PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY

COLLEGE OF AGRICULTURE, RAJENDRANAGAR, HYDERABAD-500030

COURSE NO.: GP-603

COURSE NAME: GENOMICS IN CROP IMPROVEMENT

TOPIC NAME:

STRUCTURE OF NUCLEAR GENETIC MATERIAL --

DNA, RNA AND GENE

SUBMITTED BY:

M.BHARATH KUMAR

RAD/2020-25

DEPT. OF GENETICS AND PLANT BREEDING

Introduction

It is well known fact that transmission of traits takes place from one generation to
other. The offsprings are similar to both the parents in some traits.
Gregor Johann Mendel (1866) gave the idea that transmission of traits over
generations take place through Factor or Determiner or Gene which carries information for
expression of trait or phenotype.
Genes are present on the chromosomes which are distributed equally into the two
daughter cells during cell division. The biochemical studies reveal that chromosomes are
composed of proteins (60% ) and DNA (40% ).
The resemblance of offspring to their parents depends on the precise transmission of principle
component from one generation to the next, that component is- The Genetic Material
The Genetic material must be capable of
 carrying information – Cracking the genetic code
 Must self replicate – DNA replication
 Must govern the expression of the phenotype – Gene function
 Must allow for information to change – Mutation

Is the Genetic Material Protein or DNA?

 Until 1940 Proteins were considered as genetic material as Proteins are polymer of 20
protein amino acids and present in larger quantity, encode more and variety of
information.
 DNA is polymer of only 4 different deoxyribonucleotides (ATP, CTP, GTP& TTP)
and is present in smaller quantity.
 Most geneticists focused on “transmission genetics” and passively accepted proteins
as the genetic material.
 But On the basis of certain experiments conducted from time to time, it was
ultimately demonstrated that DNA carries genetic information and not the proteins
 There are some direct evidences and some indirect evidences which prove DNA as
Genetic Material.
Direct evidences came from:
1. Frederick Griffith’s (1928) experiment on Bacterial Transformation
2. Oswald Avery, Colin Macleod and Maclyn McCarty’s (1944 ) experiment on
Transformation
 3. D. Hershey and Martha Chase (1952 ) experiment on T- Even (2,4 ) Bacteriophage

1. Griffith experiment: Transformation of bacteria

In 1928, Griffith studied the bacterium Streptococcus pneumoniae which comes in
two strains
S - Smooth (strain IIIS)
 Secretes a polysaccharide capsule (evades immune system)
 Produce smooth colonies on solid media
 Helps make the microorganism virulent, or able to cause disease.
R - Rough (strain IIR)
 Unable to secrete a capsule
 Produce colonies with a rough appearance.
 Does not cause disease as strain lacks the polysaccharide capsule

 When live, avirulent II R and heat-killed virulent III S are mixed, the heat-killed
virulent III S passed on disease-causing information to the avirulent II R making it
virulent.
 Transformation is a change in genotype caused when cells take up foreign material, as
bacteria had been Transformed from the rough to the smooth version
 Principle Component of type III S cells which induced the conversion of type II R cells
into type III S was named transforming principle.
2. Avery, MacLeod and McCarty Experiment

 In 1944 repeated Griffith’s experiment of transformation using purified cell extracts.

 Cell free extract of SIII strain Bacterium was subjected to DNase, RNase and Protease.
 Each treated extract was mixed with RII and mixture injected to mouse to see
transformation.
 In case of Protease and Rnase transformation was recorded
 In case of DNase no transformation was recorded proving the transforming material is
DNA.

 With the help of experiment they showed that DNA isolated from SIII strain Bacteria
could confer the pathogenic properties to R II strain Bacteria.
 Avery et al (1944) revealed the chemical nature of the transforming substance to be
DNA
 Two conclusion were derived
1. Active factor is DNA which can cause transformation
2. SIII strain contains the Active factor

3. Hershey and Chase’s Experiments

 1952, American biologists Alfred Hershey and Martha Chase performed an experiment
that settled the controversy
 Proved that DNA carries the genetic material by experimented with T2 bacteriophages,
viruses that attack bacteria.
 Bacteriophage virus attaches to the surface of bacteria, injects its genetic material into
the bacteria, the viral genes cause many more viruses to be made inside the bacteria until
the bacteria burst and hundreds of new viruses are released.

 The results of experiment clearly indicate that only DNA labelled with 32P entered the
bacterial cell, and not the Protein coat (35S) as it is left outside.

 The DNA entering the host cell carries all the genetic information for synthesis of new
phage particle.

 This certainly proves that DNA is the genetic material in Bacteriophage and not proteins.

INDIRECT EVIDENCES FOR DNA AS GENETIC MATERIAL

 Equal amount of DNA is present in all cells of an organism
 Nuclear Division occurs only after DNA duplication during S phase of Interphase.
 Variation in Diploid amount of DNA amongst different species.
 DNA has same physical and chemical properties in all organisms yet allow to produce
great diversity of organisms
 Indefinite number of combinations are possible with four bases A T G C
 Of all macromolecules DNA is metabolically most stable
 In prokaryotes DNA is not linked with proteins still characters are inherited.
EVIDENCE FOR RNA AS GENETIC MATERIAL
 A Gierer and G Schramm ( 1956 ) when inoculated tobacco plants with Purified RNA
isolated from TMV - Leisons appeared on leaves of healthy plant
 H Fraenkel Conrat and B Singer (1957) separated RNA from protein of TMV in 1st step.
 In second step reconstituted virus with protein from mutant strain of TMV
 Inoculated Hybrid TMV into healthy tobacco plant Tobacco Mosaic disease appeared
 TMV progeny isolated from diseased plant showed parental RNA only but not
parental proteins
 This provided evidence that RNA is the Genetic Material in TMV not but proteins.

Examples of Plant RNA viruses

 Tobbaco Mosaic Virus (TMV ) was crystallized by Stanley (1935) for the first time.
 TMV causes Tobacco mosaic disease
 TMV can also cause disease in Tomato, Pepper, Petunia, Snapdragon, Delphinium,
and Marigold.
 Papaya Ring Spot virus (PRV)
 Potato Leaf Roll Virus (PLRV)
 Holmesrib-grass Virus (HRV)
 Plantago Alfa-alfa Mosaic Virus (AMV)
DISCOVERY OF DNA
 In 1962, James Watson and Francis Crick were awarded noble prize for the discovery
of double helical structure of DNA.
 The scientific framework for their breakthrough was provided primarily by:
 Rosalind Franklin (X-ray diffraction)
 Erwin Chargaff (chemical composition)

Rosalind Franklin and Maurice Wilkins

The data of Wilkin, Franklin and others on X- ray crystallography of purified DNA
revealed following features :
 Multistranded molecule with X –shaped pattern
 Diameter – 22A
 Groups spaced at 3.4A along its length
 A repeating unit occuring at every 34A

Watson and Crick Model of DNA:

 Made of two polynucleotide strands with many deoxyribonucleotides.
 There are two asymmetrical grooves Major groove and Minor groove where proteins
can bind.
 Two strands are oriented anti-parallel i.e., 5'- end of one strand is located with 3'- end
of other strand.
 Anti parallel orientation essential for hydrogen bond formation between basepairs.
 The two strands of DNA are coiled together in a right handed helix forming the DNA
double helix
 The DNA double helix is stabilized i.e., the two strands are held together mainly by
hydrophobic forces.
 Diameter of helix -20A
 Pitch of helix- 34A
 Base pairs in one pitch -10bp
 Distance between two base pairs -3.4A
 The base pairs in a DNA are stacked between sugar and phosphate back bones.
 During replication two strands of DNA uncoil. The unpaired bases in single stranded
regions of two strands pair with complementary bases.
 Sometimes errors in base pairing may occur during replication resulting in mutations.
Hydrogen bonding:
 The formation of hydrogen bonds between A and T and between G and C is called
complementary base pairing and these bases are called complementary bases.
 The hydrogen bonds are crucial for precise replication and transcription processes.
A=T GΞC
Chargaff Rules:
Chemical analysis of DNA by chargaff and others during 1940s demonstrated following:
 Number of pyrimidine bases = Purine bases
C+T = A+G
 Percent of adenine = percent of thymine (A=T)
 Percent of cytosine = percent of guanine (C=G)
 A+C content of DNA = G+T

COMPONENTS OF DNA
DNA has three main components

1. Deoxyribose (a pentose sugar)

2. Base (there are four different ones)

3. Phosphate

Deoxy ribose and Ribose sugars

The oxygen present at the second carbon of ribose is missing in deoxyribose hence its
name 2’- deoxyribose in case of DNA

Organic Bases

 They are divided into two groups

Purines Pyrimidines
Made of a 6 member ring, fused to a 5 Made of one 6 member ring
member ring
Adenine and Guanine Thmine and Uracil In RNA, Cytosine
Nucleoside :

The H – attached to the –N at position 1 of pyrimidines or H – attached to the -N at

position 9 of purines interacts with the –OH at 1’C of pentoses and forms covalent bond
between pentose and organic base resulting in nuceloside.
 Organic base + deoxyribose = Deoxyriboside
 E.g. Deoxyadenosine, Deoxyguanosine

Nucleotide :

When a phosphate group is attached to either 3’C or the 5’C of the pentose molecule of a
nucleoside then nucleotide is formed.
 Organic base + deoxyribose+ Phosphate = Deoxyribotide
 Deoxyadenosine + Phosphate = Deoxyadenylic acid

Nucleotide Structure:
• Nucleotides are covalently linked together by phosphodiester bonds
• A phosphate connects the 5’ carbon of one nucleotide to the 3’ carbon of another
• Therefore the strand has directionality – 5’ to 3’
• The phosphates and sugar molecules form the backbone of the nucleic acid strand
Alternative Forms of DNA
 The DNA double helix can form different types of secondary structure
 The predominant form found in living cells is B-DNA
 However, under certain in vitro conditions, A-DNA and Z-DNA double helices can
form.

A-DNA
 Right-handed helix
 11 bp per turn
 Occurs under conditions of low humidity
 Little evidence to suggest that it is biologically important
 DNA.RNA heteroduplexes and RNA double helices occur in vivo in A-form
 Heteroduplex is a double helix in which two strands differ from each other.
Z-DNA
 Left-handed helix
 12 bp per turn
 The sugar –phosphate backbone follows a zig-zag path, which gives the name
Z-DNA
 Evidence from yeast suggests that it may play a role in transcription and
recombination
C-DNA
 Right-handed helix
 9.3 basepairs per turn
 It is more tightly wound than the B-form DNA
Sl.No
.
Unique Sequences Highly Repetitive sequences
01 Occur once in genome Occurs many times in a genome
 02 Long base sequences Short sequences (5-300 bases)
03 They may be genes They are not genes
04 They may be translated They are not translated
05 Small differences between individuals Can vary greatly between individuals
06 Exons are unique sequences Introns may be repetitive
07 Smaller proportion of genome Higher proportion of genome

STRUCTURE OF RNA

 RNA double helices typically right-handed (11-12 base pairs per turn)
 The primary structure of an RNA strand is much like that of a DNA strand
 RNA strands are typically several hundred to several thousand nucleotides in length
 In RNA synthesis, only one of the two strands of DNA is used as a template
 Although usually single-stranded, RNA molecules can form short double-stranded
regions
 This secondary structure is due to complementary base-pairing A to U and C to G.

TYPES OF RNA

Messenger RNA (mRNA):

 It constitutes about 5-10% of the total cellular RNA.
 a single prokaryotic mRNA molecule codes for more than one polypeptide ; such a
mRNA is known as polycistronic mRNA.
 All eukaryotic mRNAs are monocistronic i.e. coding for a single polypeptide
specified by a single cistron.
 Carry a copy of instructions from nucles to other parts of the cell.

Ribosomal RNA (rRNA):

 It constitutes about 80% of the total cellular RNA.
 The function of rRNA is binding of mRNA and tRNA to ribosomes.

Transfer RNA (tRNA):

 It is also known as soluble RNA(sRNA). It constitutes about 10-15% of total RNA of
the cell.
 Transfers amino acids(proteins) to the ribosomes to be assembled.

Differences Between RNA and DNA:

 S.No RNA DNA
Single stranded mainly except
when self complementary Double stranded (Except for certain
1) sequences are there it forms a viral DNA s which are single
double stranded structure (Hair pin stranded)
structure)
2) Ribose is the main sugar The sugar moiety is deoxy ribose
Pyrimidine components differ.
Thymine is always there but uracil is
3) Thymine is never found(Except
never found
tRNA)
Being single stranded structure- It It does follow Chargaff's rule. The total
4)
does not follow Chargaff’s rule purine = pyrimidine content.
RNA can be easily destroyed by DNA resists alkali action due to the
5)
alkalies absence of OH group at 2’ position
RNA is a relatively a labile
DNA is a stable molecule. The
6) molecule, undergoes easy and
spontaneous degradation is very 2 slow.
spontaneous degradation
Mainly found in nucleus, extra nuclear
Mainly cytoplasmic, but also
7) DNA is found in mitochondria, and
present in nucleus
plasmids etc
The base content varies from
Millions of base pairs are there
8) 100- 5000. The size is
depending upon the organism
variable.
There are various types of RNA –
DNA is always of one type and performs
mRNA, r RNA, t RNA. These
9) the function of storage and transfer of
RNAs perform different and specific
genetic information.
functions.
No variable physiological forms of
RNA are found. The different There are variable forms of DNA
10)
types of RNA do not change their (A to E and Z)
forms
RNA is synthesized from DNA, it DNA can form DNA by replication
11) can not form DNA(except by the it can also form RNA by
action of reverse transcriptase). transcription.

GENES
 The Genes are the Functional units of Heredity.
 A gene is a specific sequence of DNA containing genetic information required to
make a specific protein.
 A gene may exist in alternative forms called alleles.
 Modern definition Gene is the Unit of Genetic Information, i.e., the sequence of
DNA that specifies one polypeptide.
  Gregor Mendel assumed that each trait is determined by a pair of inherited ‘factors’
which are now called Gene.
 Johannsen coined the term ‘GENE’ in 1909
 Each gene is a segment of DNA that give rise to a protein product or RNA.
 Prokaryotic gene is uninterrupted.
 In Eukaryotic gene the coding sequences (exon) are separated by non-coding
sequences called introns.
 In complex eukaryotes, introns account for more than 10 times as much DNA as
exons.

Gene Theory:
T.H Morgan proposed the gene theory which state that:
 Chromosomes are bearers of hereditary units and each chromosome carries hundreds
or thousands of genes.
 The genes are arranged on the chromosomes in the linear order on the special regions
or locus.
Modern concept of gene
S. Benzer (1957) coined different terms for different nature of gene and genetic
material in relation to the chromosome on the basis of genetic phenomena to which they
involve.
 Cistron – Genes as a unit of transmission
 The part of DNA specifying a single polypeptide chain is termed as cistron.
 It transmits characters from one generation to other as unit of transmission.
 Recon - Genes as a unit of Recombination
 The smallest segment of DNA capable of being separated and exchange with
other chromosome is called recon.
 A recon consists of not more than two pairs of nucleotides.
 Muton – Gene as a unit of mutation
 Muton is the smallest unit of genetic material which when changed or
mutated produce a phenotypic trait.
 muton is delimited to a single nucleotide.

GENE TYPES
Types of gene based on activity
 1. House keeping genes ( genes which are always active )
2. Specific genes. (Those genes which are getting active only during some special
condition)
Types of genes based on behavior.
1. Basic genes: These are the fundamental genes that bring about expression of
particular character.
2. Lethal genes: These bring about the death their possessor.
3. Multiple gene: When two or more pairs of independent genes act together to produce
a single phenotypic trait.
4. Cumulative gene: Some genes have additive effects on the action of other genes.
These are called cumulative genes.
5. Pleiotropic genes: The genes which produce changes in more than one character is
called pleiotropic gene. E.g : Sickle cell anemia causes multiple symptoms, only one
of which is the actual sickle cell condition
6. Modifying gene: The gene which cannot produce a character by itself but interacts
with other to produce a modified effect is called modifier gene.
7. Inhibitory gene: The gene which suppresses or inhibits the expression of another
gene is called inhibitory gene.

Prokaryotic gene structure:

Prokaryotic Gene is composed of three regions:
1. Promoter region
2. RNA coding sequence
3. Terminator region
Prokaryotic gene lacks introns.
 The region 5’ of the promoter sequence is called upstream sequence and the region 3’ of
the terminator sequence is called downstream sequence.

1. Promoter region:
 This is situated on upstream of the sequence that codes for RNA.
 This is the site where RNA polymerase interact before RNA synthesis (Transcription).
 Promoter region provides the location and direction to initiate transcription.
 At -10 there is a sequence TATAAT or PRIBNOW BOX. Usually AT rich region which
requires minimum energy for strand separation or melting for initiation .
 At -35 another consensus sequence TTGACA, helps in promoter recognition.
 These two are the most important promoter elements recognized by transcription factors.

Common Bacterial Promoters used in Research

S.N Promoter Expression Description
1 T7 Constitutive but requires T7 RNA polymerase Promoter from T7 bacteriophage
2 Sp6 Constitutive but requires Sp6 RNA polymerase Promoter from Sp6 bacteriophage
3 lac Constitutive in the absense of lac repressor (lacI Promoter from Lac operon
or lacIq). Can be induced by IPTG or lactose
4 araBad Inducible by arabinose Promoter of the arabinose
metabolic operon
5 trp Repressible by tryptophan Promoter from E. coli tryptophan
operon
6 Ptac Regulated like the lac promoter Hybrid promoter of lac and trp

2. RNA coding sequence:

 The DNA sequence that will become copied into an RNA molecule (RNA transcript).
 Starts with an initiator codon (AUG, GUG, CUG). and ends with termination codon
(Amber-UAG, ochre-UAA, and opal-UGA)
 No introns (uninterrupted).
 Collinear to its mRNA.
  Any nucleotide present on the left is denoted by (-) symbol and the region is called
upstream element. E.g. -10,-20,-35 etc. Any sequence to the right of the start is
downstream elements and numbered as +10,+35 etc.).

3. Terminator region:
 The region that signal the RNA polymerase to stop transcription from DNA template.
 Transcription termination occur through Rho dependent or Rho independent manner.

Eukaryotic Gene structure

 Eukaryotic gene are complex structures compared that prokaryotic gene.
 They are composed of following regions
1. Exons
2. Introns
3. Promoter sequences
4. Terminator sequences
5. Enhancers and silencers(upstream or downstream)
6. Signals (Upstream sequence signal for addition of cap. Downstream sequences
signal for addition of poly A tail.)

1. Exons
• Coding sequence, transcribed and translated.
• Coding for amino acids in the polypeptide chain.
• Vary in number ,sequence and length. A gene starts and ends with exons.(5’ to 3’).
• Some exon includes untranslated(UTR)region.
2. Introns
• Coding sequences are separated by noncoding sequences called introns.
• They are removed when the primary transcript is processed to give the mature RNA
 • All introns share the base sequence GT in the 5’end and AG in the 3’end.
• Introns were 1st discovered in 1977 independently by Phillip Sharp and Richard
Roberts.

Significance of Introns
• Introns don't specify the synthesis of proteins but have other important cellular
activities.
• Many introns encodes RNA’s that are major regulators of gene expression.
• Contain regulatory sequences that control trancription and mRNA processing.
• Introns allow exons to be joined in different combinations(alternative splicing),
resulting in the synthesis of different proteins from the same gene.
• Important role in evolution by facilitating recombination between exons of different
genes(exon shuffling).

3. Promoters
A promoter is a regulatory region of DNA located upstream controlling gene
expression.Responsible for binding of RNA polymerase II which is used for
transcription.
E.g. TA29 promoter has tapetum specific expression
Glutelin (Gt3) promoter in rice has endosperm specific expression.

Common Eukaryotic Promoters Used in Research

S. Promoter Expression Description
N
1 CMV Constitutive Strong mammalian promoter from human
cytomegalovirus
2 EF1a Constituitve Strong mammalian promoter from human elongation
factor 1 alpha
3 CAG Constitutive Strong hybrid mammalian promoter
4 PGK Constitutive Mammalian promoter from phospholycerate kinase
gene
5 TRE Inducible Tetracycline response element promoter
6 U6 Constitutive Human U6 nuclear promoter for small RNA expression

4. Terminator
Recognized by RNA polymerase as a signal to stop transcription
5. Enhancer
Enhances the transcription of a gene upto few thousand bp upstream.
6. Silencers
Reduce or shut down the expression of a near by gene.

-Thank you-