Curs 7 - Genetica_bacteriana
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GENETICA BACTERIANA
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OBIECTIVE
• ORGANIZAREA GENOMULUI BACTERIAN
• STRUCTURA ADN
• MECANISME DE VARIABILITATE
• APLICATII
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GENOMUL BACTERIAN
• CROMOZOM
• PLASMIDE
• EPISOMI
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• 2000 m lungime
• 16 x 106 perechi de nucleotide
• 6000 gene
• Controleaza structurarea, cresterea si multiplicarea celulei
CROMOZOM
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• ADN extracromozomal, dublu catenar• Exista in 1 sau mai multe copii• Se replica autonom• Circulare (exceptie Borrelia)• Fixate la situsuri membranare de replicare• 2 Mbp (mega base pairs)• Dispensabile in economia celulei• Asigura supravietuirea celulei in conditii modificate de mediu• Codifica caractere de TULPINA
PLASMIDE
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Contin: - Determinanti genetici pentru autoreplicare
- Determinanti pt structuri si molecule exprimate in fenotipul bacterian- virulenta (hemolizine, enterotoxine, factori de colonizare)- enzime in cai metabolice particulare – degradare hidrocarburi- agenti antibacterieni (bacteriocine ex. colicine - factor Col,
antibiotice) - se transfera prin conjugare, bacteriofagi, transformare- rezistenta la UV, antibiotice – factor R (gene de rezistenta si gene
de transfer a rezistentei), se transfera prin conjugare sau prin bacteriofagi
- Determinanti pt structuri implicate in Conjugare (CONJUGONI) – factor F- Celule F-, F+, HFr, F’
• Exista plasmide criptice - nu se stie ce caractere codifica
PLASMIDE
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• Plasmide integrate liniar in cromozomul bacterian• Profagi (fagi temperati) LIZOGENIE
EPISOMI
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James Dewey WatsonFrancis Harry Compton Crick
Maurice Hugh Frederick Wilkins
The Nobel Prize in Physiology or Medicine 1962
"for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material"
STRUCTURA ADN(acid dezoxiribonucleic)
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STRUCTURA ADN
James Watson and Francis Crick with their DNA model at the Cavendish Laboratories in 1953. Photograph by C. Barrington Brown. Courtesy C. Barrington Brown. To request permission to use this photo,
please visit the Science Photo Library Web site at www.sciencephoto.com.
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STRUCTURA ADN
Macromolecula - 2 catene polinucleotidice- Antiparalele- Complementare- Rasucite in dublu helix
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STRUCTURA ADN
Macromolecula - 2 catene polinucleotidice- Antiparalele- Complementare- Rasucite in dublu helix
2 nm
- 10 perechi nucleotide- 3,4 nm
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NUCLEOTID
• Baza azotata purinica - Adenina – A
- Guanina – Gpirimidinica
- Timina - T- Citozina – C
• Pentoza (dezoxiriboza)• Acid fosforic
BAZA AZOTATA
DEZOXIRIBOZA
FOSFAT
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BAZE AZOTATE
A + TG + C
Raportul difera de la specie la specie
ADENINA GUANINA
CITOZINA TIMINA
PURINE
PIRIMIDINE
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BAZE AZOTATE
Legaturi de H2 – A si T3 – C si G
- Stabile pana la 65oC- Denaturarecompleta la 90oC
Rezulta 2 catenecomplementare
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PROPRIETATI ADN
• REPLICON – unitata genetica discreta (molecula ADN) care se replica •Ex. Cromozom, plasmid,
•AUTOREPLICARE – CONSERVAREA INFORMATIEI SI TRANSMITERE : EREDITATE si VARIABILITATE •TRANSCRIERE (urmata de TRANSLATIE) EXPRIMAREA INFORMATIEI: ADNARNm PROTEINE
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REPLICAREA SEMICONSERVATIVA
Cairns, J.P.: Cold Spring Harbor Symposia on Quantitative Biology 28:44, 1963
AUTORADIOGRAFIEReplicare cromozom E. coli
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"RNA World hypothesis" afirmă că înainte de apariţia primei celule, ARN a fost dominant şi probabil singura formă de viaţă.
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MECANISME DE VARIABILITATE – MUTATII
• SUBSTITUTII• INVERSII• INSERTII• DELETII
• SPONTANE• INDUSE
• PUNCTIFORME• EXTINSE
• RETROMUTATII– TRANSFER si RECOMBINARE GENETICA
• TRANSFORMARE• TRANSDUCTIE (BACTERIOFAGI)
– SPECIALIZATA– GENERALIZATA– CONVERSIA LIZOGENICA
• CONJUGARE
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MUTATIA
Modificare in secventa nucleotidica a unei gene in cursul replicarii GENA ALELA – ocupa acelasi locus genetic ca si gena originara
EROARE DE REPLICARE
Eveniment• RAR rata mutatiei 10-6 – 10-9 , creste sub
actiunea agentilor mutageni (radiatii, s. chimice)• IMPREVIZIBIL• SPECIFIC• INDEPENDENT• DISCONTINUU• STABIL (exceptie retromutatiile)
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SUBSTITUTII SI INVERSII- afecteaza 1 codon sunt punctiforme
- Apare un codon sinonim lant polipeptidic nemodificat- Apare un codon nou alt aminoacid
Mutatie muta
Functie modificata sau pierduta
- Apare un codon nonsens polipeptid incomplet
INSERTII SI DELETII (I/D)
- un nucleotid (micro-I/D) proteina modificata
- mai multe (macro-I/D) pierdere functie sau letale
3 NUCLEOTIDE = 1 CODON = 1 Aminoacid3 NUCLEOTIDE = 1 CODON = 1 Aminoacid
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TRANSFER si RECOMBINARE GENETICA
1. TRANSFORMARE
2. CONJUGARE
3. TRANSDUCTIE (BACTERIOFAGI)– SPECIALIZATA– GENERALIZATA– CONVERSIA LIZOGENICA
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TRANSFORMARE
• Transfer prin acceptarea de ADN liber provenit de la bacteria donor• Griffith, 1928, pneumococi virulenti la soareci• Conditii:
– celula acceptor sa fie “competenta” (sinteza molecule proteice de legare si translocare membranara a ADN)– ADN transformant bicatenar, 1% din genom
• Rezulta TRANSFORMANT
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CONJUGARE
• Transfer prin contact fizic intre 2 bacterii cu polarizare sexuala• sens unic, de la bacteria donor la cea acceptor• bacteria donor ramane viabila• DONORUL are plasmide conjugative ce contin genele tra – operon
– Replicare de transfer
– pili sexuali (la bacterii gram negative) – factor F
• se transfera conjugonul, alte plasmide neconjugative si gene cromozomale (donorii HFr)• Rezulta TRANSCONJUGANT
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TRANSDUCTIE
• Transfer ADN prin intermediul unui bacteriofag• in general fagi temperati, care includ in cursul morfogenezei capsidei alaturi de ADN fagic si ADN cromozomal (2%) sau plasmidic al bacteriei donatoare• Rezulta TRANSDUCTANT• ADN-ul transferat se integreaza in cromozomul celulei acceptoare – lizogenizare (conversie lizogenica) – apare un caracter nou codificat de gene fagice nereprimate; ex tox difterica, Ag O Salmonella
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RECOMBINAREA GENETICA
• Rearanjarea genomurilor donor si acceptor cu formarea a doua genomuri noi, hibride
• in general fagi temperati, care includ in cursul morfogenezei capsidei alaturi de ADN fagic si ADN cromozomal (2%) sau plasmidic al bacteriei donatoare• Rezulta TRANSDUCTANT• ADN-ul transferat se integreaza in cromozomul celulei acceptoare – lizogenizare (conversie lizogenica) – apare un caracter nou codificat de gene fagice nereprimate; ex tox difterica, Ag O Salmonella
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APLICATII
• HIBRIDIZAREA – se demonstreaza inrudirea sau identitatea a doua microorganisme pe baza omologiei secventelor nucelotidice taxonomia bacteriana – cu sonde de acizi nucleici marcati radioactiv
• AMPLIFICAREA GENICA PCR (Kary B. Mullis)
• INGINERIA GENETICA
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Kary B. Mullis