CSM News
Electronic Edition
Volume 2, number 20
June 4, 1994

Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu.

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through www.nwu.edu.

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Abstracts
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Regulation of Expression of the Cyclic Nucleotide Phosphodiesterase Gene
in Phosphodiesterase Inhibitor-negative Mutants of Dictyostelium discoideum

Neil R. Adames, M. Barrie Coukell and Lin Wu#

Department of Biology, York University, North York, Ontario, M3J 1P3
#Department of Anatomy and Cell Biology, College of Physicians & Surgeons,
Columbia University, New York, NY, 10032

Biochem. Cell Biol. - in press

Summary
	During early development of Dictyostelium discoideum, the enzyme
cyclic nucleotide phosphodiesterase (PD) is produced at a low rate during 
the period its specific inhibitor (PDI) is being synthesized.  In addition,
PD gene expression is derepressed in the aggregation-deficient (Agg-), Pdi-
mutant HC35.  These observations suggest that the PDI might function to 
regulate PD gene expression as well as modulate its activity.  To explore 
this idea further, five new Agg-, Pdi- mutants were isolated and analyzed.
All of the mutants produced high PD activity and overexpressed PD mRNA; 
four exhibited elevated levels of the 2,400-nt aggregative transcript, and
one overproduced the 1,900-nt vegetative transcript.  In contrast, PD 
transcripts were not elevated in two Agg-, Pdi+ mutants.  To determine if
PDI production regulates PD expression, HC35 cells were transformed with 
plasmids carrying the PDI structural gene under the control of either the
vegetative or aggregative PD promoter.  Neither expression of PDI by the 
transformants nor addition of partially purified PDI to HC35 cells affected PD 
transcription.  These results suggest that PD overexpression in the Pdi-
mutants is not a direct consequence of the inability of these cells to 
produce inhibitor.

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Serine-113 is the Site of Receptor-Mediated Phosphorylation of
the Dictyostelium G protein `-Subunit, G`2

Mei-Yu Chen@, Peter N. Devreotes@ and Robert E. Gundersen#

@ Department of Biological Chemistry, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205-2185

# Department of Biochemistry, Microbiology and Molecular
Biology University of Maine, Orono, Maine 04469-5735

J. Biol. Chem., in press.

SUMMARY

  The G protein `-subunit, G`2, is essential to the developmental
program of Dictyostelium.  G`2 is transiently phosphorylated on a
serine residue(s) following stimulation with extracellular cAMP
(Gundersen, R.E.  and Devreotes, P.N.  (1990) Science 248, 591-593).
To aid in defining the function of `-subunit phosphorylation, we
identified the site of G`2 phosphorylation.  Comparison of the
isoelectric points (pI) of the phosphorylated and nonphosphorylated
forms indicated that a single mole of phosphate is added to G`2.
Cleavage at tryptophan residues and immunoprecipitation with a
specific peptide antibody localized the phosphorylated serine in the
N-terminal 119 residues.  Analysis of a series of G`1 and G`2
`-subunit chimeras further confined the site between amino acids 33
and 215.  Site-directed mutagenesis of serines between amino acids 33
to 119 produced two mutants that were not phosphorylated, S45A and
S113A.  S113 was identified as the site by sequential Edman
degradation of 32P-radiolabeled G`2 digested with endoproteinase
Glu-C.  We have expressed the G`2 mutants S113A, S113I, S113T, and
S113D in a G`2 null cell line to examine the function of
phosphorylation.

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REMI-RFLP Mapping in the Dictyostelium Genome
	
Adam Kuspa*, and William F. Loomis

Center for Molecular Genetics, Department of Biology,
University of California, San Diego, La Jolla, CA 92093-0322
*Present address: Department of Biochemistry, Baylor College of Medicine,
One Baylor Plaza, Houston, TX 77030

Genetics, in press.

ABSTRACT

   A set of 147 Dictyostelium discoideum strains was constructed by
random integration of a vector containing rare restriction sites.  The
strains were generated by transformation using restriction enzyme
mediated integration (REMI) which results in the integration of linear
DNA fragments into randomly distributed genomic restriction sites.
Restriction fragment length polymorphism (RFLP) was generated in a
single genomic site in each strain.  These REMI-RFLP strains were used
to confirm gene linkages previously supported by two other physical
mapping techniques: Yeast Artificial Chromosome (YAC) contig
construction, and megabase-scale restriction mapping.  New linkages
were uncovered when two or more hybridization probes identified the
same RFLP fragments.  Probes for 100 genes have marked 53% of the
RFLPs, representing greater than 22 Mb of the 40 Mb Dictyostelium
genome. Alignment of these and other large fragments along each
chromosome should lead to a complete physical map of the Dictyostelium
genome.

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