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MOLECULAR BASIS OF INHERITANCE
Focus area Notes
The salient features of the Double-helix structure of DNA
(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'-->3' , the other has 3'--> 5'.
(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 . Similarly, Guanine is bonded with Cytosine
with three H-bonds.
(iv) The two chains are coiled in a right-handed fashion. The pitch of the helix
is 3.4 nm(10-9 m) and there are roughly 10 bp in Each turn. Consequently,
the distance between a bp in a helix is approximately equal to 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.
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Central dogma in molecular biology
In some viruses the flow of information is in reverse direction, that
is,from RNA to DNA. It is known as Reverse transcription.
PACKAGING OF DNA HELIX
• In eukaryotes, DNA is packed with the help of a set of positively
charged, basic proteins called histones.
• Histones are organised to form a unit of eight molecules called as
histone octamer.
• The negatively charged DNA is wrapped around the positively
charged histone octamer to form a structure called nucleosome
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• A typical nucleosome contains 200 bp of DNA helix
• Nucleosomes constitute the repeating unit of a structure in nucleus
called chromatin
• The nucleosomes in chromatin are seen as ‘beads-on-string’
structure when viewed under electron microscope (EM).
Chromatin has 2 forms:
Euchromatin:
• Loosely packed and transcriptionally active region of chromatin.
• It stains lightly
Heterochromatin:
• Densely packed and inactive region of chromatin.
• It stains darkly.
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Griffith’s Transformation Experiment
In 1928, Frederick Griffith, in a series of experiments with
Streptococcus pneumoniae (bacterium responsible for pneumonia),
Proposed transformation in bacteria.
• Two strains of Streptococcus pneumoniae (pneumococcus)
bacteria are there S strain bacteria and R strain bacteria.
• 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 heat-killed bacteria and injected into mice
• He observed that heat-killed S strain bacteria injected into mice
did not kill them.
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• 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.
Conclusion of Experiment
• 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.
‘Hershy and chase experiment’
• Alfred Hershy and Martha Chase in 1952 proved that DNA is the genetic
material.
• They worked with viruses that infect bacteria called bacteriophages.
• When a virus attaches with a bacteria, the genetic material of virus enters
into the bacterial cell.
• The bacterial cell treats the viral genetic material as its own and more
virus particles are produced.
• Hershy and Chase worked to discover whether the protein coat or DNA of
the virus enters the bacterium.
Experiment
• They grew some viruses in a radioactive medium of phosphorus(32P) and
some others in a radioactive sulfur(35S) medium .
• Viruses grown in radioactive phosphorus medium have radioactive DNA.
• Viruses grown in radioactive sulfur medium have radioactive protein.
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• These radioactive phages were allowed to attach to [Link] bacteria.
• This process is known as infection.
• During infection, genetic material of the virus enters into the bacteria.
• After infection, the viral protein coats were removed from the bacteria by
blending.
• The virus particles were separated from the bacteria by spinning them
in a centrifuge and the process is called centrifugation.
• Bacteria which were infected with viruses with radioactive protein were
not radioactive.
• But the bacteria which were infected with radioactive DNA were
radioactive indicating that DNA was the material that passed from the
virus to the bacteria.
Prepared by
Biju TL
GTHSS Poomala, Idukki
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The Machinery and the Enzymes for DNA Replication
The main enzyme of DNA replication is DNA-dependent DNA Polymerase
DNA Polymerase
• This is one of the fastest enzyme which catalyses the polymerization
of nucleotides with greatest accuracy.
• Any defect in replication leads to mutation.
• In [Link], 4.6x10 6 bp completes replication with 38 mts, ie, average
rate is 2000 bp per second.
Replication fork
•
• For long DNA molecules, the two strands of DNA cannot be separated in its
entire length due to very high energy requirement.
• In such DNAs replication occur within a small opening of the DNA helix
known as replication fork.
Leading strand
• The DNA dependent DNA Polymerases catalyses polymerization in 5`->3`
direction only.
• In 5`->3` direction , (the template with polarity 3`-> 5`) the replication is
continuous and it is known as leading strand.
•
Lagging strand
• In the other strand, template with polarity 5`->3` , the replication is
discontinuous and is called lagging strand.
• Discontinuously synthesized DNA fragments are called okazaki fragments.
DNA LIGASE
• Discontinuously synthesized DNA fragments are joined by the enzyme DNA Ligase
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Replication Fork
• The strand that has the polarity 3' →5' acts as a template, and is
referred to as template strand.
• The other strand which has the polarity (5'→3' ) and the
sequence same as RNA is referred to as coding strand.
Promoter
• Promoter is a DNA sequence that provides binding site for RNA
polymerase.
• It is located towards the 5` end(upstream) of the structural gene.
• The presence of a promoter in a transcription unit defines the template
and coding strands.
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Terminator
The terminator is located towards 3' -end (downstream) of the coding
strand and it usually defines the end of the process of transcription.
Transcription Unit and the Gene
Cistron
Cistron is a segment of DNA coding for a polypeptide.
Monocistronic unit
• The structural unit in transcription unit of eukaryotes are
monocistronic.
• Monocistronic structural genes have interrupted coding
sequences. Ie, the genes are split or the genes with coding
sequences and genes with non-coding sequences.
Exons
• The coding sequence or expressed sequences of cistron are called
exons.
Introns
• The non coding sequences of cistrons are called introns or
intervening sequence. They do not appear in the mature or
processed RNA.
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Polycistronic unit
This is the structural transcription unit of bacteria or prokaryotes
The Lac operon
• 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.
Components of Lac operon
• Regulatory gene (i gene)
Regulatory gene produces the repressor protein.
• Promoter gene (P)
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It is the region in which RNA Polymerase binds
• Operator gene (O)
It is the region in which repressor protein binds.
• Structural gene( z, y,a)
Structural gene transcribe into lac mRNA and it is translated into
lac proteins:-
• β -galactosides,
z gene codes for beta-galactosidase ( β -gal),
It converts the disaccharide, lactose into galactose and glucose.
• The y gene codes for permease
Permease increases permeability of the of the cell to β -
galactosides.
• The a gene encodes a transacetylase.
Lac operon (In absence of inducer)
• Regulatory gene transcribes into repressor mRNA.
• Repressor mRNA translates into repressor protein.
• Repressor protein binds to the Operator region.
• This binding prevents the RNA Polymerase binding to the
Promoter region.
• Thus the transcription of the structural genes is prevented.
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Lac operon (In presence of inducer)
• Regulatory gene transcribes into repressor mRNA.
• Repressor mRNA translates into repressor protein.
• inducer, such as lactose or allolactose binds with repressor
protein and it becomes inactive.
• Inactive repressor could not bind to operator. So RNA
Polymerase binds to Promoter region.
• RNA Polymerase transcribes lac mRNA
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Human Genome Project (HGP)
• Human Genome Project was the first mega project for the
sequencing of nucleotides and mapping of all the genes in human
genome.
BAC= Bacterial Artificial Chromosomes
YAC= Yeast Artificial Chromosomes
DNA FINGERPRINTING
STEPS IN DNA FINGERPRINTING
(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.
Applications of DNA Fingerprinting
• 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.
• It is used as a powerful forensic tool to solve the problems of
paternity, rape, murder etc.
• It is used in the diagnosis of genetic diseases.
• It is used in the determination of phylogenetic status of animals etc.
