CHAPTER-6

MOLECULAR BASIS OF INHERITANCE

Structure of Polynucleotide Chain

 DNA and RNA are polynucleotides. They are made of three components—
 a nitrogenous base
 a pentose sugar (ribose in case of RNA and deoxyribose in case of DNA)
 a phosphate group

 Nitrogenous Bases—Purines and Pyrimidines

 Purines—Adenine and Guanine
 Pyrimidines—Cytosine, Thymine (Present in DNA), Uracil (Present in RNA)
 A bond N-glycosidic linkage is formed between a nitroegnous base and the pentose sugar to
form a
 nucleoside.
 A phosphate group is linked to the 5’ end of the nucleoside by a phosphoester linkage to
form a nucleotide.
 Two nucleotides are then linked by a 3’-5’ phosphodiester linkage to form a dinucleotide.
 In the same manner, more and more nucleotides are linked to form a polynucleotide chain.
 In a polynucleotide chain, one end has a free phosphate molecule at 5’-end of ribose sugar
which is
 the 5’-end of polynucleotide and the other end has free –OH group at 3’-end of ribose sugar
which is the 3’-end of polynucleotide.
 In RNA, every nucleotide has an additional –OH group present at 2’-position in the ribose.
 The base pairing on the two polynucleotide strands are complementary to each other.

Chargaff’s Rules

 Purine and pyrimidine base pairs are present in equal amounts.

 adenine + guanine = thymine + cytosine
 [A + G] = [T + C]
 [A+G]/[T+C] = 1

Structure of DNA

 DNA is the largest biomolecule in the cell.

 The two polynucleotide chains of DNA have antiparallel polarity, 5'-3' in one and 3'-5' in the
other.
 The backbone of each polynucleotide chain is made of alternate sugar-phosphate groups,
with nitrogen bases projecting inwards.
 The nitrogen bases form complementary pairs.
 Adenine pairs with thymine by two hydrogen bonds, while guanine pairs with cytosine by
three
hydrogen bonds.
 The double chain is right-handed helically coiled. This produces major and minor grooves
alternately.
 The pitch of the helix is 3.4 nm with about 10 base pairs in each turn.
 Planes of adjacent base pairs are stacked over one another which confer stability to the
helical structure.
 DNA is acidic in nature.
Packaging of DNA Helix
 The long-sized DNA is accommodated in small areas through packing and compaction.
 Compaction occurs by folding and attachment of DNA with basic proteins, non-histones in
prokaryotes and histones in eukaryotes.

Packaging in Prokaryotes
 DNA is super coiled with the help of some proteins.
 The compacted mass of DNA is called nucleoid.

Packaging in Eukaryotes
 It is carried out with the help of lysine- and arginine-rich basic proteins called histones.
 Histones organize to form a unit of eight molecules called histone octamer.
 Negatively charged DNA is wrapped around a positively charged histone octamer. This
structure is called nucleosome.
 DNA connecting the two nucleosomes is called linker DNA.
 Nucleosomes consist of chromatin which is packaged by non-histone chromosomal (NHC)
proteins.
 At some places, chromatin is densely packed to form darkly staining heterochromatin. At
other places, chromatin is loosely packed to form euchromatin which is lightly stained.

Experiments on Genetic Material

 Genetic material is a substance which not only controls the inheritance of traits from one
generation to another but is also able to express its effect through the formation and
functioning of traits.
 Transformation (S. F. Griffith)

(i) Frederick Griffith (1928) carried out a series of experiments with Streptococcus pneumoniae
(bacterium causing pneumonia).

(ii) According to him, when the bacteria are grown on a culture plate, some produce smooth
shiny colonies (S), while others produce rough (R) colonies.

(iii) This is because the S-strain bacteria have a mucous (polysaccharide) coat, while R-strain
does not.

(iv) Mice infected with S-strain (virulent) die from pneumonia but mice infected with R-strain do
not develop pneumonia.

(v) Griffith killed bacteria by heating and observed that heat-killed S-strain bacteria injected into
mice did not kill them. On injecting mixture of heat-killed S and live R bacteria, the mice died.
He recovered living S-bacteria from dead mice.

(vi) From this experiment, he concluded that the ‘R-strain bacteria’ had been transformed by
the heat-killed S-strain bacteria. Some transforming principle transferred from heat-killed S-
strain, had enabled the R-strain to synthesise a smooth polysaccharide coat and become
virulent. This must be due to transfer of the genetic material.
 Biochemical Characterization of Transforming Principle: (Avery, MacLeod and McCarty)

 In 1944, Avery, MacLeod and McCarty purified biochemicals from the heat-killed S-type
bacteria into four components—DNA with DNase, DNA without DNase, carbohydrate and
protein.
 Only DNA of S-type can change R-type of bacteria into S-type. Therefore, the character or
gene of virulence is located in DNA
 Thus, it was proved that the chemical to be inherited is DNA and it forms the chemical or
molecular basis of heredity.

Transduction (Hershey and Chase)

 The experiment performed by Hershey and Chase is as summarised below:

(i) Alfred Hershey and Martha Chase (1952) gave unequivocal proof that DNA is the genetic material.

(ii) In their experiments, bacteriophages (viruses that infect bacteria) were used.

(iii) They grew some viruses on a medium that contained radioactive phosphorus and some others on
sulphur containing radioactive medium.

(iv) Viruses grown in the presence of radioactive phosphorus contained radioactive DNA but not
radioactive protein because DNA contains phosphorus but protein does not. In the same way, viruses
grown on radioactive sulphur contained radioactive protein, but not radioactive DNA because DNA does
not contain sulphur.

(v) Radioactive phages were allowed to attach to E. coli bacteria. As the infection proceeded, 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.

(vi) Bacteria which were infected with viruses that had radioactive DNA were radioactive, indicating that
DNA was the material that passed from the virus to the bacteria.

(vii) Bacteria that were infected with viruses that had radioactive proteins were not radioactive. This
indicated that the proteins did not enter the bacteria from viruses. It proved that DNA is a genetic
material that is passed from virus to bacteria.
Properties of Genetic Material

 The hereditary information must be present in the coded form in the structure of genetic
material.
 It should be able to replicate.
 The replicated genetic material must be faithfully transferred from a cell to its daughter
cells.
 It should be stable chemically and physically.
 It should be able to express its effect in the form of Mendelian characters.

RNA as a Genetic Material

 The first genetic material was RNA.

 Chemical reactions of metabolism, splicing and translation evolved around RNA.
 The first biocatalysts were RNAs. Some of the enzymes, such as ribozyme, are made of RNA.
 RNAs were effective in early unstable environmental conditions.
 By small chemical modifications, RNA gave rise to DNA as the genetic material.

Replication

 Replication is the process of the formation of two identical copies from one DNA molecule.
 According to Watson and Crick (1953), during replication, the weak hydrogen bonds
between the nitrogenous bases of nucleotides separate and the two polynucleotide chains
of DNA uncoil and separate.
 When the two strands separate one strand acts as a template and synthesises its
complementary strand.
 Each new DNA molecule will have one parental strand and one newly synthesised strand.
Hence, this method of DNA replication is called the semiconservative method.

Francis Crick proposed the central dogma in molecular biology, which states that the

genetic information flows from

 Central Dogma

Experimental Proof for Semiconservative Replication of DNA (Meselson and Stahl)

 The experimental proof for semiconservative replication of DNA was provided by Meselson
and Stahl (1958) in Escherichia coli.

(i) E. coli was grown in a medium containing 15NH4Cl as the only nitrogen source for many
generations. 15N got incorporated into newly synthesised DNA (and other nitrogen containing
compounds). This heavy DNA molecule could be distinguished from the normal DNA by
centrifugation in a cesium chloride (CsCl) density gradient.

(ii) They then transferred the cells into a medium with normal 14NH4Cl and took samples at
various definite intervals as the cells multiplied and extracted the DNA that remained as double
stranded helices. DNA samples were separated independently on CsCl gradients to measure
DNA densities.

(iii) The DNA that was extracted from the culture, one generation (after 20 min) after the
transfer from 15 N to 14N medium had a hybrid or intermediate density. DNA extracted from
the culture after another generation (after 40 min) was composed of equal amounts of this
hybrid DNA and of light DNA.

(iv) Very similar experiments were carried out by Taylor and Colleagues on Vicia faba (faba
beans) using radioactive thymidine and the same results, i.e. DNA replicates
semiconservatively, were obtained as in earlier experiments.

Process of DNA Replication in Eukaryotes

 DNA replication is initiated at a defined sequence of nucleotides called the initiation point
or origin of replication (ori C).
 Initiator protein Dna A binds to the ori C site and denatures DNA.
 Enzyme helicase then bind to each strand of unwounded DNA close to Dna A.
 The two strands of DNA begin to separate by the breakdown of hydrogen bonds between
the paired nucleotides exposing their nitrogenous bases so that each can serve as a
template for the synthesis of a new strand.
 As eukaryotic DNA is large, it has several origins of replication and an equal number of
replicons.
 During replication, each origin of replication is marked as a replication fork.
 RNA polymerase synthesises a short priming strand of RNA called RNA primer on the DNA
 template.
 DNA polymerase polymerises the growth of the new DNA chain on RNA primer.
 The new strands of DNA are formed in the 5′-3′ direction on the 3′-5′ template DNA by the
addition of deoxyribonucleotides to the 3′ end of the primer RNA. This is influenced by DNA
polymerase III in the presence of ATP.
 Once the synthesis of DNA has been initiated, it proceeds continuously in coordination with
the unwinding of DNA at the replication fork.
 Leading strand synthesis: It occurs in the 5′-3′ direction using the 3′-5′ strand of parental
DNA as a template. The leading strand is synthesised as one piece.
 Lagging strand synthesis: It occurs in the 5′-3′ direction on the 5′-3′ strand of DNA in short
segments of 1000–2000 nucleotides called Okazaki fragments. Synthesis of each Okazaki
fragment begins with the RNA primer synthesised by the enzyme primase. The process is
carried out by DNA polymerase III.
 The gaps left between the Okazaki fragments by the removal of the RNA primer are filled
with complementary deoxyribonucleotide residues by DNA polymerase-I. Finally, the
adjacent 3′ and 5′ ends of Okazaki fragments are joined by DNA ligase.
 Sometimes, wrong bases are added in the new chain during synthesis.
 The enzyme DNA polymerase-I identifies and replaces these forbidden base pairs with
correct nitrogenous base pairs.
 OR

Transcription
 Transcription is the process of copying the genetic information coded in the DNA into an
mRNA molecule.
 The segment of the DNA template which transcribes RNA is called the transcription unit. It
comprises three regions—promoter, structural gene and terminator.

Promoter region:
 It is located just upstream of the initiation codon of the structural gene towards the 5′ end
of the coding strand.
 The promoter sequence of the nitrogenous bases provides the binding site for RNA
polymerase for the initiation of transcription.

Structural Gene:
It is a segment of DNA with a specific sequence of nucleotides which code for a protein or
polypeptide required for a morphological or functional trait of the cell.
 Structural genes are of two types:
Polycistronic genes: Genes which transcribe mRNA which codes for more than one type of
polypeptide chains are called polycistronic genes. These are found only in prokaryotes.

Monocistronic genes: Genes which transcribe mRNA which codes for only one polypeptide
chain are called monocistronic genes. These are found only in eukaryotes.

Terminator Region:
 It is located downstream towards the 3′ end of the coding strand.
 It defines the end of process of transcription.

In Prokaryotes

 DNA-dependent RNA polymerase catalyses the transcription of three types of RNAs in

bacteria.
 Three types of RNA are mRNA (messenger RNA), tRNA (transfer RNA) and rRNA (ribosomal
RNA).
 RNA polymerase binds to the promoter and initiates the transcription.
 RNA polymerase associates with the initiation factor and the termination factor for the
initiation and termination of transcription.
 Polymerisation is carries out by using nucleoside triphosphates as substrate.
 Once polymerases reach the terminator site, RNA and RNA polymerase falls off which
results in termination of transcription.

In Eukaryotes

 Three RNA polymerases I, II and III synthesise rRNA, mRNA and tRNA, respectively.
 RNA polymerase I transcribes rRNAs, RNA polymerase II transcribes precursor of mRNA and
heterogenous nuclear RNA (hnRNA) and RNA polymerase III transcribes tRNA, 5srRNA and
small nuclear RNAs (snRNAs).
 The primary transcripts contain introns as well as exons. Hence, they are subjected to
splicing in which introns are removed and exons are joined.
 hnRNA undergoes capping and tailing. In capping guanosine triphosphate is added to the 5′
end of hnRNA.
 In tailing, 200–300 adenylate residues are added at the 3′ end of hnRNA.

Genetic Code

 The relationship between the sequence of amino acids in a polypeptide and nucleotide
sequence of DNA or mRNA is called the genetic code.

 Each codon should have a combination of three nitrogenous bases, i.e. a combination of three
bases would generate 64 codons (43 = 4 × 4 × 4 = 64 codons).

Salient Features of the Genetic Code

 The codon is triplet.

 There are 61 codons code for amino acids and 3 codons do not code for any amino acid. They
are called termination codons.

 One codon codes for only one amino acid. It is unambiguous and specific.

 Some amino acids are coded by more than one codon, hence the code is degenerate.

 The code is universal. A codon specifies the same amino acid in case of a human being or in
case of a tree or even in case of a virus.

 Polypeptide synthesis is signaled by two initiation codons—mostly AUG or methionine codon

and rarely GUG or valine codon.
Mutation and Genetic Code

 Gene mutations involve changes in the form of addition, deletion or rearrangement of

nitrogen bases. Types of Gene Mutations

 Insertion: A distortion of DNA by mutagen can change the base sequence of a cistron in
the reverse order. This process is called inversion.
 Substitution: In substitution, a nitrogen base is changed with another. It is also called
point mutation.
 Frame-shift mutation: Frame-shift mutations are mutations in which the reading of the
frame of the base sequence shifts laterally either in the forward direction because of
insertion of one or more nucleotides or in the backward direction because of deletion of
one or more nucleotides. Frame-shift mutations are of two types:
a. Insertion: One or more nucleotides are added in the segment of DNA representing a
cistron or gene.
b. Deletion: One or more nucleotides are lost from a segment of DNA representing a
cistron or gene.

Translation

 Translation is the process of polymerisation of amino acids to form a polypeptide chain.

Mechanism of Translation:

 Amino acids are activated in the presence of ATP.

 Once activated, they are joined to their 3′ end of their specific tRNA molecules to form
aminoacyl tRNA. This step is called charging of tRNA or aminoacylation of tRNA.

 The aminoacyl tRNA complexes are taken to the ribosomes for protein synthesis.

 The anticodon of tRNA - Met base pairs with the initiation codon AUG, the first codon on the
5′ end of the mRNA. AUG is brought to the P-site when mRNA binds to the 30S ribosomal
subunit.

 The 50S subunit ribosome then binds creating the A-site.

 Each new AA-tRNA enters the site A base pair with the codons of m-RNA.
 Peptide bonds are formed between the two consecutive amino acids and thus a polypeptide
chain is formed.

 When the site A reaches the stop codon, the process terminates and the polypeptide chain is
released from the ribosome.

OR

Diagram showing mechanism of translation

Regulation of Gene Expression

 Gene expression is the mechanism at the molecular level by which a gene is able to express
itself in the phenotype of an organism.

Levels of Gene Regulation in Eukaryotes

Gene Regulation in Prokaryotes

 In prokaryotes, the rate of transcription is controlled at the initiation of transcription using the
on–off mechanism. This is regulated by some regulatory proteins.

 Positive regulation is through activators or inducers and is called induction.

 Negative regulation takes place through repressors and is called repression.

Lac Operon System:

 The operon model for lactose catabolism is called lac operon.

It consists of three kinds of genes: Operator gene, promoter gene and regulator gene.
 Operator gene (O) lies just before the first structural gene and overlaps the promoter
sequence.
 Promoter gene (P) lies immediately adjacent to the operator gene where RNA polymerase
binds.
 Regulator gene (R) lies outside the operon and produces a repressor substance.
  In the absence of inducer lactose, the regulator gene R produces a repressor protein
which binds to the operator site and prevents transcription of structural genes.
 When inducer lactose is introduced in the medium, it binds to the repressor and
prevents it from binding to the operator. The operator then induces the RNA
polymerase to bind to the promoter and transcribe mRNA by the structural genes and
enzymes produced.

Human Genome Project

 The Human Genome Project (HGP) also called International Human Genome Sequencing
Consortium is a comprehensive international research effort of mapping the entire human
genome by determining the sequence of nucleotides in DNA of each of the 22+X and Y
chromosome and to study the functions of human genes.

 In 1988, the Human Genome Project was established as a loose but organized collaboration
between geneticists in all the parts of the world.
 The Human Genome Project officially began on 1 October 1990 and was completed in April
2003.

Goals of the Human Genome Project

 To sequence the entire genome which includes more than 3 billion base pairs.

 To store information in databases which is easily accessible to scientists all over the world.

 To identify thousands of genetic markers and map them in the genome.

 To identify 20000-25000 genes in human DNA.

 To address the ethical, legal and social implications of the results obtained from the project.
Salient

Features of the Human Genome

 The human genome contains more than 3 billion nucleotides.

 An average gene is formed of 3000 base pairs, but the size of the gene varies greatly.

 The functions of more than 50% of genes discovered so far are not known.

 Less than 2% of the genome sequences code for protein.

 Genes associated with several human diseases and particular sequences responsible for
various disorders have been identified.

 Chromosome-I is the largest human chromosome with the largest number of genes (2968),
and the Y-chromosome has the least number of genes (231).

 There are 1.4 million single base differences. These are called single nucleotide
polymorphisms (SNPs).

Strategy and Methodology of the Human Genome Project

 The genetic and physical maps of the human genome are prepared using molecular markers,
simple sequence repeats (SSRs), microsatellites and PCR amplification of certain
microsatellites.
 Two methods used for sequencing the entire human genomic DNA are:

 Expressed Sequence Tagging (EST) Method: It involves identifying genes which are
expressed as RNA. They are represented as ESTs.

 Sequence Annotation Method: It involves determining all genomic sequences including

coding and non-coding sequences and assigning functions to different regions in the
sequence.

 The total DNA obtained from the cell was broken into relatively smaller fragments and
cloned in suitable host. Cloning of the segments resulted in the amplification of the inserted
fragments. Fragments were sequenced using automated DNA sequences.

 For functional analysis of the human genome, genetic and physical maps of the genome
were generated.

Applications of the Human Genome Project:

 Describing the genetic constitution of a human being

 Diagnosis and treatment of diseases

 Establishing paternity and other family relationships

 Identifying potential suspects by DNA fingerprinting technology using hair, blood and
semen drops as sample specimens.

DNA Fingerprinting:

 DNA fingerprinting is a method of identifying DNA by locating the differences in the

arrangement of nucleotides in those specific regions of the DNA sequence which are
repeated several times. The technique is called DNA typing or DNA profiling.

 The technique was devised in 1984 by Alec Jeffreys, a British geneticist, to visually identify
nucleotide sequences in DNA. Principle of DNA Fingerprinting

 DNA fingerprinting involves identifying differences in repetitive DNA sequences.

 The repetitive DNA is called satellite DNA.

 The repetitive DNA sequences show a high degree of polymorphism and form the basis of
DNA fingerprinting.
 Variation in satellite DNA is very useful in establishing the identity of victims from blood
stains, semen stains, hair roots, tears or saliva and in solving paternity disputes.

Procedure of DNA Fingerprinting:

 In DNA fingerprinting, radiolabelled variable number of tandem repeats (VNTRs) are used
as probes.

 DNA for printing is isolated from WBCs of blood, semen, vaginal swabs, skin cells or cells
from root hair.

Applications of DNA Fingerprinting

 It helps in identification of criminals or potential suspects and relieving people wrongly

accused of crimes.

 It is used to infer blood relationships in the members of the same family.

 It is used to determine to the real parents of a given offspring.

 It is used to determine the actual parents in case of a lost child.

 It is used to determine the sex of badly damaged bodies of accident victims or of

archaeological specimens.