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Differential derepression of the genome of potato tuber cells can be initiated by slicing the tissue into disks. The consequence of this procedure on the cells of the wound surface is dedifferentiation and cell division followed by redifferentiation to a suberized phellem cell. The drift of glucose-, glucose-1-phosphate-, glucose-6-phosphate-, fructose-6-phosphate- and 6-phospho-gluconatelevels has been determined in the derepressed tissue. With the exception of 6-phospho-gluconate all intermediates so far investigated showed a rise in concentration after derepression.
This is interpreted as a consequence of altered enzymic activities which were estimated for phosphoglucomutase, hexokinase, phosphoglucoisomerase, gluco-6-phosphate- and 6-phosphogluconatedehydrogenase. The two dehydrogenases were activated after derepression, the activation represented a de-novo-synthesis, as was demonstrated with the inhibitors Actidione (translation) and p-Fluorophenyl-alanine (protein synthesis in general). Hexokinase and phosphoglucoisomerase were not severely affected by cutting the tissue. Phosphoglucomutase was degrated rapidly, the degradation being dependent on protein synthesis. The importance of an enhanced activity of the pentose phosphate shunt for the stressed cell is emphasized and the possibility of an alteration in the osmotic pressure within the cell and especially in the nucleus — a primary consequence of wounding — as a cause of derepression in potato tuber cells is discussed.
At pH 5.3 and 4.5 the half life of valyl-, threonyl-, leucyl- and seryl-tRNA from E. coli K 12 is significantly higher than at pH 6.8. While no changes were observed in the MAK elution patterns of valyl- and threonyl-tRNA, leucyl-tRNA was eluted in two peaks at pH 6.8 and 5.3 and in one broad peak at pH 4.5. Seryl-TRNA - two peaks at pH 6.8 - was separated in three peaks at pH 5.3 and 4.5. Rechromatography of these peaks at the other pH suggests the existence of at least four species of seryl-tRNA in E. coli K 12.
One of the earliest consequences of slicing plant storage organs such as potato tubers into thin disks is the formation of polysomes, which in potato slices is complete after 9 hours and is dependent on transcription. Fresh disks do not incorporate 32P, 3H-uridine or 14C-leucine into their ribosomes, whereas ribosomes and polysomes of aged disks use these precursors effectively. This development can be completely blocked by actinomycin D. Among the different RNAs synthesized during aging is 28S- and 16S—rRNA, 5S—RNA, tRNA, and a component sedimenting around 15—18S with a base-composition different from 16S—rRNA, 5S- and 4S—RNA and which supports peptide formation in an in vitro incorporation system.
It is suggested that this compound represents mRNA, which is not available immediately after slicing the tissue. These findings are consistent with the view of a derepression phenomenon in sliced storage tissue.
Whereas ribosome preparations of freshly sliced potato disks do not show appreciable activity in an in-vitro amino acid incorporation system, aging of the tissue leads to a greatly enhanced incorporation activity which reaches its maximum 24 hours after slicing. If ribosomes from freshly excised disks are provided with polyuridylic acid, their activity in the incorporation of phenylalanine is increased about 8 fold.
Moreover, an RNA-fraction can be dissociated by EDTA from ribosomes of aged potato tuber slices, which sediments at 15 —18S, has a base composition different from that of 16S — rRNA, 5S-and 4S —RNA, and is not present on ribosomes of fresh slices. Its appearance is inhibited by actinomycin D and therefore most probably dependent on transcription. This compound, purified from sucrose gradients, enhances in vitro leucine incorporation into peptide material by ribosomes of fresh potato slices.
The possibility is discussed that this fraction-among other factors-is responsible for the enhanced protein synthesis after slicing plant storage organs, and is indicative of a general derepression phenomenon in these tissues.
Resting potato tuber tissue possesses only faint activity of the two dehydrogenases of the oxidative pentose phosphate cycle, glucose-6-phosphate- and 6-phosphogluconate dehydrogenase. Slicing of the tissue, however, greatly enhances the action of both enzymes. The slicing-induced increase in activity is a consequence of intensified action of at least 5 glucose-6-phosphate dehydrogenase isozymes and a more differentiated activation/inactivation of seven 6-phosphogluconate dehydrogenase isozymes.
Using density labelling and isopycnic equilibrium centrifugation it could be demonstrated, that the bulk of both enzymes appearing after slicing the tissue is the result of de novo synthesis rather than activation of pre-existing proenzymes.
An improved method for isolation of yeast m utants auxotrophic for 5′-dTM P is presented. The procedure employs the two folic acid antagonists am inopterin and sulfanilam ide (SAA). Selectiveness of the procedure depends on concentration of SAA and time of incubation.
44 mutants auxotrophic and 3 conditionally auxotrophic for 5′-dTMP were isolated. All belong to one complementation group. The corresponding gene was designated TMP1. Tetrad dissection revealed its chromosomal nature. TMP1 is not closely linked to the genes ADE2,, LEU1, ARG 4, ILV2, HIS5, LYS1 and the mating type locus. With the centromere-linked genes ARG4 and LEU1 I gene TMP1 exhibited second division segregation frequencies of 0.42 and 0.53 respectively, indicative of centromere-linkage.
Strains auxotrophic and conditionally auxotrophic for 5′-dTM P were all respiratory deficient (petite). Genetical analysis indicates that the petite phenotype is due to loss of the rho factor in cells harbouring either tmp1 or tmp1ts alleles.
In haploid and diploid S. cerevisiae the dimer yield ratio TT̂/CT̂ is found to be 1.2/1 and 1.3/1, resp., at the UV (254 nm) unit dose 1 erg/mm2, the share of TT̂ and CT̂ in a UV (254 nm) lethal hit being 0.7 TT̂ and 0.6 CT̂. A general formulation of the UV lethal hit is given and discussed. The TT̂ + CT̂ yields obtained for S. cerevisiae are compared to those reported for other organisms. It is found that there obviously exists a directly proportional linear correlation between genome size and TT̂ + CT̂ yield for the UV dose range well below the stationary levels of the TT̂ and CT̂ formation kinetics.
Testosterone, Androst-4-en-3,17-dione, Enzyme Induction, S trep to m yces hydrogenans After cultivation of S trep to m yces hydrogenan s in the presence of 3H-labelled testosterone, radio active steroids were extracted separately from the cytosolic, ribosomal and cell wall-membrane fraction of the cells and from the culture medium, respectively.. The separation of the steroids was performed by one-and two-dimensional thin layer chromatography (TLC). The identification of the main metabolites was achieved by crystallization to constant specific radioactivity, specific staining procedures and acetylation. The oxidation of testosterone to androst-4-en-3,17-dione is by far the predominating reaction, which is almost finished after 3 h cultivation. Androst-4-en-3,17-dione is mainly transferred into the culture medium and partly accumulated within the cell wall-membrane fraction. High polar steroid metabolites and androstane derivatives are present in very small amounts only.
A screening procedure is presented which allows the isolation of yeast mutants (typ tlr) with highly efficient utilization of exogenous deoxythymidine-5′-monophosphate (5′-dTMP) (>50% ). Data are given concerning the phenomenon of 5′-dTMP utilization in general: (i) The ability of S. cerevisiae to incorporate exogenous 5′-dTMP was found to already be a wild type feature of this yeast, i. e. apparently not to be due to any mutation such as typ , tup, tmp per or tum. Consequently these mutations are interpreted as amplifiers of a pre-given wild type potency. So far eight stages of 5′-dTMP utilization were detected as classified by the optimal 5′-dTMP requirement, with 5′-dTMP biosynthesis blocked, of the corresponding mutant strains isolated. All of them fit well into a mathematical series of the type “2n × 1.5” (n = 0, 1, 2, … , 11), where the product term for n = 11 represents the 5′-dTMP requirement (μg/ml) of the best 5′-dTMP utilizing wild type strain found, (ii) Amplification of the 5′-dTMP utilizing potency obviously is due to any genetically determined alteration of the yeast 5′-dTMP uptaking principle itself or of physiological processes accompanying the monophosphate’s uptake, (iii) The functioning of 5′-dTMP uptake requires acidic (≦ pH 6) conditions in the yeast cell’s outer environment, (iv) Some yeast typ and typ tlr mutants were found to exhibit a more or less pronounced sensitivity towards exogenously offered 5′dTM P. The response of a sensitive strain towards inhibitory concentrations of the nucleotide apparently is co-conditioned by the presence or absence of thymidylate biosynthesis. With 5′-dTMP biosynthesis blocked the 5′-dTMP mediated inhibition is a permanent one and finally leads to the death of a cell. With a functioning thymidylate biosynthesis, in contrast, the inhibition is only temporary, (v) Yeast typ or typ tlr strains were observed to dephosphorylate exogenous 5′-dTMP to thymidine due to a phosphatase activity which cannot be eliminated at pH 7 + 70 mм inorganic phosphate conditions in the growth medium. This 5′-dTMP cleavage obviously occurs outside the cell and does not seem to be correlated both to the monophosphate’s uptake and to the phenomenon of 5′-dTMP sensitivity. The destruction of 5′-dTMP does not disturb (5′-dTMP) DNA-specific labelling.
Chromatin, RNA Polymerase, Potato Tuber Tissue, Aging Phenomenon The synthesis of RNA by chromatin-bound RNA polymerase (E.C. 2.7.7.6.) from white potato tubers proceeds at a low rate, which is enhanced after slicing the tissue, however. Concomitantly DNA template availability as measured with saturating amounts of Escherichia coli polymerase is diminished drastically. Nearest neighbor frequency analysis proved that the RNA synthesized on chromatin of intact tubers is different from that synthesized on chromatin of sliced tissue.
The RNA polymerase of white potato tubers is dependent on all four ribonucleoside triphos phates and a divalent metal ion such as Mg2+ or Mn2+ and totally inhibited by the presence of pyrophosphate. Actinomycin D blocks the formation of the RNA product, which could be shown to be a heteropolymer by nearest neighbour frequency technique. The Km of the chromatin-bound enzyme with regard to ATP, GTP, CTP and UTP was 5.1 X10-5 M, 1.6X10-5 M, 0.9X10-5 M and 0.45 X 10-5M/1 respectively, α-amanitin inhibits the overall activity to about 50%, which indicates the presence of equal amounts of polymerase I and polymerase If.
A thylakoid membrane preparation isolated from the blue-green alga Anacystis nidulans was freed from carboxysomes, soluble enzymes and the pigment P750 by floating in a discontinuous sucrose density gradient. In a buffer containing sucrose and the zwitterionic detergent Miranol S2M-SF the thylakoids were loaded on a linear 10-18% sucrose density gradient which also contained Miranol. The sedimentation yielded three bands, the lower two of which were green and the upper one was orange. The light green band in the middle of the gradient was the only one to show any photosystem II activity. This was measured as light-induced electron transport from diphenylcarbazide (DPC) to dichlorophenol-indophenol (DCPIP). The activity was sensitive to dichlorophenyl-dimethylurea (DCMU).
The red absorption maximum of the particles in this middle band - henceforth called photosystem II particles - was found at 672 nm and the maximum of their low temperature fluorescence emission spectrum at 685 nm upon excitation with blue light. Cytochrome b559 was the only cytochrome found in these particles; it was present at an average ratio of one molecule cytochrome per 40 -50 molecules chlorophyll a. C550 photoreduction with accompanying photooxidation of cytochrome b559 was also observed in the photosystem II particles. Good photosystem II preparations did not contain any detectable amounts of P 700.
By means of sodium dodecylsulfate polyacrylamide gel electrophoresis the polypeptide composition of the photosystem II particles was studied. Dissolution of the chlorophyll protein complexes was done under strongly denaturing conditions; consequently, no green bands were observed on the gels. The polypeptide pattern of the photosystem II particles showed two strong predominant bands of protein components with apparent molecular weights (app. mol. wts.) of about 50 000 and 48 000. These two bands are unique for photosystem II. Two other weaker bands were also found characteristic for photosystem II, the band of a polypeptide with an app. mol. wt. of 38 000 and that of a polypeptide with an app. mol. wt. of 31 000. Sometimes in addition the weak band of a polypeptide with the app. mol. wt. 27 000 was observed on the gel. The polypeptide 38 000 aggregated upon boiling of the sample in the presence of the denaturing agents prior to the electrophoresis, yielding an aggregate with an app. mol. wt. of 50 000. Additional polypeptides which were often found in the photosystem II particle preparation could be identified as subunits of the coupling factor of photophosphorylation CF1. None of the polypeptides described as characteristic for photosystem II are due to proteolytic activity.
As the observed photosystem II activity was found to be DCMU-sensitive it appears that the DCMU-binding protein is among the here described photosystem II polypeptides. Moreover, the authors have reason to believe that one of the major protein components found characteristic for photosystem II is cytochrome b559.
The growth of Synechococcus at different intensities of white and red light caused changes in the pigment composition. The ratio of chlorophyll a to phycocyanin varied from 1:8,2 in LWLI-grown cells to 1:1,4 in cells grown at HWLI and to 1:15,7 in cultures exposed to HRLI. Acyl lipids were quantitatively determ ined and fatty acids of the individual lipid classes analysed by GLC. Phycocyanin-free photosynthetic lam ellae were obtained by fractional centrifugation. No variation was found in the acyl lipid composition of the m em brane preparations. These all contained MGDG, DGDG, SQDG and PG as components. In all the lipids investigated, palmitic, hexadecenoic and octadecenoic acids m ade up to more than 90% of total fatty acids. The pattern of these major components w ithin the lipids from the different cultures depended on the light used. No large differences were detected between zones obtained from LWLI and HRLI isolated membranes, whereas density gradient centrifugation of those from HWLI-grown cells resulted in a completely different pattern of bands. The variations in lipid and fatty acid composition are discussed with respect to changes observed in lipid composition of whole cells and the results reported on tem perature dependent shifts in lipid fluidity in cyanobacteria.
A large number of staphylinid beetles are closely associated with ants and termites (for review see Wilson 1971, Kistner 1979). Those species living with ants are commonly called myremcophiles. At least a few (Atemeles, Lomechusa) have "broken" the communication code of their host species and are thereby able to become completely integrated in the social system of the ants (Hölldolber 1967, 1970, 1971). In an attempt to understand the evolutionary pathways of this highly specialized social parasitic behavior, we studied closely related staphylinid species that do not live within the ant society but instead occupy the foraging trails and garbage dumps of an ant nest. ...
Synechococcus (Anacystis nidulans, strain L 1402-1) were grown at + 37 °C in an atmosphere of 0.04 vol.% CO2 using different light conditions. Changing the culture conditions caused alterations in pigment ratios and ultrastructure of Synechococcus. In comparison to the low white and red light grown cells under strong white light the number of thylakoids decreased and an accumulation of storage carbohydrates could be observed. The number of the polyhedral bodies also varied with culture conditions. The results are discussed with reference to the pigment composition and the function of the polyhedral bodies.
14C-and 15N-Assimilation, 15N-Labelled Amino Acids, M arine D iatom s The marine diatoms Bellerochea yucatanensis and Skeletonema costatum were grown at +20 °C in 0.03 vol.% CO2 with nitrate or ammonia. The 15N -am m onia and 15N -nitrate assim ila tion and 15N -incorporation into various amino acids were studied of both diatom s during exponential growth phase in dependence of different nitrogen conditions. In all experiments the 15N -am m onia uptake was lower than the 15N -nitrate assim ilation rate up to 20-40 min photo synthesis. N itrate lim itation -cells grown in nitrate followed by growth in nitrogen-free m edium for 24 h — caused a strong 15N-label into aspartate after adding 15NH 4C1 (1 m M). In cells grown in nitrate highest enrichment of 15N was found in glutamine. Results were discussed with reference to the operating of the GS/GOGAT system and glutam ic acid dehydrogenase pathway. Photosynthetic 14CO2 fixation experiments showed a very high labelling of aspartate which was interpreted with a phosphoenolpyruvate carboxylation catalysed by phosphoenolpyruvate carb-oxykinase.
The cyanobacterium Synechococcus (Anacystis nidulans, strain L 1401-1) grown under different light conditions showed variations in pigmentation. Ratios of photosynthetic pigments and the effect on quantum requirement and oxygen evolution were studied. An increase in the ratio of chlorophyll a forms with absorption maxima in the far red regime to total chlorophyll a forms was observed in cells grown in strong white light. The quantum efficiency of orange light (637 nm) - absorbed by phycocyanin - was higher after growth of Synechococcus in white than in red light. The quantum efficiency at 677 nm increased when cells were grown in red light and decreased strongly after transfering red light grown cells to conditions of strong white light. The results show an adaptation of pigment composition to light regimes during growth and its effect on photosynthesis.
Bleaching of chlorophyll was studied in the leaves of rye seedlings (Secale cereale L.) treated with four chlorosis-inducing herbicides of different potency (weak photodestructions, group 1: aminotriazole, haloxidine; strong photodestructions, group 2: San 6706, difunone). Chlorophyll deficiency and particularly the inactivation of a chloroplast marker enzyme, NADP-dependent glyceraldehyde-3-P dehydrogenase, that occurred in the presence of group 2 herbicides were stronger in red, than in blue, light.
When grown in white light of low intensity (10 lx) herbicide-treated leaves contained chloro phyll, 70 S ribosomes and unimpaired activities of NADP-dependent glyceraldehyde-3-P de hydrogenase. At 10 lx only the leaves treated with SAN 6706 and difunone were strongly carotenoid-deficient but not those treated with group 1 herbicides. After all herbicide treatments 10 lx-grown leaf tissue was, however, not capable of photosynthetic O2-evolution indicating some disorder of photosynthetic electron transport. Leaf segments grown at 10 lx were exposed to a high light intensity of 30000 lx at either 0 ° C or 30 °C. In treatments with group 1 herbicides chlorophyll accumulation was stopped in bright light at 30 °C but breakdown was not apparent. Only at 0 °C and in the presence of high, growth-reducing, herbicide concentrations chlorophyll was slightly degraded. The RNAs o f the 70S ribosomes were, however, clearly destroyed at 30000 lx and 30 °C in aminotriazole-treated leaves. In leaves treated with group 2 herbicides chlorophyll was rapidly degraded at 30000 lx both at 0 ° C and 30 °C, however, only in the presence of O2, indicating a true photooxidative and mainly photochemical nature o f the reactions involved. This chlorophyll breakdown was accompanied by the photodestruction of 70S ribosomes and the inactivation of NADP-glyceraldehyde-3-P dehydrogenase.In treatments with group 1 herbicides photoinactivation of the latter enzyme did not occur, although it was clearly localized in the bleached plastids, as demonstrated by gradient separation of organelles.
In the presence of group 2 herbicides the chlorosis was originating from a direct photo oxidation of chlorophyll, accompanied by a massive destruction of other plastid constituents and functions. In treatments with group 1 herbicides photodestructions appeared to be much weaker and insufficient to affect chlorophyll directly. Mediated through some photodestructive inter ference with obviously more sensitive plastid components, such as their ribosomes, further chlorophyll accumulation was, however, prevented.
Pheromonal synergism and inhibition in P. flammea was further studied through electrophysiological and field trapping tests. Z11-tetradecenyl acetate and Z11-hexa - decenyl acetate, each acting upon a separate type of male sensory cell, were equally effective in synergizing attraction responses to the major pheromone component, Z9-tetradecenyl acetate. Addition of Z7-dodecenyl acetate to these lures reduced captures. Male attraction specificity markedly varied with local moth density.
Among chlorosis-inducing herbicides that interfere with carotenoid synthesis two groups of different potency can be discriminated (group 1: aminotriazole amd haloxidine; group 2 with more extensive photodestructions: pyridazinone herbicides and difunon). After application of herbicides of group 2 colored carotenoids were completely absent and preexisting chlorophyll was degraded by photochemical reactions requiring high light intensity and O2, that occurred also at 0°C. In treatments with group 1 herbicides direct photodegradation of chlorophyll was not sufficient to generate the chlorosis. Light-induced interference with constituents of the chloroplast protein synthesis apparatus being more sensitive to photooxidative damage than chlorophyll, appeared to indirectly mediate the chlorosis. In the absence of chloroplast protein synthesis further chlorophyll accumulation is prevented. Photodegradation of chlorophyll in the presence of group 2 herbicides involved the participation of O2- radicals and was accompanied by lipid peroxidation. In all herbicide treatments the catalase activity of the leaves was very low. Only in the presence of group 2 herbicides chloroplast enzymes of cytoplasmic origin (e.g. NADP-glyceraldehyde-3-phosphate dehydrogenase) were also inactivated. Rapid inactivation of catalase as well as of NADP-glyceraldehyde-3-phosphate dehydrogenase was induced by exposure of dim-light-grown herbicide-treated leaves to bright light, also at 0°C. In treatments with herbicides of group 2 also other peroxisomal enzymes (e.g. glycolate oxidate, hydroxy-pyruvate reductase) were affected. The elimination of these peroxisomal enzymes also appeared to depend on photooxidative processes of the chloroplast.