Subject: abstract: Homologous in vitro transcription

From: [email-masked] (ds106)
Date: 12 Sep 1994 10:49:46 -0700

2 abstracts relating to reconstitution of regulated transcription in
homologous mycelial extracts

abstract: in press, Current Genetics

IN VITRO TRANSCRIPTION FROM THE NECTRIA HAEMATOCOCCA PDA1
PROMOTER IN HOMOLOGOUS EXTRACT REFLECTS IN VIVO PISATIN-
RESPONSIVE REGULATION

Yijun Ruan and David C. Straney
Department of Botany and Maryland Agricultural Experiment Station, University
of Maryland, College Park Maryland 20742

The PDA1 gene of Nectria haematococca MP VI (anamorph: Fusarium solani)
encodes pisatin demethylase.  This enzyme detoxifies the isoflavanoid
phytoalexin pisatin, produced by the plant on which this fungus is pathogenic.
Expression of pisatin demethylase activity is induced in mycelium by
pretreatment with pisatin.
 We have developed a homologous in vitro transcription system which accurately
initiates transcription from the PDA1 promoter. Transcription levels in vitro
reflect the same pisatin-responsive stimulation as measured for PDA1 mRNA  in
vivo, and are dependent upon sequences in the 5' upstream region of PDA1.
Pisatin-responsive transcription from the PDA1 promoter indicates that
initiation of transcription is a major regulatory step in pisatin induction of
pisatin demethylase expression.

Another related abstract:  Gene, in press

A PCR-BASED CONSTRUCTION OF PROMOTER/G-FREE TEMPLATES FOR
IN VITRO TRANSCRIPTION ANALYSIS ALLOWS SELECTION OF PLASMIDS
WITH OPTIMAL ACTIVITY IN HOMOLOGOUS EXTRACTS

Yijun Ruan and David C. Straney
Department of Botany and Maryland Agricultural Experiment Station,
 University of Maryland, College Park, MD 20742, USA

     In vitro transcription has been used for dissecting transcriptional
controls in many eukaryotic systems.  One modification which greatly reduces
background non-specific transcription is the placement of a guanosine-free
(G-free) region of DNA immediately downstream of a promoter [Sawadogo and
Roeder, Proc. Natl. Acad. Sci. USA 82 (1985) 4394-4398]; transcription in the
presence of RNase T1 and 3' O-Me-GTP eliminates non-specific transcripts, and
produces the G-free transcripts initiated at the promoter. Restriction
site-based fusion of a G-free cassette downstream of promoters is complicated
by the requirement for G nucleotides to be excluded from the coding strand
downstream of the transcription start points.  We present an approach to add a
G-free template onto a eukaryotic promoter by combining PCR-based termini
construction and terminal deoxynucleotidyl transferase extension. The pisatin
demethylase promoter (PDA1p) of the filamentous fungus Nectria haematococca,
was used as the test promoter.  Three PDA1p/G-free constructs were tested in
heterologous Drosophila melanogaster and HeLa and homologous N. haematococca
transcription extracts. Each extract produced a PDA1p-specific transcript from
each construct, but the relative level of transcription between constructs
varied, particularly in the homologous extract. Since the choice of G-free
sequence influences transcription differently among systems, this method for
producing multiple G-free constructs should be useful for constructing and
selecting optimal promoter/G-free templates for in vitro transcription in
other homologous systems.