1. Site-specific mutagenesis of M13 clones
i. Site-specific mutagenesis involves making a
predetermined change in the DNA sequence, unlike
random 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 to
know which plaque contains the mutated DNA.
iii. In Fig. 7.16, thymines in the M13 cloning vector are
replaced with uracils by propagating the phage in a
dUTPase- and uracil-N-glycosylase-deficient (Dur-Ung-)
host.
(i) When the double-stranded RF molecules are
synthesized from such template, the newly synthesized
strand contains thymines while the template strand still
contains uracil.

2. Site-specific mutagenesis of M13 clones
(ii) When these RFs are
transfected into
Ung+ E. coli strains,
the uracil-
containing
template strands are
preferentially
degraded by uracil-
N-glycosylase,
so that most of the
phage that survive
will be descended
from the mutated
complementary
strands.

3. Site-specific mutagenesis of M13 clones

4. III. Phage DNA replication - phage T4
2. Phage T4 – a complex phage with an icosahedral head and a
filamentous tail, and a linear, double-stranded DNA.
(1) Terminally redundant DNA – A DNA, usually a phage
genome, that has repeats at both ends, that is the
sequences at both ends are the same in the direct
orientation.
(2) Cyclically permuted genome – In a Cyclically permuted
genome, there are no unique ends. If a genome of such
phage is drawn as a circle, each genome starts from
somewhere on the circle and extends around the circle
until it return to the same place, so that individual genome
have different endpoints, but contain all of the genes.

5. III. Phage DNA replication - phage T4

6. III. Phage DNA replication - phage T4

7. III. Phage DNA replication - phage T4

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, using
RNA primers as usual.
ii. In stage 2, recombinational intermediates furnish the
primers for initiation, that is the leading strand of
replication is primed by recombinational intermediates
rather than by RNAs.
(i) Repeated rounds of strand invasion and replication
lead to very long branched concatemers which can
then packaged into phage heads.
(ii) Packaging Of DNA into head is initiated by cutting the
DNA by a terminase complex which remains attached
to the end. This complex then binds to the head at an
opening called the portal, and DNA begins to be
sucked into the head.
(iii) Each packaged genome is a different cyclic
permutation with different terminal redundancies.

9. III. Phage DNA replication – phage T4

10. IV. Lysogeny
Lytic 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 host
usually involves integration of phage genome into host DNA
prophage – integrated phage genome
lysogens (lysogenic bacteria)
infected bacterial host
temperate phages
phages able to establish lysogeny

12. Life cycle of λ phage

13. λ phage double-stranded DNA phage linear genome with cohesive ends
circularizes upon entry into host

14. Genetic map of λ phage

15. Pattern of gene expression

16. Very early events
Very early after infection of E. coli, RNA polymerase
transcrubes genes N and cro from different strands of the
DNA.

17. Early events
N protein, an anti-terminator turns on the early genes to the
left of N and to the right of cro.

18. The action of N protein
1. Transcription from promoter PL, RNA polymerase will encounter
nut ( N protein utilization) site:
(1) If no N protein, RNA polymerase will ignore the nut site and fall
off the DNA, releasing the mRNA when it reaches the downstream
stop signal.
(2) In the presence of N protein, RNA polymerase will pass over nut
and ignore the downstream stop signal.

19. Late lytic events
1. Protein Cro represses the transcription from PL and PR when it
binds to the operators between these two promoters.
2. Protein Q, also an anti-terminator recognizes Qut, lies very near
the beginning of the long transcript that initiates at P’R.
3. The Q-modified RNA polymerase transcribes the late genes into
a single long transcript.

20. A short segment of the  DNA molecule
1. This segment locates between cI and cro genes shown in
previous 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 needed
for finally establising lysogeny
2. PRE: promoter for repression establishment;
Pint: promoter for the genes leftward the int gene.

22. The lysogeny decision
1. Host proteases (Hf1A ) regulate the level of CII protein. Although CIII
protein 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 the
activities of bacterial proteases:
(1) Rich medium activates the proteases;
(2) Starvation has opposite effect.

23. Establishing lysogeny
CII-stimulated transcription on int whose promoter lies
within the xis gene

24. Establishing lysogeny
Retroregulation involved in hairpin structure, RNAaseIII and other
RNAase

25. Establishing lysogeny
Integration (recombination at att) has separated sib
from int.

26. Induction process by which phage reproduction is initiated
results in switch to lytic cycle
triggered by drop in levels of lambda repressor (even only 5 %)
caused by exposure to UV light and chemicals that cause DNA damage
excisionase
binds integrase
enables integrase to reverse integration process

27. Induction Repressor is cleaved between the amino acids alanine
and glycine located in the linker between repressor’s
domains.

28. Lysogenic conversion change in host phenotype induced by lysogeny
e.g., modification of Salmonella lipopolysaccharide structure
e.g., production of diphtheria toxin by Corynebacterium diphtheriae

29. V. Generalized transduction vs specialized transduction
Transduction is the transfer of the bacterial genes by
phages.
Bacterial genes are incorporated into a phage capsid
because of errors made during the phage life cycle.
The phage containing these genes then injects them
into another bacterium, completing the transfer.
There are two different kinds of transductions:
generalized and specialized.

30. Generalized transduction

31. Specialized transduction

32. Using phage DNA molecule as a cloning vector
Lambda ZAMP® II vector (of STRATAGENE)