genome supercoiling repetetive DNA transposons pathogenecity islands

1. Prokaryotic and Eukaryotic
Genome Anatomies
Presented to : Dr. P.P. Balgir
Presented By : Sarabjot Kaur
M.Sc. Biotech 1
Roll No. 170110051

2. CONTENTS
• Genome
• Supercoiling
• Repetitive DNA and its types
• Transposons and transposase
• Pathogenecity islands
2

3. 3

4. Most prokaryotic genomes are less than 5 Mb in
size, few are larger like B. megaterium , has a
huge genome of 30 Mb.
The typical prokaryote the genome is contained
in a single circular DNA molecule, localized
within the nucleoid .
THE PHYSICAL STRUCTURE OF THE
PROKARYOTIC GENOME
4

5. Human and bacterium cell
• Eukaryotic cells have
membrane-bound
compartments, which
are absent from
prokaryotes.
• The bacterial DNA is
contained in the
structure called the
nucleoid.
5

6. GENOME CAN BE NATURALLY
COMPACTED BY SUPERCOILING IT
The first feature to be recognized was
that the circular E. coli genome
is supercoiled.
• It’s the expression of strain
over a strand i.e. over or under
winding.
• No winding in linear molecule.
• circular molecule responds by
winding around itself to form a
more compact structure .
• Enzymes : Gyrase and
Topoisomerase 1.
Supercoiling : negative supercoiling. 6

7. SUPERCOILING
7
Positive negative
• When additional
turns are added into
DNA.
• Its right handed.
• When turns are
removed from
DNA
• Its left handed.

8. MODEL FOR GENOME ORGANIZATION
Between 40 and 50 supercoiled loops of DNA radiate from the central protein core. One of the
loops is shown in circular form, indicating that a break has occurred in this segment of DNA,
resulting in a loss of the supercoiling.
8

9. • Bacterial DNA is attached to proteins that restrict its
ability to relax, so that rotation at a break site results in
loss of supercoiling.
• The current model has the E. coli DNA attached to a protein
core from which 40–50 supercoiled loops. Each loop contains
approximately 100 kb of supercoiled DNA, the amount of
DNA that becomes unwound after a single break.
9

10. THE GENETIC ORGANIZATION OF
THE PROKARYOTIC GENOME
• Bacterial genomes have compact organizations
very little space between genes.
• There is non-coding DNA in the E.
coli genome, but it accounts for only 11% of the
total.
• prokaryotic genomes have very little wasted
space. 10

11. OPERONS ARE CHARACTERISTIC
FEATURES OF PROKARYOTIC
GENOMES
An operon is a group of genes that are located
adjacent to one another in the genome,.
All the genes in an operon are expressed as a
single unit.
This type of arrangement is common in
prokaryotic genomes. Eg : lactose operon.
11

12. HUMAN GENOME
• 22 autosome pairs + 2
sex chromosomes
• 3 billion base pairs in the
haploid genome
12

13. 13

14. MUCH DNA IN LARGE GENOMES IS
NON-CODING
• Contributors to the non-coding DNA include:
• Introns in genes
• Regulatory elements of genes
• Multiple copies of genes, including pseudogenes
• Intergenic sequences
• Interspersed repeats
14

15. 15

16. • All eukaryotes have at least two chromosomes and the
DNA molecules are always linear.
• The only variability at this level of eukaryotic genome
structure lies with chromosome number, which appears to
be unrelated to the biological features of the organism.
16

17. Repetitive DNA

18. Definition:
Stretches of DNA that repeat themselves throughout a
Genome , either in tandem or interspersed along the
Genome. These stretches can comprise up to fifty
Percent or more of an organism’s DNA.
* It can have a structural function (such as telomere)
or can comprise sequences of no known function
18

19. Some of its DNA use as a useful
purpose
but significant proportional is
of uncertain purpose & may be
treated as JUNK DNA OR
SELFISH DNA
19

20. • Refers to non-coding tandem
repeating sequences.
• These are generally short
sequence repeats (upto 60
base pair long).
• These appear as small dark
bands in CsCl density
gradient analysis test.
Satellite DNA Repetitive DNA
20
• It is the non-coding DNA
with tandem or
interspersed sequences.
• These can be few base pairs
to hundreds or thousands
of base pairs long.
• In CsCl density gradient
analysis, They appear as
light bands

21. REPETITIVE DNA
QUANTITY
21

22. HOMOSAPIENS – 21%
22

23. MOUSE – 35%
23

24. DROSOPHILA – 70%
24

25. PEA – 52%
25

26. SACCHAROMYCES SEREVISIAE
-5%
26

27. E.COLI – 0.3%
27

28. REPETITIVE DNA
ELEMENT
COMPRISES A
MAJOR COMPONENT
OF PLANT GENOME
28

29. STUDY OF REPETITIVE DNA IS IMPORTANT
BECAUSE
• Repeats Drive Evolution in Diverse Ways
• Repetitive DNA are generally not found to
have any function
•Homology searches need repeat masking
• Repeat also contain information about
parentage 29

30. DENSITY GRADIENT ANALYSIS
30

31. 31
C -C DENSITY GRADIENT
ANALYSIS
S l

32. IN PROKARYOTES
DNA forms a single band in
gradient while cscl density is equal
to density of DNA having 50% of
GC base pair
32

33. IN EUKARYOTES
•Cscl density gradient analysis ususally reveals
presence of 1 large band & one to several
small bands.
•And these several bands are called satellite
band having DNA reveals repeating sequences
of various links in different organisms. 33

34. A repetitive DNA sequences will be
identified as
Satellite DNA only if sequences has base
composition
different from that of “MAINBAND DNA”
34

35. TYPES OF REPETITIVE DNA
•SATELLITE DNA
•MINI SATELLITE DNA
•MICRO SATELLITE DNA
•TRANSPOSABLE ELEMENTS
•LINES,SINES & OTHER
RETROSEQUENCES
35

36. 1. SATELLITE DNA
• Form distant bands
• Location - heterochromatin region of
chromosome
• Unit - 5 – 300 bp depending on species
• Repeat - 105 – 106 times
• There are 10 distinct types of satellite dna
Egs – centromeric DNA , telomeric DNA
36

37. 2. MINI SATELLITE DNA
• Repeat – generally 20 – 50 times
(1000 – 5000 bp long)
• Location – euchromatic region of chromosomes
• Egs – DNA finger prints (variable no. tandem repeats)
37

38. 3. MICRO SATELLITE
• Units – 2 – 4 bp
• Repeats – 10 – 100 times in a
genome
• Location – euchromatin region
of chromosome
38
 These Repetitive DNA s Can Cause Diseases:
Fragile X Syndrome – “CGG” is repeated.

39. 4. TRANSPOSABLE ELEMENTS
• These are called transposable elements because of transposons that
change their position in genome.
• Interspaced repeats have the capability to “move around” in the
genome.
• Transposition in germ cells are passed down to progeny resulting in an
accumulation in the genome.
• Transposons provide a mechanism for bringing about DNA
rearrangements throughout evolution.
• Adjacent DNA sequences sometimes mobilized
39

40. 40

41. TRANSPOSASE
41

42. 3 PRINCIPLE CLASSES OF TRANSPOSONS
1. DNA transposons:
move using cut and paste or replicative
mechanism
2. Virus-like retrotransposons
(long terminal repeat [LTR] retrotransposons):
RNA intermediate, includes retroviruses
3. Poly-A retrotransposons
(nonviral retrotransposons):
RNA intermediate
42

43. CUT AND PASTE MECHANISM OF TRANSPOSITION:
• Nonreplicative
1.Transposase (usually 2 or 4 subunits) binds
terminal inverted repeats
2.Brings 2 ends together  stable protein complex
called transpososome
3.Transposase cleaves one DNA strand at each end at
junction between transposon DNA and host DNA 
transposon sequence terminates with free 3’-OH
groups at each end
4.Other DNA strands cut by various mechanisms 
transposon excised
43

44. Cut and paste mechanism of transposition
5. 3’-OH ends of transposon DNA attack
DNA phosphodiester bonds at site of
new insertion (target DNA)
6. Nicks introduced in other target DNA
strands few nucleotides apart 
transposon joined via reaction called
DNA strand transfer
7. Few nucleotides between nicks leaves
small ss gaps  filled in by host DNA
repair polymerase  small target site
duplications on either side transposon
8. DNA ligase seals final nicks
9. Ds break where transposon left
repaired by homologous
recombination 44

45. REPLICATIVE TRANSPOSITION:
• Transposon DNA replicated during each round of
transposition
1.Transposase assembles on each end of
transposon to form transpososome
2.Transposase introduces nicks at junctions
between transposon and flanking host DNA 
generates 3’-OH ends on transposon (but
transposon NOT excised from flanking DNA)
3.3’-OH joined to target DNA by strand transfer
reaction (same mechanism as cut-and-paste) 
intermediate is double branched DNA molecule
45

46. REPLICATIVE TRANSPOSITION:
4. 3’ ends transposon covalenty linked to target DNA, but
5’ ends still linked to old flanking DNA
5. 2 branches like replication forks, DNA replication proteins
assemble at these forks, 3’-OH serves as primer
6. Replication proceeds through transposon and stops at 2nd
fork  2 copies of transposon flanked by short target site
duplications
• Frequently causes chromosomal inversions and deletions
detrimental to host
46

47. Virus-like retrotransposons and retroviruses:
1. Retrotransposon DNA transcribed into RNA by host
RNAP (transcription starts at promoter within LTR)
2. RNA reverse-transcribed (by RT)  RNA:DNA 
dsDNA (cDNA)
3. Integrase (transposase) recognizes and binds ends
of cDNA then cleaves few nucleotides off 3’ end of
each strand (just like cleavage step of DNA
transposons)
4. Integrase performs strand transfer reaction to insert
3’ ends into target DNA
5. Gap fill and ligation by host proteins
47

48. PLANT GENOMES ARE RICH IN TRANSPOSONS:
• Snapdragons: size of white
patches related to frequency
of transposition
• Maize color variation due to
chromosome breakage by
transposition
48

49. Transposon excision
49

50. 5. LINES , SINES
SINES - Short Interspersed Nuclear Elements.
do not have a reverse transcriptase gene but can still transpose, probably by
‘borrowing’ reverse transcriptase enzymes that have been synthesized by other
retroelements.
Eg – human genome Alu
• Length – 280 bp
• Repeats – 700 × 10 to 1000 × 10 (in introns)
LINES – Long Interspersed Nuclear Elements.
It contain a reverse-transcriptase-like gene probably involved in the retro
transposition process
Eg - LINE 1. (Copy no. of 60,000 – 100,000) is a non viral retro element.
50

51. Haemoglobin histones immunoglobulins
51

52. FUNCTIONS OF REPETITIVE DNA
• structural & organizational role in chromosomes.
• protection of genes as histones, rRNA, ribosomal
protein gene.
• At telomere allow linear replication to maintain &
to protect its ends.
52

53. Pathogenenicity islands are discrete genetic loci that
encode factors which make a microbe more virulent
& located on chromoses.
• A host may have more than one pathogenicity island.
• Pathogenicity islands are transferred horizontally,
through plasmids or transposons.
• The addition of a pathogenicity island to a non-
invasive species can make the non-invasive
species pathogenic.
53

54. • These mobile genetic elements may range from 10-
200 kb .
• There are adherence factors, toxins, iron uptake
systems, invasion factors, and secretion systems.
• PAIs are present in the genomes of pathogenic
organisms but absent from the genomes of
nonpathogenic organisms of the same or closely related
species
54

55. • Horizontal gene transfer (HGT) can take place by
transduction, transformation and conjugation.
Plasmids and also larger parts of the genome, like
genomic islands, can be conjugated from one
bacterium to another.
• Pathogenicity islands (PAIs) are a subgroup of
genomic islands. PAIs encode several virulence
factors such as adhesins, toxins, capsules and
siderophore systems and play a major role in the
evolution of pathogenic bacteria such as extra
intestinal e.coli.
• The species E. coli is subdivided into four major
phylogenetic groups (A, B1, B2 and D).
55

56. • PAIs in pathogenic E.
coli were the first
described. It was soon
discovered that
pathogenic bacteria
share many features of
PAIs.
• PAIs carry genes
encoding one or more
virulence factors. They
were first described in
human pathogens but
are also present in
plant pathogens
COMMON FEATURES OF
PATHOGENICITY ISLANDS
56

57. 57
• The average G+C content of bacterial DNA can range
from 25 to 75%. Most pathogenic bacterial species have
G+C contents between 40 and 60%.
• PAI are frequently located adjacent to tRNA genes.
• DR might have served as recognition sites for the
integration of bacteriophages, and their integration resulted
in the duplication of the DR.
• Mutations are cause of instability…integrases,
transposases, and IS elements, have been identified that
contribute to mobilization and as well as to instability.

58. 58
PROTEIN SECRETION SYSTEMS ENCODED
BY PAI
General requirement for pathogenic and nonpathogenic bacteria.
Secreted proteins are required for the assembly of the cell envelope,
metabolism, and defense against, and interaction with, host cells
during pathogenesis.
the presence of an outer membrane in gram-negative bacteria led to
different secretion systems.
Type I Systems
Type II Systems
Type III Systems
Type IV Systems
Type V Systems

59. As of April 2006, PAIDB contains 112 types of PAIs and 889 GenBank
accessions containing either partial or all PAI loci previously reported in the
literature, which are present in 497 strains of pathogenic bacteria.
Bacterial pathogenicity/virulence determinants that can be found in PAIs
include the type III secretion system (e.g. LEE PAI in pathogenic Escherichia
coli), superantigen (e.g. SaPI1 and SaPI2 in Staphylococcus aureus),
colonization factor (e.g. VPI in Vibrio cholerae), iron uptake system (e.g. SHI-2
in Shigella flexneri) and enterotoxin (e.g. espC PAI in E.coli ).
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60. 60
• https://www.youtube.com/watch?v=2L-OL2i7ysQ
• Slide share
• herehttps://23bios1unsoed.wordpress.com/bahan-
ajar/2-genome-anatomies/23-the-anatomy-of-the-
prokaryotic-genome/
• http://www.bio.utexas.edu/faculty/sjasp
er/bio212/eukaryotes.html
References
• file:///C:/Users/dosan/Desktop/Pathogenicity%20Islands%
20in%20Bacterial%20Pathogenesis.html

61. THANK YOU
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