Presentation on theme: "Site-specific mutagenesis of M13 clones"— Presentation transcript:
1 Site-specific mutagenesis of M13 clones i. Site-specific mutagenesis involves making apredetermined change in the DNA sequence, unlikerandom mutagenesis, where the change is made by chance.(Fig and Fig. 7.16)ii. The major difficulty for the method of Fig.7.15 is how toknow which plaque contains the mutated DNA.iii. In Fig. 7.16, thymines in the M13 cloning vector arereplaced with uracils by propagating the phage in adUTPase- and uracil-N-glycosylase-deficient (Dur-Ung-)host.(i) When the double-stranded RF molecules aresynthesized from such template, the newly synthesizedstrand contains thymines while the template strand stillcontains uracil.
2 Site-specific mutagenesis of M13 clones (ii) When these RFs aretransfected intoUng+ E. coli strains,the uracil-containingtemplate strands arepreferentiallydegraded by uracil-N-glycosylase,so that most of thephage that survivewill be descendedfrom the mutatedcomplementarystrands.
4 III. Phage DNA replication - phage T4 2. Phage T4 – a complex phage with an icosahedral head and afilamentous tail, and a linear, double-stranded DNA.(1) Terminally redundant DNA – A DNA, usually a phagegenome, that has repeats at both ends, that is thesequences at both ends are the same in the directorientation.(2) Cyclically permuted genome – In a Cyclically permutedgenome, there are no unique ends. If a genome of suchphage is drawn as a circle, each genome starts fromsomewhere on the circle and extends around the circleuntil it return to the same place, so that individual genomehave different endpoints, but contain all of the genes.
8 III. Phage DNA replication – phage T4 (3) T4 replication occurs in two stages illustrated in Fig. 7.18:i. In stage 1, replication initiates at specific origins, usingRNA primers as usual.ii. In stage 2, recombinational intermediates furnish theprimers for initiation, that is the leading strand ofreplication is primed by recombinational intermediatesrather than by RNAs.(i) Repeated rounds of strand invasion and replicationlead to very long branched concatemers which canthen packaged into phage heads.(ii) Packaging Of DNA into head is initiated by cutting theDNA by a terminase complex which remains attachedto the end. This complex then binds to the head at anopening called the portal, and DNA begins to besucked into the head.(iii) Each packaged genome is a different cyclicpermutation with different terminal redundancies.
10 IV. LysogenyLytic life cycle is not the life style of phages, some phages are able to maintain a stable relationship with host cell in which they neither multiply nor are lost from the cell. Such a phage is called lysogen-forming or temperate phage.Phage lambda (phage λ) is a kind of lysogen-forming or temperate phage.
11 Temperate Bacteriophages and Lysogeny nonlytic relationship between a phage and its hostusually involves integration of phage genome into host DNAprophage – integrated phage genomelysogens (lysogenic bacteria)infected bacterial hosttemperate phagesphages able to establish lysogeny
16 Very early eventsVery early after infection of E. coli, RNA polymerasetranscrubes genes N and cro from different strands of theDNA.
17 Early eventsN protein, an anti-terminator turns on the early genes to theleft of N and to the right of cro.
18 The action of N protein1. Transcription from promoter PL, RNA polymerase will encounternut ( N protein utilization) site:(1) If no N protein, RNA polymerase will ignore the nut site and falloff the DNA, releasing the mRNA when it reaches the downstreamstop signal.(2) In the presence of N protein, RNA polymerase will pass over nutand ignore the downstream stop signal.
19 Late lytic events1. Protein Cro represses the transcription from PL and PR when itbinds to the operators between these two promoters.2. Protein Q, also an anti-terminator recognizes Qut, lies very nearthe beginning of the long transcript that initiates at P’R.3. The Q-modified RNA polymerase transcribes the late genes intoa single long transcript.
20 A short segment of the DNA molecule 1. This segment locates between cI and cro genes shown inprevious slide.2. PRM: promoter of maintenance; PR: right promoter;cl: repressor gene; cro; cro gene; O; operator
21 Late events in establishing lysogeny 1. CII protein directs transcription of the two genes neededfor finally establising lysogeny2. PRE: promoter for repression establishment;Pint: promoter for the genes leftward the int gene.
22 The lysogeny decision1. Host proteases (Hf1A ) regulate the level of CII protein. Although CIIIprotein is not shown here, it protects CII.2. Other host proteins could regulate translation of the CII mRNA.3. Growth medium conditions of infected bacteria influence theactivities of bacterial proteases:(1) Rich medium activates the proteases;(2) Starvation has opposite effect.
23 Establishing lysogeny CII-stimulated transcription on int whose promoter lieswithin the xis gene
24 Establishing lysogeny Retroregulation involved in hairpin structure, RNAaseIII and otherRNAase
25 Establishing lysogeny Integration (recombination at att) has separated sibfrom int.
26 Induction process by which phage reproduction is initiated results in switch to lytic cycletriggered by drop in levels of lambda repressor (even only 5 %)caused by exposure to UV light and chemicals that cause DNA damageexcisionasebinds integraseenables integrase to reverse integration process
27 Induction Repressor is cleaved between the amino acids alanine and glycine located in the linker between repressor’sdomains.
28 Lysogenic conversion change in host phenotype induced by lysogeny e.g., modification of Salmonella lipopolysaccharide structuree.g., production of diphtheria toxin by Corynebacterium diphtheriae
29 V. Generalized transduction vs specialized transduction Transduction is the transfer of the bacterial genes byphages.Bacterial genes are incorporated into a phage capsidbecause of errors made during the phage life cycle.The phage containing these genes then injects theminto another bacterium, completing the transfer.There are two different kinds of transductions:generalized and specialized.