Molecular Basis of Inheritance

1. The DNA
2. The Search for Genetic Material
3. RNA World
4. Replication
5. Transcription
6. Genetic Code
7. Translation
8. Regulation of Gene Expression
9. Human Genome project
10. DNA Fingerprinting
 living systems
2 nucleic acids

÷:
Other
functions also
Deoxyribonucleic Ribonucleic acid - messenger(mostly)
acid (DNA) (RNA) adapter
structural
catalytic molecule

Genetic material Genetic material

most organisms. some viruses

 The DNA
long polymer of deoxyribonucleotides
Depends
v
no. of nucleotides

bacteriophage a ×174 5386 nucleotides

Bacteriophage lambda
=, 48502 (bp)
Escherichia coli 4.6 x 10 bp
human DNA : :
3.3 x 10 bp (haploid)
gene
- --

human DNA 6.6 x 10 bp (diploid)

-

>
 Structure of Polynucleotide Chain
RNA structure DNA structure
Purines (Ade!ne, Gua!ne)
Pyri"dines (Cytosine, Thy"ne,
✓
Uracil,) RNA
> ^
DNA

Nucleo$de
sooooo -3gal
De#yribose DNA
Ribose RNA
 Nucleoside Nucleo$de

N-glycosidic linkage

OH of 1'C
÷
↓
✓ OH of 5'C
,
Eg.RNA DNA
phosphoester linkage
adenosine deoxyadenosine,
guanosine deoxyguanosine,
cytidine. deoxycytidine
uridine. deoxythymidine
 Dinucleo$de
→
2 nucleotides linked
"

3'-5' phosphodiester linkage

gggggqg
 polynucleotide chain formation More nucleotides

free phosphate moiety at 5'-end of sugar polynucleotide chain

free sugar OH of 3'C group

sugar + phosphates = backbone

:
linked

nitrogenous bases
In RNA
every nucleotide (additional -OH group) at 2'-position

RNA DNA
(5-methyl uracil, another
uracil in place of thymine
chemical name for thymine).

X
um-isDmetuyuracil)
+
 Friedrich Meischer (1869) →
Can’t isolate

0 DNA
↓
'Nuclein'
acidic -
Nucleus ↳ DNA structure
0 unknown

James Watson & Francis Crick (1953)

I +chargafy.
proposed Based on X-ray diffraction data

-
by
DNA structure
Maurice Wilkins &
Rosalind Franklin
D%ble Hel& model
Hallmark

-
base pairing proposition b/w 2 strands of polynucleotide
 Erwin Chargaff
Ratios ( constant & equals)

AT=
b/w
=G V v

Adenine & Thymine Guanine & Cytosine A T

No. Of purines = no. Of pyrimidine C 9
Known ⑧
Predictable
↓ ↓ ↓
base pairing sequence
base pairing sequence Bcz ↓
↓ 2nd strand
1 strand ↓
complementary

-
DNA bases proposition

b genetic implication became revolutionary

 "

Or
Parental DNA
As (template) synthesis Daughter DNA
-

v
identical (parental DNA)
Figure 6.2 Double stranded polynucleotide chain

A=T T=A
G C C G
DNA salient features
1) 2 polynucleotide chains,( sugar,phosphate, bases)

2) anti-parallel polarity .one 5'->3', other 3'->5'.

3) hydrogen bond b/w bases

4) Two chains coiled right handed

pitch of helix
each turn
(3.4 nm)
(10 bp)-
distance b/w bp . (0.34 nm)
-
5) Stability of helical

⑭ one base pair

H-bonds

stacks over other

Figure 6.3 DNA double helix
 Francis Crick
e

:
-
proposed

Central dogma

-Riverse
(genetic information flows) transcription
~
reverse transcription (RNA to DNA)

O some viruses
Packaging of DNA prokaryotes
E. coli
length of DNA ⑳
1.36 mm,
>

No.of bp
-
4.6 x 10
-6
,

No defined nucleus
DNA not -> scattered
&
DNA [-]with some proteins (+) as 'nucleoid'
-

↳
-

DNA in nucleoid
Mariana I
Haqq
by proteins
w
polyamine ⑧ large loops

Mama
nucleoid

Mok
 packaging of DNA Eukaryotes
"

6.6 x 10 bp × 0.34 × 10 m/bp

÷
v
r

total no. of bp distance b/w 2 bp

DNA ⑧
2.2 metres

nucleus (10
~
m)

nucleosome

2oo bp DNA [-] with histones octamer (+)

HM3RY
 xz ⑧
basic proteins histones [+]

⑧ a~ ✓
rich in basic amino acid residues
Octamen
~
lysine & arginine
~
-
-

↓
⑧
Figure 6.4b EM picture - 'Beads-on-String
 chromatin, thread- like stained

Mmmmm

Repeated nucleosomes in

--
chromatin

BppE•
 ⑧
chromatin packaging at higher level
requires
⑤ -
(NHC) proteins

Euchr!a"n Heterochr!a"n
loosely packed densely packed

--
transcriptionally transcriptionally
active inactive

oak
(metaphase stage)
chromosomes
 The Search for Genetic Material
Meischer Or Mendel
same !me

:
fdiscovery of principles of
nuclein 9 inheritance
-

DNA (genetic material) took long discovered & proven

By 1926 genetic inheritance reached molecular level

Previous discoveries

Gregor Mendel,
Walter Sutton, narrowed search
Thomas Hunt Morgan
chromosomes in
& other scientists
nucleus
Transforming Principle
·ene
Frederick Griffith, 1928 Streptococcus pneumonia

men
 ⑪ transforming principle

(something in S cells) R cells into S cells

Can trans"rm
°

but WHAT IS IT?

--
Oswald Avery, MacCleod, & McCarty (1933-44)
decided to find out

Thought
- protein (genetic material)

0
proteins, DNA, RNA, from heat-killed S cells

To see transform live R cells into S cells.

 me
sof
-

~ RNO
&
They concluded DNA is hereditary material, but not all biologists convinced.
 The Genetic Material is DNA
Alfred Hershey & Martha Chase (1952) unequivocal
--

Bacteriophages

whether it protein or DNA from viruses entered bacteria ???………….

itnhat
Bacteriophage
Virus

Experiment 1: Testing Proteins

-

⑤
Protein coats E.coli Bacteria No radioactivity
Phage grown with Centrifuge radio labeled infected enters cells
Radioactivity in
radioactive sulfur supernatant

C#clusi#: Proteins are not gene!c material

 Experiment 2: Testing DNA

329
Phage grown with Centrifuge Radioactivity in
Phage DNA E.coli Bacteria radioactivity
radioactive Pellet radio labeled infected enters cells
phosphorus

C#clusi#: DNA gene!c material

Finally Debate ended

:
proteins DNA

Hershey-Chase experiment
super
Paterial Figure 6.5 The Hershey-Chase experiment
Properties of Genetic Material (DNA RNA)

RNA genetic material

(Eg, Tobacco Mosaic viruses, QB bacteriophage, etc.)
Also
j
As messenger & adapter etc

DNA predominant genetic material

DNA more stable storage of

Genetic information
RNA better transmission of
Genetic material DNA RNA
must fulfill
Yes Yes
Replication

structurally more stable structurally less stable

-
stable chemically &
thymine more stability uracil less stability
structurally
chemically less reactive chemically More reactive
2'-OH

slow mutation for

evolution e Slower rate faster rate

express in form of RNA proteins

DNA > RNA proteins

~
'Mendelian Characters'
--
Depend on RNA easily express
 RNA World "

- RNA first genetic material

Evolved around RNA

RNA as catalyst

E$en!al Life proce$es

A-
metabolism, translation, splicing, etc.

&
DNA evolved from RNA
with

If
chemical modifications (more stable)
double stranded (resists changes)
evolving process of repair
 Replication
Watson & Crick
immediately

double helical structure for DNA DNA replication

Orginal Quote
2 strands
"It has not escaped our notice that the
separate specific pairing we have postulated
immediately suggests a possible copying
template mechanism for the geneticmaterial"
(Watson and Crick, 1953).

one parental &

2nd newly synthesised strand.

Figure 6.6 Watson-Crick model for semiconservative DNA replication

 The Experimental Proof
Matthew Meselson & Franklin Stahl (1958)
Escherichia coli
as #ly %trogen s&rce "r
Mahboob
many genera!#s
\
15N heavy
not radioac!ve isotope÷

15NH4CL 14NH4Cl
Meselson & Stahl's Experiment

GL
-

my -

hybrid
 Taylor & colleagues (1958)
chromosomal
-
-
⑳
similar experiments (radioactive thymidine)

-
Vicia faba (faba beans)

Proved

DNA in chromosomes also replicate semiconservatively

 The Machinery & the Enzymes

⑧
E. coli replication DNA DNA
replication
At origin of
replication

¥hf↓µ→
4.6× 10 bp
•

DNA-dependent DNA polymerase E. coli 18 min

2000 bp
per sec fast &accurate
use DNA template Any mistake (mutations)
*
⑧
Deoxyribonucleoside triphosphates
For polymerisation reaction

A
( DNTP)
Gives

⑧8 substrates energy
-
Helicase.
-

TopoisomerateIgyrase)
-

SSBP i

Semi dis continuo

Semi Conservative
-
In eukaryotes
-

If polyploidy
DNA replication cell division cycle
coordinated failure
S-phase
 Transcription

0
Replication Transcription
Why not double strand ?
Copied RNA
Copied DNA v

DNA DNA
dsRNA
D&ble strands Single strand Can’t translate

ATGC AUGC codes for 2

diff proteins
 Transcription Unit in DNA
1) Promoter
2) Structural gene
3) Terminator
DNA-dependent RNA polymerase
5'end (upstream) 3'-end (downstream)
--

DNA

0
But no coding

3'-ATGCATGCATGCATGCATGCATGC-5' Template Strand

-
5'-TACGTACGTACGTACGTACGTACG-3' Coding Strand
.

-
Can you now write sequence of RNA transcribed from above DNA?
 ERNA,RNAe Listroy mRNA
Transcription Unit and the Gene
--
- -

- Segment of DNA
✓
functional unit of inheritance
i

Gene
(Express RNA)
Eukaryotes Prokaryotes
-

split
Unsplit

⑧ ⑳

All cistron are proteins

08
Not all proteins are cistron
Types of RNA and the process of Transcription

Bacteria

Provide template structural & catalytic role brings aminoacids &

during translation. reads genetic code
¥
1 DNA-dependent RNA polymerase
transcription
RNA polymerases
v

catalysing
Only elongation
uses initiation.f) Factor Initiates
associates transiently
I termination Factor > Terminates
substrate
As j nucleoside triphosphates
Bacteria many times translation
transcription & translation Same place
begin

Cated
b4 mRNA fully transcribed.
 1st difference from prokaryotes
In eukaryotes
clear divisi# of lab&r
3 RNA polymerases in nucleus (+ other organelles)
Transcribes

-RNA polymerase 1 rRNAs (28S, 18S, 5.8S)

--
precursor of mRNA,
RNApolymerase 2
- St
(hnRNA).

ing
RNApolymerase 3 tRNA, 5srRNA, & snRNAs
-
--
 2nd difference from prokaryotes
I
-ShuRNA
primary transcripts

↓
mRNA

-
non-functional.
⑧
introns removed

Sa
0-
i
splicing

-
exons joined
Translation
hnRNA
⑦ C
§
"

⑧ capping
↓
nucleotide (methyl guanosine
-
triphosphate) added 5'-end
tailing
↓
adenylate residues
-
(200-300) added 3'-end
--
Out of nucleus

fully processed hnNA 0

mRNA
-
- Called
I
I
-I -

30

Fiqure 6.11 Process of Transcription in Eukaryotes

 Genetic Code
1 -
(41) -
4
Replication Transcription Translation

:
DNA RNA Proteins
nucleic acid nucleic acid amino acids
complementarity Not complementarity

change in Lead changes in proteins

Genetic material to

genetic code required

physicists,
organic chemists,
biochemists,
geneticists.
 -
George Gamow, physicist
nucleotides.

-
organized into codons

⑧ ~
should be 20 codons For 20 amino acids

~
1 Nucleotide - 4 combinations
-2 Nucleotides - 16 combinations ~ci Ye
3 Nucleotides - 64 combinations (Most suited for 20 amino acids)
- =>

(Y)3 = -

- -
1 codon 3 nucleotides

codon is triplet.
 ~
Har Gobind Khorana 00
homopolymers & copolymers

-
Marshall Nirenberg's
de
cell-free protein synthesis

⑧
Severo Ochoa template free ( enzymatic RNA synthesis )

RN
-
INAf enzyme (polynucleotide phosphorylase)

-homopolyme
wple de ple -
ple
Table 6.1: The Codons for the Various Amino Acids
 salient features of gene!c code
stop codons > do not code for any amino acids,
61 codon 209.9.

degenerate 1 amino acids coded by many codon

--

no punctuations read in contiguous fashion

-
-
0
universal > bacteria
(phe).
to human UUU code for Phenylalanine
= --

-A
I
Some exceptions

:
mitochondrial
-
some protozoans.
-

dual functions I
AUG codes for Methionine (met),
A. Met
&
-
& also initiator
---
codon.

stop terminator > UAA, UAG, UGA

codons.
 Mutations & Genetic Code
relationships b/w genes & DNA understood by mutation studies

⑳
Point muta!# Frame-shi' muta!#s
-
-

~
Deletions & insertions
changes
Deletions & insertions
changes -

-single base pair of DNA base pairs of DNA

-Eg. sickle cell anemia

 letter addtion Must be triplet word as codon

RAM HAS RED CAP

⑧
insert B

:
vo

RAM HAS BRE DCA P

- insert I
RAM HAS BIR EDC AP
-
insert G

:
RAM HAS BIG RED CAP
-

letter deletion
RAM HAS RED CAP
Delete R
RAM HAS EDC AP
Delete E
RAM HAS DCA P :
Delete D
RAM HAS CAP
 tRNA- the Adapter Molecule
Francis Crick
Bcz can’t read
^

Mechanism read code & link to amino acids

tRNA First > As (soluble RNA)

A
00read code
bind to specific
amino acids
V
Later adapter molecule

amino acid acceptor end

complementary

0¥
amino acid specific
tRNA initiator

no (stop codons)
looks like clover-leaf
B
L
Actual inverted [ ]
 Translation
polymerisation of amino acids to polypeptide

mRNA bases amino acids polypeptide

> >
sequence sequence ↓
Bond
Peptide
↓ Activation of
<
requires energy
peptide bond

peptidy amino acids

transf
I
wast

↓
s

-
G3SRNA
A
a
Ribosome
aminoacylation
of tRNA

structural RNAs 80 different

inactive proteins
 ribosome as catalyst
1st site 2nd site T
↓ In bacteria (23S rRNA
↓
a(no acids binding Pep!de "rma!# ribozyme)

L
Complementary

←
protein translation begins
 translational unit in mRNA
start codon (AUG)
stop codon
codes for polypeptide
UTRs ( untranslated regions)

UTR UTR
5'-end (before 3'-end (after
ADAM
start codon)
Go
stop codon)
 Initiation Elongation termination

ribosome binds mRNA Ppolypeptide chain elongates by stop codon

start codon (AUG) adding amino acids polypeptide Release
ribosome dissociates
 Regulation of Gene Expression
By
Las ->
Desol
metabolic,
physiological
environmental conditions
9
IOMi
-

embryo adult organisms

genes expression regulation

7
galactose
synthesis
E. coli beta- galactosidase lactose
glucose

If no lactose No need to synthesised

Each operon Regulation of Gene Expression in prokaryotes
specific operator
specific repressor.
Regulation of Gene Expression in Eukaryotes
 The Lac operon

-
Geneticist,

-upt
Francois Jacob
Biochemist, Jacque Monod.
--

prot
t
first transcriptionally
regulated system
lac operon

Bacteria operon
-
polycistronic
structural gene
regulated
by
C common promoter & regulatory genes

Examples
-
lac operon
trp operon
ara operon -
hisoperon -
~
val operon, etc.
Lactose Lactose
e
negative regulation
(Absent) Lac operon ; off
present)
d

of On

Glucose
- ·- Lac operon ; On
de

off at
v

Lactose

"
↓
↓ .
increases cell permeability

galactose
lactose
glucose
 Human Genome Project $

launched 1990
mega project.

3 x 10 bp
9
>

Cost >
$ 3 per bp

:
Total [ 9 billion US ]

Storage 1 book 1000 letters 1000 pages ,

single human cell.

Total =3300 books

Time 13 years
 high speed data storage Bioinformatics
}
.

computational
devices
¥ retrieval, !
analysis.
→

Improve tools for data

analysis Transfer technologies to
Identify all other sectors, eg.

iii.
20,000-25,000 genes industries
in human DNA

Goals of HGP
÷ Address the ethical,
Determine sequences legal, social issues
of 3 billion chemical (ELSI) may arise from
base pairs Store information
project.
in databases
 Human Genome Project
byU.S. Department of Energy & National Institute of Health.
Welcome Trust (U.K.) major partner

additional contributions by Japan,

> France,

completed in 2003. Germany,

China
chromosome { 1 } in May >
others.
Last
2006

DNA variations effects

revolutionary new ways
diagnose
treat
someday prevent thousands of disorders
DNA sequences solving challenges in
health care,
agriculture,
energy production,
environmental remediation
Many non-human model organisms,
bacteria,
yeast,
Caenorhabditis elegans
Drosophila
plants (rice & Arabidopsis)

genetic & physical maps on genome

By using
v

polymorphism of restriction endonuclease recognition sites,

microsatellites
 "
Methodologies

÷
blind approach
Expressed Sequence Tags Sequence Annotation
(ESTs).
Only genes expressed as RNA sequencing whole set of genome

hosts yeast,
Frederick Sanger. sequenced using -
bacteria
automated DNA
sequencers
-
vectors
:
BAC
YAC

.
 Repetitive sequences 100 to
1000 times.
(no direct coding function)
Total 3164.7 million bp.
dystrophin at unknown functions 50
2.4 million bases. Chromosome 1 most % discovered genes.

÷
genes (2968), ^
& Y fewest (231).
Avg. gene of 3000
bases
Salient Features of Human Genome
< 2 % genome codes for ↓ Repeated sequences
make up large portion
proteins
1.4 million locations
('snips')
Almost (99.9 per
total no, of genes at 30,000-
cent) nucleotide bases
much lower than previous of
same in all people.
80,000 to 1,40,000 genes.
 Applications and Future Challenges
past, researchers studied one or a few genes at a time.

study all genes in genome,

all transcripts tissue

F organ
tumor,
how 10s of 1000s genes & proteins work together
 DNA Fingerprinting Alec Jeffreys

Bulk DNA Same in all

HGP
99.9%

:
0.1% Satellite DNA

DNA fingerprinting very quick

I
sat chy not
Satellite DNA 0.1% Sat DNA same -
-
AT / GC
On basis of
¥ Length
Repeating unit

- micro-satellites mini-satellites
Main band

Satellite DNA
 The technique involved Southern blot hybridisation using radiolabelled VNTR as a
probe

digestion of DNA by restriction

endonucleases,

separation of DNA fragments by

transferring (blotting) to electrophoresis,
synthetic membranes,
(nitrocellulose or nylon)

⑳ hybridisation using L

labelled VNTR probe autoradiography.

 DNA polymorphism
inheritable mutation in population at high frequency

genetic mapping of human genome

usatellite DNA shows very high degree of polymorphism

It differs from individual to individual in population except monozygotic

(identical) twins.

from every tissue

DNA
,
blood, hair-follicle,
skin, bone,
saliva, sperm etc
)→ individual show same degree of polymorphism

useful tool in forensic applications.

paternity testing
Crime scene etc
Figure 6.16 Schematic representation of DNA fingerprinting: Few representative chromosomes have been shown to contain
different copy number of VNTR. For the sake of understanding different colour schemes have been used to trace the origin of
each band in the gel. The two alleles (paternal and maternal) of a chromosome also contain different copy numbers of VNTR.
It is clear that the banding pattern of DNA from crime scene matches with individual B, and not with A