Retrovirus Vectors - Rectroviruses have

single stranded RNA genomes which are

reverse transcribed by reverse transcriptase to
yield DNA double strand copies inside the host
cells. The DNA copy integrates into the host
genome to become a provirus, which causes
permanent transfection of the cells. The
provirus genome is transcribed and
expressed; virions are formed and extruded
into the medium.

Retroviral vectors have the following three

features.

(1) The vector has viral sequences for

replication, gene expression and packaging (ψ
sequences).

(2) DNA inserts may either replace or be

located in the nonessential coding region of
the viral genome.
(3) The vector and the recombinant DNAs are
packaged into virions and used as transducing
phages.

(4) The viral proteins are usually provided by a helper

virus or a provirus.

(5) DNA copies of the retrovirus genomes are used as

vectors, generally as shuttle vectors.

A typical vector has the following sequences:

(1) pBR322 ori and a selectable marker,

(2) retroviral 5'-LTR and 3'-LTR (long terminal direct

repeats),
(3) R, US, U3, P and Pu encoding sequences
(involved in reverse transcription),

(4) ψ sequence (necessary for packaging into

virions),

(5) S sequence needed for splicing to produce

functional mRNA for envelope protein
synthesis, and

(6) at least one unique restriction site for

insertion of DNA fragment without interrupting
any of the essential sequences.

The recombinant DNA is constructed and first

cloned in E. coli; it is then isolated and
introduced into animal cells where the entire
vector, except the E. coli plasmid sequence, is
transcribed. This transcript contains RNA copy
of the DNA insert, and is packaged into
virions, which is infective to animal cells.
Since the vector does not encode the viral capsid
proteins, they has to be supplied by a helper virus or
provirus. If the provirus or helper virus lacks the ψ
sequence, its genome will not be packed into virions.
As a result, all the virions recovered from the
medium contain the recombinant DNA.
The recombinant DNA so recovered can be used in
further experiments. The recombinant DNA can
integrate as provirus into the host genome; the
transfected cells can be easily selected either as piled
up colonies of cells (if the recombinant DNA retains
an oncogene) or due to the expression of an E. coli
gene, e.g., gene neo conferring resistance to the
drug G-418.

Recombinant DNA
Technology - In nature, gene
transfers are rather imprecise,
and their range, in tenns of
species involved, is remarkably
limited. The above problems are
circumvented by the recombinant
DNA technology. A recombinant
DNA molecule is produced by
joining together two or more DNA
segments usually originating
from different organisms.

More specifically, a recombinant

DNA molecule is a vector into
which the desired DNA fragment
has been inserted to enable its
cloning in an appropriate host.
This is achieved by using specific
enzymes for cutting the DNA
(restriction enzymes) into
suitable fragments and then for
joining together the appropriate
fragments (ligation).
In this manner, a gene may be produced, which
contains the. coding region from one organism
joined to regulatory sequences from another
organism; such a gene is called chimaeric gene.
Clearly, the capability to produce recombinant DNA
molecules has given man the power and opportunity
to create novel gene functions to suit specific needs.

Recombinant DNA molecules are produced with one

of the following three objectives:
(1) to obtain a large number of copies of specific
DNA fragments,
(2) to recover large quantities of
the protein produced by the
concerned gene, or

(3) to integrate the gene in

question into the chromosome of
a target organism where it
expresses itself. Even for the
latter two objectives, it is
essential to first obtain a large
number of copies of the
concerned genes.

To achieve this, the DNA

segments are integrated into a
self-replicating DNA molecule
called vector; most commonly
used vectors are either bacterial
plasmids or DNA viruses. All
these steps concerned with
piecing together DNA segments
of diverse origin and placing
them into a suitable vector
together constitute recombinant
DNA technology.
The DNA segment to be cloned is called DNA insert.
Recombinant DNAs are introduced into a suitable
organism, usually a bacterium; this organism is
called host, while the process is called
transformation. The transformed host cells are
selected and cloned.

The recombinant DNA present in such clones would

replicate either in synchrony with or independent of
the host cell; the gene present in 'the vector mayor
may not express itself, i.e., direct the synthesis of
concerned polypeptide. The step concerned with
transformation of a suitable host with recombinant
DNA, and cloning of the transformed cells is called
DNA cloning or gene cloning.
However, often
DNA or gene
cloning is taken to
include both the
development of
recombinant DNAs
as well as their
cloning in a
suitable host.
Similarly, often the
term recombinant
DNA technology is
used as a synonym
for DNA or gene
cloning used in the
broader sense. A
rather popular
term for these
activities is genetic
engineering.

A clone consists of
asexual progeny of
a single individual
or cell, while the
process/technique
of producing a
clone is called
cloning. As a
result, all the
individuals of a
clone have the
same genotype,
which is also
identical with that
of the individual
from which the
clone was derived.

Therefore, the
genomes present
in members of a
single clone are
 also identical; this
applies to the
recombinant DNA
as well. Therefore,
gene or DNA
cloning produces
large numbers of
copies of the
gene/DNA being
cloned.

Recognition Sequences For Type II

Endonucleases - The recognition sequences for
Type II endonucleases form palindromes with
rotational symmetry. In a palindrome, the base
sequence in the second half of a DNA strand is the
mirror image of the sequence in its first half;
consequently, the complementary DNA strand of a
double helix also shows the same situation.

But in a palindrome with rotational symmetry, the

base sequence in the first half of one strand of a
DNA double helix is the mirror image of the second
half of its complementary strand. Thus in such
palindromes, the base sequence in both the strands
of a DNA duplex reads the same when read from the
same end (either 5' or 3') of both the strands.
Most of the type II restriction endonucleases have
recognition sites of 4, 5 or 6 bp (base pairs), which
are predominantly GC-rich. Longer palindromic
target sequences are also known, and so are
nonpalindromic ones (specific for some enzymes).
Some restriction enzymes have ambiguities in their
recognition sites, e.g. EcoRII, so that they may
recognise upto 4 different target sequences.

Identification of Clones Having

Recombinant DNAs- The second step consists
of identification and isolation of those clones
that are transformed by the recombinant DNAs
from among those that contain the unaltered
vector. This may be achieved in one of several
ways listed below.

1. In case the vector has two selectable

markers, e.g., pBR322, the DNA insert may be
placed within one of these markers, say, ampT
gene. The other marker, in this case, tetr, is
used for elimination of the nontransformed
cells. The transformed clones are then
replicaplated on ampicillin containing medium.
The clones containing the recombinant DNAs will be
sensitive to ampicillin due to inactivation of the gene
ampT by insertion of the DNA fragment. Such clones
are identified and isolated from the master plate.

2. Some vectors contain a gene, or sometimes only

part of a gene, which complements a function
missing in their host cells, e.g., gene lacZα in the
pUC vectors, which complements such lacZ- E. coli
strains in which lacZα is deleted. The same
combination is used for some A. vectors and M13
phage vectors. In all such cases, the DNA insert is
so placed that it disrupts the expression of lacZα.
Therefore, E. coli cells containing the
recombinant DNA are deficient in β-
galactosidase and produce white colonies or
plaques on a medium containing X-gal and
IPTG. On the other hand, tells having the
unchanged vector produce active β
-galactosidase and give rise to blue colonies or
plaques on the same medium. This allows an
easy identification of the clones containing the
recombinant DNAs.

3. When the DNA insert codes for a gene

product, which is defective in the auxotrophic
host cells, a direct selection for the
recombinant DNA is possible. The host cells are
grown on a medium lacking the compound
needed by the auxotrophic host; only those
cells, which contain the recombinant DNA can
grow and form colonies. Obviously, this
approach is limited in application.

4. Similarly, selection by suppression of

nonsense mutations present in the host also
permits a direct selection for the recombinant
DNA.
5. Some A. vectors retain the lysogenic function as
well, e.g., λgt10. In such vectors, the DNA insert
may be placed within the lysis repressor gene cI- so
that the vector becomes cI. As a result, cells
transfected by the recombinant DNA will give rise to
clear plaques, whereas those infected by the
unaltered vector will yield cloudy or turbid plaques.
Thus the recombinant DNAs are readily identified
and isolated.

6. Some vectors, e.g., A. replacement vectors and

cosmids, are much shorter than the minimum
genome length needed for their packaging within
virus particles. In such cases, the length of DNA
insert can be so adjusted as to allow the packaging
of only the recombinant DNA. This provides an
efficient selection strategy for recombinant DNA.