message

P9323611%ARIZRVAX at PUCC.PRINCETON.EDU P9323611%ARIZRVAX at PUCC.PRINCETON.EDU
Thu Feb 21 00:54:00 EST 1991


I would like to clearify a question I sent to GENBANK several days ago. I
am now trying to send the message to the proper bulletin board. The  prior
message read as follows:

Curious,

   I haven't been able to get a clear answer to a question I have had for almost

a year now. I'm curious to know why researchers et. al. haven't developed

an in Vitro transcription system that could be utilized somewhat similar to the

way the Polymerase Chain Reaction (PCR) is used.  Instead of the crude extracts

that are currently available on the market, can't the RNA polymerase from cells

be purified and then used in a test tube containing:

template DNA (containing the proper promoter sequence)
RNA nucleotides
ATP
necessary ions

this way a standardized method would be available -- in pre-prepared test tubes-

for scientists to test specific transcription factors that are required for

transcription of their particular gene.  I know that different cells regulate

their genes differently and by different mechanisms, but it seems to me that

the basic  necessary components of transcription should be available in a test

tube.  It seems like a standard system like this would supplement the current

in vivo systems and would be relatively cheap.  What is the limiting factor

that is holding this technology back. Is it the purification of the RNA

polymerase?

I did receive a reply that answered most of my questions and it was the
following message from Michael Gribskov:

John-

I won't try to give a comprehensive answer to your question but here are
a few (off the cuff) comments.

The PCR system is a heterologous and non-specific system, and can be used to rep
licate any probe that meets certain gemometric conditions of
base-pairing etc.  It could not be used, for instance, to search for
DNA replication factors in human cells, which is the analogue of what
you propose for RNA polymerase.  There is an RNA system that bears some
resemblence to PCR in which a gene of interest is cloned next to a T7
promoter, which can be used both in vitro and in vivo to produce large
amounts of a specific RNA.

Off-the-shelf preparations of bacterial RNA polymerase have been
available for fifteen or twenty years and have, as you suggest, been
very useful in elucidating transcriptional control mechanisms in
bacteria.  A great deal of effort has gone into trying to develop
similar systems for eukaryotes, with limited success.  But there are
major  problems:

Transcription is more complicated in eukaryotes which have three types
of RNA polymerase.  The polymerase which transribes most genes, pol II,
is not the most abundant, but of course has similar properties to the
others since  they are all RNA polymerases.  It is often difficult to
even obtain sufficient amounts of tissue/cells to purify the polymerase
from.  The eukaryotic polymerase II, apparently, interacts with a larger
number of weakly bound factors than does the bacterial RNA polymerase,
which requires only the sigma factor.  This means that often one has to
search for two or more factors simultaneously, all of which are unknown
and have to be present to achieve activity.  At the same time, it is
difficult to start from a functional cell extract and work by
subtraction because of the confounding activity of other RNA
polymerases, the presence of RNases, etc.  Pol II also has the
irritating property of showing much higher but non-specific activity
towards damaged DNA (e.g. nicked or restriction enzyme cut) than towards
a native template.  Furthermore, the identity of the "native" template
is open to question.  Should it be linear, or supercoiled (if so how
much).  Should it organized into nucleosomes? What about higher order
structWhat about HMG and other chromosomal proteins?

As you can see is a difficult problem.  Work is continuing however,
and it may be solved in our lifetime (hopefully much sooner).  It could
use a few more hands, though, if you are interested?

Michael Gribskov
gribskov at ncifcrf.gov

--- Micheal, if you see this message, thank you for your reply ---

Now, to clearify my questions, first, I have to present some background
information. Even though the purpose of the Polymerase Chain Reaction (PCR)
is to amplify specific pieces of DNA (e.g. a gene), it is also an elegant
way of simulating DNA replication.  Basically, PCR has made the old method
of DNA amplification (i.e. growing it up in bacteria) seem cumbersome and
time consuming in comparison.

So my questions are:

Instead of the crude extracts that are available and the in vivo systems
(i.e. the many bacteria systems and a few eukaryotic systems) that are
available for transcription, isn't it possible to develop a basic in Vitro
system
that would simulate transcription.  Is Manley trying to develop such a
system?  How many years will it take to develop?  Has anyone tried taking
the minimal number of transcriptional components and placing them in a test
tube to see what happens? or are the minimum number of transcriptional
factors (proteins) even known?
All these questions are refering to eukaryotic transcription.

If someone knows the answers to these questions or would just like to comment
on the progress of an in Vitro transcription system please send them to me.

Thanks,

John Pierpont
medical student
University of Arizona

p.s.   Sorry the message is so lengthy.  I tried to keep it as concise as
possible.



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