Download FAQs (frequently asked questions) Q.1 What are plasmids? Ans

FAQs (frequently asked questions)
Q.1
What are plasmids?
Ans.
They
are
the
extrachromosomal
naturally
occurring
DNA molecules present in
many prokaryotes and also in some lower
eukaryotes. These are usually double stranded
circular molecules.
Q.
2
What are cryptic and conjugated
plasmids?
Ans.
The naturally occurring plasmids are known as
the cryptic plasmids; while as derivatives of
cryptic
plasmids
are
referred
to
as
the
conjugative plasmids, which are commonly used
for cloning purposes.
Q.
How
do
stringent
and
relaxed
plasmids
replicate?
Ans.
The replication of stringent plasmids is coupled
with
the replication of host genome and as a
result, these are present only in low copy
number.
On
the
plasmids
can
other
continue
hand,
the
replicating
relaxed
even
in
absence of the replication of host genome.
Q.
What are the features of a plasmid vector used
for cloning?
Ans.
It should contain an origin of replication and
reduction in size of vector to a minimum to
expand the capacity of vector to clone large
fragments. Introduction of selectable markers
and synthetic cloning sites are also important.
Q.
What is pBR322?
Ans.
This was one of the first artificial cloning vectors
to be constructed, and is undoubtedly the most
widely used cloning vector till now. It is a 4.36kb double stranded cloning vector. This plasmid
vector has been put together from fragments
originating
from
three
different
naturally
occurring plasmids.
Q.
Write down the nucleotide sequence of pBR322.
Ans.
The pBR322 plasmid contains 20 unique
recognition sites for restriction enzymes. Six of
these sites, i.e. EcoRV, BamHI, Sphl, SalI,
XmaIII and Nrul, are located within the genes
coding for tetracycline resistance; two sites for
HindIII and CIa I lie within the promoter of the
tetracycline resistance gene; and three sites for
PstI, PvuI and Sca l lie within the β-lactamase
gene. Cloning of a DNA fragment into any of
these 11 sites results in the insertional inactivation of either one of the antibiotic resistance
markers.
Q.
Ans.
Q.
Ans.
What is Replicon?
The origin of replication of the plasmid along
with all
the necessary factors is known as the replicon.
What are Shuttle Vectors? Cite an example.
A
plasmid
constructed
to
have
origin
of
replication for two hosts, so that it can be used
to carry a foreign sequence in either prokaryotes
or eukaryotes. It can be, therefore, used to
shuttle the genes from one to another organism.
For example the Yep, pJDB219 a shuttle vector
with E. coli and S. cerevisiae.
Q.
Ans.
What are auxotrophic mutants?
A mutant organism that will not grow on a
minimal medium; it requires the addition of
some growth regulators.
Q.
What are the Yeast integrated plasmids?
Ans.
These are not actually yeast plasmid vectors,
because they are unable to replicate within a
yeast
cell
without
integration
into
a
chromosome. Such vectors contain a yeast
marker as the only addition to the bacterial
plasmid.
Q.
Ans.
What are the Yeast Episomal Plasmids?
It is a cloning vector for the yeast that uses 2µm
plasmid as origin of replication and is maintained
as an extrachromosomal nuclear DNA molecule.
Q.
Define ARS?
Ans.
It is an autonomous replicating sequence. Any
eukaryotic DNA sequence that initiates and
supports chromosomal replication.
Q.
What is Ti plasmid?
Ans.
Ti plasmid is a circular plasmid that oftenis a
part
of
the
genetic
equipment
that
Agrobacterium tumefaciens use to transduce its
genetic material to plants.
Q.
What is organization of Ti plasmid?
Ans.
Ti plasmids of Agrobacterium are large circular
DNA molecules, up to 200 kb in length with
molecular
weights
of
about
1.2
x
108.
Ti
plasmids have major regions for virulence, origin
of replication, conjugation, oncogenicity, and
catabolism of opines.
Q.
What are the important features of cloning
vectors?
Ans.
The important features are:
1.
The size of cloning vector should be small,
preferably 3-4 kb.
2. It should contain an origin of replication.
3.
4.
Introduction of selectable markers.
Introduction of synthetic cloning sites
termed
polylinker, restriction site
bank, or polycloning sites
that are recognized
by restriction enzymes.