Patrick O'Neil patrick at corona
Sun Apr 9 14:15:41 EST 1995

  I am working on a research proposal for the study of telomere length 
regulation.  Since telomerase activity is constant in germ cells, stem 
cells, and in a perhaps abnormal way in certain cancer cells, the 
question arises as to what prevents telomeres from extending indefinitely.
  Telomerase activity in the above mentioned cell types appears to be 
independent of cell cycle position, with telomerase activity throughout S 
and M phase.  Therefore, cell cycle regulatory proteins do not appear to 
affect telomerase transcription levels...so how are telomeres maintained 
at a rather stable length? (All within a heterogenous but rather narrow 
range of several kb)
  My thoughts:

1)  The level of telomerase transcription is under two layers of control:  
self regulated by feedback loop and developmentally regulated - for 
permanent silencing upon differentiation of a stem cell into a somatic 
tissue cell.  If telomerase levels are low in general in, say, stem 
cells, then kinetically the enzyme may be constantly operating in a 
balance with cell division so that shortening due to division and normal 
degredation is balanced by extension.  Thus telomerase is limiting.

  If this is the case, then the process is saturable so that if a lot of 
telomeric seed sequence is added to such a cell, then all telomeres 
should shorten and ultimately stabilize at a shorter length.

2)  Telomere binding factors (proteins and ribonucleoproteins) act to 
regulate telomere length.  I see that this could take place in two 
ways:  Telomerase is a multisubunit enzyme.  If any of its subunits are 
limiting, then the overall activity of the enzyme is limited by subunit 
levels as in 1 above.  Alternatively, if such telomere binding species 
act to protect telomeres from degradation, and they are produced at a 
constant level so that an equilibrium is attained, then extension of 
telomere beyond a point at which there is enough binding factors to bind 
and protect the sequence would be automatically degraded back to 
protection levels.  This would be a dynamic process in which telomerase 
constantly adds sequence but it get degraded back to set point.  This is 
closely related to 1 above.

3)  All telomeric sequence repeats from all species examined thus far 
appear capable of forming very stable G-quartets.  Numerous studies with 
different sequences and various SHORT lengths (containing 1 to 4 tandem 
repeats - vs thousands of tandem repeats in actual telomeres) confirm 
that under physiological conditions, telomeric repeats can form stable 
G-quartet structures in vitro.  In 1991, Alan M. Zahler, James R. 
Williamson, Thomas Cech, and David Prescott found that G-quartet DNA 
structures can inhibit telomerase activity - that is, telomerase cannot 
use such quartets as primer for extension.
  If telomeric sequences in vivo do form G-quartets, then it must be 
regulated somehow or as soon as 4 tandem repeats were produced, further 
extension would be inhibited since a G-quartet could form.  Obviously, 
such early inhibition doesn't occur.  
  Thus comes my research proposal.  I seek to confirm that telomeres, in
vivo, do in fact contain G-quartets (the regulation of their formation is
not the subject of _this_ particular proposal).  To confirm this, I intend
to remove and seperate telomeric DNA from chromosomes in Oxytricha (since
they contain many thousands of telomeres), treat with Proteinase K and
RNAse, and check the CD on the DNA to check for G-quartet content. 
  Another part of the study would be in S. cerevisiae in which I wish to 
knockout the TLC1 gene, which codes for the yeast ribonucleioprotein 
telomerase template component of the holoenzyme, with a version 
containing alterations to the template such that G-quartets could not 
form.  If G-quartets are important for telomere length regulation, 
then the inability to produce G-quartets should lead to increased length 
of telomeres in so treated yeast.
  Alternatively, I could use a MoMLV vector to introduce a similarly
altered telomerase template gene into a mouse cell line, overexpress it
with a CMV promoter to favor mutant holoenzyme formation, and check for
telomere length alterations vs G-quartet content (alternatively, I could
go for a knockout).  This latter (mouse) is more complicated but has the 
advantage to being more applicable to mammalian telomere systems. 

Any comments or suggestions?  Have I missed something obvious that might 
go easier or quicker?


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