Exonucleases Endonucleases
(i) They remove
nucleotides from
terminal ends of DNA
strand.They make cut at
specific positions
within the DNA except
terminal ends.
(ii) They act on single
strand of DNA or gaps
in double stranded
DNA.They cleave one strand
or both strands of
double stranded DNA.(iii) They do not cut RNA. They may cut RNA.
(b) Eco R I and Hin d III.- (a) Recognition sequence of Eco R I is :
(^) ↓
5 ′ - GAATTC-3′
3 ′ - CTTAAG-5′
↑
Such sequences are called palindromic sequences, that
are same base pair sequences when read forward or
backward direction from central axis of symmetry.
(b) Restriction endonuclease recognises the base sequence
at palindrome site in DNA duplex and cuts its strands.
- Vectors that can exist in both eukaryotic cell and Escherichia
coli are known as shuttle vectors ,e.g., yeast episomal plasmid
(YEp). Such vectors contain two types of origin of replication
and selectable marker genes, one that functions in E. coli and
other that functions in eukaryotic cell. - (a) Transgenic animals are those animals which contain a
foreign gene introduced in their genome by recombinant
DNA technology. Such a gene is called transgene.
Examples are transgenic mice, rabbit, dogs, cows, etc.
(b) Edward Jenner introduced the term vaccine. The genes
encoding antigenic proteins can be isolated from
pathogens and expressed in plants. Such transgenic
plants or their tissues producing antigens can be eaten
for vaccination/immunisation and are called as edible
vaccines.
OR
Flavr Savr is a transgenic variety of tomato. In this variety native
tomato gene, which produces enzyme polygalacturonase is
inactivated. The non-availability of this enzyme prevents over-
ripening because the enzyme is essential for degradation of
cell walls. Thus, fruit remains fresh for long time and it also
retains flavour, superior taste and higher quantity of total
soluble solids. So, it prevents post harvest and over ripening
losses, and is preferred over normal native variety.
- Insertional inactivation refers to the process where insertion
of recombinant DNA (rDNA) within the coding sequence
of an enzyme causes its inactivation. The non-recombinants
having intact functional gene, e.g., b-galactosidase
produce blue colour with chromogenic substrate but when
rDNA is inserted within the coding sequence of enzyme
b-galactosidase, recombinants do not produce any colour.
Hence, recombinants can be easily differentiated from non-
recombinants due to insertional inactivation.- (a) Escherichia coli.
(b) Origin of replication (ori) is a sequence from where
replication starts and is also responsible for controlling
the copy number of the inserted gene.
(c) The given vector contains ampR as selectable marker. It
helps in selecting transformants (host cells containing
vector) and eliminating non-transformants. Host cells
containing ampR are resistant to antibiotic ampicillin. - Gene therapy is the technique of genetic engineering to
replace a faulty gene by a normal healthy functional gene.
The first clinical gene therapy was given in 1990 to a 4
years old girl with adenosine deaminase deficiency (ADA
deficiency). This enzyme is very important for the immune
system to function. Severe combined immunodeficiency
(SCID) is caused due to defect in the gene for the enzyme
adenosine deaminase. SCID patient lacks functional
T-lymphocytes and, therefore, fails to fight the infecting
pathogens. Lymphocytes are extracted from the patient’s
bone marrow and a normal functional copy of human gene
coding for ADA is introduced into these lymphocytes with the
help of retroviral vector. The cells so treated are reintroduced
into the patient’s bone marrow. The lymphocytes produced
by these cells contain functional ADA gene which reactivate
the victim’s immune system.
Though these cells are not immortal, the patient requires
periodic infusion of such genetically engineered lymphocytes.
However, if the gene isolated from marrow cells producing
ADA is introduced into cells at early embryonic stages, it
could be a permanent cure. Steps of gene therapy can be
summarised in the given diagram.
3. Retrovirus makea DNA copys
of its RNA, this DNA carrying
the normal allele inserts into
chromosome of host cell.
Normal allele- Normal allele inserted into retrovirus
Retrovirus- Retrovirus infects lymphocyte
extracted from bone marrow of
the patient
Bone
4.Genetically engineered cells Bone marrow
injected into patient bone marrowLymphocyte