CSM News Electronic Edition Volume 2, number 8 February 19, 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. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [129.105.233.50], via Gopher at the same address, or by World Wide Web through WWW.acns.nwu.edu. ========== Abstracts ========== 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. ---------------------------------------------------------------------- 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. --------------------------------------------------------------------- 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. -------------------------------------------------------------------- [End CSM-News, volume 2, number 8]