RNA IN CYTOPLASMIC AND NUCLEAR FRACTIONS OF CELLULAR SLIME MOLD AMEBAS

A method is described for the rapid separation of cellular slime mold (Dictyostelium discoideum) cells into nuclear and cytoplasmic fractions. Sucrose density sedimentation profiles of radioactivity from cells that had been grown for long or short periods in the presence of uridine-³H indicate very low levels of cross-contamination between the fractions. The nuclear fraction contains few, if any, ribosomes. In exponentially growing cells, at least 80% of the ribosomes were associated in polysomal complexes. No loss of counts from pre-labeled rRNA was observed during 2 generations (24 hr) of logarithmic growth and, within the polysomal complexes, the distributions of the preformed material and of rRNA synthesized during the 2 generations were identical. In stationary phase cells that had entered the developmental program leading to fruiting body construction, the rRNA turned over rapidly so that by the end of development at least 75% of the ribosomes fabricated during exponential growth had disappeared and had been replaced by new ones synthesized during the morphogenetic sequence. The preformed ribosomes disappeared preferentially from the monosomal contingent; the newly synthesized ribosomes appeared exclusively in the polysomal contingent and did not appear as monosomes in appreciable numbers for at least 6 hr. The possible significance of this wholesale replacement of ribosomes is discussed.     

An increase of calcium/manganese binding sites in cell ghosts associated with the acquisition of aggregative competence in Dictyostelium discoideum.

In Dictyostelium discoideum, the formation of multicellular aggregates represents the first morphogenetic event that leads ultimately to the construction of fruiting bodies. The altered adhesive properties of the cells can be demonstrated in ghosts derived from them which consist of largely intact membranes containing a few empty vesicles and exploded mitochondria but lacking nuclei, RNA, soluble cytoplasm and ATP [4]. A cofactor requirement for the aggregation of the ghosts can be satisfied by the following divalent cations: Ca²⁺, Mn²⁺, Zn²⁺ and Cu²⁺. In this paper it is shown that associated with the acquisition of aggregative competence is a 15–20-fold increase in the ghosts of sites capable of binding either Ca²⁺ or Mn²⁺ with relatively high affinity.     

Ultrastructural Studies of Microconidium Formation in Neurospora crassa

Microconidiating cultures of “peach-fluffy” (pe, fl; Y8743m, L; FGSC #569) were fixed at various times after the initiation of growth and examined with an electron microscope. Hyphae from which microconidia form are markedly vacuolated and show a much more extensive system of rough endoplasmic reticulum than young vegetative hyphae. A bulge in the hypha presages the start of microconidium formation, followed by the rupture of the outermost wall layers. A thick collar forms around the protruding microconidium due to extensive thickening of the inner wall layer of the parent hypha. At this stage, the cytoplasm of the developing microconidium is still continuous with that of the microsporophore cell from which it arises and is contained by a wall which is derived from the thickened collar. The microconidium is finally isolated from the cytoplasm of the microsporophore by a centripetal extension of the collar. Microconidia differ from macroconidia in having a more extensive endoplasmic reticulum and fewer mitochondria, in addition to being smaller and having a single nucleus.     

Tropomodulin binding to tropomyosins. Isoform-specific differences in affinity and stoichiometry.

Tropomodulin is a human erythrocyte membrane cytoskeletal protein that binds to one end of tropomyosin molecules and inhibits tropomyosin binding to actin filaments [Fowler, V. M. (1990) J. Cell Biol. 111, 471-482]. We have characterized the interaction of erythroid and non-erythroid tropomyosins with tropomodulin by non-denaturing gel electrophoresis and by solid-phase binding assays using 125I-tropomyosin. Non-denaturing gel analysis demonstrates that all tropomodulin molecules are able to bind tropomyosin and that tropomodulin forms complexes with tropomyosin isoforms from erythrocyte, brain, platelet and skeletal muscle tissue. Scatchard analysis of binding data using tropomyosin isoforms from these tissues indicate that tropomodulin binds preferentially to erythrocyte tropomyosin. Specificity is manifested by decreases in the apparent affinity or the saturation binding capacity of tropomodulin for non-erythrocyte tropomyosins. Erythrocyte tropomyosin saturates tropomodulin at approximate stoichiometric ratios of 1:2 and 1:4 tropomyosin/tropomodulin (apparent Kd = 14 nM-1 and 5 nM-1, respectively). Brain tropomyosin saturates tropomodulin at a 1:2 ratio of tropomyosin/tropomodulin, but with a threefold lower affinity than erythrocyte tropomyosin. Platelet tropomyosin saturates tropomodulin at a tropomyosin/tropomodulin ratio of 1:4, but with a sevenfold lower affinity than erythrocyte tropomyosin at the 1:4 ratio. These results correlate with oxidative cross-linking data which indicate that tropomodulin can self-associate to form dimers and tetramers in solution. Since tropomodulin interacts with one of the ends of tropomyosin, varying interactions of tropomyosin isoforms with tropomodulin probably reflect the heterogeneity in N-terminal or C-terminal sequences characteristic of the different tropomyosin isoforms. Isoform-specific interactions of tropomodulin with tropomyosins may represent a novel mechanism for selective regulation of tropomyosin/actin interactions.     

Crystallization of a DNA tridecamer d(C-G-C-A-G-A-A-T-T-C-G-C-G)

Crystals of the DNA tridecamer d(C-G-C-A-G-A-A-T-T-C-G-C-G) have been grown by the vapor-diffusion technique with 2-methyl-2,4-pentanediol as precipitant. They are monoclinic space group C2, with a = 79.6 A, b = 43.1 A, c = 24.9 A and beta = 98.7 degrees. Previous nuclear magnetic resonance studies predicted that this tridecamer forms a duplex similar to the B DNA dodecamer, d(C-G-C-G-A-A-T-T-C-G-C-G), except for an extra adenosine residue that is stacked within the helix but remains unpaired: (formula; see text) Preliminary X-ray diffraction studies confirmed that the tridecamer is in the B DNA conformation, consistent with the nuclear magnetic resonance results.