Rhythmic changes in transcriptional activity during the development of potato tubers

Chromatin-bound, DNA-dependent RNA polymerase (EC 2.7.7.6) activity and chromatin template availability, as measured with saturating amounts of E. coli RNA polymerase, changes rhythmically during the formation, dormancy, and sprouting of potato tubers. Active growth processes coincide with the highest RNA polymerase activity as well as the greatest template accessibility, during tuberization and sprouting. Consequently, chromatin-associated RNA and protein content is highest in young developing tubers and in old tubers at the onset of sprouting. Ribosomal RNA content, in turn, is maximal in small tubers, remains constant during dormancy, and decreases when sprouting begins, probably due to the translocation of rRNA into the sprouts. The nucleolus changes its shape and size concomitantly with the process of tuberization.     

The genes for ribosomal RNA in diploid and polytene chromosomes of Drosophila melanogaster

The proportion of the Drosophila genome coding for ribosomal RNA was examined in DNA from both diploid and polytene tissues of Drosophila melanogaster by rRNA-DNA hybridization. Measurements were made on larvae with one, two, three and four nucleolus organizer regions per genome. In DNA from diploid tissues the percent rDNA (coding for 28S and 18S ribosomal DNA) was found to be in proportion to the number of nucleolus organizers present. The number of rRNA genes within a nucleolus organizer therefore does not vary in response to changes in the number of nucleolus organizers. On the other hand, in DNA from cells with polytene chromosomes the percent rDNA remained at a level of about 0.1% (two to six times lower than the diploid values), regardless of either the number of nucleolus organizers per genome or whether the nucleolus organizers were carried by the X or Y chromosomes. This independence of polytene rDNA content from the number of nucleolus organizers is presumably due to the autonomous polytenization of this region of the chromosome. When the rDNA content of DNA from whole flies is examined, both the rDNA additivity of the diploid cells and the rDNA independence of polytene cells will affect the results. This is a possible explanation for the relative rDNA increase known to occur in X0 flies, but probably not for the phenomenon of rDNA magnification. — In further studies on DNA from larval diploid tissues, the following findings were made: 1) the Ybb-chromosome carries no rDNA; 2) flies carrying four nucleolus organizers do not tend to lose rDNA, even after eleven generations, and 3) the nucleolus organizer on the wild type Y chromosome may have significantly less rDNA than does that on the corresponding X chromosome.     

High temperature — induced changes in germination,seedling vigour and the metabolic activities in rice seeds

The effects of temperature treatments on rice seeds in the early imbibitional phase were studied with respect to changes in germination percentage, seedling growth in terms of root and shoot lengths, water uptake, respiration and hydrolytic enzymes,viz. α-amylase, adenosine triphosphatase (ATPase) and phytase. As compared with the control, the treatment for 30 min at 50 °C caused a variable degree of increase in all these characters and a positive correlation existed between seedling vigour and hydrolase activities. The treatment for 30 min at 60 °C, on the other hand, elicited a retarding influence on these characters. As regards the enzyme activities, the damaging effect of 60 °C could, however, be visualized only after 72 h of germination which was preceded by an enhancement during the early hours.     

Temperature sensitivity on proliferation and morphologic alteration of human esophageal carcinoma cells in culture

Summary As basic studies of hyperthermia and hypothermia on malignant tumor, the kinetics of proliferative activity, the morphologic changes in the two cell lines, SGF-3 and SGF-5, established in our department after the change of culture temperature were examined. The results obtained were: a) A significant difference was found in the sensitivity to temperatures between the two cell lines originated from human esophageal squamous cell carcinoma. The temperature range allowing cultured cell to proliferate were from 31° to 39° C in SGF-3 and from 29° to 41° C in SGF-5. b) Minor difference occurred in the results between the two cell lines examined during the recovery of proliferative activity, but no proliferative activity was discovered after the cells were exposed to 42° C for 72 h. Two cell lines resumed their proliferation after having been exposed to 27° or 28° C for 72 h. c) Morphologic changes of the cell lines cultured at high temperature were cytoplasmic vacuolation and cell aggregation by phase contrast microscope and the increase of heterochromatin, the decrease of granular formation in nucleoli, and nucleolar vacuolation by transmission electron microscopy (TEM). At low temperatures the changes observed included cytoplasmic ballooning and circumnuclear halo formation by phase contrast microscope, and the increase of heterochromatin, nucleolar segregation, swelling of mitochondria, and dilatation of rough endoplasmic reticulum (rER) by TEM.     

Relationships between circadian rhythm of chilling resistance and acclimation to chilling in cotton seedlings

Cotton (Gossypium hirsutum L. cv. Deltapine 50) seedlings grown under light-dark cycles of 12:12h at 35°C showed rhythmic daily changes in chilling resistance. Chilling treatment (5°C, 48h) started at the beginning or middle of the daily light period resulted in a substantial growth inhibition of the seedlings upon return to 35°C whereas when chilling was started at the beginning or middle of the dark period the subsequent growth of the seedlings was much less inhibited. This rhythm in chilling resistance persisted under continuous light for three 24-h periods, indicating that it is of an endogenous nature. Seedlings grown under continuous light from germination showed no daily changes in resistance, but a rhythm was initiated by introduction of a dark period of 6h or longer. In 24-h cycles with different light and dark periods, maximal resistance was reached just before the start of dark period. Seedlings grown at 35°C could be acclimated to chilling by exposure to low, non-damaging temperatures (25–15°C). A short-term (6h) exposure to 25°C started at the resistant phase resulted in a large increase in resistance during the following otherwise sensitive phase. The resistance induced by the low temperature matched or slightly exceeded the maximal resistance reached during the resistant phase of the daily rhythm of chilling. The low-temperature-induced resistance and the daily rhythmic increase in resistance were not additive, indicating a common mechanism for the two kinds of resistances. An adaptive advantage of a combination of a rapid temperature-induced acclimation and the daily rhythmic increase in resistance is suggested.     

Characterization of freeze-thaw induced ultrastructural damage to endothelial cells in vitro

Summary The pathophysiology of endothelial cells is important to a variety of vascular conditions including coagulation and hemostasis resulting from clinical frostbite. Use of an in vitro model system demonstrated that when bovine endothelial cells were frozen at 1°C or 20°C/min and thawed immediately (20°C/min), a variety of ultrastructural alterations occurred. Membraneous structures were most extensively damaged, with mitochondria the most sensitive organelle. Low amplitude mitochondrial swelling, first evident at 0°C, progressed to high amplitude swelling by −10°C (frozen). In addition, the rough endoplasmic reticulum was dilated and formed large vesicles with a homogeneous matrix. Nuclear changes first occurred at −15°C. These included separation and distortion of the nuclear membrane, changes in chromatin distribution, and disruption of the nucleolus. Scanning electron microscopy revealed perforated plasma membranes in some cells at −10°C (frozen) and in most cells by −20°C. Cultures frozen at 20°C/min revealed mostly the same ultrastructural damage noted at 1°C/min except a higher percentage of cells exhibited alterations. Data from the recovery index and lactic dehydrogenase (LDH) release correlated well with observed ultrastructural changes. Early swelling of mitochondria and dilation of rough endoplasmic reticulum was not lethal in the absence of freezing. Increased swelling in cytoplasmic organelles coupled with nuclear alterations at −15°C resulted in a decreased survival rate and release of significant quantities of LDH by −20°C. No unique morphological changes were temperature specific, but the total number of cells that displayed alterations increased as temperature decreased. The views, opinions or findings, or both, contained in this report are those of the authros and should not be construed as indicative of an official Department of the Army position, policy, or decision unless so designated by other official documentation.     

Conditional rifampicin sensitivity of arif mutant ofEscherichia coli: rifampicin induced changes in transcription specificity

Arif mutantof Escherichia coli that exhibits medium and temperature-dependent sensitivity to rifampicin is described. In the absence of rifampicin, this strain grows in minimal and rich media at 30°C and 42°C. In its presence it is viable in rich medium at both temperatures, but in minimal medium only at 30°C. In minimal-rifampicin medium at the higher temperature, RNA synthesis is decreased. The addition of certain divalent salts (MgSO₄, CaCl₂, BaCl₂) in excess, or chelators (EDTA, EGTA, o-phenanthrolein) greatly increase viability in minimal-rifampicin medium at 42°C. Excess MgSO₄ (10 mM) also increases the rate of RNA synthesis in the same medium. A model is proposed wherein therif mutation is suggested to cause a structural change in RNA polymerase that allows the binding of rifampicin and other ligands at 42°C. Rifampicin-binding is suggested to alter the conformation of RNA polymerase, impairing its ability to express genes required for growth in minimal medium. Implicit in this view is the assumption that these genes are structurally different from those expressed in rich medium in respect of certain template features recognized by RNA polymerase.     

Analysis of the temperature-dependent temporal pattern of heat-shock-protein synthesis in fish cells

Continuous exposure of Chinook salmon embryo cells to an elevated incubation temperature of 24°C induces the transient expression of a set of heat-shock or stress proteins whereas maintenance of the cells at a higher incubation temperature of 28°C produces a continuous synthesis of these stress proteins. In vitro translation studies suggest that the temperature-dependent temporal pattern of stress-protein synthesis is correlated with the levels of stress-protein mRNA. This was verified using a recombinant-DNA probe complementary to the 70K heat-shock-protein mRNA. A transient increase in the level of the fish heat-shock 70K mRNA was observed in RNA samples isolated from cells continuously exposed at 24°C However, a constant increase in the level of this specific mRNA was found in RNA preparations obtained from cells maintained at 28°C Therefore, the temperature-dependent pattern of fish heat-shockprotein synthesis appears to be directly related to the level of heat-shock-protein mRNA.     

Differential effect of temperature on mitrochondrial activity in shoots from freshly harvested and moderately aged kernels of maize (Zea mays L.)

This paper reports on a study of mitochondrial activity in etiolated shoots of freshly harvested and moderately aged kernels of maize. Activity was investigated after incubation at a favourable temperature (25°C), sub-optimal temperature (13°C) and after a heat shock (46°C for 2h). Although impaired mitochondrial activity in shoots from moderately aged maize kernels was not detected at 25°C, deficiencies became evident under low temperature stress (13°C). State 3 oxygen uptake, cyanide-insensitive oxygen uptake and cytochrome oxidase activity were lower in mitochondria from these shoots at 13°C than in mitochondria from shoots of freshly harvested kernels at this temperature. After a heat shock, cyanide-insensitive oxygen uptake was higher, and cytochrome oxidase activity lower, in shoots of aged kernels than in shoots of fresh kernels. No significant differences in ADP: O ratio or succinate dehydrogenase activity occurred between mitochondria from shoots of the two seed lots in any of the temperature treatments.     

Cold-induced changes affect survival after exposure to vitrification solution during cryopreservation in the south-west Australian Mediterranean climate species Lomandra sonderi (Asparagaceae)

There is limited knowledge of the effects of exposure to low temperatures in the unique Mediterranean climate plant species of Western Australia. We have thus investigated the effect of low temperature on cryogenic tolerance in Lomandra sonderi, an endemic perennial species of southwest Western Australia. Lomandra sonderi plants were preconditioned in tissue culture at constant 23 °C (12 h light/dark cycle) or alternating 20/?1 °C (16 h light and 8 h dark cycle). Shoot tips from both conditions were analysed for their phospholipid, sterol and soluble sugar compositions. Shoot tips were also cryoexposed via a droplet-vitrification protocol. Survival in both preconditioning regimes for cryoexposed and non-cryoexposed samples was the same, but plants from the 20/?1 °C regime displayed an improved tolerance to the overall cryopreservation process in both cryoexposed and non-cryoexposed samples, thereby eliminating exposure to liquid nitrogen as a primary cause of reduced post-cryogenic viability. Preconditioning of in vitro shoots of L. sonderi at 20/?1 °C induced significant increases in phosphatidylcholine (from 7.30 ± 3.46 to 22.2 ± 7.80 ng mg^?1 FW) and increases in several soluble sugars (fructose, galactose, glucose, sucrose) compared to shoots incubated at 23 °C—changes consistent with known cold acclimation responses in plant species generally—but sterol content remained largely unchanged. Analysis of electrolyte leakage in shoot tips from both preconditioning regimes generated a significantly lower LT₅₀ value in the 20/?1 °C samples (?5.45 ± 0.53 °C) over the 23 °C samples (?2.5 ± 0.08 °C). Increased tolerance to cryoexposure in L. sonderi appears to lie mainly with acclimation-induced changes in membrane composition and promotion of membrane stability and hence increased resistance to freeze damage.     

Germination and mitochondrial damage in spores of Dictyostelium discoideum following supraoptimal heating

Spores of Dictyostelium discoideum may be quantitatively activated with a heat treatment of 45°C for 30 min. Heat activation at either higher temperatures or for longer duration at 45° C resulted in damaged spores. The spores showed an increased postactivation lag time at 23°C and an increased inability to respond to deactivation with 0.2 M sucrose. As the severity of supraoptimal heating increased, a greater percentage of the spores appeared to contain phase dark lesions and to lose viability. Oxygen uptake began to decrease during and after the appearance of the lesions. Using electron microscopy, the phase dark lesions were found to be mitochondria with disrupted cristae.     

Nucleolar Organizer ultrastructure in Allium cepa

The Nuoleolar Organizer Region (NOR) was studied in Allium cepa. The cells analyzed were: meristematic root tip cells, cells from the layers of the anther (exothecium, intermediate layer cells, endothecium and tapetum) and meiocytes from preleptotene to diptotene. Conventional electron microscope techniques and serial section were used in the study. Ultracytochemical tests, using preferential techniques for nucleic acids were also applied. The results show that: a) Although the NOR may have a very variable form in the different cells analyzed, in each case its fine structure is similar, being constituted of chromatin differentiated in high and low density zones. b) In each case, its cytochemical characteristics are similar; highly positively to EDTA (preferential stain for RNP) being observed in the low density zones. As scattered chromatin these would constitute the active regions of the NOR. c) In certain cases a clear relationship is seen between NOR volume and the size of the nucleolus. d) The chromatin associated to the NOR always appears as very dense zones. e) The NOR-Nucleolus relationships are established exclusively with the latter's fibrillar pars. f) The nucleolar segregation phenomenon takes place sometimes as a result of a NOR contraction and in other cases depends on the position of the NOR in relation to the nucleolus. g) In preleptotene, the NOR shows its characteristic morphology and is positive to EDTA, while from zygotene to diplotene the structure of the NOR presents no differences from the rest of the chromatin and only the lateral elements of the synaptonemal complex or its remnants are positive to EDTA. h) The synaptonemal complex runs through the NOR with no differentiation and on occasions its lateral elements show associations with the nucleolus.     

Light,Temperature, and Desiccation Effects on Photosynthetic Activity,and Drought-Induced Ultrastructural Changes in the Green Alga <Emphasis Type="Italic">Klebsormidium dissectum</Emphasis> (Streptophyta) from a High Alpine Soil Crust

Members of the cosmopolitan green algal genus Klebsormidium (Klebsormidiales, Streptophyta) are typical components of terrestrial microbiotic communities such as biological soil crusts, which have many important ecological functions. In the present study, Klebsormidium dissectum (Gay) Ettl &; Gärtner was isolated from a high alpine soil crust in the Tyrolean Alps, Austria. Physiological performance in terms of growth and photosynthesis was investigated under different controlled abiotic conditions and compared with ultrastructural changes under the treatments applied. K. dissectum showed very low light requirements as reflected in growth patterns and photosynthetic efficiency. Increasing temperatures from 5°C to 40°C led to different effects on respiratory oxygen consumption and photosynthetic oxygen evolution. While at low temperatures (5–10°C), respiration was not detectable or on a very low level, photosynthesis was relatively high, Reversely, at the highest temperature, respiration was unaffected, and photosynthesis strongly inhibited pointing to strong differences in temperature sensitivity between both physiological processes. Although photosynthetic performance of K. dissectum was strongly affected under short-term desiccation and recovered only partly after rehydration, this species was capable to survive even 3 weeks at 5% relative air humidity. K. dissectum cells have a cell width of 5.6?±?0.3 μm and a cell length of 8.4?±?2.0 μm. Desiccated cells showed a strongly reduced cell width (46% of control) and cell length (65% of control). In addition, in desiccated cells, fewer mitochondria were stained by DIOC₆, and damaged plasma membranes were detected by FM 1–43 staining. High-pressure freeze fixation as well as chemical fixation allowed visualizing ultrastructural changes caused by desiccation. In such cells, the nucleus and chloroplast were still visibly intact, but the extremely thin cell walls (75–180 nm) were substantially deformed. The cytoplasm appeared electron dense and mitochondria were altered. Although K. dissectum can be characterized as euryoecious species, all ecophysiological and ultrastructural data indicate susceptibility to desiccation. However, the steadily occurring fragmentation of filaments into smaller units leads to improved self protection and thus may represent a life strategy to better survive longer periods of drought in exposed alpine soil crusts.