 DEPARTMENT OF PLANT BIOLOGY AND PLANT

BIOTECHNOLOGY
 SEMESTER V
 COURSE: PLANT BIOTECHNOLOGY
 COURESE CODE:16UPBTC09
BY Dr.E.GAYATHIRI
GENETICS
 Experiment s Proved that DNA as Genetic
Materials

 1. Griffith experiment

 2. Avery, McCarthy and Mac Leod

 3. Herchey – Chase Experiment

 Frederick Griffith Experiment
Streptococcus pneumoniae model
o S III strain (smooth) encapsulated - virulent – Pneumonia
– mice die
o R II (Rough) non-encapsulated, non-virulent – No disease
- survive
o Heat killed S III – mice die
o Heat killed S III mixed with R II live cells - Pneumonia –
mice die

Concluded that
S cells have a
chemical
component that
can transform
live R II cells
 Avery–MacLeod–McCarty experiment
 o Tracked protein and DNA of E. coli phage T2
o Bacteriophage is a virus that infects bacteria

oRadioactive isotopes
to label cells with
32 Phospherous and
35Sulphur

oDetermined that
DNA entered bacteria
and directed virus
reproduction not
protein
 DNA  Polymer : Nucleotide  Monomer

 Each Nucleotide has 3 parts:

 1) 5 carbon sugar  Deoxyribose
 2) Phosphate group  PO4
 3) Nitrogen Base
 Nucleoside = Sugar + Base
Nucleotide = Sugar + Base + Phosphate
Nucleotide = monomers that make up DNA and RNA (Figs. 2.8)

Three components

1. Pentose (5-carbon) sugar

DNA = deoxyribose
RNA = ribose
(compare 2’ carbons)

2. Nitrogenous base

Purines (2 rings)
Adenine
Guanine

Pyrimidines (1 ring)
Cytosine
Thymine (DNA)
Uracil (RNA)

3. Phosphate group attached to 5’ carbon

Structure of DNA

James D. Watson/Francis H. Crick 1953 proposed the

Double Helix Model
based on two sources of information:

1. Base composition studies of Erwin Chargaff (Chargaff’s Rules)

• indicated double-stranded DNA consists of ~50% purines

(A,G) and ~50% pyrimidines (T, C)

• amount of A = amount of T and amount of G = amount of C

• %GC content varies from organism to organism

Examples: %A %T %G %C %GC

Homo sapiens 31.0 31.5 19.1 18.4 37.5

Zea mays 25.6 25.3 24.5 24.6 49.1
Drosophila 27.3 27.6 22.5 22.5 45.0
Aythya americana 25.8 25.8 24.2 24.2 48.4
Structure of DNA

James D. Watson/Francis H. Crick 1953 proposed the Double Helix

Model based on two sources of information:

2. X-ray diffraction studies by Rosalind Franklin & Maurice Wilkins

Conclusion-DNA is a helical structure with

distinctive regularities, 0.34 nm & 3.4 nm.
Fig. 2.11
Double Helix Model of DNA: Six main features

1. Two polynucleotide chains wound in a right-handed (clockwise)

double-helix.

2. Nucleotide chains are anti-parallel: 5’  3’

3’  5’

3. Sugar-phosphate backbones are on the outside of the double

helix, and the bases are oriented towards the central axis.

4. Complementary base pairs from opposite strands are bound

together by weak hydrogen bonds.

A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).

5’-TATTCCGA-3’
3’-ATAAGGCT-5’

5. Base pairs are 0.34 nm apart. One complete turn of the helix
requires 3.4 nm (10 bases/turn).

6. Sugar-phosphate backbones are not equally-spaced, resulting

in major and minor grooves.
Nucleotides are linked by phosphodiester bonds to form
polynucleotides.

Phosphodiester bond

Covalent bond between the phosphate group (attached to

5’ carbon) of one nucleotide and the 3’ carbon of the
sugar of another nucleotide.

This bond is very strong, and for this reason DNA is

remarkably stable. DNA can be boiled and even
autoclaved without degrading!

5’ and 3’

The ends of the DNA or RNA chain are not the same. One
end of the chain has a 5’ carbon and the other end has a
3’ carbon.
 5’ end
Fig. 2.9

3’ end
Fig. 2.13
Fig. 2.12
Type B-DNA

Other DNA forms

include:

A-DNA:
Right-handed double
helix with 11 bases
per turn; shorter and
wider at 2.2 nm
diameter. Exists in
some DNA-protein
complexes.

Z-DNA:
Left-handed double
helix with 12 bases
per turn; longer and
thinner at 1.8 nm
diameter.
Types of DNA
 RNA
RNA - ribonucleic acid - non-hereditary nucleic
acid - Except retroviruses.

a polymer of ribonucleotides

- made up of ribose sugar, phosphoric acid and

nitrogenous bases (A, U, C, G).

RNA differs from DNA in

(i) The pentose sugar in RNA is ribose sugar
while it is deoxyribose sugar in DNA,

(ii) Thymine in DNA is replaced by uracil in RNA.

All other nitrogenous bases are same
Storage/transfer of genetic information

• Genomes
• many viruses have RNA genomes
single-stranded (ssRNA)
e.g., retroviruses (HIV)
double-stranded (dsRNA)

• Transfer of genetic information

• mRNA = "coding RNA" - encodes
proteins
D Dobbs ISU - BCB
444/544X: RNA Structure &
21 Function
Structural
• e.g., rRNA, which is major structural
component of ribosomes
Catalytic
RNA in ribosome has
peptidyltransferase activity
• Enzymatic activity responsible for
peptide bond formation between amino
acids in growing peptide chain
• Also, many small RNAs are enzymes
"ribozymes”
Regulatory
Recently discovered important new roles for
RNAs
In normal cells:
• in "defense" - esp. in plants
• in normal development
e.g., siRNAs, miRNA
As tools:
• for gene therapy or to modify gene expression
• RNAi
• RNA aptamers
 Types of RNAs Primary Function(s)
mRNA - messenger translation (protein synthesis)
regulatory
rRNA - ribosomal translation (protein synthesis) <catalytic>

t-RNA - transfer translation (protein synthesis)

hnRNA - heterogeneous nuclear precursors & intermediates of mature

mRNAs & other RNAs
scRNA - small cytoplasmic signal recognition particle (SRP)
tRNA processing <catalytic>
snRNA - small nuclear mRNA processing, poly A addition <catalytic>
snoRNA - small nucleolar rRNA processing/maturation/methylation

regulatory RNAs (siRNA, regulation of transcription and translation,

miRNA, etc.) other??

mRNA constitutes about 3.5% of cellular

RNA; tRNA is about 15% and rRNA about 80%
25
When a particular gene codes for a mRNA
strand, - monocistronic or monogenic, e.g.,
eukaryotes.
When several genes (cistrons) are transcribed
into a single mRNA - polycistronic or
polygenic, e.g., prokaryotes.

Monocistronic mRNA codes for one complete

protein molecule

polycistronic mRNA codes for a number

of protein molecules.
 tRNA - transfer RNA - soluble (s) RNA -
acceptor RNA - adaptor RNA.
tRNAs are smallest, bearing 70-80 nucleotides.
Holley 1965 - alanine tRNA from yeast clover
leaf model
Klug 1974 -Three dimensional (L-shaped)
structure of tRNA was proposed by
tRNA contains 5 site
1.AA (amino acid) binding site,
2. T '¥ C loop,
3. DHU loop,
4. extra arm
5. anticodon loop.
AA - binding site or amino acid attachment site

lies opposite to the anticodon site.

tRNA molecules have unpaired (single stranded) CCA-OH

sequence at the 3' end.

Pick up the amino acid from amino acid pool for protein
synthesis
T '¥ C loop – ribosomes attachment site
contains pseudouridine.

•
DHU loop contains dihydrouridine.
It is binding site for aminoacyl synthetase
enzyme.

Extra arm - is a variable site or loop

which lies between T '¥ C loop and anticodon.
The exact role of extra arm is not known
. Anticodon loop
is made up of three nitrogen bases
for recognising and attaching to the codon
'of mRNA.

• tRNA helps to transport amino acids from

the surrounding cytoplasm to the site of
protein synthesis
DNA Packaging
Organization of DNA/RNA in chromosomes

Genome = chromosome or set of chromosomes that contains all the

DNA an organism (or organelle) possesses

DNA in chromosomes is linear

while in prokaryotes, mitochondria and chloroplast is circular.

Viral chromosomes 1. single or double-stranded DNA or RNA

2. circular or linear
3. surrounded by proteins
TMV T2 bacteriophage  bacteriophage

Prokaryotic chromosomes

1. most contain one double-stranded circular

DNA chromosome
2. others consist of one or more chromosomes
and are either circular or linear
3. typically arranged in arranged in a dense
clump in a region called the nucleoid
Prokaryotic chromosome structure---solutions:

1. Supercoiling DNA double helix is twisted in space about its

own axis, a process is controlled by
topoisomerases (enzymes).
(occurs in circular and linear DNA molecules)

Fig. 2.17
2. Looped domains

Fig. 2.18
Eukaryotic chromosome structure

Chromatin complex of DNA and chomosomal proteins

~ twice as much protein as DNA

Two major types of proteins:

1. Histones abundant, basic proteins with a positive charge

that bind to DNA
Histone occupies major groove of DNA at 30° angle.
Bond between DNA and Histone is salt linkage (Mg

5 main types: H1, H2A, H2B, H3, H4

~equal in mass to DNA
evolutionarily conserved

1. Non-histones all the other proteins associated with DNA

differ markedly in type and structure
amounts vary widely
>> 100% DNA mass
<< 50% DNA mass
Packing of DNA into chromosomes:

1. Level 1 Winding of DNA around histones to create a

nucleosome structure.

2. Level 2 Nucleosomes connected by

strands of linker DNA like
beads on a string.

3. Level 3 Packaging of nucleosomes into

30-nm chromatin fiber.

4. Level 4 Formation of looped domains.

See Fig. 2.20

Fig. 2.21 - Metaphase chromosome depleted of histones maintains its
shape with a nonhistone protein scaffold.
Two other regions of DNA you should know about:

•Centromeric DNA (CEN) Center of chromosome, specialized

sequences function with the
microtubles and spindle apparatus
during mitosis/meiosis.

•Telomeric DNA At extreme ends of the chromosome,

maintain stability, and consist of
tandem repeats. Play a role in DNA
replication and stability of DNA.

Fig. 2.25
Repeated DNA:

•Unique-sequence DNA Often referred to as single-copy and

usually code for genes.

•Repetitive-sequence DNA May be interspersed or clustered

and vary in size.

SINEs short interspersed repeated sequences (100-500 bp)

LINEs long interspersed repeated sequences (>5,000 bp)

Microsatellites short tandem repeats

<---TTATTATTATTATTATTATTA--->
More about genome size:

C value = total amount of DNA in the haploid (1N) genome

Varies widely from species to species and shows no simple

relationship to structural or organizational complexity, but there is
similarity among closely related species.

Examples C value (bp)

 48,502
T4 168,900
HIV-1 9,750
E. Coli 4,639,221
Lilium formosanum 36,000,000,000
Zea mays 5,000,000,000
Amoeba proteus 290,000,000,000
Drosophila melanogaster 180,000,000
Mus musculus 3,454,200,000
Canis familiaris 3,355,500,000
Equus caballus 3,311,000,000
Homo sapiens 3,400,000,000