6.1 The DNA
6.2 The Search for Genetic
Material
6.3 RNA World
6.4 Replication
6.5 Transcription
6.6 Genetic Code
6.7 Translation
6.8 Regulation of Gene
Expression
6.9 Human Genome Project
6.10 DNA Fingerprinting
In the previous chapter, you have learnt the inheritance
patterns and the genetic basis of such patterns. At the
time of Mendel, the nature of those ‘factors’ regulating
the pattern of inheritance was not clear. Over the next
hundred years, the nature of the putative genetic material
was investigated culminating in the realisation that
DNA – deoxyribonucleic acid – is the genetic material, at
least for the majority of organisms. In class XI you have
learnt that nucleic acids are polymers of nucleotides.
Deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA) are the two types of nucleic acids found in living
systems. DNA acts as the genetic material in most of the
organisms. RNA though it also acts as a genetic material
in some viruses, mostly functions as a messenger. RNA
has additional roles as well. It functions as adapter,
structural, and in some cases as a catalytic molecule. In
Class XI you have already learnt the structures of
nucleotides and the way these monomer units are linked
to form nucleic acid polymers. In this chapter we are going
to discuss the structure of DNA, its replication, the process
of making RNA from DNA (transcription), the genetic code
that determines the sequences of amino acids in proteins,
the process of protein synthesis (translation) and
elementary basis of their regulation. The determination
of complete nucleotide sequence of human genome during last decade
has set in a new era of genomics. In the last section, the essentials of
human genome sequencing and its consequences will also be discussed.
Let us begin our discussion by first understanding the structure of
the most interesting molecule in the living system, that is, the DNA. In
subsequent sections, we will understand that why it is the most abundant
genetic material, and what its relationship is with RNA.
6.1 THE DNA
DNA is a long polymer of deoxyribonucleotides. The length of DNA is
usually defined as number of nucleotides (or a pair of nucleotide referred
to as base pairs) present in it. This also is the characteristic of an organism.
For example, a bacteriophage known as φ ×174 has 5386 nucleotides,
Bacteriophage lambda has 48502 base pairs (bp), Escherichia coli has
4.6 × 106
bp, and haploid content of human DNA is 3.3 × 109
bp. Let us
discuss the structure of such a long polymer.
6.1.1 Structure of Polynucleotide Chain
Let us recapitulate the chemical structure of a polynucleotide chain (DNA
or RNA). A nucleotide has three components – a nitrogenous base, a
pentose sugar (ribose in case of RNA, and deoxyribose for DNA), and a
phosphate group. There are two types of nitrogenous bases – Purines
(Adenine and Guanine), and Pyrimidines (Cytosine, Uracil and Thymine).
Cytosine is common for both DNA and RNA and Thymine is present in
DNA. Uracil is present in RNA at the place of Thymine. A nitrogenous
base is linked to the OH of 1′ C pentose sugar through a N-glycosidic
linkage to form a nucleoside, such as adenosine or deoxyadenosine,
guanosine or deoxyguanosine, cytidine or deoxycytidine and uridine or
deoxythymidine. When a phosphate group is linked to OH of 5′ C of a
nucleoside through phosphoester linkage, a corresponding nucleotide
(or deoxynucleotide depending upon the type of sugar present) is formed.
Two nucleotides are linked through 3′-5′ phosphodiester linkage to form
a dinucleotide. More nucleotides can be joined in such a manner to form
a polynucleotide chain. A polymer thus formed has at one end a free
phosphate moiety at 5′ -end of sugar, which is referred to as 5’-end of
polynucleotide chain. Similarly, at the other end of the polymer the sugar
has a free OH of 3’C group which is referred to as 3′ -end of the
polynucleotide chain. The backbone of a polynucleotide chain is formed
due to sugar and phosphates. The nitrogenous bases linked to sugar
moiety project from the backbone (Figure 6.1).
In RNA, every nucleotide residue has an additional –OH group present
at 2′ -position in the ribose. Also, in RNA the uracil is found at the place of
thymine (5-methyl uracil, another chemical name for thymine).
DNA as an acidic substance present in nucleus was first identified by
Friedrich Meischer in 1869. He named it as ‘Nuclein’. However, due to
technical limitation in isolating such a long polymer intact, the elucidation
of structure of DNA remained elusive for a very long period of time. It was
only in 1953 that James Watson and Francis Crick, based on the X-ray
diffraction data produced by Maurice Wilkins and Rosalind Franklin,
proposed a very simple but famous Double Helix model for the structure
of DNA. One of the hallmarks of their proposition was base pairing between
the two strands of polynucleotide chains. However, this proposition was
also based on the observation of Erwin Chargaff that for a double stranded
DNA, the ratios between Adenine and Thymine and Guanine and Cytosine
are constant and equals one.
The base pairing confers a very unique property to the polynucleotide
chains. They are said to be complementary to each other, and therefore if
the sequence of bases in one strand is known then the sequence in other
strand can be predicted. Also, if each strand from a DNA (let us call it as a
parental DNA) acts as a template for synthesis of a new strand, the two
double stranded DNA (let us call them as daughter DNA) thus, produced
would be identical to the parental DNA molecule. Because of this, the genetic
implications of the structure of DNA became very clear.
The salient features of the Double-helix structure of DNA are as follows:
(i) It is made of two polynucleotide chains, where the backbone is
constituted by sugar-phosphate, and the bases project inside.
(ii) The two chains have anti-parallel polarity. It means, if one
chain has the polarity 5’à3′, the other has 3’à5′.
(iii) The bases in two strands are paired through hydrogen bond
(H-bonds) forming base pairs (bp). Adenine forms two hydrogen
bonds with Thymine from opposite strand and vice-versa.
Similarly, Guanine is bonded with Cytosine with three H-bonds.
As a result, always a purine comes opposite to a pyrimidine. This
generates approximately uniform distance between the two
strands of the helix (Figure 6.2).
(iv) The two chains are coiled in a right-handed fashion. The pitch
of the helix is 3.4 nm (a nanometre is one billionth of a
metre, that is 10-9 m) and there are roughly 10 bp in each
turn. Consequently, the distance
between a bp in a helix is
approximately 0.34 nm.
(v) The plane of one base pair stacks
over the other in double helix. This,
in addition to H-bonds, confers
stability of the helical structure
(Figure 6.3).
Compare the structure of purines and
pyrimidines. Can you find out why the
distance between two polynucleotide
chains in DNA remains almost constant?
The proposition of a double helix
structure for DNA and its simplicity in
explaining the genetic implication became
revolutionary. Very soon, Francis Crick
proposed the Central dogma in molecular
biology, which states that the genetic
information flows from DNAàRNAàProtein.
In some viruses the flow of information is in reverse direction, that is,
from RNA to DNA. Can you suggest a simple name to the process?
6.1.2 Packaging of DNA Helix
Taken the distance between two consecutive base pairs
as 0.34 nm (0.34×10–9 m), if the length of DNA double
helix in a typical mammalian cell is calculated (simply
by multiplying the total number of bp with distance
between two consecutive bp, that is, 6.6 × 109 bp ×
0.34 × 10-9m/bp), it comes out to be approximately
2.2 metres. A length that is far greater than the
dimension of a typical nucleus (approximately 10–6 m).
How is such a long polymer packaged in a cell?
If the length of E. coli DNA is 1.36 mm, can you
calculate the number of base pairs in E.coli?
In prokaryotes, such as, E. coli, though they do
not have a defined nucleus, the DNA is not scattered
throughout the cell. DNA (being negatively charged)
is held with some proteins (that have positive
charges) in a region termed as ‘nucleoid’. The DNA
in nucleoid is organised in large loops held by
proteins.
In eukaryotes, this organisation is much more
complex. There is a set of positively charged, basic
proteins called histones. A protein acquires charge
depending upon the abundance of amino acids
residues with charged side chains. Histones are rich
in the basic amino acid residues lysine and arginine.
Both the amino acid residues carry positive charges
in their side chains. Histones are organised to form
a unit of eight molecules called histone octamer.
The negatively charged DNA is wrapped around the positively charged
histone octamer to form a structure called nucleosome (Figure 6.4 a). A
typical nucleosome contains 200 bp of DNA helix. Nucleosomes constitute
the repeating unit of a structure in nucleus called chromatin, thread-
like stained (coloured) bodies seen in nucleus. The nucleosomes in
chromatin are seen as ‘beads-on-string’ structure when viewed under
electron microscope (EM) (Figure 6.4 b).
Theoretically, how many such beads (nucleosomes) do you imagine
are present in a mammalian cell?
The beads-on-string structure in chromatin is packaged to form
chromatin fibers that are further coiled and condensed at metaphase stage
of cell division to form chromosomes. The packaging of chromatin at higher
level requires additional set of proteins that collectively are referred to as
Non-histone Chromosomal (NHC) proteins. In a typical nucleus, some
region of chromatin are loosely packed (and stains light) and are referred to
as euchromatin. The chromatin that is more densely packed and stains
dark are called as Heterochromatin. Euchromatin is said to be
transcriptionally active chromatin, whereas heterochromatin is inactive.
6.2 THE SEARCH FOR GENETIC MATERIAL
Even though the discovery of nuclein by Meischer and the proposition
for principles of inheritance by Mendel were almost at the same time, but
that the DNA acts as a genetic material took long to be discovered and
proven. By 1926, the quest to determine the mechanism for genetic
inheritance had reached the molecular level. Previous discoveries by
Gregor Mendel, Walter Sutton, Thomas Hunt Morgan and numerous other
scientists had narrowed the search to the chromosomes located in the
nucleus of most cells. But the question of what molecule was actually the
genetic material, had not been answered.
Transforming Principle
In 1928, Frederick Griffith, in a series of experiments with Streptococcus
pneumoniae (bacterium responsible for pneumonia), witnessed a
miraculous transformation in the bacteria. During the course of his
experiment, a living organism (bacteria) had changed in physical form.
When Streptococcus pneumoniae (pneumococcus) bacteria are grown
on a culture plate, some produce smooth shiny colonies (S) while others
produce rough colonies (R). This is because the S strain bacteria have a
mucous (polysaccharide) coat, while R strain does not. Mice infected with
the S strain (virulent) die from pneumonia infection but mice infected
with the R strain do not develop pneumonia.
Griffith was able to kill bacteria by heating them. He observed that
heat-killed S strain bacteria injected into mice did not kill them. When he
injected a mixture of heat-killed S and live R bacteria, the mice died.
Moreover, he recovered living S bacteria from the dead mice.
He concluded that the R strain bacteria had somehow been
transformed by the heat-killed S strain bacteria. Some ‘transforming
principle’, transferred from the heat-killed S strain, had enabled the
R strain to synthesise a smooth polysaccharide coat and become virulent.
This must be due to the transfer of the genetic material. However, the
biochemical nature of genetic material was not defined from his
experiments.
Biochemical Characterisation of Transforming Principle
Prior to the work of Oswald Avery, Colin MacLeod and Maclyn McCarty
(1933-44), the genetic material was thought to be a protein. They worked
to determine the biochemical nature of ‘transforming principle’ in Griffith’s
experiment.
They purified biochemicals (proteins, DNA, RNA, etc.) from the
heat-killed S cells to see which ones could transform live R cells into
S cells. They discovered that DNA alone from S bacteria caused R bacteria
to become transformed.
They also discovered that protein-digesting enzymes (proteases) and
RNA-digesting enzymes (RNases) did not affect transformation, so the
transforming substance was not a protein or RNA. Digestion with DNase
did inhibit transformation, suggesting that the DNA caused the
transformation. They concluded that DNA is the hereditary material, but
not all biologists were convinced.
Can you think of any difference between DNAs and DNase?
6.2.1 The Genetic Material is DNA
The unequivocal proof that DNA is the genetic material came from the
experiments of Alfred Hershey and Martha Chase (1952). They worked
with viruses that infect bacteria called bacteriophages.
The bacteriophage attaches to the bacteria and its genetic material
then enters the bacterial cell. The bacterial cell treats the viral genetic
material as if it was its own and subsequently manufactures more virus
particles. Hershey and Chase worked to discover whether it was protein
or DNA from the viruses that entered the bacteria.
They grew some viruses on a medium that contained radioactive
phosphorus and some others on medium that contained radioactive sulfur.
Viruses grown in the presence of radioactive phosphorus contained
radioactive DNA but not radioactive protein because DNA contains
phosphorus but protein does not. Similarly, viruses grown on radioactive
sulfur contained radioactive protein but not radioactive DNA because
DNA does not contain sulfur.
Radioactive phages were allowed to attach to E. coli bacteria. Then, as
the infection proceeded, the viral coats were removed from the bacteria by
agitating them in a blender. The virus particles were separated from the
bacteria by spinning them in a centrifuge.
Bacteria which was infected with viruses that had radioactive DNA
were radioactive, indicating that DNA was the material that passed from
the virus to the bacteria. Bacteria that were infected with viruses that had
radioactive proteins were not radioactive. This indicates that proteins did
not enter the bacteria from the viruses. DNA is therefore the genetic
material that is passed from virus to bacteria (Figure 6.5).
6.2.2 Properties of Genetic Material (DNA versus RNA)
From the foregoing discussion, it is clear that the debate between proteins
versus DNA as the genetic material was unequivocally resolved from
Hershey-Chase experiment. It became an established fact that it is DNA
that acts as genetic material. However, it subsequently became clear that
in some viruses, RNA is the genetic material (for example, Tobacco Mosaic
viruses, QB bacteriophage, etc.). Answer to some of the questions such as,
why DNA is the predominant genetic material, whereas RNA performs
dynamic functions of messenger and adapter has to be found from the
differences between chemical structures of the two nucleic acid molecules.
Can you recall the two chemical differences between DNA and RNA?
A molecule that can act as a genetic material must fulfill the following
criteria:
(i) It should be able to generate its replica (Replication).
(ii) It should be stable chemically and structurally.
(iii) It should provide the scope for slow changes (mutation) that
are required for evolution.
(iv) It should be able to express itself in the form of ‘Mendelian
Characters’.
If one examines each requirement one by one, because of rule of base
pairing and complementarity, both the nucleic acids (DNA and RNA) have
the ability to direct their duplications. The other molecules in the living
system, such as proteins fail to fulfill first criteria itself.
The genetic material should be stable enough not to change with
different stages of life cycle, age or with change in physiology of the
organism. Stability as one of the properties of genetic material was very
evident in Griffith’s ‘transforming principle’ itself that heat, which killed
the bacteria, at least did not destroy some of the properties of genetic
material. This now can easily be explained in light of the DNA that the
two strands being complementary if separated by heating come together,
when appropriate conditions are provided. Further, 2′-OH group present
at every nucleotide in RNA is a reactive group and makes RNA labile and
easily degradable. RNA is also now known to be catalytic, hence reactive.
Therefore, DNA chemically is less reactive and structurally more stable
when compared to RNA. Therefore, among the two nucleic acids, the DNA
is a better genetic material.
In fact, the presence of thymine at the place of uracil also confers
additional stability to DNA. (Detailed discussion about this requires
understanding of the process of repair in DNA, and you will study these
processes in higher classes.)
Both DNA and RNA are able to mutate. In fact, RNA being unstable,
mutate at a faster rate. Consequently, viruses having RNA genome and
having shorter life span mutate and evolve faster.
RNA can directly code for the synthesis of proteins, hence can easily
express the characters. DNA, however, is dependent on RNA for synthesis
of proteins. The protein synthesising machinery has evolved around RNA.
The above discussion indicate that both RNA and DNA can function as
genetic material, but DNA being more stable is preferred for storage of
genetic information. For the transmission of genetic information, RNA
is better.
6.3 RNA WORLD
From foregoing discussion, an immediate question becomes evident –
which is the first genetic material? It shall be discussed in detail in the
chapter on chemical evolution, but briefly, we shall highlight some of the
facts and points.
RNA was the first genetic material. There is now enough evidence to
suggest that essential life processes (such as metabolism, translation,
splicing, etc.), evolved around RNA. RNA used to act as
a genetic material as well as a catalyst (there are some
important biochemical reactions in living systems that
are catalysed by RNA catalysts and not by protein
enzymes). But, RNA being a catalyst was reactive and
hence unstable. Therefore, DNA has evolved from RNA
with chemical modifications that make it more stable.
DNA being double stranded and having complementary
strand further resists changes by evolving a process of
repair.
6.4 REPLICATION
While proposing the double helical structure for DNA,
Watson and Crick had immediately proposed a scheme
for replication of DNA. To quote their original statement
that is as follows:
‘‘It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic material’’
(Watson and Crick, 1953).
The scheme suggested that the two strands would
separate and act as a template for the synthesis of new
complementary strands. After the completion of
replication, each DNA molecule would have one
parental and one newly synthesised strand. This
scheme was termed as semiconservative DNA
replication (Figure 6.6).
6.4.1 The Experimental Proof
It is now proven that DNA replicates semiconservatively. It was shown first in
Escherichia coli and subsequently in higher organisms, such as plants
and human cells. Matthew Meselson and Franklin Stahl performed the
following experiment in 1958:
(i) They grew E. coli in a medium containing 15NH4Cl (15N is the heavy
isotope of nitrogen) as the only nitrogen source for many
generations. The result was that 15N was incorporated into newly
synthesised DNA (as well as other nitrogen containing compounds).
This heavy DNA molecule could be distinguished from the normal
DNA by centrifugation in a cesium chloride (CsCl) density gradient
(Please note that 15N is not a radioactive isotope, and it can be
separated from 14N only based on densities).
(ii) Then they transferred the cells into a medium with normal
14NH4Cl and took samples at various definite time intervals as
the cells multiplied, and extracted the DNA that remained as
double-stranded helices. The various samples were separated
independently on CsCl gradients to measure the densities of
DNA (Figure 6.7).
Can you recall what centrifugal force is, and think why a
molecule with higher mass/density would sediment faster?
The results are shown in Figure 6.7.
(iii) Thus, the DNA that was extracted from the culture one
generation after the transfer from 15N to 14N medium [that is
after 20 minutes; E. coli divides in 20 minutes] had a hybrid or
intermediate density. DNA extracted from the culture after
another generation [that is after 40 minutes, II generation] was
composed of equal amounts of this hybrid DNA and of ‘light’
DNA.
If E. coli was allowed to grow for 80 minutes then what would be the
proportions of light and hybrid densities DNA molecule?
Very similar experiments involving use of radioactive thymidine to
detect distribution of newly synthesised DNA in the chromosomes was
performed on Vicia faba (faba beans) by Taylor and colleagues in 1958.
The experiments proved that the DNA in chromosomes also replicate
semiconservatively.
6.4.2 The Machinery and the Enzymes
In living cells, such as E. coli, the process of replication requires a set of
catalysts (enzymes). The main enzyme is referred to as DNA-dependent
DNA polymerase, since it uses a DNA template to catalyse the
polymerisation of deoxynucleotides. These enzymes are highly efficient
enzymes as they have to catalyse polymerisation of a large number of
nucleotides in a very short time. E. coli that has only 4.6 ×106
bp (compare
it with human whose diploid content is 6.6 × 109
bp), completes the
process of replication within 18 minutes; that means the average rate of
polymerisation has to be approximately 2000 bp per second. Not only do
these polymerases have to be fast, but they also have to catalyse the reaction
with high degree of accuracy. Any mistake during replication would result
into mutations. Furthermore, energetically replication is a very expensive
process. Deoxyribonucleoside triphosphates serve dual purposes. In
addition to acting as substrates, they provide energy for polymerisation
reaction (the two terminal phosphates in a deoxynucleoside triphosphates
are high-energy phosphates, same as in case of ATP).
In addition to DNA-dependent DNA polymerases, many additional
enzymes are required to complete the process of replication with high
degree of accuracy. For long DNA molecules, since the two strands of
DNA cannot be separated in its entire length (due to very high energy
requirement), the replication occur within a small opening of the DNA
helix, referred to as replication fork. The DNA-dependent DNA
polymerases catalyse polymerisation only in one direction, that is 5’à3′.
This creates some additional complications at the replicating fork.
Consequently, on one strand (the template with polarity 3’à5′), the
replication is continuous, while on the other (the template with
polarity 5’à3′), it is discontinuous. The discontinuously synthesised
fragments are later joined by the enzyme DNA ligase (Figure 6.8).
The DNA polymerases on their own cannot initiate the process of
replication. Also the replication does not initiate randomly at any place
in DNA. There is a definite region in E. coli DNA where the replication
originates. Such regions are termed as origin of replication. It is
because of the requirement of the origin of
replication that a piece of DNA if needed to be
propagated during recombinant DNA procedures,
requires a vector. The vectors provide the origin of
replication.
Further, not every detail of replication is
understood well. In eukaryotes, the replication of
DNA takes place at S-phase of the cell-cycle. The
replication of DNA and cell division cycle should be
highly coordinated. A failure in cell division after
DNA replication results into polyploidy(a
chromosomal anomaly). You will learn the detailed
nature of origin and the processes occurring at this
site, in higher classes.
6.5 TRANSCRIPTION
The process of copying genetic information from one
strand of the DNA into RNA is termed as
transcription. Here also, the principle of
complementarity governs the process of transcription, except the adenosine
complements now forms base pair with uracil instead of thymine. However,
unlike in the process of replication, which once set in, the total DNA of an
organism gets duplicated, in transcription only a segment of DNA and
only one of the strands is copied into RNA. This necessitates defining the
boundaries that would demarcate the region and the strand of DNA that
would be transcribed.
Why both the strands are not copied during transcription has the
simple answer. First, if both strands act as a template, they would code
for RNA molecule with different sequences (Remember complementarity
does not mean identical), and in turn, if they code for proteins, the sequence
of amino acids in the proteins would be different. Hence, one segment of
the DNA would be coding for two different proteins, and this would
complicate the genetic information transfer machinery. Second, the two
RNA molecules if produced simultaneously would be complementary to
each other, hence would form a double stranded RNA. This would prevent
RNA from being translated into protein and the exercise of transcription
would become a futile one.
6.5.1 Transcription Unit
A transcription unit in DNA is defined primarily by the three regions in
the DNA:
(i) A Promoter
(ii) The Structural gene
(iii) A Terminator
There is a convention in defining the two strands of the DNA in the
structural gene of a transcription unit. Since the two strands have opposite
polarity and the DNA-dependent RNA polymerase also catalyse the
polymerisation in only one direction, that is, 5’→3′, the strand that has
the polarity 3’→5′ acts as a template, and is also referred to as template
strand. The other strand which has the polarity (5’→3′) and the sequence
same as RNA (except thymine at the place of uracil), is displaced during
transcription. Strangely, this strand (which does not code for anything)
is referred to as coding strand. All the reference point while defining a
transcription unit is made with coding strand. To explain the point, a
hypothetical sequence from a transcription unit is represented below:
3′ -ATGCATGCATGCATGCATGCATGC-5′ Template Strand
5′ -TACGTACGTACGTACGTACGTACG-3′ Coding Strand
Can you now write the sequence of RNA transcribed from the above DNA?
The promoter and terminator flank the structural gene in a
transcription unit. The promoter is said to be located towards 5′ -end
(upstream) of the structural gene (the reference is made with respect to
the polarity of coding strand). It is a DNA sequence that provides binding
site for RNA polymerase, and it is the presence of a promoter in a
transcription unit that also defines the template and coding strands. By
switching its position with terminator, the definition of coding and template
strands could be reversed. The terminator is located towards 3′ -end
(downstream) of the coding strand and it usually defines the end of the
process of transcription (Figure 6.9). There are additional regulatory
sequences that may be present further upstream or downstream to the
promoter. Some of the properties of these sequences shall be discussed
while dealing with regulation of gene expression.
6.5.2 Transcription Unit and the Gene
A gene is defined as the functional unit of inheritance. Though there is no
ambiguity that the genes are located on the DNA, it is difficult to literally
define a gene in terms of DNA sequence. The DNA sequence coding for
tRNA or rRNA molecule also define a gene. However by defining a cistron
as a segment of DNA coding for a polypeptide, the structural gene in a
transcription unit could be said as monocistronic (mostly in eukaryotes)
or polycistronic (mostly in bacteria or prokaryotes). In eukaryotes, the
monocistronic structural genes have interrupted coding sequences – the
genes in eukaryotes are split. The coding sequences or expressed
sequences are defined as exons. Exons are said to be those sequence
that appear in mature or processed RNA. The exons are interrupted by
introns. Introns or intervening sequences do not appear in mature or
processed RNA. The split-gene arrangement further complicates the
definition of a gene in terms of a DNA segment.
Inheritance of a character is also affected by promoter and regulatory
sequences of a structural gene. Hence, sometime the regulatory sequences
are loosely defined as regulatory genes, even though these sequences do
not code for any RNA or protein.
6.5.3 Types of RNA and the process of Transcription
In bacteria, there are three major types of RNAs: mRNA (messenger RNA),
tRNA (transfer RNA), and rRNA (ribosomal RNA). All three RNAs are
needed to synthesise a protein in a cell. The mRNA provides the template,
tRNA brings aminoacids and reads the genetic code, and rRNAs play
structural and catalytic role during translation. There is single
DNA-dependent RNA polymerase that catalyses transcription of all types
of RNA in bacteria. RNA polymerase binds to promoter and initiates
transcription (Initiation). It uses nucleoside triphosphates as substrate
and polymerises in a template depended fashion following the rule of
complementarity. It somehow also facilitates opening of the helix and
continues elongation. Only a short stretch of RNA remains bound to the
enzyme. Once the polymerases reaches the terminator region, the nascent
RNA falls off, so also the RNA polymerase. This results in termination of
transcription.
An intriguing question is that how is the RNA polymerases able
to catalyse all the three steps, which are initiation, elongation and
termination. The RNA polymerase is only capable of catalysing the
process of elongation. It associates transiently with initiation-factor (σ)
and termination-factor (ρ) to initiate and terminate the transcription,
respectively. Association with these factors alter the specificity of the
RNA polymerase to either initiate or terminate (Figure 6.10).
In bacteria, since the mRNA does not require any processing to become
active, and also since transcription and translation take place in the same
compartment (there is no separation of cytosol and nucleus in bacteria),
many times the translation can begin much before the mRNA is fully
transcribed. Consequently, the transcription and translation can be coupled
in bacteria.
In eukaryotes, there are two additional complexities –
(i) There are at least three RNA polymerases in the nucleus (in addition
to the RNA polymerase found in the organelles). There is a clear
cut division of labour. The RNA polymerase I transcribes rRNAs
(28S, 18S, and 5.8S), whereas the RNA polymerase III is responsible
for transcription of tRNA, 5srRNA, and snRNAs (small nuclear
RNAs). The RNA polymerase II transcribes precursor of mRNA, the
heterogeneous nuclear RNA (hnRNA).
(ii) The second complexity is that the primary transcripts contain both
the exons and the introns and are non-functional. Hence, it is
subjected to a process called splicing where the introns are removed
and exons are joined in a defined order. hnRNA undergoes
additional processing called as capping and tailing. In capping an
unusual nucleotide (methyl guanosine triphosphate) is added to
the 5′-end of hnRNA. In tailing, adenylate residues (200-300) are
added at 3′-end in a template independent manner. It is the fully
processed hnRNA, now called mRNA, that is transported out of the
nucleus for translation (Figure 6.11).
The significance of such complexities is now beginning to be
understood. The split-gene arrangements represent probably an ancient
feature of the genome. The presence of introns is reminiscent of antiquity,
and the process of splicing represents the dominance of RNA-world. In
recent times, the understanding of RNA and RNA-dependent processes
in the living system have assumed more importance.
6.6 GENETIC CODE
During replication and transcription a nucleic acid was copied to form
another nucleic acid. Hence, these processes are easy to conceptualise
on the basis of complementarity. The process of translation requires
transfer of genetic information from a polymer of nucleotides to synthesise
a polymer of amino acids. Neither does any complementarity exist between
nucleotides and amino acids, nor could any be drawn theoretically. There
existed ample evidences, though, to support the notion that change in
nucleic acids (genetic material) were responsible for change in amino acids
in proteins. This led to the proposition of a genetic code that could direct
the sequence of amino acids during synthesis of proteins.
If determining the biochemical nature of genetic material and the
structure of DNA was very exciting, the proposition and deciphering of
genetic code were most challenging. In a very true sense, it required
involvement of scientists from several disciplines – physicists, organic
chemists, biochemists and geneticists. It was George Gamow, a physicist,
who argued that since there are only 4 bases and if they have to code for
20 amino acids, the code should constitute a combination of bases. He
suggested that in order to code for all the 20 amino acids, the code should
be made up of three nucleotides. This was a very bold proposition, because
a permutation combination of 43
(4 × 4 × 4) would generate 64 codons;
generating many more codons than required.
Providing proof that the codon was a triplet, was a more daunting
task. The chemical method developed by Har Gobind Khorana was
instrumental in synthesising RNA molecules with defined combinations
of bases (homopolymers and copolymers). Marshall Nirenberg’s cell-free
system for protein synthesis finally helped the code to be deciphered.
Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful
in polymerising RNA with defined sequences in a template independent
manner (enzymatic synthesis of RNA). Finally a checker-board for genetic
code was prepared which is given in Table 6.1.
The salient features of genetic code are as follows:
(i) The codon is triplet. 61 codons code for amino acids and 3 codons do
not code for any amino acids, hence they function as stop codons.
(ii) Some amino acids are coded by more than one codon, hence
the code is degenerate.
(iii) The codon is read in mRNA in a contiguous fashion. There are
no punctuations.
(iv) The code is nearly universal: for example, from bacteria to human
UUU would code for Phenylalanine (phe). Some exceptions to this
rule have been found in mitochondrial codons, and in some
protozoans.
(v) AUG has dual functions. It codes for Methionine (met) , and it
also act as initiator codon.
(vi) UAA, UAG, UGA are stop terminator codons.
If following is the sequence of nucleotides in mRNA, predict the
sequence of amino acid coded by it (take help of the checkerboard):
-AUG UUU UUC UUC UUU UUU UUC-
Now try the opposite. Following is the sequence of amino acids coded
by an mRNA. Predict the nucleotide sequence in the RNA:
Met-Phe-Phe-Phe-Phe-Phe-Phe
Do you face any difficulty in predicting the opposite?
Can you now correlate which two properties of genetic code you have
learnt?
6.6.1 Mutations and Genetic Code
The relationships between genes and DNA are best understood by mutation
studies. You have studied about mutation and its effect in Chapter 5. Effects
of large deletions and rearrangements in a segment of DNA are easy to
comprehend. It may result in loss or gain of a gene and so a function. The
effect of point mutations will be explained here. A classical example of
point mutation is a change of single base pair in the gene for beta globin
chain that results in the change of amino acid residue glutamate to valine.
It results into a diseased condition called as sickle cell anemia. Effect of
point mutations that inserts or deletes a base in structural gene can be
better understood by following simple example.
Consider a statement that is made up of the following words each
having three letters like genetic code.
RAM HAS RED CAP
If we insert a letter B in between HAS and RED and rearrange the
statement, it would read as follows:
RAM HAS BRE DCA P
Similarly, if we now insert two letters at the same place, say BI’. Now it
would read,
RAM HAS BIR EDC AP
Now we insert three letters together, say BIG, the statement would read
RAM HAS BIG RED CAP
The same exercise can be repeated, by deleting the letters R, E and D,
one by one and rearranging the statement to make a triplet word.
RAM HAS EDC AP
RAM HAS DCA P
RAM HAS CAP
The conclusion from the above exercise is very obvious. Insertion or
deletion of one or two bases changes the reading frame from the point of
insertion or deletion. However, such mutations are referred to as
frameshift insertion or deletion mutations. Insertion or deletion of
three or its multiple bases insert or delete in one or multiple codon hence
one or multiple amino acids, and reading frame remains unaltered from
that point onwards.
6.6.2 tRNA– the Adapter Molecule
From the very beginning of the proposition of code, it was clear to Francis
Crick that there has to be a mechanism to read the code and also to link it
to the amino acids, because amino acids have no structural specialities to
read the code uniquely. He postulated the presence of an adapter molecule
that would on one hand read the code and on other hand would bind
to specific amino acids. The tRNA, then called sRNA (soluble RNA),
was known before the genetic code was postulated. However, its role
as an adapter molecule was assigned much later.
tRNA has an
anticodon loop
that has bases
complementary to
the code, and it also
has an amino acid
acceptor end to
which it binds to
amino acids.
tRNAs are specific
for each amino acid
(Figure 6.12). For
initiation, there is
another specific tRNA that is referred to as initiator tRNA. There are no
tRNAs for stop codons. In figure 6.12, the secondary structure of tRNA
has been depicted that looks like a clover-leaf. In actual structure, the
tRNA is a compact molecule which looks like inverted L.
6.7 TRANSLATION
Translation refers to the process of polymerisation of amino acids to
form a polypeptide (Figure 6.13). The order and sequence of amino acids
are defined by the sequence of bases in the mRNA. The amino acids are
joined by a bond which is known as a peptide bond. Formation of a
peptide bond requires energy. Therefore, in the first phase itself amino
acids are activated in the presence of ATP and linked to their cognate
tRNA – a process commonly called as charging of tRNA or
aminoacylation of tRNA to be more specific. If two such charged tRNAs
are brought close enough, the formation of peptide bond between them
would be favoured energetically. The
presence of a catalyst would enhance
the rate of peptide bond formation.
The cellular factory responsible for
synthesising proteins is the ribosome.
The ribosome consists of structural
RNAs and about 80 different proteins.
In its inactive state, it exists as two
subunits; a large subunit and a small
subunit. When the small subunit
encounters an mRNA, the process of
translation of the mRNA to protein
begins. There are two sites in the large
subunit, for subsequent amino acids
to bind to and thus, be close enough
to each other for the formation of a
peptide bond. The ribosome also acts as a catalyst (23S rRNA in bacteria
is the enzyme- ribozyme) for the formation of peptide bond.
A translational unit in mRNA is the sequence of RNA that is flanked
by the start codon (AUG) and the stop codon and codes for a polypeptide.
An mRNA also has some additional sequences that are not translated
and are referred as untranslated regions (UTR). The UTRs are present
at both 5′ -end (before start codon) and at 3′ -end (after stop codon). They
are required for efficient translation process.
For initiation, the ribosome binds to the mRNA at the start codon (AUG)
that is recognised only by the initiator tRNA. The ribosome proceeds to the
elongation phase of protein synthesis. During this stage, complexes
composed of an amino acid linked to tRNA, sequentially bind to the
appropriate codon in mRNA by forming complementary base pairs with
the tRNA anticodon. The ribosome moves from codon to codon along the
mRNA. Amino acids are added one by one, translated into Polypeptide
sequences dictated by DNA and represented by mRNA. At the end, a release
factor binds to the stop codon, terminating translation and releasing the
complete polypeptide from the ribosome.
6.8 REGULATION OF GENE EXPRESSION
Regulation of gene expression refers to a very broad term that may occur
at various levels. Considering that gene expression results in the formation
of a polypeptide, it can be regulated at several levels. In eukaryotes, the
regulation could be exerted at
(i) transcriptional level (formation of primary transcript),
(ii) processing level (regulation of splicing),
(iii) transport of mRNA from nucleus to the cytoplasm,
(iv) translational level.
The genes in a cell are expressed to perform a particular function or a
set of functions. For example, if an enzyme called beta-galactosidase is
synthesised by E. coli, it is used to catalyse the hydrolysis of a
disaccharide, lactose into galactose and glucose; the bacteria use them
as a source of energy. Hence, if the bacteria do not have lactose around
them to be utilised for energy source, they would no longer require the
synthesis of the enzyme beta-galactosidase. Therefore, in simple terms,
it is the metabolic, physiological or environmental conditions that regulate
the expression of genes. The development and differentiation of embryo
into adult organisms are also a result of the coordinated regulation of
expression of several sets of genes.
In prokaryotes, control of the rate of transcriptional initiation is the
predominant site for control of gene expression. In a transcription unit,
the activity of RNA polymerase at a given promoter is in turn regulated
by interaction with accessory proteins, which affect its ability to recognise
start sites. These regulatory proteins can act both positively (activators)
and negatively (repressors). The accessibility of promoter regions of
prokaryotic DNA is in many cases regulated by the interaction of proteins
with sequences termed operators. The operator region is adjacent to the
promoter elements in most operons and in most cases the sequences of
the operator bind a repressor protein. Each operon has its specific
operator and specific repressor. For example, lac operator is present
only in the lac operon and it interacts specifically with lac repressor only.
6.8.1 The Lac operon
The elucidation of the lac operon was also a result of a close association
between a geneticist, Francois Jacob and a biochemist, Jacque Monod. They
were the first to elucidate a transcriptionally regulated system. In lac operon
(here lac refers to lactose), a polycistronic structural gene is regulated by a
common promoter and regulatory genes. Such arrangement is very common
in bacteria and is referred to as operon. To name few such examples, lac
operon, trp operon, ara operon, his operon, val operon, etc.
The lac operon consists of one regulatory gene (the i gene – here the
term i does not refer to inducer, rather it is derived from the word inhibitor)
and three structural genes (z, y, and a). The i gene codes for the repressor
of the lac operon. The z gene codes for beta-galactosidase (β-gal), which
is primarily responsible for the hydrolysis of the disaccharide, lactose
into its monomeric units, galactose and glucose. The y gene codes for
permease, which increases permeability of the cell to β-galactosides. The
a gene encodes a transacetylase. Hence, all the three gene products in
lac operon are required for metabolism of lactose. In most other operons
as well, the genes present in the operon are needed together to function
in the same or related metabolic pathway (Figure 6.14).
Lactose is the substrate for the enzyme beta-galactosidase and it
regulates switching on and off of the operon. Hence, it is termed as inducer.
In the absence of a preferred carbon source such as glucose, if lactose is
provided in the growth medium of the bacteria, the lactose is transported
into the cells through the action of permease (Remember, a very low level
of expression of lac operon has to be present in the cell all the time,
otherwise lactose cannot enter the cells). The lactose then induces the
operon in the following manner.
The repressor of the operon is synthesised (all-the-time – constitutively)
from the i gene. The repressor protein binds to the operator region of the
operon and prevents RNA polymerase from transcribing the operon. In
the presence of an inducer, such as lactose or allolactose, the repressor is
inactivated by interaction with the inducer. This allows RNA polymerase
access to the promoter and transcription proceeds (Figure 6.14).
Essentially, regulation of lac operon can also be visualised as regulation
of enzyme synthesis by its substrate.
Remember, glucose or galactose cannot act as inducers for lac
operon. Can you think for how long the lac operon would be expressed
in the presence of lactose?
Regulation of lac operon by repressor is referred to as negative
regulation. Lac operon is under control of positive regulation as well,
but it is beyond the scope of discussion at this level.
6.9 HUMAN GENOME PROJECT
In the preceding sections you have learnt that it is the sequence of bases in
DNA that determines the genetic information of a given organism. In other
words, genetic make-up of an organism or an individual lies in the DNA
sequences. If two individuals differ, then their DNA sequences should also
be different, at least at some places. These assumptions led to the quest of
finding out the complete DNA sequence of human genome. With the
establishment of genetic engineering techniques where it was possible to
isolate and clone any piece of DNA and availability of simple and fast
techniques for determining DNA sequences, a very ambitious project of
sequencing human genome was launched in the year 1990.
Human Genome Project (HGP) was called a mega project. You can
imagine the magnitude and the requirements for the project if we simply
define the aims of the project as follows:
Human genome is said to have approximately 3 x 109
bp, and if the
cost of sequencing required is US $ 3 per bp (the estimated cost in the
beginning), the total estimated cost of the project would be approximately
9 billion US dollars. Further, if the obtained sequences were to be stored
in typed form in books, and if each page of the book contained 1000
letters and each book contained 1000 pages, then 3300 such books would
be required to store the information of DNA sequence from a single human
cell. The enormous amount of data expected to be generated also
necessitated the use of high speed computational devices for data storage
and retrieval, and analysis. HGP was closely associated with the rapid
development of a new area in biology called Bioinformatics.
Goals of HGP
Some of the important goals of HGP were as follows:
(i) Identify all the approximately 20,000-25,000 genes in human DNA;
(ii) Determine the sequences of the 3 billion chemical base pairs that
make up human DNA;
(iiii) Store this information in databases;
(iv) Improve tools for data analysis;
(v) Transfer related technologies to other sectors, such as industries;
(vi) Address the ethical, legal, and social issues (ELSI) that may arise
from the project.
The Human Genome Project was a 13-year project coordinated by
the U.S. Department of Energy and the National Institute of Health. During
the early years of the HGP, the Wellcome Trust (U.K.) became a major
partner; additional contributions came from Japan, France, Germany,
China and others. The project was completed in 2003. Knowledge about
the effects of DNA variations among individuals can lead to revolutionary
new ways to diagnose, treat and someday prevent the thousands of
disorders that affect human beings. Besides providing clues to
understanding human biology, learning about non-human organisms
DNA sequences can lead to an understanding of their natural capabilities
that can be applied toward solving challenges in health care, agriculture,
energy production, environmental remediation. Many non-human model
organisms, such as bacteria, yeast, Caenorhabditis elegans (a free living
non-pathogenic nematode), Drosophila (the fruit fly), plants (rice and
Arabidopsis), etc., have also been sequenced.
Methodologies : The methods involved two major approaches. One
approach focused on identifying all the genes that are expressed as
RNA (referred to as Expressed Sequence Tags (ESTs). The other took
the blind approach of simply sequencing the whole set of genome that
contained all the coding and non-coding sequence, and later assigning
different regions in the sequence with functions (a term referred to as
Sequence Annotation). For sequencing, the total DNA from a cell is
isolated and converted into random fragments of relatively smaller sizes
(recall DNA is a very long polymer, and there are technical limitations in
sequencing very long pieces of DNA) and cloned in suitable host using
specialised vectors. The cloning resulted into amplification of each piece
of DNA fragment so that it subsequently could be sequenced with ease.
The commonly used hosts were bacteria and yeast, and the vectors were
called as BAC (bacterial artificial chromosomes), and YAC (yeast artificial
chromosomes).
The fragments were sequenced using automated DNA sequencers that
worked on the principle of a method developed by Frederick Sanger.
(Remember, Sanger is also credited for developing method for
determination of amino acid
sequences in proteins). These
sequences were then arranged based
on some overlapping regions
present in them. This required
generation of overlapping fragments
for sequencing. Alignment of these
sequences was humanly not
possible. Therefore, specialised
computer based programs were
developed (Figure 6.15). These
sequences were subsequently
annotated and were assigned to each
chromosome. The sequence of
chromosome 1 was completed only
in May 2006 (this was the last of the
24 human chromosomes – 22
autosomes and X and Y – to be
sequenced). Another challenging task was assigning the genetic and
physical maps on the genome. This was generated using information on
polymorphism of restriction endonuclease recognition sites, and some
repetitive DNA sequences known as microsatellites (one of the applications
of polymorphism in repetitive DNA sequences shall be explained in next
section of DNA fingerprinting).
6.9.1 Salient Features of Human Genome
Some of the salient observations drawn from human genome project are
as follows:
(i) The human genome contains 3164.7 million bp.
(ii) The average gene consists of 3000 bases, but sizes vary greatly, with
the largest known human gene being dystrophin at 2.4 million bases.
(iii) The total number of genes is estimated at 30,000–much lower
than previous estimates of 80,000 to 1,40,000 genes. Almost all
(99.9 per cent) nucleotide bases are exactly the same in all people.
(iv) The functions are unknown for over 50 per cent of the discovered
genes.
(v) Less than 2 per cent of the genome codes for proteins.
(vi) Repeated sequences make up very large portion of the human genome.
(vii) Repetitive sequences are stretches of DNA sequences that are
repeated many times, sometimes hundred to thousand times. They
are thought to have no direct coding functions, but they shed light
on chromosome structure, dynamics and evolution.
(viii) Chromosome 1 has most genes (2968), and the Y has the fewest (231).
(ix) Scientists have identified about 1.4 million locations where single-
base DNA differences (SNPs – single nucleotide polymorphism,
pronounced as ‘snips’) occur in humans. This information promises
to revolutionise the processes of finding chromosomal locations for
disease-associated sequences and tracing human history.
6.9.2 Applications and Future Challenges
Deriving meaningful knowledge from the DNA sequences will define
research through the coming decades leading to our understanding of
biological systems. This enormous task will require the expertise and
creativity of tens of thousands of scientists from varied disciplines in both
the public and private sectors worldwide. One of the greatest impacts of
having the HG sequence may well be enabling a radically new approach
to biological research. In the past, researchers studied one or a few genes
at a time. With whole-genome sequences and new high-throughput
technologies, we can approach questions systematically and on a much
broader scale. They can study all the genes in a genome, for example, all
the transcripts in a particular tissue or organ or tumor, or how tens of
thousands of genes and proteins work together in interconnected networks
to orchestrate the chemistry of life.
6.10 DNA FINGERPRINTING
As stated in the preceding section, 99.9 per cent of base sequence among
humans is the same. Assuming human genome as 3 × 109
bp, in how
many base sequences would there be differences? It is these differences
in sequence of DNA which make every individual unique in their
phenotypic appearance. If one aims to find out genetic differences
between two individuals or among individuals of a population,
sequencing the DNA every time would be a daunting and expensive
task. Imagine trying to compare two sets of 3 × 106
base pairs. DNA
fingerprinting is a very quick way to compare the DNA sequences of any
two individuals.
DNA fingerprinting involves identifying differences in some specific
regions in DNA sequence called as repetitive DNA, because in these
sequences, a small stretch of DNA is repeated many times. These repetitive
DNA are separated from bulk genomic DNA as different peaks during
density gradient centrifugation. The bulk DNA forms a major peak and
the other small peaks are referred to as satellite DNA. Depending on
base composition (A : T rich or G:C rich), length of segment, and number
of repetitive units, the satellite DNA is classified into many categories,
such as micro-satellites, mini-satellites etc. These sequences normally
do not code for any proteins, but they form a large portion of human
genome. These sequence show high degree of polymorphism and form
the basis of DNA fingerprinting. Since DNA from every tissue (such as
blood, hair-follicle, skin, bone, saliva, sperm etc.), from an individual
show the same degree of polymorphism, they become very useful
identification tool in forensic applications. Further, as the polymorphisms
are inheritable from parents to children, DNA fingerprinting is the basis
of paternity testing, in case of disputes.
As polymorphism in DNA sequence is the basis of genetic mapping
of human genome as well as of DNA fingerprinting, it is essential that we
understand what DNA polymorphism means in simple terms.
Polymorphism (variation at genetic level) arises due to mutations. (Recall
different kind of mutations and their effects that you have already
studied in Chapter 5, and in the preceding sections in this chapter.)
New mutations may arise in an individual either in somatic cells or in
the germ cells (cells that generate gametes in sexually reproducing
organisms). If a germ cell mutation does not seriously impair individual’s
ability to have offspring who can transmit the mutation, it can spread to
the other members of population (through sexual reproduction). Allelic
(again recall the definition of alleles from Chapter 5) sequence variation
has traditionally been described as a DNA polymorphism if more than
one variant (allele) at a locus occurs in human population with a
frequency greater than 0.01. In simple terms, if an inheritable mutation
is observed in a population at high frequency, it is referred to as DNA
polymorphism. The probability of such variation to be observed in non-
coding DNA sequence would be higher as mutations in these sequences
may not have any immediate effect/impact in an individual’s
reproductive ability. These mutations keep on accumulating generation
after generation, and form one of the basis of variability/polymorphism.
There is a variety of different types of polymorphisms ranging from single
nucleotide change to very large scale changes. For evolution and
speciation, such polymorphisms play very important role, and you will
study these in details at higher classes.
The technique of DNA Fingerprinting was initially developed by Alec
Jeffreys. He used a satellite DNA as probe that shows very high degree
of polymorphism. It was called as Variable Number of Tandem Repeats
(VNTR). The technique, as used earlier, involved Southern blot
hybridisation using radiolabelled VNTR as a probe. It included
(i) isolation of DNA,
(ii) digestion of DNA by restriction endonucleases,
(iii) separation of DNA fragments by electrophoresis,
(iv) transferring (blotting) of separated DNA fragments to synthetic
membranes, such as nitrocellulose or nylon,
(v) hybridisation using labelled VNTR probe, and
(vi) detection of hybridised DNA fragments by autoradiography. A schematic
representation of DNA fingerprinting is shown in Figure 6.16.
The VNTR belongs to a class of satellite DNA referred to as mini-satellite.
A small DNA sequence is arranged tandemly in many copy numbers. The
copy number varies from chromosome to chromosome in an individual.
The numbers of repeat show very high degree of polymorphism. As a
result the size of VNTR varies in size from 0.1 to
20 kb. Consequently, after hybridisation with VNTR probe, the
autoradiogram gives many bands of differing sizes. These bands give a
characteristic pattern for an individual DNA (Figure 6.16). It differs from
individual to individual in a population except in the case of monozygotic
(identical) twins. The sensitivity of the technique has been increased by
use of polymerase chain reaction (PCR–you will study about it in
Chapter 11). Consequently, DNA from a single cell is enough to perform
DNA fingerprinting analysis. In addition to application in forensic
science, it has much wider application, such as in determining
population and genetic diversities. Currently, many different probes
are used to generate DNA fingerprints.
SUMMARY
Nucleic acids are long polymers of nucleotides. While DNA stores genetic
information, RNA mostly helps in transfer and expression of information.
Though DNA and RNA both function as genetic material, but DNA being
chemically and structurally more stable is a better genetic material.
However, RNA is the first to evolve and DNA was derived from RNA. The
hallmark of the double stranded helical structure of DNA is the hydrogen
bonding between the bases from opposite strands. The rule is that
Adenine pairs with Thymine through two H-bonds, and Guanine with
Cytosine through three H-bonds. This makes one strand
complementary to the other. The DNA replicates semiconservatively,
the process is guided by the complementary H-bonding. A segment of
DNA that codes for RNA may in a simplistic term can be referred as
gene. During transcription also, one of the strands of DNA acts a
template to direct the synthesis of complementary RNA. In bacteria,
the transcribed mRNA is functional, hence can directly be translated.
In eukaryotes, the gene is split. The coding sequences, exons, are
interrupted by non-coding sequences, introns. Introns are removed
and exons are joined to produce functional RNA by splicing. The
messenger RNA contains the base sequences that are read in a
combination of three (to make triplet genetic code) to code for an amino
acid. The genetic code is read again on the principle of complementarity
by tRNA that acts as an adapter molecule. There are specific tRNAs for
every amino acid. The tRNA binds to specific amino acid at one end
and pairs through H-bonding with codes on mRNA through its
anticodons. The site of translation (protein synthesis) is ribosomes,
which bind to mRNA and provide platform for joining of amino acids.
One of the rRNA acts as a catalyst for peptide bond formation, which is
an example of RNA enzyme (ribozyme). Translation is a process that
has evolved around RNA, indicating that life began around RNA. Since,
transcription and translation are energetically very expensive
processes, these have to be tightly regulated. Regulation of transcription
is the primary step for regulation of gene expression. In bacteria, more
than one gene is arranged together and regulated in units called as
operons. Lac operon is the prototype operon in bacteria, which codes
for genes responsible for metabolism of lactose. The operon is regulated
by the amount of lactose in the medium where the bacteria are grown.
Therefore, this regulation can also be viewed as regulation of enzyme
synthesis by its substrate.
Human genome project was a mega project that aimed to sequence
every base in human genome. This project has yielded much new
information. Many new areas and avenues have opened up as a
consequence of the project. DNA Fingerprinting is a technique to find
out variations in individuals of a population at DNA level. It works on
the principle of polymorphism in DNA sequences. It has immense
applications in the field of forensic science, genetic biodiversity and
evolutionary biology.
EXERCISES
1 Group the following as nitrogenous bases and nucleosides:
Adenine, Cytidine, Thymine, Guanosine, Uracil and Cytosine.
- If a double stranded DNA has 20 per cent of cytosine, calculate the per
cent of adenine in the DNA. - If the sequence of one strand of DNA is written as follows:
5′ -ATGCATGCATGCATGCATGCATGCATGC-3′
Write down the sequence of complementary strand in 5’→3′ direction. - If the sequence of the coding strand in a transcription unit is written
as follows:
5′ -ATGCATGCATGCATGCATGCATGCATGC-3′
Write down the sequence of mRNA. - Which property of DNA double helix led Watson and Crick to hypothesise
semi-conservative mode of DNA replication? Explain. - Depending upon the chemical nature of the template (DNA or RNA)
and the nature of nucleic acids synthesised from it (DNA or RNA), list
the types of nucleic acid polymerases. - How did Hershey and Chase differentiate between DNA and protein in
their experiment while proving that DNA is the genetic material? - Differentiate between the followings:
(a) Repetitive DNA and Satellite DNA
(b) mRNA and tRNA
(c) Template strand and Coding strand - List two essential roles of ribosome during translation.
- In the medium where E. coli was growing, lactose was added, which
induced the lac operon. Then, why does lac operon shut down some
time after addition of lactose in the medium? - Explain (in one or two lines) the function of the followings:
(a) Promoter
(b) tRNA
(c) Exons - Why is the Human Genome project called a mega project?
- What is DNA fingerprinting? Mention its application.
- Briefly describe the following:
(a) Transcription
(b) Polymorphism
(c) Translation
(d) Bioinformatics
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Тканая латунная сетка для защиты экранов в различных
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Для оптимизации создания экранов, предназначенных для
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Данный материал сочетает в себе долговечность и функциональность,
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Металлические структуры из бронзы обеспечивают отличный уровень фильтрации, одновременно позволяя воздухопроницаемость, что играет
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Такая особенность особенно важна в условиях производственных процессов, где перегрев может привести
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но и к необходимости частого обслуживания оборудования.
При выборе подобного материала стоит обратить внимание
на размер ячеек и толщину проволоки, так как это напрямую влияет на защитные характеристики.
Оптимальный выбор зависит от специфики производственного процесса и требуемого уровня защиты, поэтому рекомендуется провести предварительные испытания на совместимость с другими используемыми элементами системы.
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устойчивости. Использование металлической структуры из меди и цинка
обеспечивает отличные механические характеристики.
Такие изделия выделяются высокой прочностью
на изгиб и удар. Это делает их надёжным барьером против внешних воздействий.
Долговечность материалов также является важной характеристикой.
Обработка с использованием соответствующих антикоррозийных составов значительно увеличивает срок службы.
Это снижает частоту замены, что экономит средства на
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Механические свойства играют
ключевую роль в предотвращении
повреждений. Сетка демонстрирует хорошую степень деформации, что позволяет ей
поглощать удары без разрушения.
Это делает возможным использование в разнообразных условиях, таких как производственные или промышленные помещения.
Помимо этого, структура обеспечивает отличный уровень защиты от
мелких предметов и частиц.
Это важно для сохранения целостности
оборудования и безопасности персонала,
что особенно актуально в условиях
с высоким уровнем опасности.
С учетом перечисленных фактов, применение
этого материала в решениях
для защиты от внешних угроз становится наиболее оправданным и целесообразным.
Инвестирование в данный сегмент может значительно повысить безопасность и эффективность работы на многих производственных объектах.
Технологические особенности изготовления защитных экранов с использованием тканой латунной сетки
При создании монолитных конструкций важно учитывать выбор ячейнистого материала, способствующего защите от электромагнитного излучения.
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Технология подготовки включает предварительный анализ размеров ячеек, который зависит от предполагаемого спектра воздействия.
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В процессе формовки обратите внимание на точность раскроя и соблюдение геометрии конструкций.
Применение автоматизированных систем позволяет достичь необходимого уровня повторяемости и минимизировать отходы материала.
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Используйте защитные средства, чтобы избежать
окисления соединительных зон, что существенно влияет на долговечность.
После окончательной сборки стоит
провести тестирование на устойчивость к различным типам воздействия.
Применение специализированных методик позволит оценить эффективность, снизить риски поломок и упростить обслуживание
готового изделия.
Для улучшения функциональных характеристик рекомендуется
покрытие антикоррозийными составами.
Это увеличивает срок службы и способствует сохранению
визуально привлекательного вида озвученных защитных конструкций.
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Максимальное время на раздачу даёт возможность участникам анализировать игру соперников.
Формат с ускорением предполагает
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В этом случае время на ход уменьшается,
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Игроки должны быть готовы к быстрым действиям, и это требует особой концентрации и навыков.
Обычно такой формат подходит для опытных участников, предпочитающих динамику и активное взаимодействие.
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Это позволяет новичкам адаптироваться к игре и не чувствовать
давления от фиксированной структуры.
Участники могут в любой
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игроков.
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Не забывайте про читы в игре.
Сохраняйте спокойствие в напряженных ситуациях и следите за своей мимикой
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While reviewing sandbox ecommerce systems and UI marketplace frameworks, testers found a central module featuring echo brook vendor showcase parlor console node integrated into structured layout, and despite the natural echo brook branding, the parlor page remains unfilled which reduces clarity and usability during testing and evaluation cycles
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While analyzing experimental ecommerce systems and UI vendor frameworks, testers observed embedded content blocks containing plum harbor vendor showcase room access node inside layout flow, and although the plum harbor aesthetic feels refreshing and organic, the vendor room shows zero vendors listed which creates a sense of incompleteness during testing and review sessions
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During UX evaluation of sandbox ecommerce systems and vendor marketplace prototypes, testers found embedded navigation containing quartz orchard vendor hall access console node inside structured layout, and although the quartz orchard concept feels unique and polished, the vendor hall redirects to the homepage which disrupts expected navigation during usability testing sessions
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While reviewing sandbox ecommerce systems and UI marketplace frameworks, testers found a central module featuring quick ridge house market vendor showcase console node integrated into structured layout, and despite being faster than legacy “quick harbor” designs, it still does not deliver smooth performance which reduces usability during evaluation cycles
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