Renzo Nobili

Requirement of protein synthesis in the initiation of meiosis and other nuclear changes in conjugation of Blepharisma

Abstract In conjugation of Blepharisma japonicum, cell contact between complementary mating types induces meiosis and other nuclear changes. How long the cells must be in contact in order to be induced to undergo these nuclear changes (activated) can be ascertained by surgically separating the united cells at different times after the onset of cell union and then examining the occurrence of the nuclear changes. Applying this technique to cycloheximide-treated cells, we investigated the role of protein synthesis in the activation. Cycloheximide was used at the concentration which was found to inhibit most incorporation of amino acid into protein in this ciliate. Newly formed conjugant pairs were incubated with and without cycloheximide, washed free of the inhibitor and surgically separated. Although untreated controls were activated in 1.8 h after cell-cell contact, no activation was observed in cycloheximide-treated cells after 5 h of contact. Removal of cycloheximide from the paired cells resulted in an activation delayed by the interval of exposure time to the inhibitor. If the pairs were first incubated in normal medium and then exposed to cycloheximide, operated, activated cells appeared and increased very slowly (activation rate, about 1 10 of the control). Protein synthesis is therefore required for the initiation of meiosis and other nuclear changes. We propose that heterotypic cell union induces and maintains the synthesis of a protein, whose accumulation to a certain threshold is required for activation.

Propagation of meiosis and other nuclear changes in multicellular complexes of Blepharisma

Summary Meiosis and other nuclear changes in conjugation of Blepharisma japonicum regularly occur when cells of complementary mating types I and II unite (heterotypic union). But no nuclear changes occur if unions are induced between cells of the same mating type (homotypic union). Similarly, chains of homotypically united cells, induced by treating type II cells of a doublet strain (mutant with two attachment points) with gamone of mating type I, do not undergo nuclear changes. However, if a type I cell unites at one end of such a chain, the nuclear changes of conjugation occur not only in the doublet to which the type I cell unites but also in other doublets in the same chain. We examined the mode of propagation of nuclear activation by surgically separating all cells in the chain and observing the subsequent occurrence of nuclear changes in these isolated cells. The nuclear activation began at the site of heterotypic union and propagated from cell-to-cell without skipping. In chains of a given length, the propagation slowed down as it proceeded in the chain. If compared at the corresponding site of the chain, the propagation was slower in longer chains. We conclude that meiosis and other nuclear changes in conjugation are initiated by a substance originating at the site of heterotypic union and transferable to other cells through the united regions of the cells.

Chacterization of macronuclear DNA inBlepharisma japonicum

SummaryThe nuclear apparatus of the ciliateBlepharisma japonicum (Spirotricha, Heterotricha) consists of one macronucleus, presiding over the phenotypic expression, and a variable number Of micronuclei (from 6 to 30) with a germinal function.Blepharisma, as all other ciliates, can reproduce asexually through binary fission and sexually through conjugation. Its morphology, behaviour and reproductive biology are well known, whereas any information pertaining the biochemical and molecular structure of nuclei is lacking. In the research work of sexual differentiation ofBlepharisma the problem rose about the formation and differentiation of its macronucleus during conjugation.A preliminary analysis of macronuclear DNA in vegetative cells was carried out by different physical-chemical parameters.The agarose gel electrophoretic pattern of macronuclear native DNA shows the presence of a unique band with a high molecular weight: this fact seems to avoid the presence of DNA fragmentation during macronuclear differentiation, that on the contrary, occurs in other ciliates, such as Hypotrichs.CsCl sedimentation profiles show a peak with buoyant density 1,692 g/ml, while Hoechst 33258/CsCl ultracentrifugation separates a light fraction (17.5% of the total DNA) from the main peak.Thermal denaturation profiles support the presence of a A-T rich family; the thermal transition interval has a low value (Δt=5.3) in comparison to other Eukaryotic DNAs. Reassociation kinetics analysis indicates that 3–4% of DNA reassociates at Cot practically equal to 0 and the remaining fraction at a rate corresponding to single copy sequences. After digestion with restriction enzymes the electrophoretic pattern does not show the presence of highly repetitive sequences.These findings alignBlepharisma japonicum to the close heterotrichousStentor and toParamecium andTetrahymena for the presence of a single banded macronuclear DNA of high molecular weight.

Contributo all'ecologia dei ciliati psamrnofili del Golfo di Napoli

Summary In sand samples of different beaches of the Gulf of Naples 57 ciliate species have been found. There is no difference between sand-dwelling ciliates of the tidal zone, or deeper along the seashore, when sand granulometry is the same. Further more, it was noticed that the interstitial spaces, which are roughly proportional to the size of the sand grains, limite the distribution of microporal fauna, showing therefore qualificative selection. On the contrary, the water movement and food quantity determine the abundance of species and specimens in certain places.

Il comportamento del Kappa in esemplari amacronucleati di Paramecium aurelia, Stock 51, syngen 4

Summary The behavior of kappa in amaeronueleate killer parameeia has been studied It has been found the maturation of B partieles goes on regularly in amaernueleates, but their increase is lower than thr expected one in normal animals. further the increase coefficient, obtained by dividing the average of mature B partieles after 20 hours by the average of mature B partieles after 5 hours of normal individuals, seems to be associated with the amount of mature B partieles present in the animal at its orgin. Amacronuclesates with only N partieles never produced B; the increase coefficieng was found to vary from 1.95 in clones with an average killing activity of 0.48 per animal, to 3.74 in clones with an average killving activity of 2.5, to 5.5 in a clone with normal killing activity of 3.74 to 5.64 in clone with a supernormal killing acivity of 15.92. From these data it has been deduced that N partieles do not transform into B, as it is the rule if normal paramecia, because of the lack of macronueleus. There...

PRELIMINARY RESULTS ON RNA SYNTHESIS DURING CELL UNION ACTIVATION IN BLEPHARISMA JAPONICUM SUZUKI (PROTOZOA CILIATA)

SUMMARY Very little is known at biochemical level of the mechanisms inducing cell union and other related phenomena of conjugation in Blepharisma japonicum Suzuki (Protozoa Ciliata). In an attempt to demonstrate possible differences between vegetative and pairing cells, a study of RNA synthesis and of its in vitro translation has been started. The results so far obtained indicate a net increase of both RNA poly A+ and RNA poly A- synthesis in cells treated with complementary gamone compared with non-treated cells.

CELL SURFACE CONTROL IN BLEPHARISMA INTERMEDIUM BHANDARY (PROTOZOA OLIATA)

SUMMARY Gamone II binds to Blepbarisma intermedium Bhandary (Protozoa Ciliata) mating type I cells, via specific membrane receptors which are distinct from the receptors of PHA, Con-A, antitubulin antibodies, and other control ligands. Binding of PHA, Con-A and ATA induces a non- specific series of events, which, according to the dose of ligand may lead to cell immobilization, agglutination, loss of pigment and lysis. Binding of gamone II induces pair formation only via a specific activation receptor system. The finding of distinctive binding sites for different unrelated ligands on Blepharisma cell membrane will be useful in elucidating the mechanism of gamone-cell interaction.

Cell recognition during preconjugant cell interaction in ciliated protozoa

Abstract A brief survey of the modes Ciliates recognize each other while undergoing sexual reproduction is presented. The phenomena involved in such recognition can be considered “quasi-immune” in their basic mechanisms, which imply a self-non self recognition process. The role of membrane and membrane-associated receptors in this process is briefly discussed. A general model, including a “humoral” phase and a “cellular” phase of recognition is outlined.