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Lecture 12, part 2 of 2
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D. Telomeres

In the first two experiments, Szostak and colleagues created what are now known as Yeast Artificial Chromosomes.
Up to now, we've been getting away with creating artificial chromosomes by making them circular. In yeast, they function as completely independent chromosomes. But eukaryotic chromosomes are linear, and Experiment C illustrates that linear chromosomes can't normally replicate without telomeres at each end.

1. Isolation of telomeric sequences


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.

Flourescent-labeled DNA 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

de Lange T (2001) Telomere capping -- one strand fits all. Science 292: 1075-1076.

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


Is telomerase active in somatic cells?

Unicellular eukaryotes - Telomerase is required in each cell division to maintain telomere length

1 - 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.

1 - 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?)

2 - 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.

5. Linear Chromosomes and Telomeres in Prokaryotes (yes, I said Prokaryotes)

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.

Invertrons (eg. 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 nucleotide.

Based on Fig. 1 from Hinnebusch and Tilly.
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last  page PLNT3140 Introductory Cytogenetics
Lecture 12, part 2 of 2
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