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Lambda bacteriophage: model
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Vikas J. Chikane
1/29/2019

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 Bacteriophage lambda: as a model system

THE STRUCTURE AND GENETIC MAP OF LAMBDA(λ) PHAGE

Virus classification
Group Group 1(dsDNA)
Order Caudovirales
Family Siphoviridae
Genus Lambdavirus
Species λ phage

1.INTRODUCTION:
Viruses are obligate intracellular parasites. Bacteriophages are viruses
infecting bacteria. They multiply inside the host system through partial or complete
utilization of the host biosynthetic machinery. Lambda phage is a temperate phage. The genes
of lambda phage make a single DNA molecule-the chromosome wrapped within a protein
coat, composed of 12-15 different proteins of all which are encoded by the lambda
chromosome. The coat is composed of icosahedral head with diameter of 64nm and a tail,
150nm in length. The head is composed of double stranded linear DNA surrounded by a
capsid made up of protein capsomers. At the 5' end of each strand are 12 nucleotide long
sequence complementary to each other. Thus on circularization, the bacteriophage DNA has
49,211 base pairs. The 5' ends are called cos sites and the opposite of cos site is att site meant
to attachment. The lambda phage attaches to the cell surface of E.coli through its tail, making
a hole in cell wall and pushes its chromosome into the bacterium E.coli leaving behind the
protein coat.

First stage of infection involves a process called Adsorption. Adsorption involve landing and
attachment. tail play critical role in this stage, tailless phage use analogous structure for
adsorption. Specific receptors on bacterial cell like proteins, lipopolysaccharides, pilli apart
from lipoproteins are exploited for phage attachment. Second stage is penetration, the sheath
of phage contracts resulting in insertion of hollow tail fiber through bacterial envelope.
Nucleic acid is inserted inside bacterial cell via hollow tail. Remaining part of the phage
outside of the bacterial cell is called ghost. Some phages upon infecting bacteria lyse the
 bacterial cell after forming their progeny. such phages are called virulent. some phages
integrate their genome into bacterial genome and can remain inside host without harming
them but under drastic conditions can become virulent and can cause host cell lysis. Such
phages under drastic conditions become lytic are called temperate phges.

Genome of bacteriophage lambda:

The λ - phage uses the maltose pore LamB for delivering its genetic material into the host
cell. The phage binds to the cells of the target E.coli and the J-protein in the tip of its tail
interacts with the LamB, gene product of E.coli (LamB is a porin molecule and is a part of
the maltose operon). Most of the E.coli K-12 mutations resistant to λ phage are located in two
genetic regions malA and malB. LamB is composed of three identical subunits, each of which
is formed by an 18-stranded antiparallel β-barrel, which forms a wide channel with a
diameter of about 2.5 nm. The phage consists of a hollow tube composed of 32 –stacked
discs, each of which has a 3nm central hole to eject its genetic material into the host. Lambda
phage uses this channel for ejecting its genetic material. After injecting the DNA into
bacteria, the double stranded linear DNA circularizes due to the presence of cos sites and site
specific nucleases cut DNA at the att site of the phage DNA.
Gene regulation in λ.

(A) The binding of repressor and RNA polymerase to various sites within the left and right
operator regions. RNA polymerase is shown bound at the three promoters in this region.
The top line shows the arrangement found in a lysogen, the bottom line shows polymerase
transcribing from PR and PL upon initial infection. In the lysogen, the repressors bound at
OR and those at OL would interact to form a DNA loop, but for clarity this is not shown here.
(B) The major component of the regulatory network required for a phage to establish
lysogeny (see text). The figure ignores the role of cro (shown in green). This gene encodes a
second repressor which binds to the same sites as λ repressor, but with different affinities
and consequences. Transcription from PR leads to the production of CII, an activator
essential for lysogeny, and Cro, which opposes that pathway (by binding to OR3 and OL3).
The resolution of this competition is important in determining whether the phage develops
lytically or establishes lysogeny. Other features of the decision-making apparatus have been
widely studied, but are not discussed here, including the following. Transcription from PL
produces CIII, a protein that protects CII from degradation and thus promotes lysogeny. CII,
in addition to activating transcription of cI, also activates transcription from two other
promoters, one directing expression of a gene required for integrating the phage genome
into the bacterial chromosome, the other an antisense RNA that opposes expression of gene
Q whose product is required for expression of the late (lytic) genes. The Q protein works as
an antiterminator of transcription; a second phage-encoded antiterminator (N) is important
in regulating early events in infection. The binding sites, promoters and genes are not to
scale.

The genes involved in the switching mechanism of the lambda phage

 Name of Function
gene/promotor/operator
Att Provides site of attachment for phage to host chromosome
cI Repressor protein
cII Coding for promotor establishment activator protein
cIII Codes for stabilizing protein
OR Operator right
PR Promotor right
OL Operator left
PL Promotor left
Cro Gene for second repressor
N Positive regulator counteracting rho dependent termination
J through U Genes encoding tail proteins
Z through A Genes encoding head proteins
Int Gene encoding integrase
Xis Encodes excisionase
O and P Encode proteins involved in replication of Lambda DNA
Q Encodes anti terminator protein

Type Name RefSeq INSDC Size (Kb) GC% Protein

Chr - - KT232076.1 49.21 49.7 68

phylogenetic tree of bacteriophage lambda by NJ method

 λ has made many contributions beyond those highlighted in the text. E. coliPhage λ
provided the selection system for the first refined fine-structurelysogenic for analysis of genes
in this case the rII genes of phage T4. This system then enabled identification of the triplet
nature of the genetic code. The rapid progress in many aspects of biology during the past 25
years owes much to recombinant DNA technology, and hence to the discovery of restriction
enzymes. — itThese were first recognised as a barrier to the transmission of phages λ and P2
was this phenomenon that gave rise to the name ‘restriction’. Experiments with λ indicated
that the ‘barrier’ was an enzyme that attacked incoming DNA in the absence of a strain-
specific modification.

λ DNA provided the substrate for the purification of the first restriction endonuclease. The
size and nature of the λ genome, and ease of purification, made it a common substrate for
detecting, purifying and analysing the enzymes, such as polymerases, endonucleases and
ligases, that underpin recombinant DNA. In the early 1970s, SV40 and λ were used in the
development of agarose and polyacrylamide gel systems for the separation and visualization of
DNA fragments. Phage λ DNA was chosen to demonstrate the feasibility of ‘shotgun’
sequencing of a genome, an achievement published in 1982. λ is a commonly used single-
molecule substrate for analysis of protein-DNA interactions, particularly those involving
translocation, and provides a system for estimating mutation frequency in transgenic rodents.

Protein Details for Escherichia virus Lambda

lambda bacteriophages used as a vector

Bacteriophage l Insertion
capacity 9-15 kb

Enzymes needed:

1.Restriction enzymes: cuts the DNA at specific sequences to generate a set of fragments

2. DNA ligase: inserts DNA restriction fragments into replicating DNA molecules to produce
recombinant DNA

steps in cloning with l :

– Isolate vector DNA and gene of interest

– Cut both with restriction enzyme(EcoRI)
– Connect two fragments of foreign DNA with DNA ligase. (recombinant DNA)
– Package DNA by adding cell extracts containing head and tail proteins
– Transfer recombinant molecules into host cell (transform)
– Grow/select transformants: check recombinant phage for the presence of
desired foreign DNA sequence by observing its genetic properties.

-PL ( promoter) for transcription for the left side of l with N and cIII

-PR (promoter) for right, including cro, cII and the genes encoding the structural proteins.

-OL and OR is short non-coding region of genome, they control the promoters.

-cI (repressor) protein of 236 a.a. which binds to OR and OL, preventing transcription of cro and
N, but allowing transcription of OL, and the other genes in the left hand end.
-cII and cIII encode activator proteins which bind to the genome.

-Cro (66 aa) protein which binds to OR and OL, blocking binding of the repressor to this site to
prevent lysogeny.

-N codes an antiterminator protein and allows transcription from PL and PR. It also allows RNA
polymerase to transcribe a number of phage genes, including those responsible for DNA
recombination and integration of the prophage, as well as cII and cIII.

-Q is an antiterminator similar to N, but only permits extended transcription from PR

-Two Termination sites- One between N and CIII and other between cro and CII.

References:
https://www.ncbi.nlm.nih.gov/genome/proteins/4416?genome_assembly_id=352019