Nuclear behavior in multinucleate Schizophyllum commune germlings

Summary The ultrastructure of a fir morphological mutant containing multinucleate cells is described in Schizophyllum commune. The germlings of basidiospores which arose from mating fir with wild-type mycelium were studied in culture by phase contrast microscopy to elucidate behavior of multinucleate cells. Nuclear division appeared synchronous from two nuclei yielding four progeny through six nuclei producing twelve products, beyond which loss of synchrony was indicated. Compensatory nuclear migration into an anucleate cell was presumed during synchronous division of nuclear aggregates in the adjacent cell of an individual germling. The migrant nucleus eventually returned to the cell of origin. However, the return route was not via the central pore of the septum but rather occurred at the juncture of the cross-wall with the germling-periphery. Ultrastructure of a partial septum in fir which could accommodate nuclear passage of this sort is described.     

Heat shock response in Escherichia coli promotes assembly of plasmid encoded RNA polymerase beta-subunit into RNA polymerase

Summary Escherichia coli cells, carrying a rifampicin sensitive RNA polymerase -subunit gene in the chromosome and a rifampicin resistant -subunit gene placed under the control of a strong promoter in a multicopy plasmid, are unable to grow in the presence of rifampicin, despite the accumulation of large quantities of the resistant subunit. A major portion of the overproduced subunit is found in an insoluble form. Conditions known to induce the heat shock proteins (hsps), e.g. elevated temperature or the presence of ethanol in the growth medium, increase the amount of the plasmid-borne -subunit which apparently assembles into active RNA polymerase and makes the plasmid bearing cells rifampicin resistant. Alternatively, plasmid-borne subunits assemble into RNA polymerase with low efficiency in rpoH mutant cells known to have reduced level of hsps. We suggest that the plasmid-borne subunit is poorly assembled into RNA polymerase and that hsps promote the assembly by interfering with -subunit aggregation.     

The macronuclear envelope ofTetrahymena pyriformis GL in different physiological states

Summary A simple formula is derived to calculate the nucleocytoplasmic RNA-Efflux per nuclear Pore complex per min (REP-rate) which is generally applicable both for growing and stationary eukaryotic cells. In actively growing cells this REP-rate is mainly dependent on the cytoplasmic RNA-pool, the number of RNA-transporting pores, and the growth constant of RNA. These parameters are determined in logarithmicTetrahymena pyriformis GL. In this organism, 45 molecules both of the larger ribosomal RNA (25s rRNA) and of the smaller (17s rRNA) are transported per pore per min from nucleus to cytoplasm. Pulse-label experiments with³H-uridine indicate that the 25s rRNA is obviously transferred more slowly to the cytoplasm than the 17s rRNA. We postulate a gating hypothesis on the regulation of the nucleocytoplasmic RNA-efflux by nuclear pore complexes. This gating hypothesis suggests that nucleopores are controlling points of secondary importance in the sequence of gene expression, and do not directly control the cytoplasmic protein synthesis in eukaryotic cells.     

The cytology of phytophthora infestans

Summary Phytophthora infestans has three kinds of somatic nuclei: an oval shaped nucleus (approx. 3.1×2.7 ) which stains diffusely except for a crescent shaped Feulgen positive cap which stains intensely; a granular nucleus whose contents are organized into a fairly constant number of stained bodies, and, a deeply staining condensed nucleus. The capped nucleus is thought to be metabolic or resting and the granular nucleus is thought to be dividing as it is most commonly found in hyphal tips. Attenuated forms of all three kinds of nuclei are found.Nuclear division is mitotic and intranuclear. Eight—ten chromosomes are seen at metaphase.Sporangia have a mean of 6.3 nuclei which is constant for age and strain of culture. Sporangia become multinucleate as a result of nuclear migration and not by division in the developing sporangium. Zoospores are usually uninucleate.The nuclear cap is persistent throughout nuclear division when it also divides. It is associated with flagella production and nuclear migration and has some of the properties of a blepharoplast.     

Spermatogonia and the cycle of the seminiferous epithelium in the nine-banded armadillo

Summary The cycle of the seminiferous epithelium of the nine-banded armadillo can be divided into ten stages. As in most mammals, only one stage is observed per tubular cross-section. The process of spermiogenesis can be divided into thirteen steps according to the development of the acrosomal system and the flagellum. Four generations of spermatogonia are observed in the germinal epithelium: 1) stem cells, 2) type A, 3) intermediate, and 4) type B spermatogonia. The stem cell is characterized by a highly irregular nucleus and the presence of glycogen in its cytoplasm. The type A spermatogonium contains an oblong nucleus with one or two shallow infoldings of the nuclear membrane. The intermediate spermatogonium contains an ovoid nucleus characterized by one or two nuclei and heterochromatin scattered in the nucleoplasm. The nucleus of the type B spermatogonium is more spherically shaped with a centrally placed nucleolus and heterochromatin associated with the nuclear envelope.The author wishes to acknowledge the technical assistance of Teri Lane     

Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli

Summary The and subunit of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme. To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed. This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control. When the structural genes encoding the components of core RNA polymerase (, and ) or holoenzyme (, , and ⁷⁰) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase. The induction of RNA polymerase overproduction is characterized by an initial large burst of synthesis followed by a gradual decrease as the concentration of RNA polymerase increases. Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of synthesis off the chromosome. These results indicate that RNA polymerase feedback regulation controls synthesis in vivo.     

Non-coordinate synthesis of RNA polymerase beta beta'' subunits in a temperature-sensitive beta''-subunit mutant of Escherichia coli

Summary On exposure to high temperature of a temperature-sensitive RNA polymerase subunit (rpoC92) mutant of Escherichia coli, selective reduction was observed in the rate of synthesis of a group of proteins including RNA polymerase subunit. The finding that the synthesis of subunit but not subunit was specifically repressed in this mutant grown at non-permissive temperature indicates that the functionally intact RNA polymerase is required for the synthesis of subunits be coordinated. In addition, the assembly of newly synthesized RNA polymerase subunits was inefficient in this mutant at the steps where altered subunit was involved, and the unassembled enzyme subunits were rapidly and preferentially degraded. During recovery to non-restricted growth, the synthesis of both and subunits was transiently enhanced in parallel leading to recovery of the intracellular concentration of functional RNA polymerase.     

Effect of temperature on nuclear membranes and nucleo-cytoplasmic RNA-transport inTetrahymena grown at different temperatures

Summary The effect of temperature on the nuclear envelope structure and the transport of total RNA and ribosomal subunits from nucleus to cytoplasm was examined inTetrahymena cells propagated at two different temperatures. Freeze-etch electron microscopy of cells grown at 23 and 18°C detects the emergence of smooth areas on the fracture faces of the nuclear membranes upon lowering the temperature below 15 and 12°C, respectively. Coincident with these freeze-etch changes, a discontinuous decrease is observed in the nucleocytoplasmic RNA-transport; this is probably not due to a cease in RNA-synthesis. Below the thermotropic discontinuity observed in the transport of total RNA in 18°-cells the nucleocytoplasmic transport of the small and large ribosomal subunits is equally retarded. Recent temperature studies on the endoplasmic reticulum membranes ofTetrahymena suggest that the freeze-etch changes in the nuclear membranes are induced by a thermotropic clustering of the membrane lipids. We conclude that this lipid clustering induces the permanent protein constituents in the nuclear envelope pore complexes to change from a relatively open into a relatively closed state thus causing the observed decrease in RNA-transport.     

Variations in the rate of synthesis of beta and beta'' RNA polymerase polypeptides under the influence of certain factors.

Summary In merodiploid cells containing a double dose of structural genes of RNA polymerase subunits-rpoBand rpoC-the rate of and subunits synthesis is 2 times higher than in haploidcells. Missence mutation rpoC1 (tsX) alters polypeptide and inducesthe and subunits synthesis at increased rate, particularly at a nonpermissive temperature. When rpoBCoperon carrying mutation rpoC1 is duplicated no dosage effect is observed. In the rpoC ⁺/rpoC1 heterodiploid the rpoC1 mutation does not significantly accelerate RNA polymerase subunits synthesis i.e. is recessive with respect to rpoC ⁺ Rifampicin causes 6-fold stimulation of RNA polymerase subunits synthesis in a sensitive wild-type strain. The rpoC1 mutation itself accelerates the synthesis of these subunits 3-fold. In the presence of rifampicin the mutant strain produces 13–22-fold faster as compared to wild-type strain without the drug. Thus, the effects of rifampicin and the mutation are multiplied suggesting that these factors act independently. Similar data have also been obtained with rifampicin-treated cells of rpoB22 (ts22) amber-mutant.After UV-irradiation of cells and synthesis is depressed much stronger than the total protein synthesis. Infection with a transducing phage rif ^d-47 which carries rpoB gene provokes a higher rate of synthesis. When pre-irradiated cells (500 erg/mm²) are infected with this phage, the rate of synthesis grows 20-fold compared to irradiated, non-infected cells and 6.5-fold compared to intact cells.The data are discussed in terms of the possible regulatory mechanisms of RNA polymerase subunit synthesis.     

Isolation and culture of anucleate protoplasts from cotton fiber; assessment of viability and analysis of regenerated wall polymers

Procedures were developed for the isolation and culture of an anucleate protoplast system from cotton fibers actively undergoing secondary wall synthesis. Because the fibers at this stage are elongated single cells (30 m × 1–2 cm), most of the cellular vesicles released in the process of isolation are anucleate. After purification, the protoplast population was nuclei-free. When transferred to culture medium, the anucleate protoplasts (cytoplasts) synthesized starch, hydrolyzed fluorescene diacetate for up to 9 days and formed cell wall material for at least 7 days. The composition of the regenerated cell walls was dependent upon the substrate supplied in the medium: -1,3-linked glucans were predominantly synthesized when 1 mM UDP[¹⁴C]glucose was supplied; -1,4-linked glucans were predominantly synthesized when 1 mM [¹⁴C]-glucose was supplied. Thus the composition of the regenerated cell walls formed by the anucleate protoplasts was similar to the secondary cell wall synthesized by intact cotton fibers under the same culture conditions.     

Mutation to rifampicin resistance at the beginning of the RNA polymerase beta subunit gene in Escherichia coli

Summary The unusual recombinant plasmid pRC19 carrying the N-terminal fragment of the Escherichia coli RNA polymerase rpoB gene was found to specify high level rifampicin resistance of E. coli cells. Sequence analysis of this plasmid revealed one substitution only: transversion GT, leading to amino acid substitution Val¹⁴⁶Phe. This mutational change marks the second domain of the subunit involved in rifampicin binding.     

Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.     

Microtubule-nucleation sites on nuclei of higher plant cells

Summary The nucleation and the elongation of microtubules from isolated nuclei of higher plant cells were investigated. Isolated intact nuclei failed to nucleate microtubules at their surface when they were incubated with purified tubulin from plant or animal sources. However, frozen and thawed nuclei or nuclear particles obtained by gentle nuclei homogenization nucleated microtubules and nucleated microtubules elongated radially from the surface of nuclei or from the nuclear particles. Microtubules radiating from the nuclear particles were very much shorter than those radiating from frozen and thawed nuclei. The washing of the nuclear particles diminished the ability of the particles to nucleate microtubules. The ability of the washed nuclear particles to nucleate microtubules was restored by the addition of the soluble fraction of a nuclear homogenate. The complexes of radiating microtubules could easily be observed under a phasecontrast microscope. Electron microscopy demonstrated that microtubules in the complexes formed bundles. The staining with a monoclonal antibody specific for plant tubulin of the complexes of radiating microtubules, prepared by successive polymerization of animal tubulin and plant tubulin, revealed that microtubules in the complex incorporated tubulin at their proximal ends. This result indicates that the mode of incorporation of tubulin onto frozen and thawed nuclei or onto the nuclear particles is different from that in pericentriolar bodies in animal cells. Mg²⁺ seems to participate in the regulatory mechanism that determines the length of microtubules on the complexes.Abbreviations MTOC microtubule-organizing center - MES 2-(N-morpholino) ethane-sulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl sulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - GTP guanosine triphosphate - NP-40 Nonidet P-40 - DMSO dimethylsulfoxide - EPC ethyl N-phenylcarbamate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - DAPI 4,6-diamidiho-2-phenylindole     

Rifampicin supersensitivity of rho strains of E. coli, and suppression by sur mutation.

Summary Escherichia coli strains with mutations rho-115, rho-ts15, rho-101 (psu-1) or rho-102 (psu-2) are more sensitive (supersensitive) to rifampicin than isogenic parent strains, as measured by growth rate in broth and colony forming efficiency on solid media with 5, 10, or 20 g of rifampicin per ml. There is no change in sensitivity of rho mutants to the antibiotics penicillin, erythromycin, chloramphenicol, or the detergent desoxycholate. The rho-101 or rho-102 mutations confer rifampicin supersensitivity at 32°C but not 42°C. Mutants of a rho-115 strain that have lost polarity suppression can be isolated by selection for rifampicin resistance. This phenotype, Sur, is not due to reversion of the original rho gene mutation but to a second mutation perhaps in the gene for rho protein or the gene for the subunit of RNA polymerase. One class of Sur mutation, occurring in rho-115 cells isolated as resistant to 20 g of rifampicin per ml, is co-transducible with the marker ilv, and the gene order is rbs-ilv-sur-38. A model suggested by this map position is that the mutations rho-115 and sur-38 define the domain of rho protein which interacts with the subunit of RNA polymerase.     

Sensitivity of the cell cycle to TGFβ1 does not correlate with transformation of a rat liver epithelial cell line

The effects of TGF1 on cell cycle events in a rat liver derived epithelial cell line (BL9) and in two in vitro transformants of this line were studied by flow cytometry. Using either ethidium bromide staining or the incorporation of bromodeoxyuridine to evaluate DNA synthesis it was shown that TGF1 prevented the entry of G0/G1 phase BL9 cells into S phase. TGF1 did not exert its inhibitory effect(s) on DNA synthesis by the modulation of early events in the cell cycle. The tumorigenic transformed BL9 cell lines gave contrasting responses to the effects of TGF1. DNA synthesis in a BL9 cell line derived by transfection with an active N-ras oncogene was unaffected by TFG1 and thus appeared refractory to its growth controlling effects. On the other hand cells from a BL9 cell line derived by in vitro transformation with activated aflatoxin B₁ retained their sensitivity to the effects of TGF1. Thus the loss of the inhibitory effect of TGF1 on DNA synthesis is not obligatory for the malignant transformation of rat liver epithelial cells.Abbreviations TGF1 transforming growth factor 1 - BSA bovine serum albumin - FBS foetal bovine serum - BrdUrd bromodeoxyuridine - PI propidium iodide - PBS phosphate buffered saline     

Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Summary Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 M CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 M CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 M or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several branched or cross over type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the aster type MT foci, from which several MTs radiated along the nuclear surface. The aster type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 M for 2 h), branched and cross over type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor aster type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of aster type MT foci that is essential for bipolar spindle development.     

Rifampicin-induced protein synthesis: A pre-requisite for increased expression of the ββ′ operon in Escherichia coli

Summary In a rif ^S/rif^R heterodiploid strain of E. coli, a 4 minute pulse of rifampicin can induce a prolonged (>60 min) increase in the rate of synthesis of the RNA polymerase subunits, and . The application of a constraint on the fidelity of protein synthesis during, but not after, the rifampicin pulse partially arrests the development of this capacity for subunit synthesis. I discuss the implications of these findings in relation to the control of the operon in E. coli.     

RNA polymerase subunit biosynthesis in Bacillus subtilis

Summary The relative rates of RNA polymerase biosynthesis in Bacillus subtilis has been examined under steady-state growth conditions. The synthesis of RNA polymerase subunits (, , , ) has been followed by subunit fractionation of immunoprecipitated [³H]-labelled samples on SDS-polyacrylamide gels. The stoichiometries of ::: subunits have been determined from cultures pulse-labelled during steady-state growth. The results suggest that an unassembled pool of the -subunit exists from which the holoenzyme is formed.Upon shift-up from acetate to glycerol containing medium, a rapid rise in the differential rate of core enzyme synthesis was observed, while the rate of synthesis of the -subunit was not stimulated. During shift-down, a concomitant reduction in the rate of synthesis of all subunits occurred for the first 20 min after the shift; thereafter, a rate of synthesis characteristic of the new growth rate was established.As cultures enter sporulation, an immediate reduction in the rate of -subunit synthesis was demonstrated.     

Blutregen und Blutschnee: Stickstoffmangel-Zellen von Haematococcus pluvialis und Chlamydomonas nivalis

Zusammenfassung Blutregen und Blutschnee sind zwei auffallende Naturerscheinungen, die in der Hauptsache durch Massenvorkommen von Aplanosporen der volvocalen Grünalgen Haematococcus pluvialis und Chlamydomonas nivalis verursacht werden. Die für die rote Farbe verantwortlichen Pigmente sind Keto-Carotinoide. Die Versuche zeigen, daß auch unter natürlichen Bedingungen für die Biosynthese dieser Polyene und für den gleichzeitig ablaufenden Abbau der Chlorophylle Stickstoffmangel der entscheidende Faktor ist.

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Blood-rain and blood-snow: Nitrogen-deficient cells of Haematococcus pluvialis and Chlamydomonas nivalis

Summary The blood-rain and the blood-snow, two phenomena caused mainly by mass occurrence of aplanospores of the volvocalean green algae Haematococcus pluvialis and Chlamydomonas nivalis, have been studied under natural conditions. The red pigments belong to the keto-carotenoids. The experiments show that also in nature the biosynthesis of these polyenes and the parallel decomposition of the chlorophylls are caused by nitrogen deficiency.