CSM News
Electronic Edition
Volume 5, number 6
August 26, 1995

Please submit abstracts of your papers as soon as they have been
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===========
 Abstracts
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TRAPPING DEVELOPMENTAL PROMOTERS IN DICTYOSTELIUM

Wen-Tsan Chang, Julian D. Gross and Peter C. Newell

Department of Biochemistry, University of Oxford, South Parks Road,
Oxford OX1 3QU,UK

PLASMID, in press.

Summary

   We have modified the REMI procedure to permit characterisation of
the promoters and structural sequences of genes that would be missed
by the standard procedure because their disruption produces no obvious
phenotype.

   A vector (designated pPTGal) carrying a promoter-less E. coli lacZ
gene was constructed so that expression of lacZ requires insertion
into an active host transcription unit.  The construct also contains a
multiple cloning site (MCS-2) adjacent to the URA gene which permits
very efficient restriction enzyme-mediated excision and rescue in
E. coli of the 5' region of the inserted vector (excluding the URA
gene) together with a segment of 5'-flanking genomic DNA.  [Note that
with vectors lacking such an internal multiple cloning site, gene
rescue requires cutting on both sides of the insertion site, (as for
normal REMI).  However, in the case of lacZ-containing vectors, very
large plasmids are thereby formed that contain (besides the inserted
flanking DNA) the complete vector together with the lacZ and URA
genes.  Although we obtained a number of lacZ-expressing Dictyostelium
transformants with such vectors lacking the MCS-2 site, rescue of the
plasmids with flanking DNA in E. coli did not prove possible, possibly
due to the size of the resulting DNA fragments].

   Using the pPTGal vector we have isolated a series of strains in which
lacZ is under the control of developmentally activated promoters,
including prestalk and prespore cell specific promoters and promoters
showing more complex patterns of lacZ activity.  We have cloned the 5'
flanking DNA adjacent to the insertion site of three of the
isolates. Sequencing of the junction between plasmid and host genome
has confirmed in-frame fusion with the lacZ gene, and reintroduction
of the cloned plasmids into parental cells has shown that the cloned
sequences do actually contain the relevant promoters.

   Not only should this technique be able to detect important
developmental genes which are present in more than one copy, or whose
function is performed by more than one gene product, it should also
give easy access to a wide range of developmentally controlled
promoters without the need to have previously identified the cell type
specific or developmental genes being regulated.  It should, for
example, facilitate the characterization of distinct classes of
promoter sequences that are controlled by different intercellular
effectors such as cyclic AMP, DIF-1 and ammonia. 

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