it will be helpful to various medical course students

1. Overview
• Just as the page you are
reading contains letters that
are arranged in discrete
information units known as
words and words are combined
into sentences, paragraphs,
chapters, and so on, DNA
contains nucleotides arranged
into genes, chromosomes, and
so on.
• Think of it this way: DNA is in
genes, genes are on
chromosomes.
Fig. Data Storage Analogy

2. Physical Organization
• The human genome is
contained within two distinct
compartments : the nucleus
and the mitochondria.
• The bulk of the genome,
containing about 20,000 to
25,000 genes encoded by
DNA, is contained within a set
of linear chromosomes within
the cell nucleus and contains
genetic material for both
maternal and paternal origin.
• In contrast, mitochondrial
DNA contains 37 genes that
are essential for normal
mitochondrial function and is
exclusively of maternal origin.
Fig. Genes are found in the nucleus and
mitochondria

3. Human genome =
nuclear genome + mitochondrial genome
NUCLEAR GENOME
24 distinct chromosomes (22
autosomal + X + Y)
3,200 Mbp
25,000 genes
Mitochondrial genome
16,569 bp
37 genes

4. DNA building blocks
• DNA contains the structural blueprint for
all genetic instructions.
• The genetic code contained within the
DNA is composed of four letters or bases.
• Two of the bases are heterocyclic
compounds or purines – adenine (A) and
guanine (G) – and two are six-member
rings known as pyrimidines – cytosine (C)
and thymidine (T).
• The famous double-helix structure of DNA
derives from its phosphate-deoxyribose
backbone.
• The backbone comprises five-carbon
sugar (pentose) molecules bound to a
nucleoside (A,G,C, or T).
• The pentose molecules are also
asymmetrically joined to phosphate
groups by phosphodiester bonds.
• Hydrogen bonds between complementary
(G:C or A:T) nucleotides (a nucleoside
linked to a sugar and one or more
phosphate groups) interact to stabilize
and form the double-helix structure. Fig. Nuclear structure of eukaryotic DNA

5. Histones
• Chromatin consists of very long
double-stranded DNA molecules,
nearly an equal mass of rather small
basic proteins termed histones, as well
as smaller amounts of nonhistones
proteins, and a small quantity of
ribonucleic acid (RNA).
• Histones are heterogenous group of
closely related arginine- and lysine-rich
basic proteins, which together make
up one fourth of amino acid residues.
• These positively charged amino acids
help histones to bind tightly to the
negatively charged sugar phosphate
backbone of DNA.
• Functionally, histones provide for the
compaction of chromatin.
Fig. Nuclear genomes packed with
proteins to form chromatin

6. Human chromosomes
23 pairs
46 chromosomes
22 pairs autosomes
1 pair
sex chromosomes
46,XY
Normal male
3 billion base
pairs in the
haploid genome

7. Human chromosomes
46,XX
Normal female

8. DNA packing
• The nucleus of a human cell is typically 6 μm in
diameter but contains DNA which at its
maximum stages of condensation is only about
1/50,000th of its linear length.
• Total uncoiled DNA within a single human cell
would stretch to more than a meter.
• At least four levels of packaging of DNA take
place in order that DNA in individual
chromosomes fits into the 1.4 μm chromosome
seen at metaphase ( a stage in mitosis in the cell
cycle where the DNA is most condensed).
• Nucleosome are the fundamental organization
upon which the higher order packing of
chromatin is built.
• Each nucleosome core consists of a complex of
8 histone proteins (two molecules each of
histone H2A, H2B, H3, and H4) with double
stranded DNA wound around it.
• 146 base pairs (bp) of DNA are associated with
the nucleosome particle and a 50 to 70bp span
of linker DNA bound by a linker histone H1
separates each nucleosome.
Fig. Structure of a nucleosome

9. Chromosome structure
• Chromosome structure varies with the cell
cycle, from the loose thread like appearance in
G1 phase to the tightly compacted state
observed during M phase.
• Chromosome require three sequence elements
for their propagation and maintenance as
individual units.
• Telomeres are hexameric DNA repeats
[(TTAGGG)n] found at the ends of chromosomes
that serve to protect the chromosome from
degradation.
• Sequence elements known as centromeres
serve “handles”, which allow mitotic spindles to
attach to the chromosome during cell division.
• As the cell progresses through the mitotic or M
phase of the cell cycle, the nuclear envelope
breaks down, and chromosome segregate into
the opposite poles of the cells (to form
daughter cells), while kinetochore forms
consisting of the centromere and mitotic
spindles.

10. Information organization
• Ploidy refers to the number of chromosome copies
within a cell.
• Most of the somatic cells within the body are
diploid, meaning that each nucleus has two copies
of each chromosome, one deriving from the
mother and other from the father.
• Germ cells are the exception to this rule, which
contain a single copy of each chromosome and are
known haploid.
• The haploid genome of each human cell consists of
3.0 x 109 bp of DNA, divided into 23 (22 somatic
and 1 sex) chromosomes.
• The entire haploid genome contains sufficient DNA
to code for nearly 1.5 million pairs of genes.
• The human genome project has shown humans to
have only about 20,000 to 25,000 genes.
• Our genome has approximately the same number
of genes as a fruit fly, yet considerably more
complex that that organism.
• The human proteome, or total number of protein
species, is five to ten times larger than that of the
fruit fly.
Fig. Organization of the
genome

11. Unique DNA sequences
• Single copy DNA or genes generally
encode information for specific
protein products.
• The 20,000 to 25,000 genes within
the human genome can be divided
into four general categories.
• Approximately 5,000 genes are
involved with genome
maintenance; nearly 5,000 with
signal transduction; and 4,000 with
general biochemical functions; the
largest portion, 9,000 genes, is
involved in other activities.
Fig. Unique (nonrepetitive) DNA
distribution with the genome

12. Repeat sequences
• Repeat sequences do not encode proteins, but they make up at least 50% of
the human genome.
• These sequences fall into two main classes: satellite DNA and LINES and SINES.
1. Satellite DNA
• These highly repetitive sequences tend to be clustered and repeated many
times in tandem ( a head-to-toe arrangement).
• They are generally not transcribed and are present in 1 to 10 million copies
per haploid genome.
• These sequences are also associated with the centromeres and telomeres of
chromosomes.
• Satellite DNA sequences are categorized according to the number of base
pairs within the repeat sequences:
• Alpha satellite – 171 bp sequence that extends several million base pairs or
more in length.
• Minisatellite - 20 to 70 bp in length and a total legth of a few thousand base
pairs.
• Microsatellite – repeat units only 2, 3, or 4 bp in length and a total length of a
few hundred.

13. 2. LINES and SINES
• these clustered sequences are found interspersed with unique sequences.
• These are also present at less than 106 copies per haploid genome.
• They are transcribed into RNA and can be according to their size.
• LINES (long interspersed elements) 7,000 bp (20 to 50,000 copies)
• SINES (short interspersed elements) 90 to 500 bp (about 100,000 copies)

14. Functional organization
• Functions within the cell are usually encoded by
genes.
• Genes are present on the chromosome and in
the mitochondria.
• Not all genes are active in all tissues and specific
alterations to these genes are necessary for
tissue-specific gene expression.
A. Genes
• A gene is the complete sequence region
necessary for generating a functional product.
• This encompasses promoters and control
regions necessary for the transcription,
processing, and, if applicable, translation of
gene.
• About 2% of the genome encodes instructions
for the synthesis of proteins.
B. Alteration to the genome
• Aside from mutation, all cells in an individual
have identical DNA content and sequence.
• However, different tissues and cells require a
specific set of genes to carry out their
functions.
Fig. chromatin structure is
tissue specific

15. Genome
• The genome is all the DNA in a cell.
– All the DNA on all the chromosomes
– Includes genes, intergenic sequences, repeats
• Specifically, it is all the DNA in an organelle.
• Eukaryotes can have 2-3 genomes
– Nuclear genome
– Mitochondrial genome
– Plastid genome
• If not specified, “genome” usually refers to the
nuclear genome.

16. Genomics
• Genomics is the study of genomes, including
large chromosomal segments containing many
genes.
• The initial phase of genomics aims to map and
sequence an initial set of entire genomes.
• Functional genomics aims to deduce
information about the function of DNA
sequences.
– Should continue long after the initial genome
sequences have been completed.

17. Genomes and Genomics
• The word “genome,” coined by German botanist
Hans Winkler in 1920, was derived simply by
combining gene and the final syllable of
chromosome.
• If not specified, “genome” usually refers to the
nuclear genome!
• An organism’s genome is defined as the complete
haploid genetic complement of a typical cell.
• The genetic content of the organelles in the cell, is
not considered part of the nuclear genome.
• In diploid organisms, sequence variations exist
between the two copies of each chromosome present
in a cell.
• The genome is the ultimate source of information

18. "Genes" are units of genetic information present
on the DNA in the chromosomes and chromatin.
" Genome" is all the DNA contained in an
organism or a cell, which includes the
chromosomes plus the DNA in mitochondria
(and DNA in the chloroplasts of plant cells).

19. • The number of genomes sequenced in their entirety is
now in the thousands and includes organisms ranging
from bacteria to mammals.
• The first complete genome to be sequenced was that of
the bacterium Haemophilus influenzae, in 1995.
• The first eukaryotic genome sequence, that of the yeast
Saccharomyces cerevisiae, followed in 1996.
• The genome sequence for the bacterium Escherichia coli
became available in 1997 .
• The much larger effort directed at the human genome was
also accelerating.

20. Prokaryotes and Eukaryotes genome
Prokaryotes Eukaryotes
Single cell Single or multi cell
No nucleus nucleus
One piece of circular DNA Chromosomes
No mRNA post
transcriptional
modification
Exons/Introns splicing

21. Karyotype
o The study of chromosomes, their structure and their
inheritance is known as Cytogenetics.
o Each species has a characteristic number of
chromosomes and this is known as karyotype.
• Bacteria 1
• Fruit fly 8
• Garden Pea 14
• Yeast 16
• Frog 26
• Cat 38
• Fox 34
• Mouse 40
• Rat 42
• Rabbit 44
• Human 46
• Chicken 78

22. o Prior to 1950's it was believed that humans
had 48 chromosomes but in 1956 it was
confirmed that each human cell has 46
chromosomes (Tjio and Levan, 1956).
o On the chromosomes the genes are situated in
a linear order.
o Each gene has a precise position or locus.
o The size of bacterial chromosomes ranges from
0.6 -10 Mbp, and the size of Archael range from
0.5 - 5.8 Mbp, whereas Eukaryotic
chromosomes range from 2.9 - 4,000 Mbp.

23. Human Genome: General Information
• Genetic material in humans is
stored in two organelles: nucleus
(about 3200 Mbp) and
mitochondria (16.6 kb).
• Human chromosomes are not of
equal sizes; the smallest,
chromosome 21, and the largest,
chromosome 1.
• Only a very small amount of
human DNA is responsible for the
differences among humans, indeed
among all organisms.

24. Number of genes in the human genome
• Number of genes at least 100,000.
• HOWEVER, the number of protein‐encoding genes is only
~20,000 to 25,000.
• How can we explain this?

25. Lecture no.
5

26. Genomics Vs. Genetics
• Genetics: study of inherited phenotypes
Peter Goodfellow (1997, Nature Genetics 16:209-210):
"...I would define genetics as the study of inheritance and
genomics as the study of genomes. The latter informs the former
and includes the sequencing of genomes. The concept of
functional genetics is a tautology (the whole point of genetics is
to link genes with phenotypes). Functional genomics is the
attachment of information about function to knowledge of DNA
sequence' paradoxically, genetics is a major tool for functional
genomics."

27. Genomics, Genetics and Biochemistry
• Genetics: study of inherited phenotypes
• Genomics: study of genomes
• Biochemistry: study of the chemistry of living
organisms and/or cells
• Revolution lauched by full genome sequencing
– Many biological problems now have finite (albeit
complex) solutions.
– New era will see an even greater interaction among
these three disciplines

28. Distinct components in complex genomes
• Highly repeated DNA
– R (repetition frequency) >100,000
– Almost no information, low complexity
• Moderately repeated DNA
– 10<R<10,000
– Little information, moderate complexity
• “Single copy” DNA
– R=1 or 2
– Much information, high complexity

29. MUTATION
DEFINTION
• A mutation can be defined as a permanent change in the DNA segment of a
gene which may be phenotypically silent or expressed.
• The substances (chemicals) which can induce mutations are collectively known
as mutagens.
CAUSES OF MUTATIONS
• Mutations are cause by
1. Error in proofreading during DNA Replication
• DNA polymerase corrects the mismatch during replication by proofreading. If
the proofreading is not effective, the lesion remains in the DNA molecule.
2. Error in DNA Repair
• If the change in base sequence occurs after DNA replication, and if the
DNA repair is defective, the lesion remains in the DNA molecule.
3. Error in DNA Recombination
• DNA recombination occurs by translocation and rearrangement of genes. If the
recombination is defective, it can lead to mutations.

30. 4. Chemical Mutagens
• Chemical mutagens alter DNA bases or structure of DNA. If these alterations
are not repaired, they lead to mutations.
• Examples of mutagens include base analogs, alkylating agents, intercalating
agents, deaminating agents, hydrazines and aflatoxin.
5. Irradiation
• Ultraviolet light or ionizing radiations can alter the structure of DNA. These
changes lead to mutations, if they are not repaired.
6. Spontaneous Mutations
• Mutations can occur without any underlying cause.
TYPES OF MUTATIONS
• Mutations are classified into two major categories, i.e.:
1. Point mutation, and
2. Frame-shift mutation.

31. 1. POINT
MUTATIONS
• The replacement of
one base pair by
another results in
point mutation.
• They are of two sub-
types.
A. Transitions:
• In this case, a purine
(or a pyrimidine) is
replaced by another.
B. Transversions:
• These are
characterized by
replacement of a
purine by a pyrimidine
or vice versa.

32. Consequences of point mutations :
• The change in a single base sequence in point mutation may cause one
of the following.
1. Silent mutation
• The codon (of mRNA) containing the changed base may code for the
same amino acid.
• For instance, UCA codes for serine and change in the third base (UCU)
still codes for serine. Therefore, there are no detectable effects in silent
mutation.
2. Missense mutation
• In this case, the changed base may code for a different amino acid.
• For example, UCA codes for serine while ACA codes for threonine.
• The mistaken (or missense) amino acid may be acceptable, partially acceptable or
unacceptable with regard to the function of protein molecule.
• Sickle-cell anemia is a classical example of missense mutation.
3. Nonsense mutation
• Sometimes, the codon with the altered base may become a termination (or
nonsense) codon.
• For instance, change in the second base of serine codon (UCA) may result in UAA.
• The altered codon acts as a stop signal and causes termination of protein synthesis,
at that point.

33. Fig. An illustration of point mutations represented by a codon of
mRNA

34. 2. Frameshift mutations
• These occur when one or more base pairs are inserted in or deleted from the
DNA, respectively, causing insertion of deletion mutations.
Consequences of frameshift mutations:
• The insertion or deletion of a base in a gene results in an altered reading of
the mRNA (hence the name frameshift).
• The machinery of mRNA (containing codons) does not recognize that a base
was missing or a new base was added.
• Since there are no punctuations in the reading of codons, translation
continues.
• The result is that the protein synthesized will have several altered amino acids
and/or prematurely terminated protein.

36. REPAIR OF DNA
INTRODUCTION
• DNA is the only macromolecule that is repaired rather than degraded.
• DNA repair is a high-priority process for the maintenance of integrity of the
genetic information and other cellular functions within the cell both, in
prokaryotes as well as eukaryotes.
• It is a very efficient process with less than 1 out of 1000 accidental changes
which may result in a mutation.
• The cell possesses an inbuilt system to repair the damaged DNA.
• This may be achieved by four distinct mechanisms:
1. Base excision repair
2. Nucleotide excision repair
3. Mismatch repair
4. Double-strand break repair

37. 1. Base excision-repair
• This type of repair eliminates modified bases
like those that have been deaminated,
methylated or modified chemically.
• The bases cytosine, adenine and guanine can
undergo spontaneous, depurination to
respectively form uracil, hypoxanthine and
xanthine.
• These altered bases do not exist in the
normal DNA, and therefore need to be
removed.
• This is carried out by base excision repair.
• A defective DNA in which cytosine is
deaminated to uracil is acted upon by the
enzyme uracil DNA glycosylase.
• This results in the removal of the defective
base uracil.
• An endonuclease cuts the backbone of DNA
strand near the defect and removes a few
bases.
• The gap so created is filled up by the action of
repair DNA polymerase and DNA ligase.

38. 2. Nucleotide excision repair
• Nucleotide excision repair is ideally suited for large-
scale defects in DNA.
• The process is activated when a bulky lesion has been
produced, such as DNA damage due to ultraviolet light,
ionizing radiation and other environmental factors,
often results in the modification of certain bases, strand
breaks, cross-linkages etc.
• After the identification of the defective piece of DNA,
the DNA double helix is unwound to expose the
damaged part.
• An excision nuclease (exinuclease) cuts the DNA on
either side ( upstream and downstream) of the
damaged DNA. This defective piece is degraded.
• The gap created by the nucleotide excision is filled up by
the DNA polymerase which gets ligated by DNA ligase.
• Xeroderma pigmentosum (XP) is a rare autosomal
recessive disease. The affected patients are
photosensitive and susceptible to skin cancers. It is now
recognized that XP is due to a defect in the nucleotide
excision repair of the damaged DNA.

39. 3. Mismatch repair
• Despite high accuracy in replication, defects do occur when the DNA is
copied.
• For instance, cytosine (instead of thymine) could be incorporated opposite to
adenine.
• Mismatch repair corrects a single mismatch base pair e.g. C to A, instead of T
to A.
• The template strand of the DNA exists in a methylated form, while the newly
synthesized strand is not methylated.
• This difference allows the recognition of the new strands.
• The enzyme GATC endonuclease cuts the strand at adjacent methylated
GATC sequence.
• This is followed by an exonuclease digestion of the defective strand, and thus
its removal.
• A new strand is now synthesized to replace the damaged one.
• Hereditary nonpolyposis colon cancer (HNPCC) is one of the most common
inherited cancers.
• This cancer is now linked with faulty mismatch repair of defective DNA.

40. Fig. A diagrammatic representation of mismatch repair of DNA

41. 4. Double-strand break
repair
• Double-strand breaks
(DSBs) in DNA are
dangerous.
• They result in genetic
recombination which may
lead to chromosomal
translocation, broken
chromosomes, and finally
cell death.
• DSBs can be repaired by
homologous
recombination or non-
homologous end joining.
• Homologous
recombination occurs in
yeasts while in mammals,
non-homologous and
joining dominates.

43. RECOMBINATION
INTRODUCTION
• Recombination basically involves the exchange of
genetic information.
• There are mainly two types of recombination.
1. Homologous recombination
2. Non-homologous recombination
1. Homologous recombination
• This is also called as general recombination, and
occurs between identical or nearly identical
chromosomes (DNA sequences).
• The best example is the recombination between the
paternal and maternal chromosomal pairs.
• It is a known fact that the chromosomes are not
passed on intact from generation to generation.
• Instead, they are inherited from both the parents.
• This is possible due to homologous recombination.

44. • Three models have been put forth to explain homologous recombination.
1. Holliday model
• Holliday model is the simplest among the homologous recombination.
• The two homologous chromosomes come closer, get properly aligned, and
form single-strand breaks.
• This results in two aligned DNA duplexes.
• Now, the strands of each duplex partly unwind and invade in the opposite
direction to form a two strands cross between the DNA molecules.
• There occurs simultaneous unwinding and rewinding of the duplexes in such a
way that there is no net change in the amount of base pairing, but the
position of crossover moves. This phenomenon referred to as branch
migration, results in the formation of heteroduplex DNA.
• The enzyme DNA ligase seals the nick.
• The two DNA duplexes (4 strands of DNA), joined by a single crossover point
can rotate to create a four stranded Holliday junction.
• Now the DNA molecules are subjected to symmetrical cuts in either of the two
directions, and the cut ends are resealed by ligase.

45. • The DNA exchange
is determined by the
direction of the
cuts, which would
be horizontal or
vertical.
• If the cross strands
are cut horizontally
(cut 1), the flanking
genes 9or markers,
i.e. Ab/ab) remain
intact, and no
recombination
occurs.
• On the other hand,
if the parental
strands are cut
vertically (cut 2), the
flanking genes get
exchanged (i.e.
Ab/aB) due to
recombination.

47. 2. Non-homologous recombination
• The recombination process without any special homologous sequences of DNA
is regarded as non-homologous recombination.
TRANSPOSITION:
• Transposition primarily involves the movement of specific pieces of DNA in the
genome.
• The mobile segments of DNA are called transposons or transposable
elements.
• They were first discovered by Barbara McClintock in maize.
• Transposons are mobile and can move almost to any place in the target
chromosome.
• There are two modes of transposition. One that involves an RNA intermediate,
and the other which does not involve RNA intermediate.
• Retrotransposition:
• Transposition involving RNA intermediate represents retrotransposition.
• By the normal process of transcription, a copy of RNA is formed from a
transposon.
• Then by the enzyme reverse transcriptase, DNA is copied from the RNA.

48. • The newly formed DNA which is a copy of the transposon gets integrated into
the genome.
• This integration may occur randomly on the same chromosome or, on a
different chromosome.
• As a result of the retrotransposition, there are now two copies of the
transposon, at different points on the genome.
• DNA transposition:
• Some transposons are capable of direct transposition of DNA to DNA.
• This may occur either by replicative transposition or conservative
transposition.
• Both the mechanisms require enzymes that are mostly coded by the genes
within the transposons.
• DNA transposons is less common than retrotransposition in case of
eukaryotes.
• However, in case of prokaryotes, DNA transposons are more important than
RNA transposons.

49. • Significance of
transposition:
• A large fraction of human genome
has resulted due to the
accumulation of transposons.
• Short interspersed elements
(SINEs) are repeats of DNA
sequences which are present in
about 500,000 copies per haploid
human genome e.g. Alu
sequences.
• Long interspersed elements
(LINEs) are also repeated DNA
sequences and are present in
about 50,000 copies in the human
genome e.g. L1 elements.