Chitin structures of the cell walls of synchronously grown virgin cells of Saccharomyces cerevisiae.

The ability of a lytic beta-glucanase of Arthrobacter GJM-1 to dissolve cell walls of Saccharomyces cerevisiae with exception of the chitin-containing fraction was employed for the isolation of chitin-rich residues of the cell walls of synchronously growing populations of virgin cells. Electron microscopical examination of such wall residues isolated from cells at various stages of the budding cycle showed that the first phase of chitin deposition in the wall corresponds to the formation of an annular structure found as a part of the bud scar after cell division. The annular chitin-rich structure could not be isolated at cell cycle stages preceding the bud emergence and at earliest stages of bud development. The observations confirmed that the annular structure (chitin ring) formed during bud growth represents a major part of total chitin present in the bud scar after septum closure.     

Macromolecular Synthesis and Degradation in Arthrobacter During Periods of Nutrient Deprivation

Cells of Arthrobacter atrocyaneus and A. crystallopoietes, harvested during their exponential phase, were starved in 0.03 M phosphate buffer (pH 7.0) for 28 days. During this time, the cells maintained 90 to 100% viability. Experimental results were similar for both organisms. Total cellular deoxyribonucleic acid was maintained. Measurable degradation rates for deoxyribonucleic acid as determined by radioisotope techniques were not observed, and only during the initial hours of starvation could a synthetic rate be determined. Total ribonucleic acid levels remained stable for the first 24 h of starvation, after which slow, continuous loss of orcinol-reactive material occurred. Synthetic and degradative rates of ribonucleic acid, as determined by radioisotope techniques, dropped quickly at the onset of starvation. Constant basal rates were attained after 24 h. In A. atrocyaneus, total cell protein was degraded continuously from the onset of starvation. In A. crystallopoietes, total cell protein remained stable for the first 24 h, after which slow continuous loss occurred. After 28 days, the total protein per cell was similar for both organisms. In the first week, amino acid pools stabilized at about 50% of the values characteristic of growth. Rates of degradation of protein decreased rapidly for the first 24 h for both organisms, but leveled to a constant basal rate thereafter. Rates of new protein synthesis dropped during the first 24 h and by 48 h achieved a constant basal rate.     

Gratuitous induction of xanthine oxidase by 5,6-diaminouracil in continuously grown cells of Arthrobacter globiformis M4

Summary 5,6-Diaminouracil (DAU), was found to be a gratuitous inducer of xanthine oxidase (XO) in Arthrobacter globiformis M4. Synthesis of urate oxidase was not induced by this compound. Preparation of a biocatalyst rich in XO could be achieved by exposing continuously grown cells to low concentrations of DAU.     

Synthesis of macromolecules and rubratoxin by Penicillium rubrum

The relationship between primary metabolism and biosynthesis of rubratoxin was studied with replacement cultures of Penicillium rubrum 3290. Synthesis of protein and RNA was measured by determining incorporation of [U¹⁴C]L-leucine and [2¹⁴C]-uridine into the respective components of the fungal biomass. Rubratoxin formation was measured by determining incorporation of [1¹⁴C]acetate into the toxin. Both protein and RNA were synthesized rapidly with synthesis increasing during 108 h of incubation and then decreasing rapidly. Rubratoxin formation increased up to 72 h, declined through 96 h, became maximal at 108 h, and then decreased rapidly. Cycloheximide, at 100 g/ml, moderately blocked accumulation of dry weight and protein synthesis by the mold; at 150 g/ml, cycloheximide completely blocked in vivo synthesis of protein. When cycloheximide was added to cultures after synthesis of toxin had begun, protein synthesis, but not toxin formation, was blocked. Inhibition of protein synthesis by cycloheximide was reversed by washing the drug out of mold cultures. Rubratoxin was formed throughout the incubation; a transitional phase, characteristic of secondary biosynthesis, was not observed.     

Synthesis of DNA polymerase alpha analyzed by immunoprecipitation from synchronously proliferating cells

Synchronously proliferating TC7 monkey and 3T3 mouse cells were pulse labeled with [35S]methionine. Radioactively labeled DNA polymerase alpha was immunoprecipitated with polymerase-specific monoclonal antibodies. The precipitated polypeptides were identified by gel electrophoresis and fluorography. The increase in DNA polymerase alpha activity during S phase was accompanied by an increased synthesis of the enzyme. Some DNA polymerase alpha was synthesized in growth-arrested TC7 cells whereas the synthesis of the large polymerase subunit in 3T3 cells was strictly coupled to the replicative phase of the cell cycle. We also found that DNA polymerase alpha was more prone to proteolysis in TC7 cells than in 3T3 cells. In 3T3 cells, a polymerase subunit with an apparent molecular weight of 186 000 was observed; this subunit was most probably associated with two smaller subunits of Mr 74 000 and 52 000. Synthesis of these three polymerase-associated polypeptides appeared to be regulated differently.     

Change in the H2 photoproduction capability in a synchronously grown aerobic nitrogen-fixing cyanobacterium,Synechococcus sp. Miami BG 043511

Synchronously growing cells of nitrogen-fixing Synechococcus sp. Miami BG 043511 were harvested periodically and the capability for hydrogen photoproduction in closed vessels was measured under hydrogen production conditions. The capability for hydrogen photoproduction in cells was correlated with that of cellular carbohydrate content. Cells with a high carbohydrate content exhibited a high capacity for hydrogen production and those with low carbohydrate content exhibited low capacity for hydrogen production. Nitrogenase activity at the onset of incubation did not coincide with a capability for the cells to produce hydrogen during the subsequent incubation period. Interestingly, when cells with a high capacity for hydrogen photoaccumulation were incubated, alternate periods of hydrogen and oxygen accumulation were observed at 12 hour intervals. About 0.5 ml of hydrogen per ml of cell suspension was accumulated in flasks during the initial 12-h incubation period. These observations indicate that the use of synchronous culture can be one of the ways of provide materials suitable not only for basic studies but also for applied aspects of hydrogen photoproduction.     

Nitrogen metabolism in the facultative methylotroph Arthrobacter P1 grown with various amines or ammonia as nitrogen sources

The metabolism of trimethylamine (TMA) and dimethylamine (DMA) in Arthrobacter P1 involved the enzymes TMA monooxygenase and trimethylamine-N-oxide (TMA-NO) demethylase, and DMA monooxygenase, respectively. The methylamine and formaldehyde produced were further metabolized via a primary amine oxidase and the ribulose monophosphate (RuMP) cycle. The amine oxidase showed activity with various aliphatic primary amines and benzylamine. The organism was able to use methylamine, ethylamine and propylamine as carbon-and nitrogen sources for growth. Butylamine and benzylamine only functioned as nitrogen sources. Growth on glucose with ethylamine, propylamine, butylamine and benzylamine resulted in accumulation of the respective aldehydes. In case of ethylamine and propylamine this was due to repression by glucose of the synthesis of the aldehyde dehydrogenase(s) required for their further metabolism. Growth on glucose/methylamine did not result in repression of the RuMP cycle enzyme hexulose-6-phosphate synthase (HPS). High levels of this enzyme were present in the cells and as a result formaldehyde did not accumulate. Ammonia assimilation in Arthrobacter P1 involved NADP-dependent glutamate dehydrogenase (GDH), NAD-dependent alanine dehydrogenase (ADH) and glutamine synthetase (GS) as key enzymes. In batch cultures both GDH and GS displayed highest levels during growth on acetate with methylamine as the nitrogen source. A further increase in the levels of GS, but not GDH, was observed under ammonia-limited growth conditions in continuous cultures with acetate or glucose as carbon sources.Abbreviations HPS hexulose-6-phosphate synthase - RuMP ribulose monophosphate - DMA dimethylamine - TMA trimethylamine - TMA-NO trimethylamine-N-oxide - ICL isocitrate lyase - GS glutamine synthetase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOGAT glutamate synthase     

Vacuolar dynamics in synchronously budding yeast

Summary A simple and rapid method for obtaining synchronously budding cultures of Saccharomyces cerevisiae is described. Synchronous cultures were started with homogeneous cell fractions isolated from exponentially growing cultures by isopycnic centrifugation in osmotically inactive media. The technique of fractionation is based on changes of cell density throughout the budding cycle. These changes are correlated with vacuolar changes observed in the light and electron microscope. During bud initiation the large vacuoles in late budding cells shrink and fragment into small vacuoles. Simultaneously the density of the cells increases. Later stages of the budding cycle are characterized by the distribution of the small vacuoles between mother and daughter cell, followed by their fusion and expansion, and by a decreasing density of the cells. The relative changes in cell density and dry weight and in the content of different macromolecules during the budding cycle suggest a cyclic change between utilization of endogenous and exogenous substrates. This is discussed in terms of a cyclic consumption and accumulation of vacuolar pools.     

Myceloid growth of Arthrobacter globiformis and other Arthrobacter species.

Transitory myceloid growth occurs in certain complex media with Arthrobacter globiformis strain ATCC 8010. This type of growth, however, was not observed in a medium which contained an array of metal ions but did not contain agents able to complex metal ions. Addition of metal-complexing agents to this medium caused an interruption in the life cycle of strain 8010 so that growth occurred only as the myceloid form. It appeared that manganese was the critical metal that was removed by the metal-complexing agents. During growth, the myceloid cells started to fragment, but wall septation was incomplete. A. globiformis strain ATCC 4336 and several other Arthrobacter species and soil isolates, but not Arthrobacter crystallopoietes, responded to metal-complexing agents as did strain 8010. Biotin and vitamin B12 were not involved in this myceloid growth.     

Synthesis of macromolecules during microcyst germination in the cellular slime mold Polysphondylium pallidum

Microcyst germination in the cellular slime mold Polysphondylium pallidum is a useful model for studying macromolecular changes necessary for or coincident with the transition from one cell type (cyst) to another (amoebae). Protein synthesis starts soon after cysts are incubated under permissive conditions, as evidenced by the incorporation of precursors and the appearance of polysomes. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins made at intervals during germination shows that protein synthesis is developmentally regulated during this process. RNA synthesis also begins early during germination. Cysts contain polyadenylated RNA that can stimulate the incorporation of radioactive amino acids into protein in an in vitro wheat germ protein synthesizing system. The concentration of poly(A)-containing RNA increases during germination and during inhibition of protein synthesis by cycloheximide.     

Synthesis, assembly and function of plant cell wall macromolecules.

The plant cell wall consists of a structurally intricate network of polysaccharide and protein whose biosynthesis, assembly and functions are still poorly understood. Recent research has shown how cell wall macromolecules, and fragments thereof, appear to be involved in processes such as cell growth, cell and tissue differentiation and the control of pathogenesis.     

Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells

How viral infections affect host cell mitochondrial functions is largely unknown. In this study, uptake of radiolabeled precursors was used to assess how a herpes simplex virus type 1 (HSV 1) infection influences synthesis of macromolecules comprising Vero cell mitochondria. Total macromolecular synthesis in infected cells was determined for comparative purposes. Mitochondrial and total cellular DNA syntheses were approximately halved at 1-2.5 h postinfection (PI). Mitochondrial DNA synthesis in infected cells then rose to 3.5-fold that in control cells at 3-4.5 h PI. Total DNA synthesis in infected cells also rose, but more slowly, reaching threefold that for control cells at 5-6.5 h PI. Mitochondrial and total RNA synthesis in infected cells were both decreased by approximately 40% at 1-3 h PI. Over the next 4 h, total RNA synthesis in infected cells slowly continued to decrease, while that in mitochondria recovered to control levels. Synthesis of mitochondrial proteins in infected cells decreased progressively, dropping to about 60% of control levels by 5-6.5 h PI. With the metabolic inhibitors ethidium bromide and cycloheximide, it was determined that nuclear DNA and mitochondrial DNA and mitochondrial DNA directed synthesis of mitochondrial proteins were each partially inhibited in infected cells. Total cellular protein synthesis was decreased by 30% at 1-2.5 h PI and then recovered to control levels by 5-6.5 h PI. Finally, phospholipid synthesis in mitochondria from infected cells was elevated 2.3-fold at 1-5 h PI, but dropped to 14% below control levels during 4-8 h PI.(ABSTRACT TRUNCATED AT 250 WORDS)