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Physarum amoebae possess an extranuclear spindle-organizing centre (abbreviated SPOC), located in a typical centrosome with a pair of associated centrioles while plasmodia possess an intranuclear SPOC without centrioles. In order to ascertain whether, during conjugation, the plasmodial SPOC is derived from the amoebal one or is not related to it, we have constructed amoebal strains possessing two and three SPOCs and we have used as a genetic marker the frequency of polycentric metaphases in order to evaluate the number of SPOCs in the plasmodia. The results of both symmetrical crosses, i.e. between amoebae possessing the same number of SPOCs, and asymmetrical crosses, i.e. between amoebae possessing a different number of SPOCs, show that: (1) the number of SPOCs in plasmodia is dependent upon the number of SPOCs in either one of the two parental amoeba; (2) in no cross does the number of plasmodial SPOCs equal the sum of the parental amoebal SPOCs, but it corresponds to that of only one parent without any polarity of transmission in asymmetrical crosses. These results are consistent with the following model: (1) plasmodial SPOCs are derived from the amoebal ones; and (2) one set of parental SPOCs is lost, destroyed or inactivated in the zygote.

The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cytoskeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodium-specific beta 2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit beta 2-tubulin at earlier or only at later stages of development. Amoeba-specific acetylated alpha 3-tubulin disappears gradually during development. Individual cells differ in the timing of loss of this isotype: alpha 3-tubulin is present in the majority of uninucleate cells, in a fraction of binucleate and quadrinucleate cells, and is absent from larger multinucleate cells. Cytoplasmic microtubules in uninucleate cells are organized by a single microtubule-organizing center (MTOC) juxtaposed to the nucleus. Binucleate cells and quadrinucleate cells exhibit variable numbers of MTOCs. Cytoplasmic microtubules persist during the APT until the stage of plasmodia containing at least 100 nuclei. The lack of a strict correlation between the changes in tubulin composition and changes in organization of microtubular structures indicates that accumulation of beta 2-tubulin and disappearance of alpha 3-tubulin isotypes are not sufficient to bring about reorganization of microtubules during development. Individual cells in a developing population differ not only in the succession of accumulation and loss of developmentally regulated tubulins, but also in the sequences of other cellular changes occurring during the APT.

Hydroxyurea, at concentrations of 40-60 mM, selectively and effectively blocked incorporation of thymidine into DNA. Inhibition occurred within 5-10 min of application of the agent when DNA synthesis was in progress, while the onset of replication at the beginning of S-phase and DNA synthesis in G2 phase were not affected. Uridine incorporation into TCA-precipitable material, in the presence of hydroxyurea, was significantly (up to 70%) inhibited in early S-phase of the cell cycle. Selective inhibition of RNA synthesis was confirmed for RNA separated into rRNA-rich and poly(A)-rich RNA fractions and analysed by the 2 kinds of DNA-RNA hybridization reactions. Uridine incorporation into poly (A) RNA was also inhibited under conditions where cycloheximide prevented maturation of nascent DNA molecules in early S-phase. We assume that chromatin which is replicating early DNA sequences may be a more competent template for transcription.

Possible mechanisms coordinating the control of mitosis and DNA synthesis with growth were experimentally tested in Physarum polycephalum by the response of plasmodia to 2 different kinds of perturbation of the DNA:mass ratio. Mitosis and DNA synthesis were delayed without stopping growth either by the use of fluorodeoxyuridine (FUdR) or puromycin, in both cases the delayed mitosis was followed by a single shortened intermitotic period, as predicted by all the mechanisms considered and substantiating a basic assumption made that the time of mitosis is homeostatically controlled to maintain a constant DNA:mass ratio. When more than 50% of the DNA was destroyed by ultraviolet irradiation 2 consecutive mitoses could occur even when growth was completely stopped by starvation. This result can only be accounted for by 2 of the 5 models considered, a finding which agrees with the results of previous experiments which were also consistent with only these 2 mechanisms.

The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence with the constitutively expressed beta 1B-tubulin and the myxamoeba-specific beta 1A-tubulin isotypes. A polyclonal antibody specific for beta 2-tubulin was used to monitor the subcellular distribution of the beta 2-tubulin antigen in the mitotic spindle of the mature plasmodium - the sole microtubular array in that stage of Physarum. By immunofluorescence, the beta 2-tubulin antigen was detected throughout this anastral mitotic spindle, at all stages of mitosis. Physarum myxamoebae contain astral mitotic spindles and cytoskeletal microtubules. No beta 2-tubulin antigen was detected in the myxamoebal stage. However, as cultures of myxamoebae developed into plasmodia, the beta 2-tubulin antigen was found in the astral mitotic spindles and cytoskeletons in developing cells. Thus, the presence of the plasmodial beta 2-tubulin isotype in a mitotic spindle does not determine a closed, anastral mitosis.

Nuclear DNA content and ploidy have been determined at different stages of the life cycle of the Colonia strain of the myxomycete Physarum polycephalum. Analyses at the plasmodial stage showed that (a) Burton and Fuelgen DNA analyses agreed within 15% with strains which ranged from 0-6 to 3-6 pg of DNA per nucleus; (b) S-phase in Colonia is during the early part of interphase as in the Wisconsin strain; (c) in heterothallic and heterothallic x Colonia crossed strains there are 1-0-1-2 pg of DNA and 70 chromosomes per nucleus and in Colonia 0-6 pg of DNA and 40 chromosomes. Germinating spores of all strains contained one population of cells with about 0-5 pg of DNA and 40 chromosomes and another of larger cells with up to 2-5 pg of DNA and 200 chromosomes. The polyploid nuclei comprised 2-20% of the total in heterothallic strains, 2-65% in heterothallic x Colonia crosses and 25-75% in Colonia. A method was devised for making chromosome spreads of amoebae grown on bacterial lawns. Cells were first exposed to dilute formaldehyde at 26 degrees C for 30 min, then spread on slides with hot lactic acid and strained. Such spreads of CLd (Colonia) and RSD4 (heterothallic) amoebae both contained about 40 chromosomes. The data are consistent with the view that Colonia is haploid throughout its life cycle and that chromosome number is neither halved during sporulation nor doubled during plasmoidal formation. However, the possibility exists that an alternance of ploidy occurs by way of the few diploid nuclei present in the plasmodium.