Macromolecular synthesis accompanying the transition from spores to vegetative forms ofStreptomyces granaticolor

The rates of RNA, protein and DNA synthesis were estimated in synchronously germinating spores ofStreptomyces granaticolor. Rapid uptake of labelled precursors of RNA and proteins was observed after 20 s. The germination process took place through a sequence of time + ordered events. RNA synthesis started after 3 min of germination, protein synthesis began at 4 min and net DNA synthesis at 60–70 min of germination. A characteristic feature of germination was the biphasic pattern in the rate of RNA and protein synthesis. Spores ofStreptomyces granaticolor were sensitive to actinomycin D, rifampicin and chloramphenicol even at the start of germination. Protein synthesis during germination was dependent on new mRNA synthesis and was independent during the first 60–70 min on replication of the spore genome.     

Nature of Deoxyribonucleic Acid Synthesis and Its Relationship to Protein Synthesis During Outgrowth of Bacillus cereus T

Deoxyribonucleic acid (DNA) synthesis during early outgrowth of spores of Bacillus cereus T (thy(-)) has been examined. (14)C-thymidine incorporated begins 2 to 5 min after germination and continues at a slow rate up to 30 min, after which the rate of (14)C-thymidine incorporation increases considerably. Early DNA synthesis up to 30 min after germination is dependent upon simultaneous protein synthesis. The examination of the stability of proteins synthesized soon after germination shows that they are susceptible to intracellular degradation. The evidence provided here indicates that protein degradation is the cause of observed dependence of DNA synthesis on simultaneous protein synthesis. The DNA synthesis occurring soon after germination is primarily a repair type synthesis which is followed by the onset of normal replication approximately 30 min after germination.     

The physiological effects of restrictive environmental conditions on Dictyostelium discoideum spore germination.

Spores may be reversibly activated by the application of heat, dimethyl sulfoxide, urea, or ethylene glucol. Severe changes in four environmental variables (high osmotic pressure, low oxygen tension, low or high pH, and low or high temperature) interfere with the germination process. Spores at the end of the postactivation lag phase of germination were usually deactivated if exposed to severe environmental conditions and thus did not swell; spores in the swelling and oxygen uptake which began during spore activation was primarily attributable to a cyanide-sensitive pathway and secondarily to a salicylhydroxamic acid (SHAM) sensitive pathway. Inhibition of the SHAM-sensitive pathway did not cause spore deactivation while the addition of cyanide resulted in rapid spore deactivation. Treatment of activated spores with azide or environmental shifts also resulted in inhibition of oxygen uptake and spore deactivation. Deactivating spores did not demonstrate the amino acid incorporation, uridine incorporation, and expression of trehalase activity which is found in the later stages of germinating control spores. Protein synthesis inhibitors did not cause spore deactivation or a decrease in oxygen uptake but they inhibited amino acid incorporation and the expression trehalase activity in swollen spores. It is concluded that control of respiratory activity is involved in regulation of reversible activation.     

Protein and nucleic acid synthesis during germination of the asexual spores of the aquatic fungus, Aphanomyces astaci

Macromolecular synthesis of asexual spores of Aphanomyces astaci Schikora, strain is, was studied during the germination phase. Germination of the spores was completely inhibited by aclinomycin D (20 ug/ml) or cycloheximide (100 ug/ml) as was incorporation of labeled uridine and leucine, respectively. During spore germination protein synthesis was almost linear, whereas incorporation of [3Hl-uridine and [3H1-thymidine showed lag phases. Initial protein synthesis is therefore suggested to take place without concomitant RNA-synthesis in this species. Germlings were not developmentally competent to sporulate before 8 h of germination.     

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.     

Activation and expression of proteins during synchronous germination of aerial spores of Streptomyces granaticolor

Synchronously germinating aerial spores of Streptomyces granaticolor were used to study protein activation and expression during the transition from dormant to metabolically active vegetative forms. The first phase of protein activation is associated with the solubility of proteins. Three major chaperones, DnaK, Trigger factor, and GroEL, were identified in spores. Enhancement in rate of protein synthesis during germination was accompanied by the association of TF and DnaK with ribosomes. During germination, the chaperones TF, GroEL, and DnaK undergo reversible phosphorylation. GroEL was phosphorylated on both Ser and Thr, whereas phosphorylation of DnaK and TF was detected on Thr only. A proteomic approach was used to gain more information on protein expression during germination on two types of media differing in the ability of cells to produce antibiotic granaticin. To obtain an overview of the metabolic activity of germinating spores, glycolytic enzymes, enzymes of citric acid cycle, metabolism of amino acids and nucleic acids, and components of the protein synthesis system were identified and analyzed using the proteomic database. The results were deposited on the SWICZ proteomic server and are accessible on http://proteom.biomed.cas.cz.     

Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.     

Cell Morphogenesis and Macromolecule Synthesis during Phytochrome-controlled Germination of Spores of the Fern, Pteris vittata

The object of this study was to characterize the pattern ofcell morphogenesis and synthesis of nucleic acids and proteinsduring phytochrome-controlled germination of spores of the fern,Pteris vittata. Phytochrome activation and germination wereinitiated in fully imbibed spores by exposure to a saturatingdose of red light. At timed intervals thereafter, spores werefixed in acrolein and embedded in glycol methacrylate for examinationin the light microscope. The first sign of germination, visiblein sections of the spore 12 h after irradiation, was the hydrolysisof storage protein granules. This was followed by a migrationof the nucleus from its central location to one side of thespore. Subsequently, the protoplast enlarged at the site ofthe nucleus and appeared outside the exine as a papillate structure.An asymmetrical division of the protoplast gave rise to a smallcolourless rhizoid cell and a large, chloroplast-containingprotonemal cell. During the early phase of germination, DNAwas synthesized both in the nucleus and cytoplasm as judgedby autoradiography of [³H]thymidine incorporation. [³H]Uridine,a precursor of RNA synthesis, was incorporated into the nucleolusand the rest of the nuclear material of germinating spores.Protein synthesis monitored by [³H]leucine incorporation occurredboth in the nucleus and cytoplasm during the early stage ofgermination, although a strictly cytoplasmic protein synthesiswas observed later. Addition of cycloheximide completely inhibitedgermination of photoinduced spores and incorporation of labelledprecursors of macromolecule synthesis into cellular components.Actinomycin D was much less effective as an inhibitor of germinationand, even in high concentrations of the drug which effectivelyinhibited DNA and RNA synthesis in spores, proteolysis and proteinsynthesis appeared normal. These findings are discussed withrespect to the regulation of nucleic acid and protein synthesisduring spore germination and the role of phytochrome in theprocess.     

Photocontrol of the Germination of Onoclea Spores: III. Analysis of Germination Processes by Means of Cycloheximide

Possible involvement of protein synthesis in the germination of Onoclea sensibilis spores was investigated by temporarily applying 0.1 mm cycloheximide before and after photoinduction. Cycloheximide was shown to inhibit protein synthesis, but not to act as an uncoupler of respiration. When cycloheximide was added before or shortly after photoinduction, spore germination was inhibited with the half-maximal inhibition attained in 30 to 45 minutes and the maximal inhibition in 2 hours of incubation. When the time of the inhibitor treatment was delayed after photoinduction, the spores escape from the inhibitory effect of cycloheximide slowly during the first 8 hours and abruptly thereafter with a half-maximal time of 10 hours. If spores are washed free of exogenous cycloheximide and subsequently irradiated, their ability to germinate can be reinstated in distilled water with a half-maximal time of 12 hours. The kinetics of recovery were identical and of apparent first order, regardless of whether cycloheximide treatments were given before or after photoinduction. These results are interpreted to indicate that the normal course of germination of Onoclea spores requires the continuous synthesis of a short lived enzyme that functions in the germination processes at about 10 hours after photoinduction. The cycloheximide-sensitive step follows in the germination processes an anaerobiosis-sensitive step, but precedes the time of acetocarmine uptake or visible signs of protrusion.     

Spores of microorganisms

Spores ofBacillus cereus were germinated in a germination limited medium (GL-medium) which facilitates only germination but not the postgerminative development of spores. Under these conditions a limited protein synthesis occurs. However, this protein synthesis is stopped after a short time interval. The rate of synthesis of new proteins, as well as their total amount, is influenced by the length of the activation heat shock. Synthesis of the wall material continues for several hours and thick-walled cells with a changed ultrastructure are formed. Synthesis of the diaminopimelic acid (dap) containing material of the cell wall is sensitive to actinomycin D and relatively resistant to chloramphenicol. Similarly, protein synthesis is relatively chloramphenicol-resistant but is fully inhibited by azauracil or spiramycin. Whereas RNA formed in the control culture is partially decomposed after 30 min of incubation, chloramphenicol accelerates its synthesis and prevents its decay. Exudate components apparently stimulate synthesis of ribonucleic acid, proteins and the wall material. The¹⁴C-dap containing material released by prelabelled spores in the form of the exudate during the germination is not re-utilized by the spores germinated in the GL-medium. The results are discussed with respect to the atypical primary synthetic activities of spores under conditions when the postgerminative development is prevented and from the point of view of participation of the germination exudate during these syntheses.     

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.     

Molecular biology of the germination of Bacillus spores

The review deals with recent results and problems of gene expression during germination of Bacillus spores. Three problems were selected: 1. The activation of metabolism as a prerequisite for the synthesis of nucleic acids and proteins. 2. The activation of nucleic acid and protein synthesis during germination. 3. The gene expression programme of germinating spores. Using the highly sensitive two-dimensional polyacrylamide gel analysis three major classes of proteins were distinguished, depending on the time of onset and duration of their syntheses: a) proteins made throughout germination (main class), b) proteins whose synthesis started only after a lag phase and then continued throughout germination, and c) proteins which are synthesized only during the early phases of germination. The programme of protein synthesis is an indicator for the control of gene expression during germination. The regulation of expression of these major gene groups during spore outgrowth is discussed.     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA     

Biogenesis of mitochondrial membranes in Neurospora crassa. Mitochondrial protein synthesis during conidial germination.

The conidia of Neurospora crassa entered logarithmic growth after a 1-h lag period at 30 degrees C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3-h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide-sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production. The rate of protein synthesis in vitro by mitochondria isolated from 0-8-h-old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 mug cycloheximide/ml, increased from low levels in the cinidia to peak levels at 3-4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0-4-h-old cells, but declined after 15 min of incorporation in 6 and 8-h-old cells. The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12 200 to 80 000). Within the high-molecular-weight class, proteins of between 12 000 and 21 500 molecular weight were preferentially lavelled early in germination, whereas after 8 h of growth proteins of 27 500 to 80 000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000-molecular-weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination.

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.     

Spore Germination and Rhizoid Differentiation in Onoclea sensibilis: A Two-Dimensional Electrophoretic Analysis of the Extant Soluble Proteins

Following a geometrically asymmetrical cell division during germination of spores of the fern Onoclea sensibilis L., the small cell differentiates into a rhizoid and the large cell divides to form the protonema. Using silver-staining of two-dimensional gels, we have examined the soluble proteins of spores during germination and of separated rhizoid protoplasts and protonemal cells. Of over 500 polypeptides followed, nearly 25% increased or decreased in prominence during spore germination and the initial phases of rhizoid elongation. Soluble proteins from purified protoplasts of young rhizoids were quantitatively different from those of protonemal cells and germinated spores. Nine polypeptides which appeared after cell division were substantially more prominent in rhizoid protoplasts than in whole germinated spores and have been putatively designated rhizoid-specific polypeptides. The differences in the soluble protein composition of young rhizoids and protonemal cells probably reflect the differential organelle distribution between the two cells as well as differential net protein synthesis in the cytoplasms of the two cells.     

Compositional changes in germinating spores ofAdiantum capillus-veneris L.

Spores ofAdiantum capillus-veneris L. incubated at 25 C for 3 days in the dark were irradiated with continuous red light to induce spore germination and cell growth during following 7 days. A portion of spores were cultured for 8 days in the dark as non-irradiated control. Rhizoidal and protonemal cells were observed at 3 days after transferring spores to the irradiation conditions. During 10 days of the experimental period, changes in the contents of following cell constituents were investigated: total lipid, total soluble sugar, reducing sugar, insoluble glucan, organic acid, protein, soluble α-amino N, and major free amino acids. A large part of nutrient reserves of spores was found to be lipid, whose content decreased markedly as spores germinated. Soluble and insoluble carbohydrates also provided carbon and energy sources during imbibition and germination. Two main reserve proteins were detected by SDS-polyacrylamide gel electrophoresis. These proteins disappeared mostly during germination. Major free amino acids could be assorted into three groups by their patterns of fluctuation during the germination.