The isolation of DNA from agarose gels by electrophoretic elution onto malachite green-polyacrylamide columns

Electrophoretic elution of DNA coupled with direct adsorption onto malachite green-polyacrylamide columns was used to isolate double- and single-stranded DNA from agarose gels. Subsequently, DNA was eluted with a high salt buffer and filtered through Sephadex which permitted recovery of the DNA in a low salt buffer at concentrations suitable for heteroduplex analysis by electron microscopy. This method was tested by examining hetero-duplexes formed from the isolated complementary single strands of T7 wild type DNA and a T7 deletion mutant. More than 80% of the reannealed molecules were intact heteroduplexes showing the deletion loop. Irradiation of single-stranded DNA with 254 nm light resulted in distorted, convoluted heteroduplexes while 366 nm light did not show this effect.     

Epithelial cell volume modulation and regulation

Summary Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cell swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cell shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described, and a number of experiments are discussed in which cell volume determination provided significant new information about the dynamic behavior of epithelia. The mechanism of volume regulation of epithelial cells exposed to anisotonic bathing solution is discussed and shown to involve the transient stimulation of normally dormant ion exchangers in the cell membrane.     

Seasonal changes in adventitious root formation in hypocotyl cuttings of Pinus sylvestris: Influence of Photoperiod during stock plant growth and of indolebutyric acid treatment of cuttings

Seedlings of Pinus sylvestris were grown for 6 weeks under natural light conditions in a temperature controlled environment room. Cuttings from these plants were rooted in tap water or in indolebutyric acid (IBA) solutions for 60 days at an irradiance of 16 W m^-2. Experiments were performed at 3-week intervals during two growth seasons. — Seasonal changes in root formation were found in control cuttings as well as in IBA treated cuttings. The number of roots and the percentage of cuttings that rooted were high during early spring and autumn. During the summer period hardly any roots were formed. Stimulation of root formation by IBA occurred manily during spring and autumn when cuttings already possessed the ability to form roots. — The influence of photoperiod during stock plant growth was also investigated. Shorter photoperiod resulted in an increase in the number of roots and rooting percentage. The period during summer where rooting was inhibited under natural light conditions was considerably shortened when stock plants were grown at a photoperiod of only 4 h. The results demonstrate the importance of the growing conditions for stock plants for subsequent root formation. The results are discussed with special reference to the role of irradiance.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

Action of red light on indole-3-acetic-acid status and growth in coleoptiles of etiolated maize seedlings

Brief irradiation of intact etiolated seedlings of maize (Zea mays L.) with red light (R; 30 W cm^-2, 10 min) reduces the amounts of diffusible and free (solvent-extractable) indole-3-acetic acid (IAA) obtainable from excised coleoptile tips. The effect is transient, the lowest level (30% of the dark control) occurring at about 3 h after irradiation. The free-IAA content of the whole coleoptile and the diffusible-IAA yield from the base of the same organ are similarly reduced, whereas the conjugated-IAA content of the coleoptile is not affected. These results support the view that R inhibits the production of IAA at the coleoptile tip. It is further shown that R inhibits biosynthesis of [³H]IAA from [³H]tryptophan supplied to the coleoptile tip. The shapes of the fluence-response curves obtained for the reduction of the diffusible-IAA yield by R and far-red light (FR) indicate the participation of two photoreactive systems. One has thresholds at 10^-3 W s cm² of R, five orders of magnitude less than the minimum required for the appearance of spectrophotometrically measurable far-red-absorbing form of phytochrome (Pfr) in vivo, and 10^-1 W s cm^-2 of FR; its response is linear to the logarithm of fluence exceeding five orders of magnitude. The other system is seen above 10² W s cm^-2 as an increase in the slope of the fluenceresponse curve; its response is FR reversible and related to the Pfr level of total photoreversible phytochrome. Both systems inhibit biosynthesis of IAA from tryptophan. Elongation of the coleoptile is stimulated by R; the stimulation is most apparent in the apical region, and is saturated with a fluence at which bo detectable pfr is formed. Farred light can also saturate this response. Since the endogenous IAA concentration in the coleoptile appears not to be in the inhibitory range, it is concluded that the stimulation of coleoptile elongation is not the result of changes in free-IAA levels.Abbreviations FR far-red light - IAA indole-3-acetic acid - Pfr phytochrome in the far-red-absorbing form - Pr phytochrome in the red-absorbing form - R red light     

Fusion of phosphatidylserine and mixed phosphatidylserine-phosphatidylcholine vesicles Dependence on calcium concentration and temperature

Dynamic light scattering has been used to study the temperature dependence of Ca²⁺-induced fusion of phosphatidylserine vesicles and mixed vesicles containing phosphatidylserine and different phosphatidylcholines. The final vesicle size after Ca²⁺ and EDTA incubation serves as a measure of the extent of fusion. With phosphatidylserine vesicles, the extent of fusion shows a sharp maximum at an incubation temperature which depends on the Ca²⁺ concentration between 0.8 and 2 mM. The shift in the fusion peak temperature with Ca²⁺ concentration is similar to the typical shift in the phase transition temperature with divalent cation concentration in acidic phospholipids. The results suggest a direct correlation between the fusion peak temperature and the phase transition temperature in the presence of Ca²⁺ prior to fusion. With mixed vesicles containing up to 33% of a phosphatidylcholine in at least 2 mM Ca²⁺, the extent of fusion as a function of incubation temperature also shows a maximum. The fusion peak temperature is essentially independent of the quantity and type of phosphatidylcholine and the Ca²⁺ concentration, and identical to that with pure phosphatidylserine in excess Ca²⁺. The results imply that Ca²⁺-induced molecular segregation occurs first, and fusion subsequently takes place between pure phosphatidylserine domains.     

Fluence response relationship of carotenogenesis inNeurospora crassa

The fluence response of the blue light induced carotenoid synthesis inNeurospora is biphasic. Using fluence rates between 0.3 and 40 Wm^-2, increasing illumination times beyond 16 min (at 20°C) result in a second rise of the amount of carotenoids synthesized in the subsequent dark period. On altering the temperature, the transition point to the second phase of the response is shifted to shorter/longer illumination periods with increasing/decreasing temperature, respectively. The transition point can also be shifted by administering high fluence rates of near UV light: The start of the second phase is already triggered after an irradiation time of 2 min. The findings suggest that elements of the transduction sequence become depleted and senstivity recovers in a temperature-dependent process. The biphasic response and the effects of UV light are discussed in relation to the transduction mechanism and to the ecological significance.Presented in part at the meeting of the Deutsche Botanische Gesellschaft, September 1978, Marburg, Federal Republic of Germany     

Relationship of photocarotenogenesis to other behavioural and regulatory responses inPhycomyces

The effect of light on carotene accumulation was studied by analyzing the -carotene content of 4--old mycelia continuously exposed to illumination of different intensities. The wild-type, mutants defective in phototropism, mutants defective in carotene regulation, and newpic mutants specifically defective for photocarotenogenesis were examined. The results indicate that photocarotenogenesis depends on a single sensory pathway which shares its earlier steps (governed by genesmadA andmadB) with the sensory pathway for phototropism. It shares its later steps (probably governed by genescarA andpicB) with one of the pathways for carotene regulation, and includes at least one specific step (governed by genepicA) not known to be involved in other responses.     

Effects of nitrogen deficiency and light of high intensity onCryptomonas rufescens (Cryptophyceae)

Summary C.rufescens excystment, experimentally induced, corresponds to a general metabolism recovery of the cell, previously in a resting phase. The cytoplasm changes without any polarity, and organelles like gullet and flagella redifferentiate. The thylakoids develop mainly from the stored lipidic compounds which then disappear. Phycoerythrin immediately fills the intrathylakoidal lumen. Pigment synthesis seems closely associated with the development of membranes. The activated cell divides and the cyst wall breaks down. The destruction of the wall begins in the median layer and is followed by a mechanical rupture of the external and internal layers. Each germinative cyst releases two or four fully differentiated cells. There is an exact symmetry between excystment and encystment, all the transformations of theC. rufescens cell being reversible.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.