CSM News
Electronic Edition
Volume 2, number 8
February 19, 1994

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Abstracts
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The promoter of a gene encoding a novel Dictyostelium spore coat
protein

Bradley K. Yoder and Daphne D. Blumberg

Department of Biological Sciences, University of Maryland Baltimore
County Baltimore, Maryland, USA 21228

Developmental Biology, in press.

Summary

   The cAMP inducible prespore gene, PL3, encodes a protein which is a
novel component of the spore coat. Unlike the well characterized spore
coat proteins (SP60, SP70 and SP96) which are found in the outer layer
of the coat, the PL3 gene product is localized to a subregion of the
coat beneath the outer proteinaceous layer. Moreover a substantial
portion of the PL3 protein is tightly associated with the spore coat
and not released under the conditions that led to the identification
of the other coat proteins. The promoter for this novel spore coat
gene is described. Unlike the other coat protein gene promoters, it
lacks the extensive CA type elements. It contains two short CA boxes
and five prominent G rich regions. Sequential deletions from the 5'
end of the promoter which remove both CA boxes as well as two of the G
rich regions reduce the level of expression but do not alter the
spatial regulation of expression. Despite the sequence differences,
the PL3 promoter still confers correct spatial, temporal and cell type
specific regulation on a reporter gene.  E.  coli FE-galactosidase
enzyme activity expressed under the control of this PL3 promoter first
appears in randomly isolated cells at the loose mound stage. Because
of the sensitivity of the assay, FE-galactosidase activity is
detectable prior to the appearance of the PL3 protein on western blots
and by immunofluorescence. Later the number of cells staining for
FE-galactosidase activity and the intensity of staining increases.
During tipped mound, slug and culminant stages, cells expressing
FE-galactosidase under the control of the PL3 promoter are localized
to prespore regions and are spatially coincident with cells expressing
the PL3 protein.

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IDENTIFICATION OF A NEW SPORE COAT PROTEIN GENE IN THE CELLULAR SLIME
MOLD DICTYOSTELIUM DISCOIDEUM


Bradley K. Yoder1, Jie Mao2, Gregory W. Erdos3, Christopher M. West2
and Daphne D. Blumberg1

1Department of Biological Sciences, University of Maryland Baltimore
County Baltimore, Maryland 21228 and 2Department of Anatomy and Cell
Biology, Health Sciences Center, University of Florida College of
Medicine, Gainesville, Florida 32610-0235 and 3Department of
Microbiology and Cell Science, Institute for Food and Agricultural
Sciences, University of Florida, Gainesville, Florida 32611

Developmental Biology, in press.

Summary

      Genomic DNA encoding the prespore cell specific PL3 cDNA was
cloned and sequenced, revealing that the gene consists of three exons
separated by short 100 base pair introns. The single long open reading
frame predicts a primary translation product of 70kD after removal of
a cleavable signal peptide, two- thirds of which consists of four
kinds of amino acid repeat elements, including two found in other
spore coat proteins. The 85kD PL3 protein synthesized in vivo
accumulates specifically in regulated secretory vesicles of prespore
cells (prespore vesicles), as determined microscopically using
antibody against a PL3 gene fusion product expressed in E. coli. The
protein later accumulates extracellularly in the spore coat, which is
formed during sporulation, as determined ultrastructurally and by
Western blot analysis of SDS- PAGE gels. In addition to its high
proportion of repeat elements, the PL3 protein has the following
properties which distinguish it from other spore coat proteins: 1) it
is located at the outer extent of the middle layer, beneath the outer
layer, 2) its dissociation from the coat requires the presence of
protein denaturants and reducing agents at elevated temperature and 3)
a large proportion of the protein is not dissociated from the coat
even under these conditions, as determined by ultrastructural analysis
of the extracted coat. The PL3 protein may contribute to the structure
of the coat at the interface between the middle, cellulosic layer and
the outer, electron-dense, proteinaceous layer.

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ANALYSIS OF A NOVEL CYCLIC AMP INDUCIBLE PRESPORE GENE IN
DICTYOSTELIUM DISCOIDEUM: EVIDENCE FOR DIFFERENT PATTERNS OF cAMP
REGULATION

Ameeta Agarwal, Marcia S. Sloger, Masakazu Oyama* and Daphne D.
Blumberg

Department of Biological Sciences, University of Maryland Baltimore
County, Baltimore, Maryland, USA, 21228 and *Laboratory of Biology,
Faculty of Science, Himeji Institute of Technology, Shosha 2167 Himeji
671-22, Japan20

Differentiation, in press.

Summary

      The D7 cDNA clone hybridizes to a 2.8 Kb mRNA which first
appears at the mound stage of development in the cellular slime mold
Dictyostelium discoideum.  This gene which is cAMP inducible and is
expressed specifically in the prespore cells contains an open reading
frame interrupted by only one intron. The predicted amino acid
sequence indicates a novel prespore protein which differs from all of
the previously described prespore proteins in that it contains no
internal repeats and does not share any homology with any of the other
prespore genes. The amino acid sequence predicts a protein of 850
amino acids with a molecular weight of 95,343 daltons and an
isoelectric point of 4.25. The protein is very rich in glutamine
(13.8%), asparagine (10.6%) and glutamic acid (10.4%) with one
potential glycosylation site and 28 possible sites for
phosphorylation.  The amino terminus is hydrophobic with
characteristics of a signal sequence while the entire carboxyl half of
the protein is notable for its hydrophilicity.  Comparison of cAMP
regulation of the D7 gene with the regulation of two other cAMP
regulated prespore genes, the PL3(SP87) gene and the Psa(D19), reveals
some striking differences. Disaggregation in the presence of cAMP
results in transient degradation of mRNA for all three genes. The
transcription rate for the D7 and PsA(D19) genes remains relatively
unaffected by disaggregation but there is a rapid although transient
decline in the transcription rate of the PL3 (SP87) gene.  Although
the accumulation of all three mRNAs is first detectable at mound
stage, transcription of the D7 and PsA(D19) genes is detected earlier
in development, at rippling aggregate stage several hours prior to the
earliest time when transcription of the PL3(SP87) gene is detected.
Analysis of the promoter region of the D7 gene reveals 3 CA like boxes
flanked by direct repeats as well as 4 G rich regions that may serve
as regulatory elements.
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[End CSM-News, volume 2, number 8]