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Phix174 (NC)

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11 views28 pages

Phix174 (NC)

Uploaded by

trung hiếu trần
Copyright
© © All Rights Reserved
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Structure and Replication of

ɸX174

Dr. Nidhi S Chandra


Assistant Professor
Department of Microbiology
Ram Lal Anand College
Outline
• Classification
• Morphology of ΦX174
• Genetic Map of ϕX 174
• Overlapping Gene
• The Replication Cycle of ϕX174
Classification
• Phi X 174 is a virus that infects the
bacterium E. coli Hence phi X 174 is a
bacteriophage.
• Group II (ssDNA)
• Family : Microviridae
• Genus : Microvirus
• Species : φX174
Electron Micrograph of ΦX174
Morphology of ΦX174
• The capsid is icosahedral i.e. spherical.
• The genome consists of circular ssDNA.
• Genome size ranges from 4.6 to 6.1 kb.
• Diameter is 25 - 27 nm.
• Tailless icosahedral bacteriophage.
• Microviridae provided the first evidence
of overlapping genes.
• Studies on replication of these phages led
to the discovery of rolling circle replication
• 60 molecules of major
coat protein F(48.4KD)
form the capsid (25-27nm
in diameter).
• 5 molecules of G protein
(19.0KD) and 1 molecule
of H protein (35.8KD) form
spikes.
• Protein J (4.0KD) binds
to the phage genome for
condensation of DNA
during packeging.
Genetic Map of ϕX 174
The genome is a circular
ssDNA of 5386
nucleotides, coding for 11
proteins
• It contains 5386 nucleotides.
• These nucleotide encodes 11 proteins.
• Proteins A, A*, B, C, D, E, F, G, H, J, K are encoded.
• The 11 proteins encoded by phi X 174 DNA range in
the size from the A protein which contains 513 amino
acids, to the J protein, which contain only 38.
• The 11 protein together contain a total of 1986 amino
acids.
• It is encoded with 10 genes but generates 11 proteins.
This is because of overlapping gene.
Overlapping Gene
The gene is organized in such a way if one gene ends in a
particular position, the succeeding gene starts with few
nucleotide overlapping the terminal region of the first
gene. This is called overlapping genes, where reading of
genes are overlapped in their sequence.
For example,
the sequence …GAGCCGCAACTTC… Can be read in
three different reading frames-
…GAG CCG CAA CTT C … which encodes ….
...Glu-Pro- Gln-Leu… …G AGC CGC AAC TTC…
which encodes…. …Ser-Arg-Asn-Phe… …GA GCC
GCA ACT TC…which encodes …. …Ala-Ala-Thr…
• Gene A (viral genome replication) contains an internal
translation initiation site to encode protein A* (shutting
down host DNA synthesis).
• B ( capsid morphogenesis) is encoded within A in
different reading frame.
• K is at the end of gene A and extend into gene C (DNA
maturation).
• E (host cell lysis) is totally within D.
• The termination codon of D overlaps initiation codon
for J.
A Replication initiation,
A* Termination of host
DNA replication,
B Capsid morphogenesis,
C Phage maturation,
D Phage assembly,
E Host cell lysis
F Major coat protein,
G Major spike protein
H Minor spike protein
J Core protein
K Growth of phage
Applications
• It has been used as a model organism in many evolution
experiments.
• ΦX174 is regularly used as a positive control in DNA
sequencing due to its relatively small genome size in
comparison to other organisms, its relatively balanced
nucleotide content
• ΦX174 is also used to test the resistance of personal
protective equipment to blood borne viruses
• ΦX174 has also been modified to enable peptide display
(phage display) from the viral capsid G protein
• The ΦX174 genome was the first phage to be cloned in
yeast, which provides a convenient dry dock for genome
modifications
Subunit Composition of DNA Polymerase III Holoenzyme
ATTACHMENT OF PHAGE ΦX174
Phage φX174 recognizes the receptor lipopolysaccharide in the outer membrane
of rough strains of Enterobacteriaceae , such as E.coli and Salmonella
typhimurium , by the minor coat protein H.
ENTRY OF PHAGE ΦX174 GENOME
• The terminal spike protein, gp H spans the capsid. The outer part recognizes
the LPS receptor. The inner part of the H protein is responsible for the
injection of genome into the host cell.
• At least one H protein enters into the host cell with the viral DNA.
The Replication Cycle of ϕX174
Replication of ϕX174

• Replication of ϕX174 genome occurs in 3


stage
Stage 1
• Synthesis of (-) strand complementary to the
(+) strand of to form the replicative form (RF)
by host enzymes.
Stage 1
Replication of ϕX174
Stage 2
• Replication of the RF involves rolling circle
replication and requires phage encoded protein
A to synthesize new plus strands. These then
serve as a templates for minus strand synthesis
to generate the new RFs.
The RF to RF Pathway

https://www.youtube.com/watch?v=ZDqsojQ8A5k
Replication of ϕX174
Stage 3
• Asymmetric replication of progeny ssDNA
plus strand.
• RF synthesis continuous until sufficient
structural proteins have been synthesized and
assembled into empty precursor particle.
DNA Packaging
Lysis of Host Cell
The Replication Proteins of E. coli used by ϕX174

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