logging in or signing up MRINAL suvichar Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 579 Category: Science & Tech.. License: All Rights Reserved Like it (1) Dislike it (0) Added: April 06, 2010 This Presentation is Public Favorites: 0 Presentation Description ABOUT A TYPE OF VIRUS CALLED LAMBDA BACTERIOPHAGE Comments Posting comment... By: hasankamal (21 month(s) ago) ma'm i m unable to download it.just sent me the ppt copy to my email id.-miracle.hasan@gmail.com. i ll be very thakful to u. Saving..... Post Reply Close Saving..... Edit Comment Close By: hasankamal (21 month(s) ago) ma'm plz do allow me to download this ppt .i need this urgently.plz. send this to my id miracle.hasan@gmail.com Saving..... Post Reply Close Saving..... Edit Comment Close Premium member Presentation Transcript A seminar onLIFE CYCLE OF LAMBDA PHAGE : 1 A seminar onLIFE CYCLE OF LAMBDA PHAGE BY: Ms. MRINAL NIKAM M.Sc. I DATE: 9 / 2 / 08 General properties of phages : 2 General properties of phages There are three basic phage structures: 1) filamentous 2) icosahederal head with no tail 3) icosahederal head with a tail Some phage have fewer than 10 genes and depend entirely on cellular functions, whereas, others have 30-100 genes which encode most of their proteins. A bacteriophage is an obligate bacterial parasite Each phage performs some minimal functions for survival - : 3 Each phage performs some minimal functions for survival - 1)Protection of its nucleic acid from environmental chemicals that could alter the molecule (break molecule or cause mutation) 2)Delivery of its nucleic acid to the inside of a bacterium. 3)Conversion of an infected bacterium into a phage -producing system , which yields a large number of progeny phage. 4)Release of progeny phage from an infected bacterium. Slide 4: 4 Phage life cycles fit into two distinct categories: lytic and lysogenic cycles During a lytic cycle , the viral genes immediately turn host cell into a virus-producing factory, and the cell soon lyses and releases its viral products. In the lysogenic cycle , the phage genome replicates without destroying the host cell Within the host, the virus’ circular DNA engages in either the lytic or lysogenic cycle. Temperate phages , like phage lambda, use both lytic and lysogenic cycles. Regardless of the type of virus, the parasite diverts the host cell’s resources for viral production : 5 Regardless of the type of virus, the parasite diverts the host cell’s resources for viral production A phage uses the protein synthesizing system, amino acids, and energy generating systems of host cells, hence can multiply only in a metabolizing bacteria. The host cell provides: Nucleotides for nucleic acid production Enzymes Ribosomes tRNA Amino acids ATP The phage Lambda : 6 The phage Lambda The lambda phage has an icosahederal head with a long tail & single tail fiber. Lambda has a ds DNA of 48,502 bp for ~ 55 proteins, injected into E.coli cell as linear molecule. Total genes-46, 14 are non-essential for lytic cycle, 7 are non-essential for both cycles. DNA has "sticky ends". ~20 bases at each end are single stranded. The two ends are complementary, DNA can circularize on entry inside E.coli cell. Lambda phage is a temperate phage & has two possible outcomes: lytic infection or lysogeny. Slide 7: 7 Lambda has 3 promoter sites. Two of these allow transcription of lytic genes. Other promoter leads to transcription of a repressor protein (Lambda repressor) that can bind to the two lytic promoters, block all lytic genes. Repressor does not block its own promoter, so cell continues to synthesize small quantity of repressor (~10-20 copies/cell).One early lambda protein is Integrase; causes specific recombination event at region where both Lambda DNA and host DNA have same 13 base pairs. (homologous sequence). If Lambda repressor is expressed before transcription of late lytic pathway genes occurs, then Lambda remains in host DNA indefinitely, gets replicated just like host genes. Induction: under "nasty" environmental conditions where DNA damage occurs (e.g. UV light or certain chemicals), can mutate repressor, make it ineffective. If this occurs, lytic promoters are no longer blocked, lytic genes get transcribed and translated, and cell becomes phage factory, leads to lytic production of lambda viruses. Lambda reproduction : 8 Lambda reproduction Infects an E. coli cell by injecting its DNA The lambda DNA molecule forms a circle. Lytic or lysogenic cycles begin In a lytic cycle, the cell is turned into a lambda producing factory, the cell lyses and releases its products. In a lysogenic cycle, the viral genome is incorporated into by genetic recombination into a specific site on the host cell’s chromosome. It is now known as a prophage. Every time the E. coli divides, it replicates the phage DNA and passes it along to the daughter cells. This enables the phage to replicate without destroying the host. The phages may at some point in time become active phages that lyse their host cell and releasing infectious particles. Slide 9: 9 The lytic cycle in lambda phage Virus-producing factory : Virus-producing factory Slide 11: 11 The lambda phage demonstrates the cycles of a temperate phage. Slide 12: 12 Phage lambda early genes Immediate early genes N -Antiterminator -Acts at nut sites -Allows transcription to proceed to delayed early genes Cro -Prevents synthesis of repressor (lytic cycle) -Turns off expression of immediate early genes Slide 13: 13 Map of lambda phage Slide 14: 14 Slide 15: 15 Repressor determines lysogeny and Cro determines lytic cycle, both Cro and repressor are expressed at delayed early stage Slide 16: 16 The structure of the Lambda DNA (a) in the phage capsid and (b) after circularization in the cytoplasm. The DNA circularizes via the cos site. Slide 17: 17 The location of the six major promoters on the Lambda genome and the direction in which they specify mRNA production. Slide 18: 18 (a) Transcription from PL leads to the production of N protein. Transcription from PR leads to Cro protein. From PL, N and CIII proteins will be produced. From PR; Cro, CII, O, P, and Q proteins will be produced. (c) N binds to the nutL site on the DNA. In conjunction with four bacterial proteins, NusA, NusB, NusD, and NusE, N allows RNA polymerase to read through the terminator tL. The first transcription and translation events that take place on the lambda genome after infection Slide 19: 19 At this point, all of the players needed to make the lytic–lysogenic decision have been made. CII and CIII are needed for lysogenic growth. Cro and Q are needed for lytic growth. The O and P proteins are used for replicating the Lambda DNA. Slide 20: 20 (a) CI leads to lysogeny and Cro leads to lytic growth. (b) Both CI and Cro bind to two operator regions, OR and OL. OR overlaps with both PR and PRM. OL overlaps with PL. (c) OR is required for the switch between developmental pathways. It is composed of three 17 base pair sequences called OR1, OR2, and OR3. (d) CI binds to OR1 first then OR2. When CI binds to OR, it represses transcription from PR and activates it from PRM. CI binding to OR is actually required for PRM to be activated. CI binding leads to lysogeny. (e) Cro also binds to OR1, OR2, and OR3 but in the opposite order from CI. Cro binding to OR3 inhibits PRM and leads to lytic growth.. Lytic-lysogeny decision Slide 21: 21 The CII protein is the major player in the switch between lytic and lysogenic growth. CII is unstable and rapidly degraded by the host encoded HflA protease. Inactive CII leads to lytic growth. CII can be protected by the phage encoded CIII protein. Active CII leads to lysogenic growth. Lysogenic pathway Slide 22: 22 Lambda recombines into the chromosome using a specific site on the phage called attP and a specific site on the bacterial chromosome called attB. When the lambda DNA is in the chromosome, it is bounded by attL and attR, which are hybrid attP/attB sites. Site specific recombination Slide 23: 23 If enough of the Q protein accumulates in the cell, RNA polymerase will continue its transcription from a third promoter, PR’ , located in front of the Q gene .This extends transcription into the late genes located downstream of Q. The Q protein which is made from PR’ when N is present is a second anti-termination protein. It acts on the qut site and allows transcription through tR’. Q is necessary for synthesis of the head and tail genes. The lytic pathway Slide 24: 24 Lambda has two modes of DNA replication: (a) Theta replication and (b) rolling circle replication. Theta replication occurs early in infection and rolling circle replication occurs late in infection. Rolling circle replication produces concatamers for packaging into phage heads. DNA replication during lytic pathway Lytic and lysogenic pathways are interlocked : 25 Lytic and lysogenic pathways are interlocked Slide 26: 26 The assembly pathway (a) The initiator structure for the head is composed of the B, C, and Nu3 proteins. (b) E, the major head protein, is added to this structure. Nu3 is degraded, B is cleaved to a smaller form (B*), and E and C are fused and cleaved at a new position to form X1 and X2. This forms the immature phage head. (c) The immature phage head is now ready for taking a DNA from a concatamer. The D protein is added to the capsid at this point. (d) Packaging starts at a cos site and proceeds to the next cos site. (e) The DNA is inserted into the capsid and sealed inside by the W and FII proteins. (f) Tails are added to the full capsid to form a phage. Slide 27: 27 Induction of lambda When a lambda lysogen is treated with ultraviolet light ~45 minutes later the cells lyse and release phage!! Slide 28: 28 Genetic strategies were developed to increase the yield of recombinant phage in DNA libraries to make up for the low ratio of insert to vector DNA used in the initial ligation reactions. SIGNIFICANCE : 29 SIGNIFICANCE Lambda phage is used in Cloning cDNA Libraries Genomic Libraries Screening for Carcinogens Slide 30: 30 Insertional cloning into the cI gene of the lambda-gt10 cDNA cloning vector (DNA inserts of ~1-5 kb) can be selected in hfl (high frequency of lysogeny) mutant strains of E. coli. In hflA strains of E. coli, expression of the lambda cII gene is elevated, resulting in transcriptional induction of the lambda cI repressor gene which promotes lysogeny. Disruption of the lambda cI coding sequence by DNA insertion into the unique EcoRI site of the lambda gt10 cDNA cloning vector, blocks the lysogenic pathway leading to cell lysis and plaque formation. Strategy for cDNA libraries of lamda Slide 31: 31 Replacement of lambda DNA containing the red and gam genes in the lambda EMBL3 genomic DNA cloning vector with BamHI compatible DNA inserts of ~10-20 kb, permits lytic growth of recombinant phage in E. coli strains containing the P2 bacteriophage lysogen. Strategy for genomic DNA libraries References: : 32 References: Maloy, S.R., Cronan, J.E. & Freifelder, D.(1994) Lytic growth of Phage in: Microbial Genetics, 2nd edition, Narosa Publishing House, New Delhi. P. 375-388 Maloy, S.R., Cronan, J.E. & Freifelder, D.(1994) Lysogeny in: Microbial Genetics, 2nd edition, Narosa Publishing House, New Delhi. P.392-416 Maloy, S.R., Cronan, J.E. & Freifelder,D.(1994) Phage biology in: Microbial Genetics, 2nd edition, Narosa Publishing House, New Delhi. P. 92-108 Trun, N. & Trempy, J. (2001) Bacteriophage in: Fundamental bacterial genetics, International edition, Blackwell Publishing House. NY. P.105-118 Websites: www.amgarizona.com www.blackwellpublishing.com You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
MRINAL suvichar Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 579 Category: Science & Tech.. License: All Rights Reserved Like it (1) Dislike it (0) Added: April 06, 2010 This Presentation is Public Favorites: 0 Presentation Description ABOUT A TYPE OF VIRUS CALLED LAMBDA BACTERIOPHAGE Comments Posting comment... By: hasankamal (21 month(s) ago) ma'm i m unable to download it.just sent me the ppt copy to my email id.-miracle.hasan@gmail.com. i ll be very thakful to u. Saving..... Post Reply Close Saving..... Edit Comment Close By: hasankamal (21 month(s) ago) ma'm plz do allow me to download this ppt .i need this urgently.plz. send this to my id miracle.hasan@gmail.com Saving..... Post Reply Close Saving..... Edit Comment Close Premium member Presentation Transcript A seminar onLIFE CYCLE OF LAMBDA PHAGE : 1 A seminar onLIFE CYCLE OF LAMBDA PHAGE BY: Ms. MRINAL NIKAM M.Sc. I DATE: 9 / 2 / 08 General properties of phages : 2 General properties of phages There are three basic phage structures: 1) filamentous 2) icosahederal head with no tail 3) icosahederal head with a tail Some phage have fewer than 10 genes and depend entirely on cellular functions, whereas, others have 30-100 genes which encode most of their proteins. A bacteriophage is an obligate bacterial parasite Each phage performs some minimal functions for survival - : 3 Each phage performs some minimal functions for survival - 1)Protection of its nucleic acid from environmental chemicals that could alter the molecule (break molecule or cause mutation) 2)Delivery of its nucleic acid to the inside of a bacterium. 3)Conversion of an infected bacterium into a phage -producing system , which yields a large number of progeny phage. 4)Release of progeny phage from an infected bacterium. Slide 4: 4 Phage life cycles fit into two distinct categories: lytic and lysogenic cycles During a lytic cycle , the viral genes immediately turn host cell into a virus-producing factory, and the cell soon lyses and releases its viral products. In the lysogenic cycle , the phage genome replicates without destroying the host cell Within the host, the virus’ circular DNA engages in either the lytic or lysogenic cycle. Temperate phages , like phage lambda, use both lytic and lysogenic cycles. Regardless of the type of virus, the parasite diverts the host cell’s resources for viral production : 5 Regardless of the type of virus, the parasite diverts the host cell’s resources for viral production A phage uses the protein synthesizing system, amino acids, and energy generating systems of host cells, hence can multiply only in a metabolizing bacteria. The host cell provides: Nucleotides for nucleic acid production Enzymes Ribosomes tRNA Amino acids ATP The phage Lambda : 6 The phage Lambda The lambda phage has an icosahederal head with a long tail & single tail fiber. Lambda has a ds DNA of 48,502 bp for ~ 55 proteins, injected into E.coli cell as linear molecule. Total genes-46, 14 are non-essential for lytic cycle, 7 are non-essential for both cycles. DNA has "sticky ends". ~20 bases at each end are single stranded. The two ends are complementary, DNA can circularize on entry inside E.coli cell. Lambda phage is a temperate phage & has two possible outcomes: lytic infection or lysogeny. Slide 7: 7 Lambda has 3 promoter sites. Two of these allow transcription of lytic genes. Other promoter leads to transcription of a repressor protein (Lambda repressor) that can bind to the two lytic promoters, block all lytic genes. Repressor does not block its own promoter, so cell continues to synthesize small quantity of repressor (~10-20 copies/cell).One early lambda protein is Integrase; causes specific recombination event at region where both Lambda DNA and host DNA have same 13 base pairs. (homologous sequence). If Lambda repressor is expressed before transcription of late lytic pathway genes occurs, then Lambda remains in host DNA indefinitely, gets replicated just like host genes. Induction: under "nasty" environmental conditions where DNA damage occurs (e.g. UV light or certain chemicals), can mutate repressor, make it ineffective. If this occurs, lytic promoters are no longer blocked, lytic genes get transcribed and translated, and cell becomes phage factory, leads to lytic production of lambda viruses. Lambda reproduction : 8 Lambda reproduction Infects an E. coli cell by injecting its DNA The lambda DNA molecule forms a circle. Lytic or lysogenic cycles begin In a lytic cycle, the cell is turned into a lambda producing factory, the cell lyses and releases its products. In a lysogenic cycle, the viral genome is incorporated into by genetic recombination into a specific site on the host cell’s chromosome. It is now known as a prophage. Every time the E. coli divides, it replicates the phage DNA and passes it along to the daughter cells. This enables the phage to replicate without destroying the host. The phages may at some point in time become active phages that lyse their host cell and releasing infectious particles. Slide 9: 9 The lytic cycle in lambda phage Virus-producing factory : Virus-producing factory Slide 11: 11 The lambda phage demonstrates the cycles of a temperate phage. Slide 12: 12 Phage lambda early genes Immediate early genes N -Antiterminator -Acts at nut sites -Allows transcription to proceed to delayed early genes Cro -Prevents synthesis of repressor (lytic cycle) -Turns off expression of immediate early genes Slide 13: 13 Map of lambda phage Slide 14: 14 Slide 15: 15 Repressor determines lysogeny and Cro determines lytic cycle, both Cro and repressor are expressed at delayed early stage Slide 16: 16 The structure of the Lambda DNA (a) in the phage capsid and (b) after circularization in the cytoplasm. The DNA circularizes via the cos site. Slide 17: 17 The location of the six major promoters on the Lambda genome and the direction in which they specify mRNA production. Slide 18: 18 (a) Transcription from PL leads to the production of N protein. Transcription from PR leads to Cro protein. From PL, N and CIII proteins will be produced. From PR; Cro, CII, O, P, and Q proteins will be produced. (c) N binds to the nutL site on the DNA. In conjunction with four bacterial proteins, NusA, NusB, NusD, and NusE, N allows RNA polymerase to read through the terminator tL. The first transcription and translation events that take place on the lambda genome after infection Slide 19: 19 At this point, all of the players needed to make the lytic–lysogenic decision have been made. CII and CIII are needed for lysogenic growth. Cro and Q are needed for lytic growth. The O and P proteins are used for replicating the Lambda DNA. Slide 20: 20 (a) CI leads to lysogeny and Cro leads to lytic growth. (b) Both CI and Cro bind to two operator regions, OR and OL. OR overlaps with both PR and PRM. OL overlaps with PL. (c) OR is required for the switch between developmental pathways. It is composed of three 17 base pair sequences called OR1, OR2, and OR3. (d) CI binds to OR1 first then OR2. When CI binds to OR, it represses transcription from PR and activates it from PRM. CI binding to OR is actually required for PRM to be activated. CI binding leads to lysogeny. (e) Cro also binds to OR1, OR2, and OR3 but in the opposite order from CI. Cro binding to OR3 inhibits PRM and leads to lytic growth.. Lytic-lysogeny decision Slide 21: 21 The CII protein is the major player in the switch between lytic and lysogenic growth. CII is unstable and rapidly degraded by the host encoded HflA protease. Inactive CII leads to lytic growth. CII can be protected by the phage encoded CIII protein. Active CII leads to lysogenic growth. Lysogenic pathway Slide 22: 22 Lambda recombines into the chromosome using a specific site on the phage called attP and a specific site on the bacterial chromosome called attB. When the lambda DNA is in the chromosome, it is bounded by attL and attR, which are hybrid attP/attB sites. Site specific recombination Slide 23: 23 If enough of the Q protein accumulates in the cell, RNA polymerase will continue its transcription from a third promoter, PR’ , located in front of the Q gene .This extends transcription into the late genes located downstream of Q. The Q protein which is made from PR’ when N is present is a second anti-termination protein. It acts on the qut site and allows transcription through tR’. Q is necessary for synthesis of the head and tail genes. The lytic pathway Slide 24: 24 Lambda has two modes of DNA replication: (a) Theta replication and (b) rolling circle replication. Theta replication occurs early in infection and rolling circle replication occurs late in infection. Rolling circle replication produces concatamers for packaging into phage heads. DNA replication during lytic pathway Lytic and lysogenic pathways are interlocked : 25 Lytic and lysogenic pathways are interlocked Slide 26: 26 The assembly pathway (a) The initiator structure for the head is composed of the B, C, and Nu3 proteins. (b) E, the major head protein, is added to this structure. Nu3 is degraded, B is cleaved to a smaller form (B*), and E and C are fused and cleaved at a new position to form X1 and X2. This forms the immature phage head. (c) The immature phage head is now ready for taking a DNA from a concatamer. The D protein is added to the capsid at this point. (d) Packaging starts at a cos site and proceeds to the next cos site. (e) The DNA is inserted into the capsid and sealed inside by the W and FII proteins. (f) Tails are added to the full capsid to form a phage. Slide 27: 27 Induction of lambda When a lambda lysogen is treated with ultraviolet light ~45 minutes later the cells lyse and release phage!! Slide 28: 28 Genetic strategies were developed to increase the yield of recombinant phage in DNA libraries to make up for the low ratio of insert to vector DNA used in the initial ligation reactions. SIGNIFICANCE : 29 SIGNIFICANCE Lambda phage is used in Cloning cDNA Libraries Genomic Libraries Screening for Carcinogens Slide 30: 30 Insertional cloning into the cI gene of the lambda-gt10 cDNA cloning vector (DNA inserts of ~1-5 kb) can be selected in hfl (high frequency of lysogeny) mutant strains of E. coli. In hflA strains of E. coli, expression of the lambda cII gene is elevated, resulting in transcriptional induction of the lambda cI repressor gene which promotes lysogeny. Disruption of the lambda cI coding sequence by DNA insertion into the unique EcoRI site of the lambda gt10 cDNA cloning vector, blocks the lysogenic pathway leading to cell lysis and plaque formation. Strategy for cDNA libraries of lamda Slide 31: 31 Replacement of lambda DNA containing the red and gam genes in the lambda EMBL3 genomic DNA cloning vector with BamHI compatible DNA inserts of ~10-20 kb, permits lytic growth of recombinant phage in E. coli strains containing the P2 bacteriophage lysogen. Strategy for genomic DNA libraries References: : 32 References: Maloy, S.R., Cronan, J.E. & Freifelder, D.(1994) Lytic growth of Phage in: Microbial Genetics, 2nd edition, Narosa Publishing House, New Delhi. P. 375-388 Maloy, S.R., Cronan, J.E. & Freifelder, D.(1994) Lysogeny in: Microbial Genetics, 2nd edition, Narosa Publishing House, New Delhi. P.392-416 Maloy, S.R., Cronan, J.E. & Freifelder,D.(1994) Phage biology in: Microbial Genetics, 2nd edition, Narosa Publishing House, New Delhi. P. 92-108 Trun, N. & Trempy, J. (2001) Bacteriophage in: Fundamental bacterial genetics, International edition, Blackwell Publishing House. NY. P.105-118 Websites: www.amgarizona.com www.blackwellpublishing.com