1) The document discusses the history and biology of telomeres, which are protective structures at the ends of chromosomes. It describes how telomeres shorten with each cell division due to the end replication problem.
2) Telomerase is an enzyme that can rebuild telomeres and counteract their shortening. It is active in stem cells and cancer cells but not most somatic cells. Telomerase expression allows cancer cells to divide indefinitely by maintaining telomere length.
3) The document examines telomere length and telomerase activity in different plant parts and telomerase-deficient Arabidopsis mutants. It finds that telomerase knockout leads to progressive telomere shortening and chromosome f
4. HISTORY
1970 Alexei Olovnikov -chromosomes are not completely
replicating their ends. & DNA sequences are lost every time
DNA replication.
In 1975–1977, Elizabeth Blackburn unusual nature of
telomeres.
Elizabeth Blackburn, Carol Greider and Jack
Szostak. 2009 Nobel Prize in Medicine - "for the discovery of
how chromosomes are protected by telomeres and the enzyme
telomerase." 4
5. Telomere biologyTelomere biologyTelomere biologyTelomere biology
HERMAN J MULLER (1938)
Telo –Telo – “end”“end” and merosand meros “part”“part”
Barbara McClintock(1941): chromosomes
lacking end parts became "sticky"
5
7. Telomere Length Changes in Human Aging
In newborn humans, telomeres are approximately 15-
20kb in length and shorten gradually throught life,
suggesting that telomere length may serve as a surrogate
marker for aging.
In newborn humans, telomeres are approximately 15-
20kb in length and shorten gradually throught life,
suggesting that telomere length may serve as a surrogate
marker for aging.
8. Alexei Olovnikov (1971)Alexei Olovnikov (1971) --
Problem of how chromosomes could replicate
right to the tip, as such was impossible with
replication in a 5’ to 3’ direction.
James Watson (1972)James Watson (1972) realized
that lagging-strand synthesis could
not completely replicate the 3’ end of
the linear T4 phage molecule.
9. TelomeresTelomeres
• Telomeres are chromatin structures that cap and protect the
end of chromosomes. It is the end of eukaryote linear
chromosomes, consisting of tandem arrays of G-rich repeats
that protect the genome from degradation.
• Telomere plays a crucial role in aging. In human somatic cells,
shortening of telomeres occurs with each cell division
• Progressive telomere shortening affects the chromosomal cap,
which further leads to chromosomal instability, end-to-end
fusion and ultimately death
9
Blackburn, E.H., 2001Blackburn, E.H., 2001
10. GroupGroup OrganismOrganism Telomeric repeat (5' to 3' toward the end)Telomeric repeat (5' to 3' toward the end)
Vertebrates Human, mouse, Xenopus TTAGGG
Filamentous fungi Neurospora crassa TTAGGG
Slime moulds Physarum Didymium TTAGGG
Dictyostelium AG(1-8)
Kinetoplastid
protozoa
Trypanosoma, Crithidia TTAGGG
Ciliate protozoa Tetrahymena, Glaucoma TTGGGG
Paramecium TTGGG(T/G)
Oxytricha Stylonychia,
Euplotes
TTTTGGGG
Apicomplexan
protozoa
Plasmodium TTAGGG(T/C)
Higher plants Arabidopsis thaliana TTTAGGG
Green algae Chlamydomonas TTTTAGGG
Insects Bombyx mori TTAGG
Roundworms Ascaris lumbricoides TTAGGC
Some known telomere sequencesSome known telomere sequences
11. Hayflick Limit
• According to Hayflick, cell division stops after
a certain number of cycles after which they
enter into a stage of irreversible cell arrest or
senescence .
• Senescence of the cells is triggered through
telomere signaling pathway, which involves
p53 and RB protein factor
11
Hayflick L., 1965.Hayflick L., 1965.
14. • Absence of p53 pathway primary cell lines
demonstrate the occurrence of a crisis in those
cells.
• A period when such cells continue to grow,
divide and cross the Hayflick’s limit and end
up with extreme shortening of telomere
• which further leads to genetic instability and
finally massive apoptosis occurs.
14
Aravind and grace., 2011
15. T loopT loop
• G-overhangs are also essential for the
formation of a t-loop at the chromosome end
and the loop formation includes invasion by
folding back of the G-overhang into duplex
DNA of telomere
15
function of the t-loop is to play an important role in
chromosome end protection by sequestering the G-
overhang and the telomeres from deleterious activities.
function of the t-loop is to play an important role in
chromosome end protection by sequestering the G-
overhang and the telomeres from deleterious activities.
Conomos, D. et al., 2013Conomos, D. et al., 2013
16. Telomere-binding ProteinsTelomere-binding Proteins
Shelterin
•Shelterin is a six protien complex
TRF1- telomeric repeat binding factors-1
TRF2- telomeric repeat binding factors-2
POT1-protection of telomeres
RAP1- transcription repressor/ activator protein
TIN2-TRF1 interacting protein
TPP1-POT1and TIN2-organizing protein
16
Gomez, D.E.,2012Gomez, D.E.,2012
18. Disruption of TRF-2 leads to
DNA damage signal, telomere homology
Homologous recombination process
sister-chromatid exchange
Disruption POT1 leads to
activate ataxia, telangiectasia and Rad3 protein
factors (ATR) formation
ATR cause DNA damage and eventually lead to
telomeric fusions
18
19. Role Of ShelterinRole Of Shelterin
• Acts as protective capping of chromosomal end
• Shelterin along with accessory protiens factors
restrict telomeric DNA from being recognized as
damaged DNA and promote proper telomeric
replication.
• Most importantly, shelterin fosters the formation of
T-loop and thus displays fusion- inhibitory activity
by inhibiting the fusion of chromosomal end
19
20. G-quadruplexesG-quadruplexes
• Single-stranded telomere with 3’ G-overhang
causes the formation of other higher order
structure G-quadruplexes.
• The G-quadruplexes are highly stable secondary
coplanar structures and forms anywhere along the
long G-rich strand.
• This involves the presence of four guanine bases
interconnected with the pairs of hoogsteen
hydrogen bonds and stacked with each other to
form G-tetrad structure
20
Nandakumar, J. et al.2013Nandakumar, J. et al.2013
22. • To the centre of tetrad ions like Na+ ions or K+
ions binds , which invokes high stability to the G-
quadruplex structures .
• Formation of the G-quadruplex structure causes
the unavailability of free 3’ telomeric end region
to bind with the RNA template of telomerase and
thus hinders the catalytic activity of the enzyme
complex
• Unfolding of G-quadruplex structures is a must
for telomerase activity.
22
23. • The factors like small molecules, which favor
the stability, prove to be a selective target of
immortalized cells wherein active telomerase
is found to be quite high.
• This approach of imparting stability has been
used to develop anti-cancer therapeutic agents.
ex:BRACO-19. RHPS4, TELOMESTATIN
23
24. TelomeraseTelomerase
• Human telomerase is a ribonucleoprotein
holoenzyme complex which functions to extend
telomeric DNA and acts to compensate telomere
loss.
• Telomerase comprises of a catalytic protein
subunit which belongs to the family of reverse
transcriptase (TERT) encoded by TERT gene.
• It also includes hTR region, encoded by the
TERC gene which works as a template during
telomere maintenance process
24
Rhodes and Giraldo., 1995Rhodes and Giraldo., 1995Rhodes and Giraldo., 1995Rhodes and Giraldo., 1995
25. Where these telomerase is presentWhere these telomerase is present
Mammals : stem cells,
germ cells ,lineage
progenitor cells.
Plants: reproductive
organs, meristems,
undifferentiated cells.
26. 26
Objective : Examining the telomerase activity in
different plant parts
Objective : Examining the telomerase activity in
different plant parts
Preparation of plant extracts
Telomere Repeat Amplification Protocol (TRAP)
Assays.
Preparation of plant extracts
Telomere Repeat Amplification Protocol (TRAP)
Assays.
28. Telomerase complexTelomerase complex
• There are four functional regions of hTR viz.,
CR4-CR5 domain, Pseudoknot region, Box
H/ACA and CR7 domain, CR4-CR5 domain
and Pseudoknot region bind with the TERT
protein and are required for enzymatic activity
of the complex.
• Telomerase complex formation involves
Scaffold formation (pontin, reptin & dyskerin)
Stabilising of ribonucleoprotien complex.
28
Cawthon et al.,2003Cawthon et al.,2003
29. Dyskerin, NOP-10 and
NHP2-provide stability
Dyskerin, NOP10, NHP2, GAR1-
Assembly and function of telomerase
complex
Scaffold formation- Recruitment of
hTR and accessory protien factors
29
30. Function of telomeraseFunction of telomerase
• Telomerase compensates the loss of telomere
• It adds the telomeric sequence at 3’OH end
• The template sequence of 11 complementary nucleotides
located inside the RNA domain of the telomerase
ribonucleoprotein complex gets hybridized with the
complementary two telomeric repeats found at the 3’ extreme
of telomeric DNA
• followed by the synthesis of nucleotide sequences by hTERT.
• synthesized strand translocation in 5’ direction, which causes
the formation of a new gap. The new gap is filled with the
same steps and the cycle continues to get repeated to attain a
specific length.
30
32. 32
Plant Growth and Callus Induction
Preparation of Telomerase Extracts and Telomere Repeat Amplification
Protocol (TRAP) Assays.
Cloning and Sequence Analysis of the AtTERT Genomic Clone and
cDNA.
RNA Extraction and RT-PCR Analysis of AtTERT mRNA
Identification of an AtTERT Null Plant.
Plant Growth and Callus Induction
Preparation of Telomerase Extracts and Telomere Repeat Amplification
Protocol (TRAP) Assays.
Cloning and Sequence Analysis of the AtTERT Genomic Clone and
cDNA.
RNA Extraction and RT-PCR Analysis of AtTERT mRNA
Identification of an AtTERT Null Plant.
Objective : To show that addition of homozygous T-DNA
abolishes telomerase activity.
Objective : To show that addition of homozygous T-DNA
abolishes telomerase activity.
33. 33
Schematic representation of AtTERT geneSchematic representation of AtTERT geneAtTERT mRNA levels correlate with
telomerase activity. (A) Telomerase
regulation In Arabidopsis. TRAP results
from different organs. Elongation ladders
correspond to the addition of TTTAGGG
repeats. Silque (seedpod); inflor.
(inflorescence) bolt.
AtTERT mRNA levels correlate with
telomerase activity. (A) Telomerase
regulation In Arabidopsis. TRAP results
from different organs. Elongation ladders
correspond to the addition of TTTAGGG
repeats. Silque (seedpod); inflor.
(inflorescence) bolt.
34. • Plants with a homozygous disruption in the
AtTERT gene lack telomerase. TRAP assays
were performed on the floral buds from plants
that are wild type (1y1), heterozygous (1y2),
or homozygous (2y2) for the T-DNA
disruption in AtTERT.
34
35. • Progressive telomere shortening in telomerase-deficient plants. (A)
TRF analysis. Shown are results with Tru9I digestion of DNAfrom
rosette leaves (lane 1) or floral buds and siliques (lane 2) of wild-type
plants and rosette leaves of heterozygous plants (lane 5). Results with
rosette leaf DNA from first-generation (G1) and second-generation
(G2) plants homozygous AtTERT disruption are shown in lanes 3 and
4.
35
36. 36
End-to-end chromosome fusions in late-
generation telomerase-deficient mutants
End-to-end chromosome fusions in late-
generation telomerase-deficient mutants
37. 37
Overview of phenotypes in telomerase-deficient Arabidopsis.
Placement of mutants into four classes: WT-like, I, II, and T .
Overview of phenotypes in telomerase-deficient Arabidopsis.
Placement of mutants into four classes: WT-like, I, II, and T .
38. TELOMERES AND CLONINGTELOMERES AND CLONING
38
• Dolly was cloned using a nucleus taken from an adult
sheep cell that had been growing in culture
• The cell donor was 6 years old, and its cells had been
growing in culture for several weeks
• Dolly's cells reveals that they were only 80% as long
telomeres as in a normal one-year-old sheep
• But medical problems probably unrelated to her
telomeres ended with her being euthanized at a
relatively young age
• But her short telomeres do add another question to the
debate about cloning mammals from adult cells.
39. Telomerase: Path towards Tumor
genesis
Telomerase: Path towards Tumor
genesis
39
TELOMERE
DYSFUNCTION
40. TumorigenesisTumorigenesis
• Cells are able to undergo replications only finite number of
times
• After few replications cells stops dividing due to p53
mediated pathway , cell enters senescence
• If further cells divides beyond Hayflick limit leads to loss of
tumor suppressor alleles, ultimately leading to
tumorigenesis
• Those cells continuously proliferate become immortalized
• Most of the cancer cells show telomere maintenance
mechanism (TMM). In these cells telomerase is reactivated
• Only some 5-10% cancer cells show ALT(alternate
lengthening of telomere) .
40
Bidzinska, J. et al.,2013Bidzinska, J. et al.,2013
41. • Among most human tumors, chromosomal gain and gene
amplification involving chromosome arm 3q is found to be
more frequent and signifies the involvement of telomerase-
associated subunits in malignancies formation
• In humans hTERT gene is expressed during early tumor
devolopment, this helps to bypass replicative senescence
• This leads to cancerous growth and other malignansis
• hTERT can also be used as tumor marker in case of humans
• Mutation in promoter region of hTERT genes leads to various
cancers
Ex : Human melanoma(70%), primary glioblastoma(80.3%)
41
42. 42
Six tomato varieties were used in this
study: L. esculentum cvs. Rio Grande PtoR, San Marzano,
TA55-VF36, UC82B, Vendor Tm2a, and VFNT Cherry.
Pulsed-Field Gel Electrophoresis
Hybridization.(probes were-Arabidopsis telomeric repeat , the tomato
macrosatellite probe TGRI , the 33.6 and 33.15 human minisatellite probes, and
a set of 15 cloned low-copy-number ( RFLP mapping).
Computation of Genetic Distances Among Accessions
43. 43
D- value
Low copy number RFLP- 0.04
M13- 0.00
Human minisatellite 33.6 - 0.14
Human minisatellite 33.15 -0.28
TEL- 0.92
TGRI- 0.65
D- value
Low copy number RFLP- 0.04
M13- 0.00
Human minisatellite 33.6 - 0.14
Human minisatellite 33.15 -0.28
TEL- 0.92
TGRI- 0.65
44. Conclusion
• Values for TEL and TGRI are significantly
higher than those of the other probes tested.
• TEL and TGRI show, 2.5 to 6.6 times more
polymorphism than the human minisatellite
probes .
• 16 times more variation than M13 or standard
single-copy RFLP probes
44
