Lecture schedule 2: Genetic engineering-gene concept-DNA and RNA genetic codeorganization of genetic material in virus

Genetic Engineering :
Genetic Engineering primarily involves the manipulation of genetic material
(DNA) to achieve the desired goal in a pre-determined way.
Boyer and Cohen (1973) successfully recombined two plasmids (pSC 101 and
pSC 102 ) and cloned the new plasmid in E.coli . pSC 101 possesses a gene resistant to
antibiotic tetracycline and pSC 102 contains gene resistant to kanamycin. The newly
developed recombinant plasmid when incorporated into E.coli exhibited resistance to
both tetracycline and kanamycin.
In another experiment, they isolated a gene encoding a protein (required to form
rRNA) from the cells of African clawed frog Xeroph laevis, by using ECOR1 (restriction
endonuclease enzyme). The same ECOR1 was used to cut open pSC 10 1. Frog DNA
fragments and plasmid DNA fragments were mixed, and pairs. By the addition of DNA
ligase, a recombined plasmid DNA was developed. These new plasmids, when introduced
into E.coli ,and grown on a nutrient medium resulted in the production of an extra protein
(i.e.the frog protein). Thus the genes of the frog could be successfully transplanted and
expressed in E.coli. this laid foundations for the present day molecular biology.
The basic principles of recombinant DNA technology are reasonably simple, and
broadly involve the following stages.
Generation of DNA fragments and selection of the desired piece of DNA (eg. a
human gene).
Insertion of the selected DNA into a cloning vector (eg. a plasmid) to create a
recombinant or chimeric DNA (chimera is a monster in Greek mythology that
has a lions head, a goats body and serpents tail. This may be comparable to
Narasimha in Indian mythology).
Introduction of the recombinant vectors into host cells (eg. bacteria)
Multiplication and selection of clones containing the recombinant molecules.
Expression of a gene to produce the desired product.
Molecular tools of Genetic Engineering
Restriction endonucleases-DNA cutting enzymes
They are one of the most important groups of enzymes for the
manipulation of DNA. These are the bacterial enzymes that can cut/ split DNA
from any source at specific sites. They were first discovered in E.coli restricting
thereplication of bacteriophages, by cutting the viral DNA. (The host E.coli DNA
is protected from cleavage by addition of methyl groups). Thus, the enzymes that
restrict the viral replication are known as restriction enzymes orn restriction
endonucleases.

Nomenclature
The first letter (in italics) of the enzymes indicates the genus name, followed by
the first two letters (also in italics) of the species, then comes the strain of the organism
and finally a Roman numeral indicating the order of discovery.
Eg. ECORI is from Escherichia (E) coli(co), strain ry13 (R) and first
endonuclease(1) to be discovered HIND III is from Haemophilus (H) influenzae (in)
strain Rd (d ) and the third endonucleases (III) to be discovered.
Types of endonucleases
There are 4 types viz., TYPE I, TYPE II, TYPE III, and TYPE 11s. Among these,
TYPE II enzymes are most commonly used in gene cloning.
Type
Salient features
I
A single enzyme with 3 subunits for recognition, cleavage and methylation. It can
cleave up to 1000 bp from recognition site.
II
Two different enzymes either to cleave or modify the recognition sequence.
Cleavage site is the same or close to recognition site.
III
A single enzyme with 2 subunits for recognition and cleavage. Clevage site is 2426 bp from recognition site.
11s
Two different enzymes, cleavage site is up to 20 bp from recognition site.
Enzyme
ECORI
E.coli

Recognition sequence
5G-A-A-T-T-C3
3C-T-T-A-A-G5

Products
A-A-T-T-C-G
..G
C-T-T-A-A
G-A-T-C-C1
G-

Bamh
Bacillus
amyloliquefacien
s
Hae III
Haemophilus
aegypticus

5G-G-A-T-C-C3
3C-C-T-A-G-G5

HIND III
Haemophilus
influenzae
Not 1
Nocardia otitidis
*Bluent

5A-A-G-C-T-T3
3T-T-C-G-A-A5

..G
C-C-T-A-G
* C-C
G-G
..*G-G
.. C-C
A-G-C-T-T..
A..
..A
..T-T-C-T-G-A
G-G-C-C-G-C-C-G
..C-G.
..C-G-C-C-G-G

5G-G-C-C3
3C-C-G-G5

5G-G-C-C3
3C-C-G-G5

Recognition sequence
The site where the DNA is cut by restriction endonucleases. They can specifically
recognize DNA with a particular sequence of 4-8 nucleotides and cleave. Each
recognition sequence has 2 field rotational symmetry i.e. the same nucleotide sequence
occurs on both strands of DNA which seen in opposite directions. Such sequences are
referred to as palindromes since they read similar in both directions (forwards and
backwards)
Cleavage patterns
Mostly sticky ends (cohesive ends) or blunt ends. DNA fragments with sticky
ends are particularly useful for rRNA experiments. This is because the single stranded
sticky DNA ends can easily pair with any other DNA fragment having complementary
sticky ends.
DNA ligase
The cut DNA fragments are covalently joined together by DNA ligases.through
they are originally isolated from viruses, they are also occur in E .coli and eukaryotic
cells. The action of DNA ligase is the ultimate step in the formation of a recombinant
DNA molecule.
Homopolymer tailing
The complementary DNA strands can be joined together by annealing. This
principle is utilized in homopolymer tailing. The technique involves the addition of oligo
(dA) to 3- ends of some DNA molecules and the addition of oligo (dT) to 3- ends of
other molecules. The homopolymer extensions (by adding 10-40 residues) can be
synthesized by using terminal deoxynucleotidyltransferase (of calt thymus).
Linkers and adaptors
They are Chemically synthesized, short, double stranded DNA molecules.
Linkers have restriction enzymes cleavage sites. They can be ligased to blunt ends of any
DNA molecule and cut with specific restriction enzymes to produce DNA fragments with
sticky ends.
Adaptors have preformed sticky or cohesive ends. They are useful to be ligated to DNA
fragments with blunt ends.
The DNA fragments held to linkers or adaptors are finally ligated to vector DNA
molecules.
Alkaline phosphatase
The removal of phosphate groups. This enzyme is useful to prevent the unwanted
ligation of DNA molecules which is a frequent problem encountered in cloning
experiments.
When the linear vector plasmid DNA is treated with alkaline phosphtase, the 5
terminal phosphate is removed. This prevents both recirculation and plasmid DNA dinner
formation. It is now possible to insert the foreign DNA through DNA ligase.

DNA modifying enzymes

These enzymes represent cutting and joining functions in DNA manipulations.
They are broadly classified as nucleases, polymerases and enzymes modifying ends of
DNA molecules.
Nucleases
Are the enzymes that break the phospho diester bonds.______
____________
_____ __ ___________
exonucleases degrade DNA from the terminal ends.
_ __________________
_ _ _____________ _ _
Endonucleases
1. nuclease S1 specifically acts on single stranded DNA or RNA molecules
_______________ _____nuclease S1 _______ _______ _______ ____
2. deoxyribose nuclease1 (DNAase 1) cuts eitherrrr single stranded or double
stranded at random sites.
DNAse
Double stranded DNA

_____ _____ _____ _____

_____ _____ _____ _____
Exonucleases
1. Exonucleases III Cuts DNA and generates molecules with protruding ends.
Exonuclease III 5
_____

_____ _____ _____ _____

_____
_____ _____ _____ _____
2. Nucleases Bal 31 is a fast acting 3 exonuclease .
ds DNA
_____________
_____________

Bal 31

___________
___________

RNA specific nucleases are generally refered as ribonuclease ( RNAse)

Table. The most commonly used enzymes in rDNA technology / Genetic Engineering

Enzyme
Aalkaline phosphatase
Bal 31Nuclease
DNA ligase
DNA polymerase 1
DNase 1
Exonuclease III
Exo nuclease
Polynucleotide kinase
Restriction enzymes
Reverse Transcriptase
RNase A
RNase H
Tag DNA polymerase
S1 nuclease
Terminal transferase

Use / reaction
Removes phosphate groups from 5-ends of
ds/ss DNA or RNA.
For the progressive shortening of DNA.
Joins DNA molecules by forming phospho
diester linkage between DNA sedments.
Synthesizes DNA complementary to a
DNA template.
Produces single stranded nicks in DNA
Removes nucleotides from 3 end of DNA
Removes nucleotides from 5 end of DNA
Transfer phosphate from ATP to 5 OH
ends of DNA or RNA
Cat ds DNA with a specific recognition site
Synthesizes DNA from RNA
Cleaves and digest RNA
Cleaves and digest RNA strand of RNA
DNA heteroduplex
Used in poloymerase chain reaction (PCR)
Degrades ss DNA and RNA
Adds nucleotides to the 3 ends of DNA or
RNA useful in homoploymer tailing.

Polymerases:
The group of enzymes that catalyse the synthesis of nucleic acid molecules are
collectively referred to as polymerases.
-DNA dependent DNA polymerase that cop8es DNA from DNA
-RNA dependent DNA polymerase that synthesizes DNA from RNA (Reverse
transcriptase)
-DNA dependent RNA polymerase that produces RNA from DNA
Enzymes modifying the ends of DNA:
Alkaline phosphatase- that removes the terminal phosphate group
Polynucleotide kinase-that adds phosphate groups
Terminal transferase (terminal deoxynucleotide transferase) repeatedly
add nucleotides to any available 3 terminal ends (protruding 3-ends).
This enzyme is particularly useful to add homopolymer tails prior to the
construction of rDNA molecules.

Host Cells:
They are the living system or cells in which the carrier recombinant DNA molecule or
vector can be propagated. Some of the host cells used in genetic engineering are
Prokaryotic
Bacteria - E.coli
limitation
Bacillus subtilis
cannot perform post translational
Streptomyces sp.
Modification.
Eukaryotic
Fungi - Saccharomyces cerevisiae
Aspergillus nidulans
Animals - Insect cells
Oocytes
Mammalian cells
Whole organisms
Plants
- Protoplasts
Intact cells
Whole plants.
Vectors - The cloning vehicles
Vectors are the DNA molecules, which can carry a foreign DNA fragment to be
cloned.They are self replicating in an appropriate host cell.
Eg. Plasmids, bacteriophage, cosmids and phagemids.
Ideal vector should be small in size, with a single restriction endonuclease site, an origin
of replication and 1-2 genetic markers (to identify recipient cells carrying vectors)
Plasmids are extrachromosomal, double stranded covalently closed circular (ccc) self
replicating DNA molecules. Almost all bacteria have plasmids with a low copy number
(1-4/cell) or high copy number (10-100/cell). The size of the plasmids varies from 1500kb. Usually plasmids contribute 0.5-5% of the total DNA of bacteria.
A few bacteria viz. Streptomyces sp, Borelia burgdoferi have linear plasmids.
Types of plasmids
Conjugative
carry transfer genes (tragenes) for bacterial conjugation. They are
plasmids
large, show stringent control of DNA replication and are present in
low number.
Non-conjugative
plasmids
Stringent
plasmids
Relaxed
plasmids
F-plasmid

Do not carry tra genes they are small, show relaxed control of DNA
replication and are present in high number.
Present in a limited number i.e., low copy-1-2/cell.

R- plasmid

Carry genes for antibiotic resistance

High copy- more number per cell.

Have genes for their own transfer from one cell to another cell.

Nomenclature
It is a common practice to designate plasmid by a lower case p, followed by the
first letter(s) of researcher (s) name and the numerical number given by them.
PBR322 - plasmid discovered by Bolivar and Rodriguez who designated it as 322.
Puc - Plasmid from university of california PBR322 of E.cloi is the most popular and
widely used plasmid vector. It has a DNA sequence of 4,361bp. It carries genes resistance
for ampicillin (Ampr ) and tetracycline (Tetr) that serves as markers for the identification
if clones carrying plasmids. It has a unique recognition sites for the action of restriction
ebdonucleases viz, ECOR1, Hind III, Bam H1, Sal 1 and Pst1.
Other vectors
1. Derivatives of PBR322 - PBR325, PBR328, PBR329
2. PUC19
2,686 bp with Ampr , gene.
Bacteriophage are the viruses that replicate within the bacteria. Phage vectors can take
up larger DNA segment than plasmids. Eg. *Phage
Phage M13 is a single stranded DNA phage of E.coli. It is a useful for
sequencing DNA through samgers method.
Cosmids- Vectors having the characters of both plasmid and bacteriophage * . They can
carry larger fragments of foreign DNA compared to plasmids.
Phagemids - Combination of plasmids and phage and can function as either plasmid or
phage. They have functional origin of replication of both.
Artificial chromosome vector
Human artificial chromosome (HAC) H.Willard, 1997.
It is a synthetically produced vector DNA, having the characteristics of human
chromosome. It Is a self replicating microchromosome. It is a self replicating
microchromosome with a size ranging form Y 10th to Y 5th of a human chromosome. The
advantage with HAC is that it can carry human genes that are too long.
YACs : (M.olson, 1987) It is a synthetic DNA that can accept large fragments of foreign
DNA. They have centrometric and telomeric region and represent the largest capacity
vectors available.
BACs : accepts DNA inserts around 300 kb. The construction is based on one F plasmid
which is larger than the other plasmids used as cloning vectors.
Shuttle vectors: The plasmid vectors that are specifically designed to replicate in two
different hosts (E.coli & Steptomyces). The origin of replication for 2 hosts are combined
in one plasmid, Hence any foreign DNA fragment introduced into the vector can be
expressed in either host. They can be grown in one host and then shiffed to another host.
A good number of eukaryotic vectors are shuttle vectors.The success of cloning depends
on t he sizw of foreign DNA and the choice of the vectors.

The size of the DNA that can be accepted by different vectors are
Vector

Host

Foreign DNA insert size

Phage *

E. coli

5.25 kb

Cosmid *

E. coli

35-45 kb

Plasmid artificial
chromosome (PAC)

E. coli

100-300 kb

BAC

E. coli

100-300 kb

Yeast chromosome

S. cerevisiae

200-2000 kb

Method of gene transfer

Transformation
Conjugation
Electroporation
Lipofection
Direct transfer of DNA
1. microinjection
2. particle bombardment
transduction
Genetic code: The total set of 64 codons that code for 20 aminoacids, and 3
termination codon. The 3 nucleotide (triplet) base sequence in mRNA that act as code
words for aminoacids in protein constitute the genetic code or codons. Codons
derermines the sequence of aminoacids in proteins. Codons have 4 bases (AUGC)
that produce 64 different combinations (43). 61 codons code for 20 aminoacids and
the other 3 codons are stop codons viz, UAG (amber) UAA(ochre) and UGA (opal).
AUG and GUG are initiating codons.
The genetic code is universal, specific, nonover lapping and degenetate.
Universality : The same codons are used to code for the same aminoacids in all the
living organisms. Eg. AUG code for methionine universality.
Exception : AUA code for methionine in mitochondria and isoleucine in cytoplasm.
Specificity : a particular codon always codes for the same aminoacid. Eg. UGG code
for tryptophan (Trp.)
Non overlapping : The genetic code is read from a fixed point as a continous base
sequence. It is non- overlapping, commaless and without any punctuations. Eg.
UUUCUUAGA is read as UUU/CUU/AGA/GGG.

Degeneracy : most of the aminoacids have more than one codon. Eg. glycine has 4
codons. The codons that designate the same aminoacid are called synonyms. Most of
the synonyms differ only in the 3rd base of the codon.
GGU, GGC, GGA and GGG- glycine differ in 3rd base.
The genetic code along with respective amino acids