P9323611%ARIZRVAX@PUCC.PRINCETON.EDU (02/21/91)
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@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.