David Francis

Changes in protein synthesis during alternate pathways of differentiation in the cellular slime mold Polysphondylium pallidum.

Abstract Acrylamide gel electrophoresis in the presence of sodium lauryl sulfate and autoradiography of dried gels were used to detect incorporation of [ 14 C]acetate into specific polypeptides of developing cells. Twenty-four polypeptides showed changes in net rate of synthesis during light-induced aggregation and sorogenesis, and 15 bands changed during KCl-induced encystment. Most of the changes during aggregation were common to three strains of P. pallidum , but different from those exhibited by D. discoideum . They failed to occur if the cells were maintained in darkness, and the pattern of changes was altered by sublethal high temperature. A few changes were common to both aggregating and encysting cells, and to stalkforming cells induced by cyclic AMP. A pulse-chase experiment indicated that all detectable increases in net synthesis were caused by alterations in actual synthesis rather than in degradation. It is suggested that the observed changes in synthesis may be essential to the correlated pathways of cellular differentiation.

Evolution of Promoter Sequences: Elements of a Canonical Promoter for Prespore Genes of Dictyostelium

An attempt is made to define a minimal prespore promoter which contains all elements essential for correct regulation of expression of a prespore gene. The prespore genes ofDictyostelium are coregulated during development. Most begin transcription at the same early stage, and activity of all is restricted to prespore tissue during the later slug stage. Sequences 5′ to the coding sequences of eight prespore genes were searched for all elements proposed to control transcription and for new elements. The meaningfulness of occurrences of elements and pairs of elements in prespore promoters was evaluated by comparison with frequencies of occurrences in promoters of other, nonprespore genes. These comparisons resulted in definition of a canonical prespore promoter, a stretch of about 200 nucleotides containing at least one of each of three elements. Certain limitations were found on the spacing of elements. Orientation of elements with respect to each other appeared unrestricted. All elements often occurred in multiple copies. This structure suggests that individual copies of each element are not conserved during evolution, but instead continually appear and disappear.

Heat shock response in a cellular slime mold, Polysphondylium pallidum

Abstract Cells of Polysphondylium pallidum were exposed to a heat shock by raising the temperature from 25 to 31°C. A set of four major polypeptides of approximate molecular weights 105,000, 87,000, 74,000, and 33,000 incorporated [1-14C]acetate when pulse labeled during the first hour after heat shock. The response resembles the heat shock response of Drosophila in occurring in cells at different stages of development (early in aggregation, late in aggregation, and during microcyst formation) and in being triggered by a threshold high temperature rather than a minimal change in temperature.

Synthesis of developmental proteins in morphogenetic mutants of Polysphondylium pallidum

Abstract Changes in labeling of 9 polypeptides were followed during development of 17 morphogenetic mutants using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pulse-labeled cultures. In most cases the change in labeling of a particular polypeptide either occurred normally or failed to occur altogether in a particular mutant, and cases where the change was partial or of a pattern different from the wild type were rare. From a table showing labeling changes in polypeptides during development of the several mutants a diagram was constructed which implies causal relations among the labeling events.

A mutant strain of Polysphondylium pallidum deficient in production of cyclic AMP

Abstract A mutant strain (PN507) of the cellular slime mold Polysphondylium pallidum is described which: (a) is morphogenetically abnormal in stalk formation; (b) secretes unusually low quantities of cyclic AMP; (c) responds to exogenous cyclic AMP in the same manner as wild type, by differentiating stalk cells and synthesizing several specific proteins; (d) complements with other morphogenetic mutants secreting normal amounts of cyclic AMP to produce fruiting structures resembling wild type. The tentative conclusion is that the critical defect of PN507 is low production of cyclic AMP.

An increase of cAMP-dependent protein kinase during development in Polysphondylium pallidum☆

Polysphondylium pallidum is a cellular slime mold in which, unlike in Dictyostelium discoideum, cAMP is not the chemotactic agent. The occurrence of a cAMP-dependent protein kinase in D. discoideum was demonstrated earlier and we suggested that it may mediate the intracellular effects of cAMP on the development of the organism, particularly since an increase in the amount of the enzyme during development was noted. In D. discoideum cAMP plays a dual role insofar as it serves both as chemotactic agent and as second messenger; it was of interest therefore, to determine whether a cAMP-dependent protein kinase occurred in P. pallidum. We found a cAMP-dependent protein kinase in P. pallidum using Kemptide as substrate. The regulatory subunit of the enzyme has an apparent molecular weight of 41,000 and seems to be similar in its properties with that isolated earlier from D. discoideum. The cAMP-dependent protein kinase catalytic subunits from the two species are also similar. Furthermore, there is a developmentally regulated, parallel, two- to threefold increase in the two subunits of the cAMP-dependent protein kinase in P. pallidum. The increase occurs before aggregates are formed. These findings are compatible with a role of the intracellular cAMP and of the cAMP-dependent protein kinase in the development of P. pallidum.