The Gene 273 by T70zwduQ


									                                       The Gene
                                      Booklet No. 273
                                   Biotechnology: BTS - 3
I.      Introduction
II.     The Gene Concept
               A. Genetic material
               B. Evidence
               C. Unraveling of gene concept
III.    Nucleic Acids
               A. Deoxyribo nucleic acid
               B. Ribo nucleic acid
IV. Protein Synthesis
               A. Flow of genetic information
               B. Role of amino acids
               C. Mechanism of protein synthesis
V. Genetic code
               A. Triplet code
               B. Properties
               C. Deciphering of genetic code
               D. Contribution of Dr .Khurana
VI. Gene Functioning
               A. Concept of cistron, muton, recon
               B. Operon concept
               C. Genes in action
               D. Regulation of gene activity
               E. Gene mutation

         Biotechnology is making inroads into the mysteries of gene exploring new possibilities of
utilizing the genetic knowledge in agriculture, animal husbandry and ecology. This booklet
covers some basics in gene theory.

Dr. K. T. Chandy, Agricultural & environmental Education

I. Introduction
        Genes are responsible for the various characteristics in a living being. So also the
various economic characters of crops and animals are governed by genes. Hence some
understanding of the basic structure and functions of genes is necessary for the proper study of
crop and animal characteristics.

       This booklet is a prerequisite for learning breeding of crops and animals. It begins with
the concept of gene and goes on to nucleic acid, protein synthesis, genetic code and functioning
of genes.

        Due to practical reasons the number of diagrams have been reduced to minimum. The
trainer has to however use as many visual -aids as possible during training to make the
participants understand the structure and functioning of genes.
II. The Gene Concept

       The advancements in methodology and instrumentation applied to chromosomes, led to
a progressive understanding of the structure and behaviour of genes. Genes located linearly on
the chromosomes, are the ultimate units of heredity. There may be as many as 1000 to 10,000
genes on a chromosome depending upon the length of the chromosome.

        Various attempts were made by early molecular geneticists to identify the physical and
chemical nature of genes. But the genes were found to be so minute in structure that their
physical identity remained almost impossible. However, the extensive chemical analysis of
chromosomes of different organisms have revealed that chromosomes contain protein and
nucleic acids (DNA and RNA) and it was thought that genes might have either protein or nucleic
acids as their component molecules. In 1953, it was universally accepted that DNA is the
genetic material. Later on, RNA was also found to be the genetic material in some viruses.

A. Genetic material
      The genetic material is of key importance to cell function and it must, therefore, fulfill a
number of basic requirements as mentioned here.

1. It must contain the information for cell structure, function, and reproduction in a stable form.
This information, is encoded in the sequence of basic building blocks of the genetic material.
2. It must be possible to replicate the genetic material accurately so that the same genetic
information is present in descendant cells and in successive generations.
3. This information coded in the genetic material must be able to be decoded to produce the
molecules essential for the structure and function of cells.
4. The genetic material must be capable of (infrequent) variation. Specifically, mutation and
recombination of the genetic material are the foundation for the evolutionary process. The
nucleic acids viz. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), mect all these

B. Evidence
        All of the evidence of recent genetic research point to DNA as the primary substance of
which genes are composed. Genetic diversity is possible because the DNA molecule is very
long and capable of many different sequences of its four basic components along its length.
There are several evidences from various sources to prove DNA to be the actual genetic
material of which genes are made. They are: evidences from studies of cell division, bacterial
transformation, virus research and biochemical evidence. These are briefly explained here.

1. Studies of cell division
        When chromosomes are clearly seen in the early prophase of mitosis, they are already
double in nature. Thus the actual gene duplication must take place some time before this state.
It was found that the DNA quantity remained rather constant for about 8 hours after a cell had
divided and then the quantity of DNA was doubled. About 5 hours after this duplication was
visible under the microscope. The DNA free portion of the chromosomes, on the other hand,
showed considerable variation in quantity and no such regular doubling.

2. Bacterial transformation
       The bacteria Pneumococci can be grouped into different types depending on their ability
to synthesize specific polysaccharides. Under particular conditions a specific strain of
Pneumococci lost its property of synthesising the corresponding polyssaccharide. As a result,
this colony appeared rough in contrast to other glistening colonies formed in vitro by the other
strains which bore their gummy polysaccharide capsules. A living culture of such 'rough' strain
(R-strain) was injected into mice with a dead culture of the capsula led strain (S-strain). On
examination of the organisms subsequently recovered from the mice, they were found to be
living virulent S-strain pneumococci. Evidently therefore, the non- capsulated pneumococci (R-
strain) have acquired the property of synthesizing polysaccharides to form capsules from some
material in the dead S-strain pneumococci.

        Later, the same pneumococcal transformation was carried out in vitro by adding a cell
free extract of the killed capsulated strain (S-strain) to a culture of R-strain pneumococci. Such
transformed pneumococci did not revert to the original type. DNA extracted from the capsulated
S-strain could transform the R-strain to the capsulated one which maintained the property
through generation and produced identical DNA. It, therefore, showed the properties of re-
duplication and of induction of the inheritable capacity of capsule synthesis -two characteristics
attributed commonly to the gene. The phenomenon of transformation was later observed in a
wide range of bacteria. Other characters than capsule formation could also be transferred,
including drug resistance to various antibiotics.

3. Virus research
        Further evidence of the DNA nature of the gene was provided by a study of viral
multiplication. Bacteria are attacked by specific types of viruses called bacteriophage. A
bacteriophage has a very peculiar shape. It resembles an injection syringe, with a long hollow
body and' a pointed hollow tail. The whole body is made up of protein and the comparable fluid
inside the body consists of DNA. The various types of phages are distinguished by the
composition of their protein coats. When a bacterium is infected, the phage attaches its pointed
end to the outer wall of the bacterium, the wall between the phage and the bacterium dissolves
and the body fluid of the phage containing DNA alone is injected into the body of the bacterium.
After sometime numerous new phages are formed through multiplication inside the bacterium.
They come out through lysis of the bacterium resulting in the death of the latter. Thus, the new
phages their DNA fluid, and protein body coat are all formed out of the substance injected into
the bacterium which contains only DNA. Hence, DNA may be regarded as the substance
responsible for hereditary transmission.

4. Biochemical evidence
a. The amount of DNA in any given species of cell or organism is remarkably constant and
cannot be altered by environmental circumstances or by changes in the nutrition. Sperms and
eggs, on the other hand, have only about one - half as much DNA as the other body cells. This
is in agreement with the fact that these gametes have only half as many genes as are found in
somatic cells.
b. The amount of DNA per cell appears to be in proportion to the complexity of the cell and the
amount of genetic information it contains. The amount of DNA per cell ranges anywhere from
0.00024 pg/cell (1 picogram =1 x 10-12 gm) found in bacteriophage -T4 to 0.02 -0.17 pg/cell in
fungi and 6 pg/cell in higher animals. Since bacteria and viruses contain relatively little genetic
information and so relatively few genes an consequently small amount of Deoxyribo-nucleic

C. Unraveling of gene concept
       The principal landmarks in the unraveling of gene concept, as a sequel to the
discoveries initiated by the advances in technology, can be grouped into three main phases.

1. Determination of the chemical composition of the chromosome.
2; Identification of the gene substance responsible for heredity.
3. Understanding the pattern of behaviour and regulation of the gene.

III. Nucleic Acids

        Subsequent to the isolation of nuclear material by Miescher (1874) which he termed
"nuclein", now known to be nucleoprotein, nucleic acids and proteins were observed to be
normal constituents of all cell nuclei.

      Nucleic acids are present in all living organisms in the form of deoxyribonucleic acids
(DNA) and ribonucleic acid (RNA). Some viruses, like tobacco mosaic and poliomyelitis virus
may contain only RNA in contrast to the bacteriophages and adeno-viruses which have only
DNA. Both DNA and RNA are present in bacteria and higher organisms.

        Within the nucleus, DNA occurs in combination with proteins. It has also been recorded
in mitochondria, chloroplasts and probably other self-replicating organelles. RNA is located in
the nucleolus, nucleoplasm and chromosomes within the nucleus and in the ribosomes within
the cytoplasm.

          Both nucleic acids are long poly-nucleotides, formed of a series of individual nucleotides
linked together. Each nucleotide is further composed of:
i. one molecule of phosphoric acid;
ii. one molecule of a pentose or deoxypentose sugar; and
iii. a nitrogenous base (purine or pyrimidine).

        The combination of a pentose sugar with a nitrogenous base is called a nucleotide. The
nucleotides are joined by the phosphoric acid, which links the pentose of two consecutive
nucleosides through an ester-phosphate bond. These bonds attach carbon 3' of one nucleoside
to the carbon 5' in the next one. The remaining free acid group is used to form ionic bonds with
the basic proteins (which enable their staining with basic dyes).

        The pentose sugar is ribose in RNA and deoxyribose in DNA. In DNA, the oxygen on
carbon 2' is missing. The principal pyrimidine bases present are cytosine (C), thymine (T) and,
uracil (U). Thymine is exclusive to DNA and uracil to RNA, while cytosine is common to both.
The purine bases observed in both DNA and RNA are adenine (A) and guanine (G). The
alternating double and single bonds between the carbon atoms in the nitrogenous bases can
interchange continuously, as a result of which the bases absorb ultraviolet light at 2600 AD.

       Since adenine pairs only with thymine and guanine with cytosine, their respective ratios
are always 1:1. However, the AT /GC ratio in the DNA of different species is variable. Adenine
and Thymine exceeds Guanine and Cytosine in higher plants and animals. Variation is much
greater in viruses, bacteria and lower' plants.

A. Deoxyribonucleic acid (DNA)
        DNA is found in the cells of all living organisms except plant viruses where RNA form the
genetic material. In bacteriophages and viruses there is a single molecule of DNA, which
remains coiled and is enclosed in the protein coat. In bacteria, mitochondria and plastids of
eukaryotic cells, DNA is circular and lies naked in the cytoplasm. In the nuclei of cukaryotic
cells, DNA occurs in the form of long spirally coiled and unbranched threads. The number of
DNA molecules is equivalent to the number of chromosomes per cell.
1. Structure
       DNA is a polymer: the monomers of which are the deoxy-ribonucleoside mono-
phosphates. Most DNAs have high molecular weight, varying from 106 to 109 or more.

        A double helix model was prepared for the spatial molecular structure of DNA by J.D.
Watson and F.H.C. Crick. According to this model, two long polynucleotides chains form a DNA
molecule. The chains run in opposite directions, forming a double helix around a central axis.
The nucleosides are arranged perpendicular to the polynucleotides chains. The latter are held
together by mobile hydrogen bonds between pairs of bases facing each other. The diameter of
the helix is about 20 Ao and each chain makes a complete turn every 34 Angstroms. There are
10 nucleotides on each chain in every turn of the helix. The two helixes are coiled in such a way
that they cannot be separated without unwinding. The base pairing between opposing
nuclcotides is specific due to this distance, one purine base being able to pair with only one
pyrimidine and vice versa. Adenine may only pair with thyamine (AT) and guanine with cytosine
(GC), the hydrogen bonds attaching them being two and three, respectively.

        When a DNA chain replicates, the two strands separate. On each strand, another poly-
nucleotide is synthesized which, in its base composition, is exactly complementary to the
original one serving as a template.

2. Biological replication
        During the replication, the DNA duplicates exactly into two chains. In the beginning both
the strands (there are nicks in the strands) of double stranded DNA separate from each other by
the dissolution of hydrogen bonds and thus uncoiling of the chains occurs. And then new
chains, "the complementary chains" are built on them so that the newly formed daughter chains
are the mirror images of each other. The entire process of DNA replication involves following

a. Initiation point
         Replication of DNA begins at a specific point, called initiation point. Specific initiator
proteins are required to recognize the initiation point in DNA. The initiator protein along with
DNA - directed -RNA polymerase initiates the synthesis of RNA primer for the formation of new
DNA chain.

b. Unwinding of DNA
       The unwinding proteins bind to the nicked strand of the duplex and open up a loop or
bubble, separating the two strands of DNA duplex. The endonuclease enzyme attacks one of
the two sides of phosphodicstre linkages, thus helping in breaking the hydrogen bonds between
the bases with the aid of exonuclease enzyme.

c. RNA priming
        The DNA- directed-RNA polymerase now synthesizes the primer strands of RNA (RNA
primer). The priming RNA strands are complementary to the two strand of DNA and are formed
of 50 to 100 nucleotidcs.

d. Formation or DNA OD RNA primers
        The new strands of DNA are formed in the 5' -3' direction from the 3'- 5' template
(model) DNA by the addition of dooxy-ribonucleotide to the 3' end of RNA primer. The addition
is affected by DNA polymerase III in the presence of ATP. The unwinding proteins separate the
duplex stands ahead of the replication fork. The leading strand of DNA is synthesized in 5'-3'
direction. The following strand of DNA is formed in its opposite direction," The DNA strands are
synthesized in short segments, consisting of 1,000 to 2,000 nucleotides. These segments are
calIed 'Okazaki fragments'.

e. Excision of RNA primers
       Once a small segment of an Okazaki fragments has been formed, the nucleotides of
RNA primer are removed from the 5' end one by one by the action of 5'- 3' exo-nuclease activity
of DNA polymerase l.

f. Joining of okazaki fragments
        The gaps left between okazaki fragments are filled with deoxy-ribonucleotide residues by
DNA polymerase 1. Finally the adjacent 5' and 3' ends are joined by DNA ligase.

       Three different types of replication of DNA have been discovered, namely, (a) semi-
conservative, (b) conservative, and (c) dispersive. However, only the semi-conservative type of
DNA replication is acceptable.

        In semi-conservative replication, the replication of DNA involves the unwinding the
double helix, but no rupture of the separated polynucleotide strands. Thus, the primary DNA is
conserved during the replication process. According to this mechanism, the unwinding of the
original strands would be accompanied or followed by the guided synthesis of the
complementary strands of each of the two separated poly-nucleotide strands. The linear
assembly, catalyzed by enzymes, of the nucleotides of the complementary sequence is guided
by their pairing with those on the original strands. In this manner, the proper nucleotide
sequence would be automatically assumed. The DNA helix would regenerate each of the strand
of the original helical duplex uniting with its freshly synthesized complement to form a total of
two new doub1e stranded structures. The semi-conservative model thus predicts that entire
polynucleotide strands of the parental DNA will be passed on to the progeny in intact form. Each
molecule of progeny DNA will receive either one half or none of its nucleotides from the parental

      In summary, the basic biochemical characteristics of the double stranded DNA are
enumerated as follows.

(i) DNA consists of two different purines (guanine, adenine) and two different pyrimidincs
(thiaminc, cytosine).
(ii) A nucleotide pair consists of one purine and one pyrimidine (adenine/thymine (AT) or
guanine/cytosine (GC)).
(iii) Nucleotide pairs are connected into a double helix molecule by sugar phosphate backbone
linkages and hydrogen bonding.
(iv) The AT base pair is held by two hydrogen bonds, while the GC pair is held by three.
(v) The distance between each base pair in a molecule is 3.4 producing 10 nucleotide pairs per
turn of the DNA helix.
(vi) The number of adenine molecules must equal the number of thymine molecules in a DNA
molecule. The same relationship exists for guanine and cytosine molecules. However, the ratio
of AT to GC base pairs may vary in DNA from species to species.
(vii) The two strands of the double helix are complementary and antiparallel with respect to the
polarity of the two sugar- phosphate backbones, one strand being 3'- 5' and the other being 5'-
3' with respect to the terminal OH group on the ribose sugar.
(viii) DNA replicates by a semi-conservative method in which the two strands separate and each
is used as a temperature for the synthesis of a new complementary strand.
(ix) The rate of DNA nucleotide polymerisation during replication is approximately 600
nucleotides per second. The helix must unwind to form templates at a rate of 3600 rpm to
accommodate this replication rate.

3. Biological significance
       There are four general requirements that genetic material is expected to fulfill.

a. That it replicates itself accurately during cell growth and duplication.
b. That its structure be sufficiently stable so that heritable changes and mutations occur only
very rarely.
c. That it has potentiality to carryall kinds of necessary biological information.
d. That it transmits the informations to the cell.

B. Ribonucleic acid (RNA)
       RNA usually occurs in nature as long unbranched polymeric molecule made up of single
chain or helix of polynucleotide strand. RNA is generally present in the cytoplasm as well as in

       RNA is composed of pentose sugars, phosphoric acid and nitrogenous bases. The
pentose sugar of RNA is ribose sugar, and pyrimidine bases comprise uracil (instead of
thymine) and cytosine. Excepting these differences, RNA has a structure similar to DNA

1. Types of RNA
       Four types of ribonuclcic acids have been discovered, they are as follows:

8. m-RNA (messenger RNA)
       Jacob and Monad (1961.) first proposed this name, believed that a "messenger" is
necessary to convey they message of the genetic code from the DNA of the nucleus to the
cytoplasm. This .'messenger is known as m-RNA, and it acts as a template for the translation of
the DNA code into a specific protein. Thus, it helps in determining the structure of protein.
m-RNA molecules exist in a large variety of lengths, depending on the length of the polypeptide
chain for which they code. Its molecular weight and sedimentation value vary considerably.
Generally, the lifespan of m-RNA is relatively short, although in some eukaryotic cells there is
evidence of some degree of stability. M-RNA is always complementary to a portion of strand of
DNA with the exception that uracil replace thymine.

       When the code has been transcribed from DNA on to m-RNA, the latter leaves the
nucleus, which passes through the nuclear membrane into the cytoplasm. Here, it moves to the
ribosomes, the site of protein synthesis m-RNA molecules attaches reversibly to the surface of
ribosome always binding to the smaller subunits. The Mg++is involved in the formation of a
complex between m-RNA and ribosomes.

        In eukaryotic cells, m-RNA is associated with protein forming ribonucleo protein
complexes. Some of these complexes remain free in the cytoplasm without being attached to
the polyribosomes. These are named as informosomes, and are very stable and can remain in
the cell cytoplasm for several days. These are used by the cell in protein synthesis only when
there is a delay in translation. The stability is attributed by the presence of protein sheath
around m-RNA molecule. In informosomes, proteins and m-RNA as found in the ratio of 4 : 1.

b. t-RNA (Transfer RNA)
        The t-RNA forms about 10 -15% of the total RNA in most of 1the cells. Each t-RNA
contains approximately 80 nucleotides. According to the Holley (1965) the t-RNA is a
polynucleotide chain which is folded upon itself to form five arms. As a result of the folding the 3'
and 5' ends of the chain come near each other. An arm consists of a stem and a loop. Bases
are arranged in pairs in arms. There is no base pairing in the loops. Four different regions or
special sites can be recognized in the molecule of t-RNA. These are described below.

i. Amino acid uttachment site
       It occurs at the 3' end of the t-RNA chain and has OH group of ribose molecule free to
pair. This -OH group combines with the specific amino acid in the presence of ATP forming
amino-acyl t-RNA.

ii. Recognition site
        It contains a specific base sequence which dictates the, attachment of correct amino
acid to the t-RNA molecule. It matches with the amino acid activating enzyme through which
attachment of amino acid to t-RNA takes place.

iii. Anti-codon or codon recognition site
         This site has three unpaired base (triplet of base) whose sequence is complementary
with a codon (triplet) in m RNA. Therefore, it determines the pairing of t-RNA with the specific
codon (triplet) of m-RNA. lt is, therefore, the most specific region of t-RNA molecule.

iv. Ribosome recognition site
       This helps in the attachment of t-RNA to the ribosome. This site is common to all the
molecules of t-RNA.

c. r-RNA (Ribosomal ribonucleic acid)
        The ribosome particles contain about 60% r-RNA and 40% protein. It is generally agreed
that cytoplasmic ribosomes or their sub units are made in the nucleus and are subsequently
transferred to cytoplasm. It is synthesized on the part of the chromosome traversing the
nucleolus (the nucleolar organizer) or in some cases, on DNA detached from the chromosome
and associated with the nucleolus r-RNA differs in size and base contents from t-tRNA and from
other RNA classes of most cells it is relatively rich in guanine and cytosine.

       The function of r-RNA in protein synthesis is not yet completely clear. The unpaired
bases in the molecule may bind m-RNA and t-RNA to ribosomes possible by Mg+ + linkages
between phosphate groups on the two molecules.

d. v-RNA (Viral RNA)
       Viruses may contain either DNA or RNA. However, each particular virus generally
contains nucleic acid of only one type. Some examples of RNA viruses are influenza, turnip
yellow mosaic, tomato bushy stunt, tobacco necrosis and the tobacco mosaic virus.

2. Biosynthesis
        Four kinds of RNA, though structurally alike, differ considerably in molecular weight and
are distributed in different parts of the cells. These are biosynthesized or replicated as follows.

a. Biosynthesis of r-RNA
        The two types of r-RNA (28s and 18s) are transcribed from the nucleolar DNA as a
single elongated unit of 45s. Inside the nucleus the 45s RNA is methylated and complex with
protein. Finally, via a number of steps, it is cleared into 32s and 18s segments. The 18s RNA
gets associated with basic proteins to form the small sub-units of ribosome. The 32s segment is
further separated and finally changed to 28s RNA. This 28s RNA also gets associated with
proteins and forms the large subunit of ribosome. These units then come out into the cytoplasm.

b. Biosynthesis of m-RNA
        m-RNA was used to describe a form of RNA that would be made in the cell nucleus, but
would be transferred to the, cytoplasm, during protein synthesis. During protein synthesis, the,
amino acid sequence is determined by DNA which is mainly in the nucleus. The information
from DNA in the form of a specific sequence of bases had to be transferred somehow to the
cytoplasm. The notion of am RNA demanded that DNA acts as template for RNA synthesis. The
principles of base pairs still applies here, because uracil in RNA has the same dimension as
thymine in DNA, and one can substitute for the other. In this manner a DNA strand could
function as template for RNA synthesis.

C. Biosynthesis of t- RNA
       The details of r-RNA synthesis are not known. It is known that t-RNA is synthesized on
DNA templates by steps similar to those of other (ypes of RNA.

IV. Protein Synthesis

        The proteins are highly organized and complex structures and playa significant role in
the structural and functional organisation of the cell. The structural proteins constitute various
cellular components and some extra-cellular parts such as cuticle and fibers. The functional
(such as enzymatic and hormonal) proteins control almost all metabolic, biosynthetic,
bioenergetic, growth regulating sensory and reproductive activities of the cell. All the proteins
which are required by the cell for its different purposes are synthesized by the cell itself.

       Chemically, proteins are polymers of amino acids. The amino acid molecules are linked
together in linear fashion forming long polypeptide chain. The adjacent amino acids are joined
together by covalent peptide bond between the carboxy group of the amino acid and the amino
group of the other.

        DNA initiates the mechanism since it caries the genetic message for protein synthesis.
This genetic message from one DNA strand is transferred to a complementary strand of
messenger RNA. m-RNA then passes it to the nucleus and carry the message to the ribosomes
where it is used to synthesize the particular type of protein molecule. The first step is called
transcription because it involves copying of the genetic information from DNA into m-RNA which
transmits the coded information from DNA to ribosomes, the site of protein synthesis. The
second step is termed as translation as it involves the translation of the information enclosed in
m-RNA with newly synthesized protein molecule.

A. Flow of genetic information
       There is a particular relationship between DNA, RNA and protein. Three theories have
been put forward to describe the flow of genetic information.

1. One way flow
       Crick (1958) suggested that DNA transmits the genetic information for protein synthesis
by transcribing m-RNA (transcription) and other types of RNA. These RNAs thcn translate the
nucleotide sequence into the sequence of amino acids (translation). This is known as one way
now of information or ‘central dogma'.
2. Circular flow
       Barry Commoner (1968) suggested the circular flow of information. The DNA transcribcs
RNA (m-RNA, t-RNA etc.) then RNA translates the information into protein; it then directs the
synthesis of RNA; and RNA dictates the synthesis of DNA.

3. Inverse now
       Temin (1970) described the presence of an enzyme-RNA dependent DNA- polymerase,
which controls the synthesis of DNA from a single strand of RNA. D. Baltimore (1970) described
the presence of this enzyme in certain RNA tumor viruses. In tumour viruses, the RNA is
genetic RNA, but in certain cases the non-genetic RNA synthesized under the control of DNA
may also control the synthesis of DNA by inverse flow of information. This is known as "central
dogma reverse".

8. Role of amino acids
        Amino acids are the units from which the protein molecules are made. In the cytoplasm
of every ccll a number of amino acids are present. Out of these, 20 amino acids lake important
part in protein synthesis. The common ones are: glycine, alanine, valine, leucine, isoleucine,
serine, threonine, phenylalaminc, tyrosine, tryptophan, cystine, cysteine, methionine, proline,
hydroxyproline, aspartic acid, glutamic acid, asparagine, histidine, arginine, lysine and

       The amino acids are distributed throughout the cytoplasm which are collected by specific
t-RNA. t-RNA brings the amino acids to the actual site of protein synthesis i.e. to ribosomes
where the synthesis of protein takes place.

C. Mechanism of protein synthesis
      Protein synthesis consists of two events, namely, (1) transcription and (2) translation.

1. Transcription
       The formation of m-RNA is called transcription. During the process an enzyme RNA
polymerase is needed. The enzyme is then attached to the DNA at the initiation site, where a
short segment of the DNA molecule is opened up. The complementary nuclcotides developed
from one of the DNA strands, from m-RNA. The RNA polymerase moves along the DNA
strands, and the growing m-RNA strand is peeled off till a termination site is reached.

        The enzyme RNA-polymcrase consists of five different polypeptide chains of which four
collectively form the core enzyme while the fifth is called the sigma chain.

        The sigma chain recognizes start signals along the DNA molecules, A specific protein
called the 'rho' factor stops the formation of the RNA chain. Once RNA synthesis is initiated, the
sigma factor dissociate and core enzymes bring about elongation of m-RNA.The m-RNA makes
its way through the pores in the nuclear membrane lo the cytoplasm lo start the next stage
which is translation.

2. Translation
        It involves the translation of the language of DNA into the language of protein i.e. the
formation of protein takes place under the control of m-RNA. This process is studied under the
following heads.

a. Activation of amino acids
        The first step in protein synthesis is the activation of amino acids with ATP. The reaction
is catalyzed by a specific enzyme i.e. amino acyl synthetase by the first product i.e. amino acid
adenylate is formed in which the energy is consumed for the requirement of next step.

Amino acid + ATP + Amino -----> Enzyme bound +
                                     2P-acyl-synthtase amino acid adenylate

b. Attachment of activated amino acid with t-RNA
         In this step the activated amino acids are attached with the t-RNA. The adenylate
remains bound to the enzyme unlit it is transformed lo the t-RNA and this transfer is again
catalyzed by the same enzyme.
Amino acyl + t-RNA ---------------- Amino acid + Adenosine monophosphate
adenylate                        Aminoacyl          t-RNA
c. Initiation of protein synthesis
         Initiation of protein synthesis in the ribosome is a complex step involving m-RNA, formyl
methionine, t-RNA (F-met-t-RNA), the initiator factors, GTP and the small subunit (of ribosome).
The small subunit recognizes the initiation codon on m RNA which is always AUG (base
sequence) in both prokaryotes and eukaryotcs. F-met-t-RNA attaches to the small subunit –m-
RNA complex. Now the larger subunit combines with this complex to form the complete
ribosome. There are three Initiator factors Fl, F2 and F3 which are loosely associated with the
small ribosomal subunit. Fl, and F2 along with GTP are required to bind F-met-t RNA to the
small, subunit in response to the codon AUG factor stimulates the binding of m RNA to the
initiation site of the small subunit.

         In eukaryotes, met-t-RNA is involved instead of F-met-t-RNA. Secondly, in them the
initiation factors are M1, M2 and M3 which are homologous to Fl, F2 and F3, of prokaryotes.

d. Chain elongation
Chain elongation involves the following four sub-phases:
i. Elongation factors- Elongation of the polypeptide chain requires elongation factor (EF). These
are EF- Tu, EF- Ts and EF-G in prokaryotes and EF -1 and EF-2 in eukaryotes.

ii. Binduzg of amuzoacyl t-RNA to the A site -The second amino acid RNA complex (ala –t-RNA)
now occupies the acceptor site (A site) of the 50s subunit of the ribosome.

iii. Peptide bond formtrion- The next step includes the formation of a peptide bond between the
amino acid and the growing polypeptide of E-met-t-RNA, if the initiation has just been
compteted. The polypeptide chain is transferred from its position at the P-site to the amino acyl
t-RNA, located at the A-site. The polypcptide is first cleaned from the t-RNA at the P-site and
becomes attached to the aminoacyl t-RNA at the A-site.

iv. Translocatioll of polypeptide -The next step comprises the discharge of the unloaded t-RNA
from the P-site. This translocation of the polypeptide from the A-site to the P-site requires the
supernatant G factor, which is ribosome dependent GTPase. GTP is also required. As
translocation occurs, the peptidyl t-RNA is moved from the A-site to the P-site, and the m-RNA
moves one codon. The uncharged t-RNA from the p-site is discharged.

e. Chain termination
       The polypeptide chain elongates, until a termination codon on m-RNA is reached. This
may be VAA, VAG or VGA. The termination codon provides signals to the ribosomes for the
attachment of release factors. In prokaryotes, there are three release factors, RF-l, RF-2 and
RF-3. In eukaryotes, a single release factor (RF) recognizes all three termination codons.

       The release factor interact with peptidyl transference and cause hydro1ysis of the
polypeptide chain at the site, thus the chain is released from the t-RNA molecule. The residual t-
RNA is discharged from the P-site. The ribosome dissociates into 30s and 50s subunits. The
polypeptide chain may undergo further processing. Secretory polypeptides pass through a
tunnel between the two ribosomal subunits into the endoplasmic cisternae. Structural proteins
are incorporated into the cell membrane.

V. Genetic Code

        Living things depend on proteins for existence, the latter produce enzymes necessary for
all chemical reactions. Structural information required to specify the synthesis of any given
protein resides in the molecule of DNA. The linear sequence of bases in DNA constitutes an
alphabet (hereditary lettering, generally considered to be of four letters-A, T, G, C) which 'codes'
for another linear structure, a protein, written in another alphabet of twenty amino acids. The
actual transfer of information is, however, indirect, DNA is a 'template' for the formation of RNA,
which are incorporated into ribosomes and in turn act as templates for protein synthesis. All
properties of protein, including its secondary and tertiary structure, are ultimately determined by
chromosomal DNA, and all biological properties are, in turn, determined by the amino acid
sequence of the proteins within an organism, either through protein structure or enzymic activity.
The term coding implies the relationship between DNA and protein. By coding, the hereditary
lettering carried in the four alphabets of DNA is ultimately converted into the protein language
composed of twenty alphabets of amino acids.

A. Triplet code
        DNA contains four kinds of nucleotides (A, T ,G and C) and protein are synthesized from
20 different types of amino acids A basic problem regarding the genetic code was as to how
many bases of DNA specify one amino acid. In a single code each base or letter would specify
one amino acid. Only 4 of the 20 types of amino acids would be coded unambiguously by a
single code. On a two letter or a double code two bases would specify one amino acid. Here
16(4 " 4) of the amino acids can be specified, but there would be ambiguous determination of a
number of amino acids.

         A triplet code or three letters code specify one amino acid. Thus, 64 (4 x 4 x 4) distinct
triplets of purine and or pyrimidine bases determine the 20 amino acids. These triplets are
called codons. Since there are 64 codons and only 20 amino acids, it is obvious that there are
many more codons than there are amino acids. The codons are mentioned here.

B. Properties
       Some properties of the genetic code are mentioned here in table 1

                         Table 1: Some properties of the genetic codes

                           1     AAA         GAA       CAA      TAA
                           2     AAG         GAG       CAG      TAG
                           3     AAC         GAC       CAC      TAC
                           4       AAT       GAT      CAT       TAT
                           5       AGA       GGA      CGA       TGA
                           6       AGG       GGG      CGG       TGG
                           7       AGC       GGA      CGC       TGC
                           8       AGT       GGT      CGT       TGT
                           9       ACA       GCA      CCA       TCA
                           10      ACG       GCG      CCG       TCG
                           11      ACC       GCC      CCC       TCC
                           12      ACT       GCT      CCT       TCT
                           13      ATA       GTA      CTA       TTA
                           14      ATG       GTG      CTG       TTG
                           15      ATC       GTC      CTC       TTC
                           16      ATT       GTT      CTT       TTT

1. Non overlapping
        The evidence for a non-overlapping triplet code was given by Khurana and his co-
workers in 1965. On the surface it appears wasteful for 64 triplets to stand for 20 amino acid, so
attempts were made to hypothesize a triplet code in which the code was overlapping and so to
reduce the number of codons to about 20. But a lot of shortcomings are there in this proposal.
Let us examine the case of lysine, whose codons appear to be AGG and AAA, and that of
phenylalamine, whose codons appear to be UUU and UUC Since the code letters of these two
amino acids are mutually exclusive, lysine could not follow phenylalamine, or vice-versa, in any
protein. This would constitute a forbidden combinations of aminoacids in proteins. Any two or
three or more amino acid combinations are possible. Therefore, it can be concluded that the
genetic code is non-overlapping.

2. Degenerate
        The code contains many synonyms, in that almost all amino acids are represented by
more than one codon. For example, the three amino acids- arginine, serine and leucine each
has six synonymous codons. However, for many of the synonym codons specifying the same
amino acid, the first two bases of the triplet are constant, where as the third can vary. For
example, all codons starting with CC specify proline (CCU, CCC, CCA and CCG) and all codons
starting with AC specify threonine.

3. Commaless
       A commaless code means that no punctuations are needed between any two words. In
other words, after one amino acid is coded, the second amino acid will be automatically coded
by the net three letters and that no letters are wasted for telling that one amino acid has been
coded and that now second should be coded.

4. Universality
       Since the composition of DNA in all organisms, from viruses to human beings, has been
found to be the same, the code was regarded as universal.

5. Polarity
        If a gene is to specify the same protein repeatedly it is essential that the code must be
read between fixed start and end points. These points are the initiation and the termination
codons, respectively. It is also essential that the code must be read in a fixed direction i.e. the
code must have polarity. It is obvious that if the code is read in opposite directions, it would
specify two different proteins, since the codons would have reserved base sequences.
6. Initiation codons
         The starting amino acids in the synthesis of most protein chains are methionine in
eukaryotes and N-formyl methionine in prokaryotes. Methionyl of N-formyl methionyl t-RNA
specifically binds to initiation sites containing the AUG codon. The codon is, therefore, called the
initiation codons. Less often, GUG also serves as the initiation codon in bacterial protein

7. Non-sense codons
         The UAA, UAG and UDC codons do not specify any amino acid, so are called non-
sense codons. They are also called termination codons, because these codons are used by the
cell to signal the natural end of translation of a particular polypeptide. However, their inclusion in
any m-RNA results in the abrupt termination of the message at the point of their location even
though polypeptide chain has not been completed.

C. Deciphering of genetic code
       Most of our current knowledge of the general nature of the genetic code and of the
nucleotide composition and sequences of the various codons has been obtained from five main
types of experimental approaches. These techniques ultimately resulted in determining the code
words for each of the 20 proteinogenic amino acids. However, only Khurana's direct method of
deciphering the code is described here briefly.

        Khurana's dircct mcthod of confirming the genetic code was devised by the Nobcl prize
winner Dr. H.G. Khorana. This method consists of in vitro chemical synthesis of short segments
of DNA of known base sequence with the help of DNA polymerase. From this synthetic DNA, an
RNA of strictly defined base sequence is transcribed under the catalytic influence of RNA
polymerase. A polypeptide is then synthesized under the direction of RNA as represented in the
following schematic diagram.

       The amino acid sequence of the polypeptide so formed is then! determined and
corrected with the base sequence of DNA and RNA. These studies of Khurana and his co-
workers with chemically defined messengers proved very conclusively that:
1. the base sequence in DNA specifics the sequence of amino acids in proteins.
2. the information contained in DNA is conveyed through RNA; and,
3. the genetic code is triplet and non-overlapping in nature.

         By this technique, Khurana not only worked out and confirmed the exact sequence of
code works for all the amino acids but also clarified the roles of the 5' and 3' terminals of m-RNA
molecule. He showed that the translation of m-RNA proceeded from its 5' hydroxyl end towards
its 3' hydroxyl end. His investigation also led to the artificial production of small segments of
DNA molecule, thereby paving the way for the synthesis of artificial genes which could function
in a living cell.

       It appears that scientists are very close to synthesizing artificial genes which, when
introduced into a cell, could function like a natural gene and alter wide field of genetic
engineering with the possibility of finding a cure for genetic disorders of metabolism and of
producing artificial genes into the body. But this also caries the risk of artificial genes producing
undesirable traits in the organisms in which they are introduced.

D. Contribution of Dr. Khurana
       Indian born biochemist Dr. khurana has devised an ingenious technique for artificially
synthesizing m-RNA with repeated sequences of known nucleotides. For this valuable
contribution he was awarded Nobel prize in 1970.

        By using synthetic DNA, khurana and his co-workers prepared chains of
polyribonucleotides with known repeating sequences of two or three nucleotides as follows. I

1. Poly CUC UCU CUC UCU ……………
2. Poly CUA CUA CUA CUA …………….

VI. Gene Functioning

        An attempt is made here to present in simple language the very complicated process of
gene functioning. But prior to that the concept of cistron, muton and recon has to be understood.
A. Concept of cistron, muton and recon
        We know that DNA is the genetic material. The question now is, what is the relationship
of the genes to the long double coils of DNA? How much of the helix makes up a gene? Our
classical concept of the gene held it to be the smallest unit which can be recognized for
recombination, mutation and function. Modern research has shown that these are not
necessarily the same units. Pseudo alleles (genes which are so close together on the
chromosome that they ordinarily move and act as one gene) show that recombination can take
place within a gene because it is a single functioning unit. In microbial genetics it has been
possible to show that the units of recombination, mutation and function are different. Three
terms have been coined to designate these three units where such distinctions are necessary.

       A 'recon' is the smallest element which is interchangeable through genetic
recombination. Extremely delicate studies of recombination in microbes indicate that a recon
consists of not more than two pairs of nucleotides, may be only one.

        A 'muton' is the smallest part which when altered, can give rise to a mutation. Even an
alteration of a single nucleotide pair can result in mutation.
        A 'cistron' is the term applied to a functional unit and conforms more closely to what we
commonly think of as a gene. It has been observed that there can be over a hundred points
within a functional unit wherein a mutation can take place and cause a detectable phenotypic
effect. This means that a cistron is over a hundred nucleotide pairs in length and there is good
evidence that some cistrons may be as long as 30,000 nucleotide pairs. In general, we refer to
the functional unit i.e. cistron, when we use the word "gene" without qualification,

B. Operon concept
       Jacob and Monod formulated the concept of operon-unit of operation in a chromosome.
They observed that the three enzymes -B- galactosidase, acetylase and permease can be
induced to appear in bacterium E. coli by the addition of an inducer in the medium. These
enzymes act in a co-ordinated manner in heredity. A mutation was later discovered in which all
these three enzymes were found to be produced in a large amount in the absence of an
inducer. All of them are affected by genes that map within the lac locus of E. coli in the
sequence: Z (Bita-galactosidase) -y (permease) -a(acetylase).

         On the basis of these observations supplemented by similar behaviour in various other
bacteria, the operon concept was postulated. It visulizes that in a chromosome, the structural
genes are responsible for the synthesis of enzymes e.g. z, y and a. In other words structural
genes are those genes which produce the m-RNA which in turn governs protein production by
the ribosomes in the cytoplasm. They behave in a co-ordinated way and are controlled by an
operator gene which act as switches to stimulate the synthesis of RNA by structural genes. Its
activity, in turn, is inhibited by a repressor gene which may not necessarily be located adjacent
to it under normal conditions. The represser produces an inhibitory substance which checks the
activity of the operator so that the structural genes are unable to function. Following a mutation,
the repressor may be inactivated and as such the operator will be able to operate freely.

       The inducible substance or inducer may directly bind with the represser or regulator
gene at the DNA level or may combine wither its product so that it cannot reach the operator.

        The entire complex is termed on operon. The operon may be mono-cistronic or poly-
cistronic depending on the number of structural genes. The operon complex controls the
synthesis of m RNA by RNA polymerase. The presence of a repressor at the operator locus in
the lac system thus blocks transcription of the operon by RNA polymerase. On the other hand,
an appropriate inducer removes the repressor, thus permitting transcription. Constitutive
enzyme synthesis, which requires continued transcription of structural genes uninhibited by
repressor, may occur through a mutation (i) either of the regulator gene to produce an inactive
repressor unable to bind to the operator, (ii) or of the operator making it incapable of binding the
repressor. The simplicity of the operon concept, as a set of structural genes controlled by an
adjacent site, has led to its becoming one of the central concepts of genetics.

C. Genes in action
        Under this heading we will study the ways in which the genes operate to bring about the
phenotypic effect. For example, why should a person with two genes for albinism fail to develop
pigment in his skin or hair, whereas another person with one or two dominant alleles of this
gene will have normal pigmentation. What is there about the dominant allele which causes the
formation of the pigment? How can one gene produce a person with normal mentality, while a
variant of this .same gene produces a person who is mentally defective? Let us examine some
of these cases in lower forms of animals and plants.

1. Eye colour in Drosophila
        Most of the genes exert their influence by means of enzymes whose specificity they
control. In Drosophila (a fruit-fly), some substances are present which are concerned with the
development of eye pigments. The eye-colour of the normal wild-type of Drosophila is a
particular shade of red which is produced by a blending of an orange-red and a brown pigment
in the facets of the eye. A different substance is necessary for the production of each of these
pigments. Whenever flies are homozygous for a certain recessive gene the substance
vermillion, which forms the brown pigment, is not produced. As a result, the eye is orange-red,
since there is no blending with the brown pigment.

        The many variation in eye-colour of drosophila result from variations in the quality of one
or both of the two pigments described, variation in acidity which alter the colour of red pigment,
and various degrees of oxidation or reduction of the brown pigment which cause changes in its
colour. Enzymes, under the control of genes, govern these various reactions.

2. Genes and enzymes in Neurospora
       Neurospora is a mould which has the ability to live on culture medium containing
minimum nutrients. A minimal medium containing a few simple salts, sugar, ammonia and of the
B vitamins, biotin, is all that is necessary. However, analysis of the mould shows that it contains
about 20 amino acids and the purines and pyrimidines needed for construction of DNA and
RNA. Since these substances are not provided in the minimal medium, it is obvious that the
mould must synthesize each of these substances from the materials that are present in the
minimial medium. This synthesis is brought about by enzymes.

D. Regulaton of gene activity
        It is obvious that all genes do not function at all times. These are genes for the
development of eyes in all the nucleated cells of the body, yet we do not have eyes on the
bottom of the feet or at other unconventional places. That means genes function at the time and
place where they are supposed to function and suspend their operations at places where they
are not supposed to function. Studies of the giant chromosomes from the salivary glands of
Drosophila have been significant in this regard. At certain regions of these chromosomes there
are swellings or puffs which look distinctly different from the other parts of the chromosomes.
Analysis of these puffed regions shows amounts of RNA much larger than those found at non-
puffed regions, thus indicating that the puffs must be in the places where RNA is being
synthesized in high quantities. It also indicates the genes in the puffs are those connected with
the activities of the salivary glands, whereas the genes in the more condensed regions of the
chromosomes are related to activities in other parts of the body. Thus, it is clear that certain
proteins (such as histone) and antibiotics are responsible for regulating the RNA out put from
genes, and also some other activities.

        When the female hormones, the estrogen of higher animals are added to cells of the
female uterus growing in a culture, they cause a marked reduction in the ratio of histones to
DNA and a rise in the output of messenger RNA. Likewise, the male hormones, have a similar
effect on tissue from prostrate gland. In plants, the flowering hormone, florigen, when applied to
plant buds will greatly increase protein synthesis and soon flowers bloom out of the buds. In
insects, the presence of the molting hormone, ecdysone, causes a stimulation of chromosome
puffing and of RNA output.

E. Gene mutation
       The possibility that new types inherited characteristics may appear suddenly, without
any previous indication of their presence in the race, was first suggested by Hugo de Vries in
1901 as a result of his experiments and observations on the evening primrose, Oenothera
lamarckina. The details will be dealt with in (booklet No: -274.1) on genetic inheritance. Here we
limit ourselves to effect of mutation of gene regulation.

        The term mutation is sometimes used to refer to all forms of changes which result in
altered patterns of heredity. Gene mutations are mutations where only one point or locus on the
chromosome is involved in each case, in contrast to chromosomal aberrations which affect
larger portions of the chromosomes.

         The way through which mutation in operator or 'regulator' genes could be determined
remained problematic for some years. In the first possibility where mutation may involve the
regulator gene, one of the repressors will be ineffective, whereas the other one will be emitting
the inhibitory signal to be take up by the two operator genes. This would result in the
continuation of the normal repressing activity and as such the mutation would be recessive or
have no effect on the diploid level. On the other hand, if the mutation is in the operator gene,
one of the operator genes will be ineffective an as such the structural gene will not receive the
inhibiting signal. The result would be that they would be functional. Therefore, a mutation in an
operator level will immediately lead to its visible expression and the effect would be positive at
the diploid level.


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