|last page||PLNT3140 Introductory Cytogenetics
Lecture 12, part 2 of 2
FUNCTIONAL CHROMOSOMAL ELEMENTS: EXPERIMENT C - Telomeres
When Tetrahymena telomeric sequences were added to the ends of linearized yeast artificial chromosomes, the YAC's were able to replicate and segregate stably in yeast.
Other examples:These telomeric sequences are oriented so that the C-rich strand always runs 5'-->3' from the end towards the interior of the chromosome. In some cases, there are nicks, or gaps in the C-rich strand.
H. sapiens CCCTAA
Example: Oxytricha (ciliated protozoan):
end of chromosome to centromere -->
fragments containing telomeric sequences were hybridized
to metaphase chromosomes. Telomeric probe is visualized in
white. Chromosomal DNA is counterstained with DAPI (blue).
Image displayed by hypertext link to The New Genetics, Chapter 2
RNA and DNA Revealed: New Roles, New Rules
Natl. Inst. of General Medical Science
a. Telomeres prevent degradation,
repair and recombination of chromosome ends.
de Lange T (2001) Telomere capping -- one
strand fits all. Science
Greider CW (1999) Telomeres do D-loop-T-loop. Cell 97:419-422.Eukaryotic cells have mechanisms for identifying and repairing damaged DNA. Typically, the end of a linear DNA molecule would be recognized as a damaged piece of DNA and "repaired", resulting in loss of sequence from the ends of chromosomes, or fusion with other double-stranded DNAs. Eukaryotic cells have mechanisms for protecting chromosome ends from repair enzymes, which vary among eukaryotes. For example, telomeres of ciliates and fungi are protected by telomere binding proteins which effectively hide the telomeres from repair machinery. Mammals have a more elaborate D-loop structure, in which the double-stranded telomere DNA opens up to form a single-stranded D-loop (or T-Loop). The 3' protruding end can loop back to form a t-loop. The end of the t-loop can base pair with internal repeat units by non-Watson-Crick base pairing. The D-loop is maintained through additional proteins that bind both the t loop and the D-loop seen in the figure.
Electron micrograph of telomeric D-Loop
For more on the details of
non-Watson-Crick base pairing in telomeres see 3 Structural
aspect of DNA at The Open University. http://www.open.edu/openlearn/science-maths-technology/science/biology/nucleic-acids-and-chromatin/content-section-3.4.1
b. Telomerases carry a small RNA
molecule which acts as a template for elongation of
Telomere terminal transferases can
add these repeat units to the 3' ends of chromosomes.
ELONGATION OF 3' ENDS BY TELOMERASE. Functionally, the telomere is a 3' protruding end which can not be elongated by DNA polymerase. Telomere terminal transferase (telomerase) allows the telomere to function as a 3' recessed end in the following way: The telomerase enzyme carries an RNA molecule (blue) whose sequence is the complement of the telomeric repeat. When the telomere (red) base pairs with this RNA, the 5' end of the RNA extends beyond the end of the telomere. This allows the RNA to act as template for the addition of nucleotides (green)to the telomeric 3' end, which acts as a primer. In the example, an RNA template with the sequence 5'(ccccaaaa)n3' codes for the actual telomeric repeat 5'(ttttgggg)n3'.
So, by extending the 3' ends of
linear chromosomes with each round of DNA synthesis,
telomerase provides a longer template for the lagging strand,
which offsets the inevitable loss of DNA from the ends.
|Is telomerase active in
Unicellular eukaryotes - Telomerase is required in each cell division to maintain telomere length
Humans1 - Telomerase activity is usually only seen in stem cells or germline cells, and telomerase activity is usually not found in somatic cells. It is hypothesized that because of the long human lifespan, somatic suppression of telomerase activity occurs as a check on cell proliferation, which could otherwise result in cancer. This is not a perfect control, because ultimately as telomeres are lost, oncogenes near the telomeres begin to be lost as well, resulting in cancer.
Mice1 - Telomerase activity is often found in somatic cells in mice. This observation makes sense in contrast to the lact of telomerase activity in human somatic cells, because mice have very short life spans, and therefore would have less need for suppressing telomerase activity as a way of suppressing cancer.
Drosophila3 - doesn't use traditional short telomeric repeats elongated by telomerase. Instead, two retrotransposons, HeT-A and TART transpose specifically to chromosome ends, elongating the array of transposon repeats at the telomeres. (Weird or what?)
Tobacco2 - High levels of telomerase activity was seen in actively dividing cells (roots and flowers), with low levels of activity in stems, and no detectable activity in mature leaves. This is consistent with the hypothesis that telomerase activity is needed in rapidly dividing tissues.
1 Wong JMY and Collins K (2003) Telomere maintanence and disease. The Lancet 362:983-988.
2 Yang SW, Jin ES, Chung IK, Kim WT (2001) Expression of telomerase activity is closely correlated with the capacity for cell division in tobacco plants. J. Plant Biol. 44:168.
3 Danilevskaya ON, Traverse KL, Hogan NC, DeBaryshe GP and Pardue ML (1999) The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol. Cell. Biol. 19:873-881.
Hinnebusch J, Tilly K
(1993) Linear plasmids and chromosomes in bacteria. Mol. Microbiol.
Volff J-N, Altenbuchner J
(2000) A new beginning with new ends: linearisation of
circular chromosomes during bacterial evolution. FEMS Microbiology
Ravin NV, Kuprianov VV,
Gilcrease EB, Casjens SR (2003) Bidirectional replication
from an internal ori site of the linear N15 plasmid
Acids Res. 31. DOI: 10.1093/nar/gkg856.
In biology, every rule has
exceptions. There are a number of cases in which prokaryotes
have either linear chromosomes, or linear plasmids. One
well-known example is bacteriophage Lambda, which is linear in
its encapsidated form, but circularizes, by annealing of its
"sticky ends", prior to replication as a circular molecule.
However, there are now many
examples of linear chromosomes and plasmids, including the
the actinomycete Streptomyces,
and the plant pathogen Agrobacterium.
see Figure 1 from Volff and
linear chromosomes or plasmids are rare amongst prokaryotes,
their occurence in widely diverse species suggests that
linearity has arisen independently numerous times over the
course of microbial evolution.
schemes have been seen for linear chromosome replication,
hairpins and invertrons.
Hairpins (eg. Borellia) -
Normally linear chromosomes contain inverted repeats at
each end, which are capable of forming hairpin loop by
intra-strand base pairing. When the leading strand from
an internal replication origin arrives at the hairpin,
the hairpin allows the template strand to be replicated
in much the same way as a circular plasmid, such that
the leading strand is redirected to "follow behind" the
lagging strand. Thus, there is always a polymerase
complex upstream from each lagging strand.
Streptomyces) - Linear chromosomes contain
inverted repeat units at both ends. Inverted repeats are
bound by terminal proteins (TP) which bind to the 5' end
of the repeats. The terminal proteins themselves act as
primers, binding DNA polymerase. The first
nucleotide to be added to the template is covalently
bound to the TP, and the chain is elongated by further
addition of nucleotides to the 3' end of that
Based on Fig. 1 from Hinnebusch and Tilly.
And, if you thought this was all
you needed to know about telomeres, check out
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|last page||PLNT3140 Introductory Cytogenetics
Lecture 12, part 2 of 2