H. G. Schweiger

Cytoplasmatische RNS-synthese in kernlosen acetabularien☆

Abstract According ti Richter s experiments we found, that under “normal” conditions after removal of the nucleus, Acetabularia are no longer able to increase substantially their RNA-content. But removing the nucleus of such plants which have been kept in darkness for ten days does not abolish the ability of RNA-net-synthesis. The extent of incorporation of [ 32 P]orthophosphate into the nucleotides of RNA is indicating that de novo-synthesis of RNA takes place. The following conclusions have been drawn: 1. 1. Cytoplasma is able to synthesize RNA. This ability has been demonstrated in anucleate cells. 2. 2. Cytoplasmic RNA-synthesis is nucleus-dependent. Substances promoting cytoplasmic RNA-synthesis are probably delivered by the nucleus. These substances are accumulated in the cytoplasma of nucleate plants during darkness and consumed at light.

Morphology of the nucleo-cytoplasmic interactions during the development ofAcetabularia cells

SummaryThe ultrastructure of the growing and maturing primary nucleus ofAcetabularia mediterranea andAcetabularia major has been studied with the use of various fixation procedures. Particular interest has been focused on the details of the nuclear periphery and the perinuclear region. It is demonstrated that early in nuclear growth a characteristic perinuclear structural complex is formed which is, among the eukaryotic cells, unique toAcetabularia and related genera. This perinuclear system consists essentially ofa)the nuclear envelope with a very high pore frequency and various pore complex associations with granular and/or threadlike structures some of which are continuous with the nucleolus;b)an approximately 100 nm thick intermediate zone densely filled with a filamentous material and occasional small membraneous structures from which the typical cytoplasmic and nuclear organelles and particles are excluded;c)an adjacent lacunar labyrinthum which is interrupted by many plasmatic junction channels between the intermediate zone and the free cytoplasm;d)numerous dense perinuclear bodies in the juxtanuclear cytoplasm which are especially frequent at the junction channels and reveal a composition of aggregated fibrillar and granular structures;e)very dense exclusively fibrillar aggregates which occur either in association with the perinuclear region of the lacunar labyrinthum or, somewhat further out, in the cytoplasmic strands between the branches of the lacunar labyrinthum in the form of slender, characteristic rods or “sausages”. A variety of other membraneous and non-membraneous structures characteristic of the juxtanuclear cytoplasm is described. The organization of the individual components in this special complex is summarized in a model drawing. The dynamic and transitory nature of this perinuclear complex apparatus is emphasized and its possible role in nuclear functions and in regulating nucleocytoplasmic interactions is discussed.

Somatic hybridization of two selected single cells

Two single mesophyll-protoplasts of Nicotiana tabacum cv. xanthi were selected into a 100 nl microdroplet of 0.4 M mannitol. Two cylindrical platinum electrodes were inserted into the microdroplet to align the two single cells via dielectrophoresis in an AC-field (1 MHz, about 120 V X cm-1). A single square DC-pulse of about 1.5 KV X cm-1 was applied to induce protoplast fusion.

Enhancer-controlled expression of the simian virus 40 T-antigen in the green alga Acetabularia.

Nuclei from Acetabularia mediterranea were isolated, microinjected with simian virus 40 (SV40) DNA and fused with cytoplasts from the same species. Various times after fusion of the injected nuclei the fusion products were screened for expression of the T‐antigen by indirect immunofluorescence. One and two days after injection a bright fluorescence could be observed in the nuclei of Acetabularia. On the basis of this immunofluorescence we conclude that in Acetabularia cells the T‐antigen is expressed and accumulated in the nucleus. Moreover, evidence is presented that the Acetabularia cell recognizes the SV40 enhancer sequence. The expression product of the SV40 DNA appears significantly earlier than the expression products of other foreign genes in Acetabularia. The results suggest that the well characterized SV40 can be used as a vector system for the introduction and expression of foreign genes in Acetabularia.

RNA-synthesis in Acetabularia. I. RNA-synthesis in enucleated cells.

SummaryIncorporation of precursors of RNA into the enucleated cells ofAcetabularia mediterranea andPolyphysa cliftonii has been studied under conditions which exclude the possibility of errors due to contamination of the preparations by nuclei, nuclear debris or microorganisms.Radioactive RNA has been isolated from the chloroplast, mitochondrial and supernatant cytoplasm fractions of nucleated and enucleated cells. Sucrose density gradient centrifugation of the isolated labelled RNA produces a sedimentation profile of radioactivity which is similar to that of RNA isolated fromE. coli ribosomes.Anion exchange chromatography of alkaline hydrolysates of the 23 s and 16 s ribosomal RNA fractions shows incorporation of labelled uracil into RNA in the form of 2′(3′)-UMP and 2′(3′)-CMP. Labelled guanosine is incorporated only as 2′(3′)-GMP.A slowly sedimenting radioactivity peak has been chromatographed on a Hershey column and found to correspond to cold t-RNA.

Nuclear control of lacate dehydrogenase in Acetabularia

ZusammenfassungIn kernhaltigen, aber auch in kernlosen Zellen vonAcetabularia wird wÄhrend des Wachstums die LDH-AktivitÄt bis zum Beginn der Hutbildung vermehrt. Verschiedene Arten vonAcetabularia weisen bezüglich der LDH entweder durch Zahl der Banden oder Position der Banden Unterschiede voneinander auf. Heterologe Transplantate bzw. Implantate lassen einige Wochen nach der Operation erkennen, da\ die artspezifischen Isozymbanden des Cytoplasmas verschwinden und die vom Kern determinierten erscheinen. Zumindest ein Teil der LDH-AktivitÄt ist an die Chloroplasten gebunden. Dieses wurde mit Hilfe der Saccharose-Dichtegradienten-Zentrifugation und auf histochemischem Wege nachgewiesen.SummaryIn nucleate as well as in anucleate cells ofAcetabularia LDH activity is increased until the cap is formed. Different species ofAcetabularia can be distinguished by the electrophoretic behavior of the LDH. Heterologous grafts of nucleus containing rhizoids or of isolated nuclei to anucleate cytoplasm result in the disappearance of the LDH pattern of the cytoplasm and in the appearance of a new one which is specific for the species of the nucleus. At least part of the enzyme activity is linked to the chloroplasts as was shown by sucrose density gradient centrifugation and by histochemical techniques.

RNA-synthesis inAcetabularia

Summary1.The kinetics of RNA synthesis in nucleate and enucleated cells ofAcetabularia mediterranea have been compared.2.Nucleate cells and cells 24 hours after enucleation show no differences in their kinetics nor in the sedimentation behavior of the newly-synthesized RNA isolated after various short intervals (≦120 min) of incubation in the presence of tritiated ribonucleosides which can be attributed to enucleation. This fact is explained by predominant extranuclear RNA synthesis.3.The kinetics of the synthesis of the 23 s and 16 s ribosomal RNA peaks suggest that each of these peaks may be composed of two components. The possible origin of these components is discussed.

Synchronization of protoplasts from Glycine max (L.) Merr. and Brassica napus (L.)

Cells of Glycine max originating in a suspension culture and cells of Brassica napus prepared from hypocotyls were synchronized. Synchronization was achieved by preparing protoplasts in the usual way and subsequently letting the protoplasts regenerate into cells by removing the cell-wall-digesting enzymes. More than 70% of the cells had divided synchronously at the end of the first cycle as determined by the mitotic index. The high frequency of mitosis critically depended on the osmolality of the medium. The duration of the S-phase was estimated by measuring the activity of thymidylate kinase as well as incorporation of [3H]deoxythymidine into acid-insoluble material. The data indicate that synchronization is induced by resetting the cell cycle.

80 S ribosomes inAcetabularia major distribution and transportation within the cell

SummaryThe 80 S ribosomes of the unicellular green algaAcetabularia are evenly distributed throughout the cell. In pulse-labeling experiments it has been ascertained that newly-formed ribosomes are entirely restricted to the basal part of the cell. They first appear as free polyribosomes and become largely membrane-bound within 5 to 7 days. Labeled ribosomes are not observed in the apical half of the cell until ten days after their formation. Two or three weeks later labeled ribosomes are primarily concentrated in the apical half of the cell. These results favor a directed mechanism of transportation. The half-life of the 80 S ribosomal RNA was estimated to be about 80 days. On the average each cell yields 200 ng of 80 S ribosomes, an amount which is synthesized within a period of 2 months. From this rate of synthesis it can be concluded that the genes for the RNA of the 80 S ribosomes have a redundancy of more than 10,000.

80 S ribosomes inAcetabularia major redundancy of rRNA cistrons

SummaryHigh redundancy of rRNA cistrons inAcetabularia has been postulated from an estimation based on the rate of synthesis and on the long half life of rRNA in this organism. This hypothesis was proved inAcetabularia major by means of Millers spreading technique. The rRNA cistrons in this giant single cell alga have been shown to be highly redundant. The redundancy seems to be correlated to the length of the cell indicating that the amplification takes place during at least the greater part of the vegetative phase. Up to 52 cistrons have been detected on one DNA matrix. The length of the rRNA cistron corresponds quite closely to what would be expected for a DNA coding for RNA of the molecular weight 2.1×106 daltons. This value indicates that there is little if any loss of nucleotide material during the maturation of the 18 S and 26.5 S rRNAs inAcetabularia.