New Product for Gene Regulation Studies - FREE!

Simon Sims ssims at genosys.com
Tue Jan 6 17:33:18 EST 1998


Genosys Biotechnologies, Inc. has developed a research kit for studying
eukaryotic promoter activity, in vitro. For a limited period, Genosys is
offering this product free-of-charge to labs that wish to evaluate the kit
and provide us with feedback. If you are studying gene promoter activity,
simply clone you promoter into one of the "RiboCOP" plasmids. The circlular
plasmids are used in a transcription assay using HeLa nuclear extract and
radioactively labeled rNTPs. The gene promoter initiates transcription of
"ribozyme" sequences within the plasmid. The Ribozyme transcript
self-cleaves to generate a labeled RNA fragment of predictable size. If
interested, please reply to this message. More details of the kit are given
below.


Overview:
The transcription of genes is regulated by a “promoter” region, upstream of
the gene sequence. The promoter DNA domain consists of a number of
cis-acting transcription factor binding sites or “sequence motifs”. Studies
of promoter regulation of transcription typically involve defining the DNA
sequences necessary for the specific expression of a particular gene. These
studies may be performed in vitro or in vivo. In vitro studies of mammalian
transcription may involve testing the activity of a promoter in a
transcriptionally competent protein extract derived from the nuclei of HeLa
cells. Such nuclear extracts contain the essential nuclear components for
accurate transcription of mRNAs by RNA polymerase II. A typical in vitro
transcription assay consists of a HeLa nuclear extract, labeled rNTPs,
appropriate salts and buffers, and a linearized plasmid containing the
promoter to be studied. The promoter initiates transcription, incorporating
the labeled nucleotides into the growing RNA chain. As the plasmid is
linear, the RNA polymerase falls of the end of the template DNA, resulting
in a labeled RNA transcript of defined size. These analyses are called
run-off transcription reactions. The labeled RNA transcripts are analyzed by
polyacrylamide gel electrophoresis followed by autoradiography.
Transcription from a linear template is less efficient than from a circular,
torsionally constrained template. However, in vitro transcription of
circular templates is challenging as mammalian transcription termination is
not well defined. Transcription initiated on circular plasmids continues
around the template without defined termination. The resulting products
generate a smear after electrophoretic separation, whereas transcripts from
a linear template give a distinct sized fragment. The pRiboCOP plasmid was
designed to allow transcription from a circular DNA template, giving
transcripts of a defined size.

Product Description:
The RiboCOP In Vitro Transcription System from Genosys was developed to
allow analysis of mammalian promoters, driven by RNA polymerase II. First,
the promoter of interest is cloned upstream of the ribozyme in one of the
ampicillin-resistant plasmids provided. The resulting plasmid is then tested
for transcriptional activity with the HeLa nuclear extract provided. The
advantages of this system include: 1) the assay is performed with a circular
template, rather than a transcriptionally less efficient linear template, 2)
the resulting transcripts are self-cleaved by the ribozyme structure, giving
products of a defined size for simple analysis by PAGE, 3) plasmids are
provided that generate different sized transcripts, allowing more than one
promoter to be distinguished in one reaction, and 4) the multiple cloning
site contains a wide selection of restriction sites that enable easy
generation of exonuclease III-based nested deletions of a cloned promoter.
The pRiboCOP plasmids are named according to the approximate length of the
transcript generated. The “actual” length of the transcripts will depend on
the location of the transcription initiation site of the cloned promoter.
The kit includes positive control plasmids that contain the human CMV
immediate early promoter upstream of the ribozyme.

Features:
Allows in vitro transcription from a circular DNA template. Circular
templates transcribe more efficiently than linear templates.

Transcripts from the circular template are self-cleaved by the ribozyme
structure, yielding products of defined size(s).

Extensive range of restriction sites in “multiple cloning site” for cloning
purposes.

Multiple cloning site contains useful sites that generate 5’- and 3’-
overhangs for exonuclease-based deletion of either end of putative promoter
fragments – valuable for defining minimal promoter elements.

Kit contains positive control DNA template.


Applications:
    Define basal or transcription factor regulated transcription from a
putative
    promoter.

    Perform 5’ and/or 3’ deletions of promoter to define motifs required for
    transcription.

    Assay for promoter strength.

    Map mRNA transcription initiation sites.

    Determine transcription factor involvement through add-back or
    complementation assays.






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