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BMB400, 4_4_04 1 1 Regulation of Gene Expression in Bacteriophage BACTERIOPHAGE 2 Phage – A virus, it is one kind of vector. Bacteriophage – a phage (virus)

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Presentasi berjudul: "BMB400, 4_4_04 1 1 Regulation of Gene Expression in Bacteriophage BACTERIOPHAGE 2 Phage – A virus, it is one kind of vector. Bacteriophage – a phage (virus)"— Transcript presentasi:

1 BMB400, 4_4_04 1 1 Regulation of Gene Expression in Bacteriophage BACTERIOPHAGE 2 Phage – A virus, it is one kind of vector. Bacteriophage – a phage (virus) that infects bacteria Simple “organism” – Basic structure consists of DNA or RNA surrounded by a protein coat – Vary greatly in shape and size

2 BMB400, 4_4_04 2 3 4

3 Pre-infection Post-infection 5 BMB400, 4_4_04 Visible effects on DNA during viral infection T4 phage DNA Phage cannot replicate on their own – Infect a host cell Once inside the host cell, they either remain quiescent (as prophage) or use the cell’s replication machinery to produce many copies of themselves – This replication may or may not lead to destruction of the host cell (lytic) 6 3

4 BMB400, 4_4_04 4 7 Why are phage important? – They cause diseases – They are useful tools in molecular biology Phage are often used as “replacement vectors” - Part of their DNA is removed and replaced with other DNA of interest (e.g., human DNA) - This recombinant DNA is repackaged into phage; phage infect host bacteria - Phage use bacteria to replicate, therefore replicating the DNA of interest Bacteriophage 8  bacteriophage as a replacement vector

5 9 toxin prophage Corynebacterium diptheriaea Phage produces diptheria toxin This is what makes people sick insertion site C.diptheriaea without phage strain produces no toxin Does not cause diptheria BMB400, 4_4_04 Phage conversion Dormant prophage – integrated bacteriophage – carries genes that alter the phenotype of the microbe - best examples are pathogens and toxin production Lysis or Lysogeny Lysis: Infection by phage produces many progeny and breaks open (lyses) the host bacterium (bacteriophage T4) Lysogeny: After infection, the phage DNA integrates into the host genome and resides there passively – No progeny – No lysis of the host Bacteriophage lambda can do either. 10 5

6 BMB400, 4_4_04 6 11 The lytic pathway of bacteriophage infection 12 Phage life cycle: Lytic vs. Lysogenic pathways

7 7 13 BMB400, 4_4_04 Elements of lysogeny The phage genome integrated into the host bacterial genome is a prophage. Bacterium carrying the prophage is a lysogen. Lysogens are immune to further infection by similar phage because the phage functions are repressed in trans. Induction of the lysogen leads to excision of the prophage, replication of the phage DNA, and lysis of the host bacterium. Site-specific recombination in bacteriophage. X-Ray structure of integration host factor (IHF) in complex with a 35-bp target DNA. 14

8 BMB400, 4_4_04 8 15 Early translation – products are usually enzymes to shut down host and synthesize viral nucleic acids Late transcription/translation produces structural proteins The Bacteriophage T4 16 Lytic virus T4 has linear DNA with specific hydroxymethylcytosine, instead of cytosine, resistent to endonuclease of the host Produce enzyme in DNA replication that similar to those host enzyme in large amount Encode three group protein (early, middle and late proteins)

9 BMB400, 4_4_04 9 17 A genetic map of bacteriophage. 18 The Bacteriophage lambdha Lytic and lysogenic virus Genome: double strand DNA, has cohesive ends (cos) Early transcription begin on PL and PR to give the product of N and Cro protein (regulator) N antiterminator (produce O, P (induce replication), Cll and Clll The antiterminator is not effective before termination before the Q gene (little Q protein is produced, Q is antiterminator to transcribe the late genes)) Cro regulator between lytic dand lysogenic pathways by blocking PL and PR (binding OL and OR (site 1, 2, 3) CII (aktivator protein by activate PE to express CI and integrase) and CIII is stabilization of CII.

10 10 20 BMB400, 4_4_04 lytic pathway lysogeny pathway 19 Lytic and lysogeny pathways Transcription is regulated by Repression (initiation) Activation (initiation) Anti-termination (elongation)

11 21 operator oR1 oR2 BMB400, 4_4_04 Repressor structure  repressor (CI) is a dimer; monomer has 236 amino acids. C-terminal domain: protein-protein interaction; dimerization and cooperativity Connector N-terminus: DNA binding; Helix-Turn-Helix motif operator  repressor can bind cooperatively to operator sub-sites. Cro structure Cro is a dimer Monomer has 66 amino acids Has only one protein domain Does NOT display cooperativity DNA binding; Helix-Turn-Helix motif; also dimerization operator oR3 22 11

12 Page 1409 Voet Biochemistry 3e© 2004 John Wiley & Sons, Inc. BMB400, 4_4_04 12 Theoretical repression curves for  p R (right) and a simple repressor–operator system such as that of the lac operon (left). 24 Bacteriophage : Events leading to lysis lysis or lysogeny (cI or Cro?) ? Both lysis and lysogeny: – P R, P L, active : synthesize N, Cro – antitermination by N : synthesize cIII, cII, Q Lysis: – Low [Cro] : binds O R 3, shuts off P RM (cI) – High [Cro] : shuts off P R and P L – antitermination by N protein to produce Q Protein which activate late genes

13 BMB400, 4_4_04 13 25 Factors favoring lysogeny High multiplicity of infection – More templates produce more of the CII protein, which stimulates P RE. – Phage sense that it is too crowded. Poor nutrient conditions for host – Low [glucose] leads to increase in [cAMP]. – Increased [cAMP] will repress the host gene hflA. – Less HflA (a protease) leads to less degradation of the CII protein. 26 MOLECULAR GENETIK ANALYSIS

14 BMB400, 4_4_04 14 27 Material Genetik Cetak biru dari semua jenis makhluk hidup sebagian besar berupa DNA 28 QuickTimeý Dz TIFFÅiLZWÅj êLí£ÉvÉçÉOÉâÉÄ Ç™Ç±ÇÃÉsÉNÉ`ÉÉǾå©ÇÈǞǽDžÇÕïKóvÇ­Ç ÅB Untai tunggal DNA adalah rangkaian nukleotida dimana untai baru selalu dilekatkan pada posisi 3`

15 15 BMB400, 4_4_04 SEKUENSING Prinsip Sekuensing Sintesis DNA in vitro dengan DNA Polymerase dan penggunaan 2`, 3` dideoxynucleotida. Hilangnya molekul 3`-OH menyebabkan berthentinya reaksi sintesis untai DNA pada point dimana molekul dideoxynukleotida disintesis, karena tidak ada reaksi yang bisa dilakukan tanpa adanya molekul tersebut Sintesis untai DNA baru diawali pada posisi spesifik dengan menggunakan primer yang menempel pada ujung 3` pada daerah yang akan disekuensing Reaksi sintesis DNA menggunakan deoxynucleotida dan dideoxynucleotida akan menghasilkan beberapa fragment DNA yang memiliki residu nukcleotida yang serupa Perbedaan ukuran fragment DNA dapat dianalisis dengan polyacrilamide gel elektroforesis Dua Teknik Sekuensing: Metode Enzymatic (Metode Sanger) Metode Degradasi Kimiawi (Metode Maxam - Gilbert) 29 30 5`3` 5`3` DNA Utas tunggal Primer 4 dNTP DNA Polymerase ddATP ddGTPddCTPddTTP Primer DNA baru dideoxynukleotida Sekuensing DNA Hasil PCR Kloning fragment PCR pada vektor Sekuensing PCR fragmen dengan Primer Universal yang komplemen dengan vektor Vektor UniversalPri mer

16 BMB400, 4_4_04 AGCT QuickTimeýDz TIFFÅiLZWÅjêLí£ÉvÉçÉOÉâÉÄ Ç™Ç±ÇÃÉsÉNÉ`ÉÉǾå©ÇÈǞǽDžÇÕïKóvÇ­ÇÅB 31 Sekuensing Fragmen DNA berukuran panjang Kloning fragment DNA pada vektor Sekuensing PCR fragmen dengan Primer Universal yang komplemen dengan vektor Sekuensing dengan penyusunan primer baru yang kompleme dengan hasil sekuensing dengan primer universal UniversalPri Vektor mer Primer 1 Primer 2 Vektor Primer 3 Vektor 32 16

17 BMB400, 4_4_04 17 33 Sekuensing Fragmen DNA Secara Random Purifikasi fragment DNA Digesti Fragmen DNA dengan sonikasi atau enzim restriksi Ligasi Fragment DNA pada Vektor Sekuensing DNA dengan universal primer Alignment sekuen fragmen DNA dari DNA target UniversalPri mer Purifikasi DNA Digesti Ligasi dan Sekuensing Alignment

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