $omplex enzyme•

RecBCD has:. 6ndon#$lease s#b#nits ) recBCD * that $#tone D+ strand $lose to !hi se #en$e.

/. D+ heli$ase a$tivity ) recBC s#b#nit* and0. &he 4e$B!D $omplex is $omposed o% the

proteins Re,B ( "0 )D7 80 a-in% a,ids#4Re,C ( 30 )D7 a-in% a,ids#4 and Re,D

(9: )D7 908 a-in% a,ids#;;. D+ -dependent &(ase a$tivity – #n"inds D+ to generate regions

$tivity / and 0 lin8ed


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RecA• 0 8Da protein that polymerizes onto D+ ?’-0’• !atalyzes strand ex$hange, also an &(ase• lso binds D D+ , b#t not as strongly as


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RecA


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RuvA and RuvB

• D+ heli$ase that $atalyzes bran$h migration• RuvA tetramer binds to = )ea$h D+ helix

bet"een s#b#nits*• RuvB is a hexamer ring, has heli$ase E &(ase

a$tivity• / $opies o% ruvB bind at the = )to ruvA and / o%

the D+ heli$es*• Bran$h migration is in the dire$tion o% recA

mediated strand-ex$hange


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http:>>""".sds$.ed#>'o#rnals>mbb>r#va.html

http://www.sdsc.edu/journals/mbb/ruva.htmlhttp://www.sdsc.edu/journals/mbb/ruva.html


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RuvC : resolvase

• 6ndon#$lease that $#ts / strands o% =•

Binds to = as a dimer• !onsens#s se #en$e: ) >&*&& )3>!*

- o$$#rs %re #ently in E. coli genome- bran$h migration needed to rea$h$onsens#s se #en$eF


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&ransposable 6lements)&ransposons*

• D+ elements $apable o% moving )GtransposingG*aro#nd the genome

• Dis$overed by Barbara M$!linto$8, largely %rom

$ytogeneti$ st#dies in maize, b#t sin$e %o#nd inmost organisms• he "as st#dying GvariegationG or se$toring in

leaves and seeds• he li8ed to $all them G$ontrolling elements“

be$a#se they e%%e$ted gene expression in myriad"ays


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M#tant Hernel (henotypes

• (igmentation m#tants – a%%e$t antho$yanin path"ay – elements '#mp in>o#t o% trans$ription

%a$tor genes )! or 4* –

se$toring phenotype - somati$ m#tations – "hole 8ernel e%%e$ted - germ line

m#tation

• tar$h synthesis m#tants- stain star$h "ith iodine, see se$toring in

endosperm


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tart "ith m#tant 8ernels de%e$tive in star$h synthesis )endospermphenotypes* or antho$yanin synthesis )ale#rone and peri$arpphenotypes*.

ome maize phenotypes $a#sed by transposableelements ex$ising in somati$ tiss#es.


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omati$ 6x$ision o% Ds %romC


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5ther !hara$teristi$s o% M$!linto$8Is6lements

• Anstable m#tations that revert %re #ently b#t o%tenpartially, giving ne" phenotypes.

• ome elements )e.g., Ds* $orrelated "ith$hromosome brea8s.

• 6lements o%ten move d#ring meiosis and

mitosis.• 6lement movement a$$elerated by genomedamage.


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Mole$#lar analysis o% transposons•

&ransposons isolated by %irst $loning a gene thatthey invaded. n#mber have been $loned this "ay,via G&ransposon trapping“.

•

ome $ommon mole$#lar %eat#res: – 6xist as m#ltiple $opies in the genome – 2nsertion site o% element does not have extensive

homology to the transposon –

&ermini are an inverted repeat – 6n$ode “transposases” that promote movement – short, dire$t repeat o% genomi$ D+ o%ten

%lan8s the transposon : “


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Ac and Ds

• Ds is derived %rom Ac by internal deletions• Ds is not a#tonomo#s, re #ires Ac to move• 6lement termini are an imper%e$t 24• Ac en$odes a protein that promotes

movement - &ransposase• &ransposase ex$ises element at 24, and also

$#ts the target

t #$t# % A d D d l ti


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tr#$t#re o% Ac and Ds deletionderivatives


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D#pli$ationremains "henelement ex$ises,th#s the


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Mutator ) 4etrotransposon*

• Dis$overed in maize7 di%%ers signi%i$antly %rom Ac by str#$t#re and transposing me$hanism

• #tonomo#s and non-a#tonomo#s versions7many $opies per $ell

• $ontains a long terminal 24 )J/@@ bp*• transposes via a repli$ative me$hanism,

instead o% a gain>loss me$hanism•

“retrotransposon” – imilarities "ith retrovir#ses – move via an 4+ intermediate – en$ode a reverse trans$riptase a$tivity


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Fig. 7.34 in Buchanan et al.


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!ontrol o% &ransposons

• #toreg#lation: ome transposases aretrans$riptional repressors o% their o"npromoter)s*

•

e.g., TpnA o% the Spm element

• &rans$riptional silen$ing : me$hanism not "ell#nderstood b#t important7 $orrelates "ithmethylation o% the promoter


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Biologi$al igni%i$an$e o%&ransposons

• &hey provide a means %or genomi$ $hange andvariation, parti$#larly in response to stress)M$!linto$8’s GstressG hypothesis*

) 9 0 +obel le$t#re, $ien$e // :K9/*• or '#st Gsel%ish D+ GL• +o 8no"n examples o% an element playing a

normal role in development.


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T AN+ ER< M=C

Recombination & Cloning of Dna

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