Observations on the marginal ruffles of an established fibroblast-like cell line

Summary LW13K2 cells, a clone of a spontaneously in vitro transformed derivative of embryonic Lewis rat fibroblastic cells, were studied by phase contrast cine-light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ruffles found at the advancing edge of cells grown on glass substrates in vitro form and recede in a period of less than one min if they do not make an attachment of the substrate. If they fail to make an attachment they may form pinocytotic channels near the leading edge as described by Price (1972) and/or collapse, generally backwards, towards the cell body. The spines which appear to reinforce the membranous ruffles are the last structures to disappear, and accumulate in an irregular array behind the ruffling edge; this area is behind that in which pinocytosis occurs. In comparison with the sparse numbers of ribosomes found in the trailing edge, they are present in notable concentrations near the leading, ruffling edge of the cell. No membrane vesicles have been found in or near the ruffling edges at the ruffle-spine concentration zone.     

Kinetics of uridine uptake and incorporation into RNA in tobacco pollen culture

The capacity of pollen tubes to utilize exogenous uridine during 8 h of cultivation in shaken suspension in a sugar-mineral medium was examined by continuous and pulse labelling with³H-uridine. The increase of uptake with increasing concentration of the nucleoside indicated a saturable transport system with an approximate K_m of 9.4 × 10⁻⁶ M and 12.5 × 10⁻⁶M as determined in 1-h and 6-h cultures, respectively. Maximal uptake took place at the beginning of germination reaching a rate of about 2 nmol h⁻¹ per 1 mg of dry pollen at 0.1 mM external uridine. The uptake activity decreased with the time of pollen tube growth to less than one third during the 8-h cultivation period. Moreover, the level of radioactivity taken up initially decreased later on during continuous cultivation in the presence of³H-uridine. The uptake took place against a concentration difference and the onset and rate of uridine release depended on its exogenous concentration. The activity of the nucleoside incorporation into RNA increased during the first 4 h of cultivation, decreasing later on. The proportion of RNA radioactivity in continuously labelled pollen tubes grew steadily during 6 h and reached 2.5% with respect to soluble pool at 0.4 μM uridine. The time course of RNA labelling was independent of uridine concentration within the range of 0.4 μM to 40 μM but this concentration rise resulted in an about fiftyfold increase of the total amount of external uridine incorporated.     

Purification and characterization of an exon 2-deleted human beta-tropomyosin constructed by the polymerase chain reaction.

We deleted exon 2 in human skeletal beta-tropomyosin (h beta-SK tropomyosin) using an improved adaptation of polymerase chain reaction (PCR) technology. The first PCR product was used to prime the full-length cDNA, leading to an exon 2-deleted h beta-SK tropomyosin. This new protein, des-(39-80)-tropomyosin, could then be expressed in Escherichia coli and purified to homogeneity. At the nucleotide level, the junction between exons 1 and 3 has been precisely made in the PCR product. The mutated protein was purified using high-performance liquid chromatography. Des-(39-80)-tropomyosin revealed new immunological properties but was still recognized by certain antitropomyosin antibodies. Furthermore, the structural characteristics of the mutated tropomyosin fit those of the full-length tropomyosin. This new adaptation of PCR technology appears to be suitable for every kind of mutation inside a cloned DNA molecule, and one mutation primer per mutation is sufficient.     

Transformation ofStreptomyces lincolnensis protoplasts with plasmid vectors

A method for the preparation and regeneration of protoplasts of Streptomyces lincolnensis is described. Mycelium in the early exponential phase appeared to be most suitable for this purpose and yielded up to 25% regenerated intact cells. Transformation of S. lincolnensis protoplasts was achieved using broad-host-range streptomycete plasmid vectors pIJ622, pMP66, pRS410 and pIJ943 constructed from replicons pIJ101, pSLG33 and SCP2. The efficiency of transformation was 3.10(3) transformants per micrograms plasmid DNA when (2-5).10(7) recipient protoplasts were used. Interspecific transformations showed that there is no efficient restriction system in S. lincolnensis that would limit the transfer of genetic information from S. lividans or E. coli.     

The effect of mineral nutrition on the growth and maintenance components of respiration during heterotrophic growth of barley seedlings

Spring barley seedling were grown in the dark for 21 d and respiration rates of the whole plant (including the seed), of the shoots, and of the roots were determined. A function describing the growth and maintenance components of respiration was interpolated through the experimental points and its parameters in plants under different mineral nutrition were compared. The plants grown in a complete nutrient solution showed the highest growth rate in the initial phase of development and thus reached the maximum respiration rate earlier than plants in the other variants. The highest proportion of substrate was respired in the shoot. Plants grown under deficiency of phosphorus and magnesium had a slower respiration rate than plants grown in the complete nutrient solution (NP), whereas the amount of respired substrate in plant parts was similar to that recorded in the NP plants. Plants grown in distilled water showed the lowest growth efficiency and respirated the highest proportion of substrate in the root.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

Immunostaining of human spermatozoa with tubulin domain-specific monoclonal antibodies. Recognition of a unique beta-tubulin epitope in the sperm head

Summary Four monoclonal antibodies that discriminate between structural domains of alpha-(TU-01, TU-04) or beta-(TU-06, TU-12) tubulin and a polyclonal anti-tubulin antibody were used for immunostaining of human spermatozoa using immunofluorescence microscopy. Specificity of antibodies was confirmed by immunoblotting experiments. Antibodies TU-01 and TU-06 uniformly stained the whole tail and the neck, whereas antibodies TU-04, TU-12 showed differential distribution of corresponding epitopes in the stable arrays of flagellar microtubules. Of the monoclonal antibodies used, only TU-12 against the antigenic determinant on C-terminal domain of -tubulin showed strong reactivity with the equatorial segment of the head. The results document a differential exposure of tubulin epitopes at the single-cell level and suggest the existence of distinct tubulin populations in various structural compartments of the human spermatozoon.     

Oenanthe aquatica (L.)Poir.: Seed reproduction,population structure,habitat conditions and distribution in Czechoslovakia

The paper summarizes data concerning the biology and ecology ofOenanthe aquatica. The species is commonly distributed all over Czechoslovakia, from lowlands to the submontane belt, especially in fishpond and river basins.O. aquatica shows no special relationship to the chemical and physical soil properties, and is well adapted to habitats with a changing water level. The reproduction ofO. aquatica depends on emerging of the bottom. The most developed populations are formed by biennial plants and arise in the year following summer or autumn drainage.