DEFINITIONS AND
TERMS
1.
Genetic
Material Genetic
Material is that substance which controls the inheritance of traits from one
generation to next .
2.
Gene Gene is a segment of DNA (Deoxyribonucleic
Acid), that codes for RNA (Ribonucleic Acid and thereby a polypeptide.The term
gene was coined by Johanssen(1909).
3.
Nucleosome Nucleosome is a structure formed when the
negatively charged DNA is wrapped around the positively charged histone
octamer.
4.
Bacteriophage A virus that infects a bacterium is
called a bacteriophage.
5.
Replication Self duplicating
property of DNA is called Replication.
6.
Replication
Fork The Y-shaped structure formed when the
double-stranded DNA is unwound upto a point during its replication, is called
replication fork.
7.
Transformation Transformation is the phenomenon by
which the DNA isolated from one type of cell, when introduced into another
type, is able to bestow some of the properties of the former to the latter.
8.
Transcription Transcription is the process of formation of
RNA from DNA.
9.
Translation Translation is the process of polymerization
of amino acids to form a polypeptide dictated by the messenger RNA.Simply it is
the biosynthesis of proteins.
10.
Operon All the genes
controlling a metabolic pathway, constitute an operon.
11.
Constitutive
Genes Constitutive
genes are those genes which are constantly expressed and whose products are
continuously needed for the cellular activity.
12.
Exons Exons are the regions of a gene, which become
part of mRNA and code for the different regions of proteins.
13.
Introns Introns are the regions of a gene,
which do not form part of mRNA and are removed during the processing of mRNA.
14.
Splicing It is a process in eukaryotic genes,
whereby the introns are removed and the exons are joined together to form mRNA.
15.
Codon
Codons a sequence of three
nitrogenous bases on mRNA, that codes for a particular amino acid.
16.
Anticodon Anticodon is a sequence of three
nitrogenous bases on tRNA, that is complementary to the codon for the
particular amino acid.
17.
Split
genes The
genes carrying genetic information in stretches rather than continuously, are
called split genes. Eukaryotic genes having coding and non-coding sequences
together are called split genes.
18.
Satellite
DNA Satellite DNA refers to the repetitive DNA
sequences which do not code for any proteins, but form a large portion of human
genome; they show high degree of polymorphism.
19.
DNA
polymorphism DNA
polymorphism refers to the variation at genetic level, where an inheritable
mutation is observed in a population in a frequency greater than 0.01
20.
Operon
system An
operon is a part of genetic material which act as a single single regulated
unit having one or more structural genes, an operator gene, a promoter gene and
a regulator gene.
21.
Father
of DNA fingerprinting Alec
Jaffereys
22.
Father
of Indian Dna fingerprinting Lalji
Singh
23.
Actinomycin
D inhibits
transcription
24.
Puromycin inhibits translation
25.
Largest
Genome A
very slow growing herb Paris japonica
has largest genome (about 50 times the human genome)
26.
Cistron The term cistron
was proposed for the unit of function. Presently a cistron is equivalent to a
gene. It is that amount of DNA which can code for one functional polypeptide
chain. Both cistron and gene refer to the same entity.
27.
Nucleosides Nitrogenous base +sugar
28.
Nucleotides Nitrogenous base
+sugar+phosphate
29.
Pribnow
box A sequence of
six nucleotides TATATT in prokaryotic promoters.
30.
TATA
box A DNA sequence
in promoter region of eukaryotic genes analogous to the prinbox of prokaryotes.
IMPORTANT
CONCEPTS
1.
Deoxyribonucleic
Acid (DNA)
·
DNA constitute the genetic material of
all the organisms with the exception of riboviruses.
·
DNA is a long polymer of
deoxyribonucleotides, whose length is defined as the number of nuleotides or
base pairs (bp).
·
The number of base pairs is
characteristic of every organism/species, e.g.,
Φ 174 phage - 5386 bp
Lambda phage - 48502 bp
Escherichia coli - 4.6
106 bp
106 bp
Human beings - 3.3 109 bp (haploid number)
109 bp (haploid number)
·
DNA was discovered by Frederich Meischer
(1869) as an acidic substance in the nucleus; he called it ‘nuclein’.
(A)
Structure
of a Polynucleotide Chain of DNA.
-
Each nucleotide has three components,
---- a nitrogenous base,
---- a deoxyribose (pentose) sugar
---- a phosphate group
-
Nitrogenous bases are of two types:
ð Purines
(adenine and guanine) and
ð Pyrimidines
(cytosine and thymine).
-
A nitrogenous base is linked to the
pentose deoxyribose sugar through a N-glycosidic linkage to form a nucleoside.
-
When a phosphate group is attached to
5’-OH of a nucleoside through phospho-ester linkage, a corresponding nucleotide
is formed.
-
Two nucleotides are linked through 3’-5’
phosphodiester linkage to form a dinucleotide and when many nucleotides are
linked in this manner, a polynucleotide is formed
-
The polylnucleotide chain has at the
5’-end of the sugar a free phosphate moiety (it is called 5’-end) and at the
3’-end a OH group (it is called 3’ end).
-
The backbone of the polynucleotide is
formed by the sugar and phosphates, while the nitrogen base project from the
backbone.
 |
| A Polynucleotide Chain |
(B)
Double
Helix – Model of DNA
-
Watson and Francis Crick (1953) proposed
a double-helix model of DNA, based on the X-ray diffraction data, produced by
Maurice Wilkins and Rosalind Franklin.
-
One of the important features of this
model is the complementary base-pairing.
-
Later Erwin Chargaff observed that in a
double-stranded DNA, the ratios between adenine and thymine and that between
guanine and cytosine are constant, i.e., A : T = 1 and G :C = 1
-
The salient features of the double
helical model are given below:
(I) DNA
is made of two polynucleotide chains, where the backbone is constituted by
sugar-phosphate and the nitrogen bases project inside.
(II) The base in the two strands are held together by hydrogen
bonds forming base pairs, Adenine pairs with thymine through two hydrogen bonds
and guanine with cytosine through three hydrogen bonds.
(III) The two chains have an anti-parallel polarity, i.e., one chain has the polarity 5’Ã
3’, the other has 3’ Ã
5’polarity.
(IV) The two chains are coiled in a right-handed fashion and the
pitch of the helix is 3.4 nm; there are about 10 base pairs in each turn with
0.34 nm between two base pairs.
(V) The
plane of one base pair stacks over the other in the double helix. This, in
addition to H-bonds, confers the stability of the helical structure. The length
of DNA in E. coli is approximately 1.36 mm, while that of humans 2.2 m.
 |
| DNA double helix |
 |
Double stranded polynucleotide chain |
CENTRAL
DOGMA OF MOLECULAR BIOLOGY
Francis
crick proposed the central dogma of molecular biology, which states that the
genetic information flows from DNAÃ RNAÃ
Protein.
In some viruses the flow of information is in
reverse direction ,that is, from RNA to DNA, This is called reverse transcription or teminism. It
was discovered by TEMIN AND BALTIMORE in 1970
(C) Packaging of DNA
¾ In
prokaryotes, with no well-defined nucleus, the DNA is organised in large loops
held by certain positively charged proteins, in a region called nucleoid.
¾ In
eukaryotes, the DNA is wrapped around positively charged histone octamere into
a structure called nucleosome.
¾ A
typicl nucleosome consists of 200 bp of
DNA helix
¾ The
nucleosomes are the repeating unit that form chromatin fibres.
¾ The
chromatin fibres condense at metaphase stage of cell division to form
chromosomes.
¾ The
packaging of chromatin at higher level requires additional set of proteins
called non-histone chromosomal (NHC) proteins.
¾ In
a nucleus, certain regions of the chromatin are loosely packed and they stain
lighter than the other regions; these are called euchromatin.
¾ The
other regions are tightly packed and they stain darker and are called
heterochromatin.
¾ Euchromatin
is transcriptionally more active than heterochromatin.
 |
| Nucleosome |
2. Transformation and the Transforming
Principle.
·
Frederick Griffith (1928) conducted as
series of experiments with Streptococcus pneumoniae, (Pneumococcus), the
bacterium causing pneumonia.
·
He observed two strains of this
bacterium; one forming smooth colonies with capsule (S-type) and the other
forming rough colonies without capsule (R-type).
·
When live S-type cells were injected
into the mice, they produced pneumonia (pathogenic/virulent) and the mice died.
·
When heat-killed S-type cells were
injected into the mice, the disease did not appear.
·
When heat-killed S-strain cells were
mixed with live R-strain cells and injected into the mice, the mice died and he
could isolate live S-strain cells from the body of the mice.
·
He concluded that the R-strain bacteria
had somehow been transformed by the heat-killed S-strain bacteria, which must
be due to the transfer of the genetic material (transforming principle).
·
They also discovered that proteases and
RNase did not affect transformation, while DNase inhibited the process; this
proves that DNA is the transforming principle.
3. DNA is the Genetic Material
·
The proof for DNA as the genetic
material came from the experiments of Alfred Hershey and Martha Chase (1952),
who worked with bacteriophages.
·
The bacteriophage on infection injects
only the DNA into the bacterial cell and not the protein coat; the bacterial
cell treats the viral DNA as its own and subsequently manufactures more virus
particles.
·
They made two different preparations of
the phage; in one, the DNA was made radioactive with 32p and in the
other, the protein coat was made radioactive with 35S.
·
These two phage preparations were gently
agitated in a blender to separate the adhering protein coat of the virus from
the bacterial cells.
·
The culture was also centrifuged to
separate the viral coat and the bacterial cells.
·
It was found that when the phage
containing radioactive DNA was used to infect the bacteria, its radioactivity
was found in the bacterial cells (in the sediment) indicating that the DNA has
been injected into the bacterial cell.
·
So, DNA is the genetic material and not
proteins.
 |
| The Hershey-Chase experiment |
4. Characteristics of Genetic Material.
·
A molecule that can act as genetic
material must have the following properties:
(I)
It should be able to generate its
replica.
(II)
It should be chemically and structurally
stable.
(III)
It should provide the scope for slow
changes (mutation) that are necessary for evolution.
(IV)
It should be able to express itself in the
form of Mendelian characters.
·
Nucleic acids ie. DNA and RNA can
replicate, but not protein.
·
The predominant genetic material is DNA,
while few viruses like tobacco mosaic virus have RNA as the genetic material.
·
The 2’-OH group in the nucleotides of
RNA is a reactive group and makes RNA labile and easily degradable; RNA as a
catalyst is also more reactive and hence DNA has the property to be the genetic
material.
5. RNA-World
·
Ribonucleic acid (RNA) was the first
genetic material.
·
The 2’-OH group of ribonucleotides is a
reactive group that makes RNA a catalyst.
·
It is evident that essential life
processes such as metabolism, translation, splicing etc, have evolved around
RNA, even before DNA has evolved as a genetic material.
·
Structure
of a Polynucleotide Chain (RNA)
¾ In
RNA also, each nucleotide has three components as in DNA
¾ The
nitrogen bases are of two types:
Þ Purines
– (Adenine and Guanine)
Þ Pyrimidines
– (Cytosine and Uracil).
·
There are three types of RNA:
(A)
Messenger
RNA (mRNA)
¾ About
3-5% of total RNA of the cell is m-RNA.
¾ It
brings the genetic information of DNA transcribed on it for protein synthesis.
¾ It
is single-stranded.
(B) Transfer/Soluble RNA (tRNA/sRNA)
¾ It
makes about 15% of the total RNA of the cell and having on average 80
nucleotides per molecule. It is the smallest of all the RNAs.
¾ It
acts as an adapter molecule that reads the code on one hand and binds to the
specific amino acid on the other hand.
¾ tRNA
has a clover leaf like secondary structure but actually it is an inverted
L-shaped compact molecule.
¾ It
has an ‘amino acid acceptor end’ (3’-end) and an ‘anticodon-loop’, where the
three bases are complementary to the bases of the codon of the particular amino
acid.
¾
(C) Ribosomal RNA (rRNA)
¾ Ribosomal
RNA was tha first RNA to be identified, and it constitutes approximately 80% of
the total RNA of the cell.
¾ It
forms the structure of ribosomes.
¾ It
also plays a catalytic role during translation.
6. Replication of DNA
·
Watson and Crick had proposed a scheme
for replication of DNA, when they proposed the double helical structure for
DNA.
·
The scheme suggested that the two
strands would separate and each acts as a template for the synthesis of a new
complementary strand.
·
After complete replication, each DNA
molecule would have one parental and one newly synthesized strand.
 |
| Watson-Crick model for semiconservative DNA replication |
(A) Proof for Semiconservative mode of replication
¾ Mathew
Meselson and Franklin Stahl have performed an experiment using Escherichia coli
to prove that DNA replication is semiconservative.
¾ They
grew E.coli in a medium containing15NH4Cl unitil 15N
was incorporated in the two strands of newly synthesised DNA; this heavy DNA
can be separated from the normal (14N) DNA by centrifugation in Cesium
Chloride (CsCl) density gradient.
¾ Then
they transferred the cells into a medium with normal 14NH4Cl
and took out samples at various time intervals and extracted DNAs and
centrifuged them to measure their densities.
¾ The
DNA extracted from the cells after one generation of transfer from the 15N
medium to 14N medium (i.e., after 20 minutes) had an intermediate/hybrid
density.
¾ Similar
experiments were conducted by Taylor et al in 1958, by using radioactive
ethymidine; they proved that DNA on the chromosomes replicates in a
semiconservative manner.
 |
| Messelson and Stahl’s Experiment |
(B) The Process of Replication of DNA
¾ The
process involves a number of enzymes/catalysts, of which the main enzyme is
DNA-dependent DNA-polymerase, that catalyses of the polymerization of the
deoxynucleotides at a rate of approximately 200 bp per second.
¾ The
process is also an energy-expensive process; deoxyribonucleoside triphosphates
serve the dual purpose of (I) acting as substrate and (II) providing energy
(from the two terminal phosphates).
¾ The
intertwined strands of DNA separate from a particular point called origin of
replication.
¾ Since
the two strands cannot be separated in its entire length, replication occurs
with small opening of the DNA- helix; the Y-shaped structure formed, is called
replication fork.
¾ The
DNA-dependent DNA-polymerases catalyse polymerisation of the nucleotides only
in 5’-3’ direction.
¾ Consequently,
one of the template strands (with 3’-5’polarity) the synthesis of DNA is
continuous, while on the other template strand (with polarity 5’-3’), the synthesis
of DNA is discontinuous, i.e., short stretches of DNA are synthesised.
¾ The
discontinuously synthesisted strands are later joined together by the enzyme
DNA-ligase.
 |
| Replicating Fork |
7. Transcription
·
Transcription is also governed by the
complementarity of bases as in DNA. (Except that uracil in place of thymine is
complementary to ademine).
·
Only one of the strands of the DNA acts
as the template for RNA synthesis for the following reasons:
(I) If
both the strands code for RNA, two different (complementary) RNA molecules and
two different proteins would be formed; hence the genetic information-transfer
machinery would become complicated.
(II) Since the two RNA molecules produced would be complementary to
each other, they would wind together to form a double-stranded RNA without
carrying out translation; that means the process of transcription would become
futile.
·
A transcription unit in DNA has three
regions:
(I) A Promoter (II) Structural and (III) A terminator.
 |
| Schematic structure of transcriptional unit |
·
The process is catalysed by DNA-dependent
RNA-polymerase, which catalyses the polymerisation of nucleotides only in 5’-3’
direction.
·
The DNA strand with 3’-5’ polarity is
called ‘template strand’, while the other strand with 5’-3’ polarity is called
‘coding strand’.
·
The coding strand is displaced and does
not code for RNA, but reference points regarding transcription are made in
relation to it.
·
The promoter refers to a particular
sequence of DNA located towards the 5’ end (upstream) of the coding strand,
where the RNA polymerase becomes bound for transcription.
·
The terminator is a sequence of DNA
located towards the 3’ end (downstream) of the coding strand, where the process
of transcription would stop.
·
There are additional regulatory
sequences that may be present upstream or downstream to the promoter.
(A) Transcription in prokaryotes
¾ In
prokaryotes, the structural genes are polycistronic and continuous.
¾ In
prokaryotes, there is a single DNA-dependent RNA polymerase, that catalyses the
transcription of all the three types of RNA (mRNA, tRNA, rRNA).
¾ RNA
polymerase binds to the promoter and initiates the process along with certain
initiation factors (
) (sigma).
) (sigma).
¾ It
uses ribonucleoside triphosphates (also called ribonucleotides) for
polymerisation on a DNA template following complementarity of bases.
¾ The
enzyme facilitates the opening of the DNA-helix and elongation continues.
¾ Once
the RNA polymerase reaches the terminator, the nascent RNA falls off and the
RNA polymerase also separates; it is called termination of transcription and is
facilitated by certain termination factors (
) (Rho).
) (Rho).
¾ In
prokaryotes, the mRNA synthesised does not required any processing to become
active and both transcription and translation occur in the same cytosol;
translation can start much before the mRNA is fully transcribed, i.e.,
transcription and translation can be
coupled.
 |
| Process of transcription in Bacteria |
(B) Transcription in Eukaryotes.
¾ In
eukaryotes, the structural genes are monocistronic and ‘split’.
¾ They
have coding sequences called exons that form part of mRNA and non-coding
sequences, called introns, that do not form part of the mRNA and are removed
during splicing.
¾ In
eukaryotes, there are atleast three different RNA polymerases in the nucleus,
apart from the RNA polymerase in the organelles, which function as follows:
 |
| Process of transcription in Eukaryotes |
Þ RNA
polymerase I transcribes rRNAs (26S, 18S and 5.8S),
Þ RNA
polymerase II transcribes the precursor of mRNA (called as heterogenous nuclear
RNA (hnRNA) and
Þ RNA-polymerase
III catalyses transcription of tRNA.
¾ The
primary transcript contains both exons and introns and it is subjected to a
process, called splicing, where the introns are removed and the exons are
joined in a definite order to form mRNA.
¾ The
hnRNA undergoes two additional processes called ‘capping’ and ‘tailing’.
¾ In
capping, methyl guanosine triphosphate is added to the 5’ end of hnRNA.
¾ In
tailing, adenylate residues (about 200-300) are added at the 3’-end of hnRNA.
¾ The
fully processed hnRNA is called mRNA and is released from the nucleus into the
cytoplasm.
8. Genetic Code
·
Genetic code refers to the relationship
between the sequence of nucleotides on mRNA and the sequence of amino acids in
the polypeptide.
·
It was George Gamow, who suggested that
the code must be made up of three bases, in order to code for the twenty
different amino acids; with only four bases (A, T, G, C), there would be (43)
or (4 x 4 x 4) = 64 triplet condons.
·
Har Gobind Khorana Could synthesise RNA
molecules with definite combinations of bases. (homopolymers and copolymers).
·
Marshal Nirenberg made a cell-free
system for protein synthesis, that helped in deciphering the genetic code.
·
Severo Ochoa discovered enzyme
polynucleotide phosphorylase, that could polymerise RNA with a definite
sequence in template-independent manner.
·
The checkerboard pattern of genetic code
was prepared as given below:
 |
| The Codons for various amino acids |
·
This
salient features of genetic code are given below:
(I) The
codons are triplets and there are sixty-four codons; sixty-one codons code for
twenty amino acids and three codons (UAA, UAG, UGA) do not code for any amino
acid, but function as stop/termination codons. UAA, UAG and UGA are also known
as ochre, amber and opal.
(II) Each codon codes for only one/particular amino acid and so the
genetic code ‘unambiguous’ and ‘specific’.
(III) Since some amino acids are coded by more than one codon, the
genetic code is said to be ‘degenerate’.
(IV) The codons are read in a contiguous manner in the 5’-3’ direction
and have no punctuations (commaless).
(V) The
genetic code is universal, i.e., the codons code for the same amino acid in any
organism, be it a bacterium or a human being.
(VI) AUG has dual functions of coding for methionine and acting as
initiation codon.
(VII) Wobble hypothesis : It
is believed that third base of a codon is not very important and that the
specificity of a codon is particularly determined by first two bases. Same tRNA
can recognize more than one codons differing only at third position.
9. Mutations
(A) Point Mutation
¾ This
type of mutation involves a change in single base pair.
¾ An
example of point mutation is a change of single base pair in the gene for beta
globin chain (of haemoglobin) that results in substitution of glutamate by
valine; it causes a disease called sickle-cell anaemia.
(B) Frameshift mutation
¾ It
is the type of mutation where addition/insertion or deletion of one or two
bases changes the reading frame from the site of mutation, resulting in a
protein with a different set of amino acids.
¾ This
also forms the basis of proof that codon is a triplet and codons are read in a
contiguous manner.
¾ Insertion
or deletion of three or its multiples of bases does not alter the reading
frame, but one/more amino acids are added or deleted in the protein translated.
(C) Silent mutation If a
base change in a codon does not alter the amino acid coded, the mutation is said to be silent mutation.
10. Translation
·
In this process, amino acids become
joined together by peptide bonds, to form polypeptides.
·
The formation of peptide bonds requires
energy and hence in the first phase, the amino acids are activated.
(A) Activation of amino acids
¾ In
this process, a particular amino acid becomes activated and attached to the 3’
end of a specific tRNA molecule.
¾ The
reaction is catalysed by the enzyme amino –acyl-tRNA synthetase.
Amino acid (AA) + ATP Enzyme AA – AmP – ENZ + PPi 
AA-AMP – ENZ + tRNA AA –tRNA + AMP + PPi
(B) Initiation of polypeptide synthesis
¾ The
ribosome, in its inactive state exits as two subunits – a large subunit and a
small subunit.
¾ When
the small subunit encounters the mRNA, translation begins.
¾ The
mRNA binds to the small subunit of ribosome, following base pair rule between
the bases of mRNA and those on rRNA; it is catalysed by ‘initiation factors’.
¾ There
are two sites on the larger subunit, the P-site and the A-site
¾ The
small subunit (with the mRNA) attaches to the large subunit in such a way that
the initiation codon (AUG) comes on the P-site.
¾ The
anticodon UAC of initiator tRNA (methionyl tRNA) carrying amino acid
methionine(AUG) undergoes pairing with the initiation codon AUG of mRNA through
hydrogen bonds at P site .
 |
| tRNA- the adapter molecule |
(C) Elongation of polypeptide chain
¾ A
second tRNA charged with an appropriate amino acid binds to the A-site of the
ribosome.
 |
| Translation |
¾ A
peptide (CO-NH) bond is formed between the carboxyl group of methionine and the
amino group of the second amino acid; this reaction is catalysed by the enzyme
peptidyl transferase.
¾ The
ribosome moves from codon to codon along the RNA in the 5’-3’ direction.
¾ Amino
acids are added one by one in the sequence of the codons and become joined
together to form a polypeptide.
(D) Regulation of Gene Expression
¾ When
one of the termination codons comes at the A-site, it does not code for any
amino acid and there is no tRNA molecule for it.
¾ As
a result, the polypeptide synthesis (or elongation of polypeptide) stops.
¾ The
polypeptide synthesised is released from the ribosome, catalysed by a ‘release
factor’.
11. Regulation of polypeptide synthesis
·
In prokaryotes, the gene expression is
controlled by the initiation of transcription.
·
In eukaryotes, the regulation can be
exerted at four levels:
(I) Transcriptional level (formation of
primary transcript)
(II) Processing level (splicing)
(III) Transport of mRNA from nucleus to cytoplasm
and
(IV) Translational level
·
The regulation at transcriptional level
was elucidated by Jacob and Monod.
·
All the genes controlling one metabolic
pathway constitute an operon.
·
A few examples are lac-operon,
try-operon, val-operon, his-operon, etc. in E.coli.
·
An operon consists of the following
components:
(A) Structural Gene (s)
¾ They
transcribe the mRNA for the amino acid sequence of proteins (enzymes).
(B) Promoter Gene
¾ The
promoter gene is a sequence of DNA, where the RNA polymerase binds and
initiates transcription
(C) Operator
¾ The
operator is a sequence of bases on DNA adjacent to the promoter.
¾ The
accessibility of promoter gene for RNA polymerase is regulated by proteins,
called repressor.
¾ In
most cases a specific repressor protein binds to the operator.
(D) Regulator Gene
¾ This
codes for the repressor protein that binds to the operator and ‘switches off
the transcription unit.
¾ So
the regulatory gene is also represented as ‘i’
gene (inhibitory gene).
(E) Inducer
¾ The
substance/substrate that prevents the repressor from binding to the operator,
is called an inducer; it keeps the switch ‘on’ and transcription continues.
12. Lac-operon in Escherichia coli
¾ It
is an inducible operon, where Lactose is the inducer; it is the substrate for
the enzyme 
-galactosidase.
-galactosidase.
¾ The
components of lac-operon and their functions are as follows:
(A) Structural Genes
¾ There
are three structural genes (z, y, a) which transcribe a polycistronic mRNA.
¾ Gene
z codes for enzyme -galactosidase (b-gal),
that catalyses the hydrolysis of lactose into galactose and glucose.
-galactosidase (b-gal),
that catalyses the hydrolysis of lactose into galactose and glucose.
¾ Gene
y codes for permease, which increases the permeability of the cell to enzyme -galactosidase
(lactose).
-galactosidase
(lactose).
¾ Gene
a codes for transacetylase, that catalyses the transacetylation of lactose into
its active form.
(B) Promoter
¾ It
is a sequence of bases near to the structural genes; it is the site where
RNA-polymerase binds for transcription.
(C) Operator
¾ It
is a sequence of base of DNA near the promoter, where a repressor always binds.
¾ It
functions as a switch for the operon.
(D) Repressor
¾ It
is a protein coded by i gene,
sythesised all the time constitutively.
¾ It
binds to the operator and prevents the RNA polymerase from transcribing.
¾ Lactose
is the inducer that inactivates the repressor and prevents it from binding to
the operator.
¾ This
allows an access for the RNA polymerase to the promoter and transcription
continues.
 |
| The lac Operon |
13. Human Genome Project (HGP)
·
Human Genome Project was a 13-year project,
that was launched in the year 1990 and completed in 2003.
·
This project was coordinated by the U.S.
Department of Energy and the National Institute of Health.
·
During the early years of the project,
the Wellcome Trust (U.K) became a major partner; other countries like Japan,
Germany, China and France contributed significantly.
·
Its aim was to find out the complete DNA
sequences for the human genome.
·
The two factors that made this possible
are:
(I) Genetic engineering techniques, with
which it was possible to isolate and clone any segment of DNA
(II) Availability of simple and fast
techniques, for determining the DNA sequences.
·
Human Genome Project was called a mega
project for the following facts:
(I) The
human genome has approximately 3.3 109 bp; if the cost of sequencing
is US $3 per bp, the approximate cost is about US $9 billions.
109 bp; if the cost of sequencing
is US $3 per bp, the approximate cost is about US $9 billions.
(II) If the sequence obtained were to be stored in typed form in
books and if each page contained 1000 letters and each book contained 1000
pages, then 3300 such books would be needed to store the complete information.
(III) The enormous quantity of data expected to be generated also
necessitates the use of high speed computational devices for data storage,
retrieval and analysis.
·
The project was closely associated with
a new branch of biology, called bioinformatics.
(A) Goals of HGP
·
Some major/important goals of HGP are
to:
(I) Identity all the genes (approximately
20000-25000) in human DNA.
(II) Determine the sequences of the three
billion base pairs present in human DNA.
(III) Store this information in data bases.
(IV) Improve the tools for data analysis.
(V) Transfer the technologies to other
sectors (like industries).
(VI) Address the ethical, legal and social
issues (ELSI), that may arise from this project.
(B) Advantages/Uses of HGP
(I) Knowledge
of the effects of variations of DNA among individuals can revolutionise the ways
to diagnose, treat and even prevent a number of diseases/disorders that affect
human beings.
(II) It provides clues to the understanding of human biology.
(C) Methodologies of HGP
¾ The
methods involved two major approaches:
(I) One
approach, called Expressed Sequence Tags (ESTs), focused on identifying all the
genes that expressed as RNA.
(II) Second approach, called Sequence Annotation, was to simply
sequence the whole set of genome, that included all the coding and non-coding
sequence and later assigning functions to different regions in the sequence.
¾ The
total DNA from the cells is isolated and converted into random fragments of
relatively smaller used hosts are bacteria and yeast and the vectors are
bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC).
¾ The
fragments are then sequenced using automated DNA sequences, which work on the
principle developed by Frederick Sanger.
¾ The
sequences were them arranged on the basis of certain overlapping regions
present in them; this required the generation of overlapping fragments for
sequencing.
¾ Specialised
computer based programmes were developed for alignment of the sequences.
¾ These
sequences were annotated and assigned to the respective chromosomes.
¾ The
next task was to assign the genetic and physical maps on the genome; this was
generated using the information on polymorphism of restriction endonuclease
recognition sites and certain repetitive DNA sequences, called microsatellites.
(D) Salient features of Human Genome
¾ Following
are some of the salient observations derived from HGP.
(I) The human genome contains 3164.7 million
nucleotides (base pairs).
(II) The size of the genes varies; an average gene consists of 3000
bases, while the largest gene, dystrophin consists of 2.4 million bases.
(III) The total number of genes is estimated as 30000 and 99.9% of the
nucleotides are the same in all humans.
(IV) The functions of over 50% of the discovered genes are not known.
(V) Only
less than 2% of the genome codes for proteins.
(VI) Repetitive sequences make up a large portion of the human
genome.
(VII) Repetitive sequences throw light on chromosome structure and
dynamics and evolution, thought they are thought to have no direct coding
functions.
(VIII) Chromosome 1 has 2968 genes (the maximum) and the Y-chromosome has
231 genes (the least).
(IX) Scientists have identified about 1.4 million locations, where
DNA differs in single base in human beings; these are called single nucleotide
polymorphisms (SNPs).
(E) Applications/Future challenges of HGP
(I) Having
the complete sequence of human genome, will enable a radically new approach to
biological research, i.e., a systematic approach on a much broader scale.
(II) All the genes in a genome or all the transcripts in a
particular tissue/organ/tumor can be studied.
(III) It will be possible to understand how the enormous number of
genes and projects work together in interconnected networks in the chemistry of
life.
14. DNA-Fingerprinting
·
The technique of DNA-fingerprinting was
developed by Dr. Alec Jaffreys, in an attempt to find out markers for inherited
disease; the process is also known as DNA-typing or DNA-profiling.
·
DNA-fingerprinting involves identifying
differences in some specific short nucleotide repeats, called Variable Number
Tandem Repeats (VNTRs), that vary in number from person to person and are
inherited.
·
The VNTRs of two persons may be of same
length and sequence at certain sites, but very at others.
·
The procedure of DNA-fingerprinting
includes the following major steps:
(I) Extraction:- DNA
is extracted from the cells in a high-speed, refrigerated centrifuge.
(II) Amplification:- Many copies of the extracted DNA are
made by polymerase chain reaction (PCR).
(III) Restriction Digestion:- DNA is cut into fragments with
restriction enzymes into precise reproducible sequences.
(IV) Separation of DNA sequences/restriction
fragments:- The
cut DNA fragments are introduced and passed through electrophoresis set-up
containing agarose polymer gel; the separated fragments can be visualized by
straining them with a dry that show fluorescence under ultraviolet radiation.
(V) Southern Blotting:- The separated DNA sequences are transferred
onto a nitrocellulose or nylon membrane or sheet placed over the gel.
(VI) Hybridisation:- The nylon membrane is immersed in a
bath and radioactive probes (DNA segments of known sequence) are added; these
probes target a specific nucleotide sequence that is complementary to them.
(VII) Autoradiography:- The nylon membrane is pressed on to an
x-ray film and dark bands develop at the probe sites which resemble the
bar-codes.
·
DNA-fingerprinting
technique is use for the following purposes:
(I) To identify criminals in the forensic
laboratories.
(II) To determine the real parents, i.e., to
identify the true biological father or mother in cause of disputes.
(III) To verify whether an immigrant is really a
close relative (as he/she claims) of mentioned resident.
(IV) To identify racial groups to rewrite the
biological evolution.
·
GENE
BANK
: With a view towards rapidly proceeding
genetic erosion, it is important to conserve
germplasm because it is difficult to say which gene may prove to be
lifesaving in future. Also, plant breeders require a variety of genes for
developing new varieties. To serve these purposes, the plant species are
searched for variations and are stored safely in a gene bank, in the form of
seeds, other propagules, or even tissues.
GENE
BANKS are
included in ex-situ conservation
strategies and aim at static
conservation, that is to retain, as far as possible, the genetic structure
of theoriginal population in the same pattern. In this type, conservation is
carried out by reducing the life processes to a low level and is the safest and
cheapest method for conserving genetic information.
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