Bakul Dna Mobile-2016
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Transcript of Bakul Dna Mobile-2016
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DNA BERPINDAH-PINDAH (MOBILE DNA)
DNA yg mampu berpindah dari satu “tempat” ke “tempat lain”,misal plasmid, phage dan transposon; dpt bertindak sebagai
“vehicle” pd proses strains improvement
Plasmid
Transposo
Phage
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1PLA!MID
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Plasmid – Extrakromosom, benang ganda,biasanya supercoiled
– Ditemukan pada bakt G+ dan G- ;
satu sel individu sering harborsmembebaskan sejumlah plasmid
– DNA adalah berada di luar ekromosom; replikon plasmid
terpisah dari anakan sel dandipelihara di dalam sel anakannya
– Plasmid manunjukkan sedikithomologi (bila ada) thd sequenkromosom DNA inang
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Plasmids are not usually essential
• Plasmid biasanya tdk mengkode gen-gen esensial; wl dmkn, gen-gen plasmid bisa memberi suatukeuntungan selektif bagi bakteri, misal:
– Produksi bacteriocin – Degradasi seny2 toksik
– Simbiosis dg jasad eukariot (fiksasi nitrogen)
– Produksi antibiotik
– Resisten thd antibiotik
– Enterotoxins (E. coli) – Hemolysins
– Mhslk Faktor adherence & colonization (pili)
– Mhslk proteins serum resistensi
– Mhslk patwai katabolik dan anabolik
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• Fertility plasmids--F plasmids(mating/conjugation).
• Resistance plasmids--R plasmids
(antibiotic resistance--see handout)• Col plasmids--encode colicins(bacteroicins)
•
Virulence plasmids--encode determinantsrequired for pathogenesis
• Metabolic plasmids-mediate degradationof toxic compounds
Types of Plasmids
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99kb F (Fertility) Plasmid Genetic Map ( E. coli)
Insertion sequences (IS) assist in the
integration of transposons (Tn) into
homologus sites of recipients genome.
Different Hfr strains are produced as aresult
Replication & segregation genes of F plasmid
Genes for conjugative transfer
Origin of transfer
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Understanding Plasmids
4. Types of plasmids & their biological significance
Remember- essential host functions not part of plasmidgenes
Pseudomonas- entire metabolic degradation pathways ofunique compounds – camphor napthalene.
!ryptic plasmids – we "now little of the plasmid functions
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Understanding Plasmids
#. Resistance Plasmids $R plasmids%
R'' carries resistance for sulfonamdes tetracylinechloramphenicol spectinomycin mercury. (road enteric hostrange- )scherichia Proteus *lebsiella +almonella +higella
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Understanding Plasmids
,. Toins & other irulence characteristics
• /bility to attach and colonise a hosto ). coli !olonisation 0actor /ntigen $!0/% assists inattaching to intestinal epithelia
• production of toins en1ymes that damage the host
o ) coli hemolysin- lyse R(+ & ). coli enterotoin- inducesalt and water secretion into the bowel
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Understanding Plasmids
2. (acteriocins
(acterocins inhibit or "ill related species or different strains ofthe same species $limited inhibitory spectrum to antibiotics%
). coli plasmids- !ol
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3nsertion +equences Transposons & 3ntegrons
obilised ia plasmids.
Promote changes to the host 56/- Rearrange and or delete
genes3ntegrons are 3+ elements or transposons which create andmoe large gene clusters as a single unit - P/3
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R plasmid
Plasmids: Examples
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pBR322 (ColEI replicon)
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Analysis and Classification
– Replikon diklasifikasikan oleh gugus "incompatibilitas"Kompatibel plasmid memp sistem replicasi & segregasiberbeda. Incompatibel plasmid mengg sistem replikasi &
segrgasi yg sama. Sepertinya mrk tdk dpt dibedakan satu dg yglain selama proses-proses ini, keduanya tdk dpt dipertahankan
– Jumlah kopi plasmid/sel bs tinggi (30 or lbh) or rendah (1-2).
– Replicasinya adl“stringent” or “relaxed”• Stringent: mereplikasi sejalan dg khromosom (low copy #)
• Relaxed: mereplicasi scr tdk terpengaaruh kromosom (high copy #)
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Panel A: coexistence of 2plasmids from differentincompatibility (Inc) groups-both maintained
Panel B: “curing” of oneplasmid. In the absence ofselection, asymmetricpartitioning results in theloss of one of each plasmid
-ultimately cellscontain oneplasmids of the otherbut not both
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– Isolated ascovalently closed circular(ccc) DNA; sizedby agarose gel electrophoresis.
– Restriction enzymes used to obtain a fragment profile
useful in epidemiology.
– DNA hybridization used to detect sequencerelationships.
–
Many plasmids remaincryptic; confer an undefinedphenotype.
Analisis dan Klasifikasi (cont.)
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ReplicasiPlasmid
Mengikuti aturan replikasi DNA kromosom, bilareplikasi mrpk stringent (low copy # plasmids;e.g., F plasmids).
High copy # plasmids (i.e., “relaxed” plasmids)Mengikuti aturannya sdr; mekanisme replikasidistinct“amplification” of plasmid DNA
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Replication: “theta” or “sigma”
Unidirectional
Bidirectional
Rolling circle
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Plasmid transmission
• Sequen DNA Plasmid dpt ditransmisikan diantr bakteri dg caratransformasi, transduksi, or konjugasi. Krn plasmids berreplikasi scrindependen, rekombinasi tdk dibutuhkan unt menurunkan sifat-sifat
• Konjugatif (self-transmissable) plasmids encode a conjugation system.
"Mobilizable" plasmids transfer through the cell contacts established byconjugative plasmids. Typically, the frequency of transfer is low. (The Fplasmid transfer system is depressed).
• "Wide host range" conjugative plasmids are able to replicate in manyorganisms; "narrow host range" plasmids, in only a few. However, DNAcan be delivered to many organisms in either case; such DNA can then
be maintained in the recipient through integration with other replicons(transposition; cointegrate formation).
• Properties disseminated by plasmids: As plasmids move from host tohost they interact with other host replicons (including the chromosome)& acquire genes by recombination or transposition.
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Plasmids: Pathogenesis
Examples: Shigella (A) and
Yersinia (B) both have virulence
plasmids that are required for
optimal virulence of these
bacteria. Other factors also
contribute to pathogenesis that
are not plasmid-encoded (i.e.,
shiga toxin). Strains “cured” ofthese plasmids have an attenuated
virulence phebnotype.
A
B
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" PHA#E
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Sifat umumPhage
• Phages adalah parasit bakteri.
• Tdk dpt berreplikasi di luar sel inang
• Mengg “mesin” inang (ribosomes,enzymes) unt mensintesis komponen virus
• Mengg sistem enersi sel inang ( karenanyahanya dpt mengreplikasi pd bakt hdp)
• Ribuan tipe beda diketh namun hanyasedikit species/strain yg spesifik
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T$ %dan T" BA&TERIO'A#
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T2 PA!" #"N!$N%"&'$ (A&T")$
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Struktur Phage – 1. Genome
•single or double- benang DNA, sirkuler or linier.
•linier, benang tunggal RNA.
– 2. Kapsid
•protein shell ("coat").•pelindung asam nukleat genome.
•Bentuknya icosohedral or filamentous
– 3. Ekor
•
Struktur protein yg nempel pd kapsid.• Struktur fisiknya sgt kompleks.
• Terlibat dlm penempelan phage pd sel inang danpemindahan DNAphage ke sel inang
•Tidak semua phages memiliki ekor
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Kehidupan siklus litik
• Fase reproduktif yg mhslk anakan phagebaru
• Biasanya (tdk selalu) mhslk kematian & lisissel inang
•Phage yg hanya mampu tumbuh liti dikenalsbg virulen
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Tingkatan dlm siklus litik
• Adsorpsi: penempelan phage ke sel inang via reseptor spesifik
• Penetrasi: introduktsi DNA phage ke dlm sel inang; mekanismesering tdk jelas
• Ekspresi: transkripsi/translasi genom phage ke protein spesifikpenghasil virus; two temporal stages, early and late. protein awalbiasanya unt replikasi & transkripsi, protein akhir unt perakitan
• Replikasi: sintesis genome phage baru
• Perakitan: pengorganisasian anakan genom phage ke dlm kapsid
• Pelepasan: anakan hsl rakitan ninggalkan sel, dg melisiskan selinang ("burst").
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Lisogeni
• Only observed for phage with double strandedDNA genomes (phages capable of lysogeny arecalled temperate).
• Phage genome becomes part of host cellchromosome (integrates into host DNA, e.g.Lambda) or is maintained as a low copy numberplasmid (e.g. P1 phage).
• Integrated phage genome called a prophage.
• Bacterium with prophage called a lysogen.
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Stages in Lysogeny
• Adsorption
• Introduction of phage DNA into host chromosome
(recombination-dependent event).
• Expression: only of genes required for lysogeny.
• Maintenance: prophage replicates along with bacterialgenome.
• Induction: disruption of the lysogenic state and initiation ofthe lytic phase.
• Replication, Assembly and Release: as in the lytic cycle
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Lytic Phages
• RNA Phages (f2, MS2, R17, Qß) – Simplest of phages.
– Genome is single-stranded, linear RNA (3000-4000bases), (+) strand.
– Encode three proteins; CP (coat protein), A (attachmentprotein), and Rep (RNA replicase).
– All utilize F-pili as receptors for attachment.
–
Rep copies (+) strand into (-) strands then uses (-)strands to make new (+) strands for packaging.
– Lyse cells and release 10-20,000 phage per cell;mechanism of lysis unknown.
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T4• 2. Genome Structure
–Double stranded (ds), linear (≈170Kb,200 genes).
–DNA is replicated bidirectionally andforms long concatemers.
–Concatemers are cut and packaged to
form a "headful" unit. –Resultant DNA in phage is terminallyredundant and circularly permuted.
–Concatemer:
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3. Life Cycle
-Infection with T4 causes shut-off of hostcell protein, DNA, and RNA synthesis.
-Progeny phage assembled within 15-20min.
-Cells lyse within 20-25 min releasing
≈300 progeny phage.
T4
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Lambda( )
–Immunity:
• Lysogenic strains of bacteria cannot be
infected with a phage of the same type as theprophage.
–Resistance tosuperinfection
• In the lysogen, the cI repressor is dominant,so an incoming phage is immediatelyrepressed and cannot replicate.
MAP #ENOM T$ 'A#
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MAP #ENOM T$-'A#
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T
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Transposons
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Mobile Genetic Elements
Transposons or Transposable elements
(TEs)
move around the genome
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Transposable elements in
prokaryotes
Insertion sequence (IS) elements
Transposons (Tn)
Bacteriophage Mu
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Insertion sequence (IS)
elements
Simplest type of transposable element found inbacterial chromosomes and plasmids
Encode only genes for mobiliation and insertion
!ange in sie from "#$ bp to % kb
IS& first identified in E. coli 's glactose operon is"#$ bp long and is present ith *&+ copies in the
E. coli chromosome
Ends of all knon IS elements sho in,ertedterminal repeats (IT!s)
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Three different mechanisms
for transposition
0onser,ati,e transposition
!eplicati,e transposition
!etrotransposition
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Three different mechanisms
for transposition 0onser,ati,e transposition: elemen itu sdr
berpindah dari TK donor ke dalam TK target
!eplicati,e transposition: element moves acopy itself a copy of itself to a new site via a DNA
intermediate (lement memindahkan satu kopiny
sdr ke TK yg baru via DNA !antara")
!etrotransposition: The element makes an #NA
copy of itself which is re,ersed*transcribed into a
DNA co which is then inserted cDNA
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0onser,ati,e transposition
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!eplicati,e transposition
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!etrotransposition
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$eneration of short direct repeats flanking the
newly inserted element
This results for a staggered cut being made in
the DNA strands at the site of insertion
common feature of mobile elements
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Transposons (Tn)
Similar to IS elements but are morecomple1 structurally and carry additionalgenes
2 types of transposons3
0omposite transposons
.oncomposite transposons
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0omposite
transposons
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%&'# is an autonomous element while %&'* is non+autonomous
http://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.html
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99kb F (Fertility) Plasmid Genetic Map ( E. coli)
Insertion sequences (IS) assist in the
integration of transposons (Tn) into
homologus sites of recipients genome.
Different Hfr strains are produced as aresult
Replication & segregation genes of F plasmid
Genes for conjugative transfer
Origin of transfer
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0omposite Transposons
Tetracycline resistance is carried by atransposable element
The transposon is a composite transposoncomposed of %&+elements flanking an includedse,uence in this case containing an antibioticresistance gene
%&'# is an autonomous element while %&'* is non+autonomous
-omposite transposons probably evolved from %&elements by the chance location of a pair in closepro.imity to one another/ %nactivation of oneelement by mutation would not harm ability totranspose and would assure continued
http://opbs.okstate.edu/~melcher/mg/MGW3/MG32221.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32211.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://www.ndsu.nodak.edu/instruct/mcclean/plsc431/transelem/trans5.htmhttp://www.ndsu.nodak.edu/instruct/mcclean/plsc431/transelem/trans5.htmhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32211.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32221.html