Transcript Site-specific mutagenesis of M13 clones

Site-specific mutagenesis of M13
clones
(5) 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. 7.15 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.
Site-specific mutagenesis of M13 clones
(ii) When these RFs are
transfected into
Ung+ E. coli strains,
the uracilcontaining
template strands are
preferentially
degraded by uracilN-glycosylase,
so that most of the
phage that survive
will be descended
from the mutated
complementary
strands.
Site-specific mutagenesis of M13
clones
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.
III. Phage DNA replication - phage T4
III. Phage DNA replication - phage T4
III. Phage DNA replication - phage T4
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.
III. Phage DNA replication – phage T4
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.
Temperate Bacteriophages and
Lysogeny
• 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
Life cycle of λ phage
λ phage
• doublestranded DNA
phage
• linear genome
with cohesive
ends
– circularizes
upon entry into
host
Genetic map of λ phage
Pattern of gene expression
Very early events
Very early after infection of E. coli, RNA polymerase
transcrubes genes N and cro from different strands of the
DNA.
Early events
N protein, an anti-terminator turns on the early genes to the
left of N and to the right of cro.
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.
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.
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
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.
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.
Establishing lysogeny
CII-stimulated transcription on int whose promoter lies
within the xis gene
Establishing lysogeny
Retroregulation involved in hairpin structure, RNAaseIII and other
RNAase
Establishing lysogeny
Integration (recombination at att) has separated sib
from int.
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
Induction
Repressor is cleaved between the amino acids alanine
and glycine located in the linker between repressor’s
domains.
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
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.
Generalized transduction
Specialized transduction
Using phage DNA molecule as a cloning
vector
Lambda ZAMP® II vector (of STRATAGENE)