Visualization of cytoskeletal changes through the life cycle inAcetabularia

Summary The cytoskeleton in the siphonous, marine green algaAcetabularia is visualized by immunocytochemistry using antibodies against plant alfa tubulin and animal smooth muscle actin. In the vegetative phase of the life cycle, when the cell grows a cylindrical stalk and until the reproductive cap is completed, actin forms continuous, parallel bundles that extend through the entire length of the stalk and cap rays respectively. Microtubules (MTs) cannot be detected until the primary nucleus, located in the rhizoid of the giant cell, divides to form thousands of secondary nuclei. MTs can then be seen radiating from each secondary nucleus that is encountered in the stalk on its migration upwards into the cap rays. They are oriented mostly parallel to the long axis of the cell. At arrival in the cap rays up to the white spot stage, when nuclei assume equidistant positions in the cap ray cytoplasm, a radiating system of MTs forms around each nucleus and dramatically increases until impressive radial arrays have developed. This phase coincides with a disappearance of actin bundles in the cap rays, but they are retained in the stalk cytoplasm. Shortly after that additional MTs appear around the disk like partitions of cap ray cytoplasm. Concomitantly, bundles of actin reappear colinearly with the circumferrential MTs eventually forming complete rings around each disk of cap ray cytoplasm. During this process the compartments of the future cysts are gradually bulging outwards and simultaneously the rings of actin sink inwards until domes are formed with the nuclei fixed in the top centers of the domes. At this stage the peripheral areas of the radiating MT systems around the nuclei start to break down, whereas the circumferrential MT systems remain intact. Subsequently, the rings of both actin and MTs decrease in diameter, and finally contract to a spot opposite the nucleus, while the cysts continue to develop their oval shape. After the cysts have become separated, they round up and enter several rounds of nuclear divisions. MTs form short radial arrays around each nucleus with minor changes due to a reduction of MTs during division followed by a reappearance after completion of each division. Actin is rearranged in the cysts to a cortical network of randomly oriented, short bundles, that is maintained until gamete formation sets in.These findings accentuate the involvement of Cytoskeletal elements in the key steps of morphogenesis inAcetabularia to an extent that is unknown in higher plants.     

Effect of oxygen limitation on the formation of the electron transport system of the phytopathogenic fluorescent bacteriumPseudomonas cichorii

The composition of the membrane-bound electron transport system of the phytopathogenic bacteriumPseudomonas cichorii underwent modification in response to oxygen supply. Growth adaptation to low oxygen concentrations was characterized by repression of cytochromes involved in ubiquinol-cyt.c oxidoreductase and cyt.c oxidase activities. By contrast, cyto.o, i.e., the alternative cyanide-insensitive oxidase ofP. cichorii, was unaffected by low oxygen tension. Noa-type cytochromes could be detected at any stage of growth.     

Phagocytosis of different matrix components by different cell types at bone-forming sites in cultured mouse calvariae

Summary Sites of bone formation on fragments of parietal bone of fetal-mice cultured for 10 days were examined by electron microscopy after addition of either ruthenium red or ferrocyanide to the postfixation fluid. Osteoclasts, osteoblast-like cells, and macrophages were the principal active cells at these formation sites. The mononuclear cells (osteoblast-like cells and macrophages) in the osteoid tissue showed evidence of having incorporated elements of calcified tissue. Osteoblast-like cells had phagocytized collagen fibrils and calcified bone matrix. This occurred more frequently in the calcifying area. Mononuclear macrophages showed not only phagocytosis and digestion of cellular debris and bone spicules in the osteoid, but also active incorporation of calcified bone matrix that had been detached from its surroundings by its pseudopod-like projections from long cytoplasmic processes. Collagen fibrils were seldom observed within the macrophages. These observations suggest that in our culture system osteoblast-like cells and macrophages at bone formation sites have a phagocytic role in bone remodeling.This study was supported in part by a grant from the Ministry of Education. Science and Culture of Japan (No. 59771321)     

Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity

Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6-P₂) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P₂ from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa.Abbreviations ALD Fru-1,6-P₂ aldolse - DHAP dihydroxyacetone phosphate - F6P fructose 6-phosphate - G6P glucose 6-phosphate - Gly3P glyceraldehyde 3-phosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - PFK fructose 6-phosphate kinase - PGI phosphoglucose isomerase - 6PG 6-phosphogluconate     

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [³H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [³H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.Abbreviations CV(s) coated vesicle(s) - Da dalton - ER endoplasmic reticulum - GSI,II glucan synthase I and II, respecitively Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA     

Pattern formation in fragmented eggs of the short germ insect Schistocerca gregaria

Summary The effect of transverse fragmentation on the segment pattern of the short germ embryo of the locust Schistocerca gregaria has been investigated at two stages subsequent to the formation of the germ anlage. Following fragmentation both anterior and posterior partial embryos were observed, although rarely in a single egg. Anterior partial patterns usually terminated with a segment visible at the time of fragmentation or with the next segment due to appear. Posterior partial patterns began with a wide range of segments depending on the level of fragmentation.Anterior and posterior partial patterns developing in a single egg were usually not complementary and the segments missing sometimes included some segments visible when the embryo was fragmented. Non-complementary patterns resulted following fragmentation in all regions, while complementary patterns only occurred after fragmentation in the visibly-segmented region.The results suggest that following fragmentation isolated posterior portions of the embryo continue to form segments, while isolated anterior regions usually do not. This effect could result from variable damage to an existing pattern of unequally-sized segment primordia, or from the disruption of a process of sequential segmentation in the elongating posterior region of the embryo. The results are broadly compatible with the progress zone model proposed by Summerbell et al. (1973).     

Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo

Summary Mutations in seven different maternal-effect loci on the second chromosome of Drosophila melanogaster all cause alterations in the anterior-posterior pattern of the embryo. Mutations in torso (tor) and trunk (trk) delete the anterior- and posterior-most structures of the embryo. At the same time they shift cellular fates which are normally found in the subterminal regions of the embryo towards the poles. Mutations in vasa (vas), valois (vls), staufen (stau) and tudor (tud) cause two embryonic defects. For one they result in absence of polar plasm, polar granules and pole cells in all eggs produced by mutant females. Secondly, embryos developing inside such eggs show deletions of abdominal segments. In addition, embryos derived from staufen mothers lack anterior head structures, embryos derived from valois mothers frequently fail to cellularize properly. Mutations in exuperantia (exu) cause deletions of anterior head structures, similar to torso, trunk and staufen. However in exu, these head structures are replaced by an inverted posterior end which comprises posterior midgut, proctodeal region, and often malpighian tubules.The effects of all mutations can be traced back to the beginning stages of gastrulation, indicating that the alterations in cellular fates have probably taken place by that time. Analysis of embryos derived from double mutant mothers suggests that these three phenotypic groups of mutants interfere with three different, independent pathways. All three pathways seem to act additively on the system which specifies anterior-posterior cellular fates within the egg.     

Effects of terbium on the hemocyanin pore formation rate in phosphatidylcholine planar bilayers

Megathura crenulata hemocyanin forms ionic channels in planar lipid bilayer membranes. It was found that hemocyanin is more potent as a channel former if TbCl₃ is added to the bathing solution. Furthermore membranes separating symmetrical TbCl₃ solutions show a pore formation rate which depends exponentially on the applied voltage, positive potentials favouring the insertion of new channels. The slope of this voltage dependence, which gives a measure of the effective charge displaced during the incorporation of one channel, increases and saturates with TbCl₃ concentration. The dose response curve indicates that binding of Tb³⁺ to the phosphatidylcholine bilayer is involved in creating the effective charge.     

Physical conditions of propagation media and their influence on the rooting of cuttings

Summary The formation and subsequent growth of roots by cuttings of poinsettia, hydrangea, rose and azalea in various propagation media, Jiffy-7, Jiffy-9 and Grodan under different conditions of aeration was investigated. The interrelationships of the effects of air content of the media, temperature and light intensity on the rooting of poinsettia cuttings was also studied.With low air contents (0 cm moisture tension) in the propagation media the formation and growth of roots was strongly inhibited. The rooting performance of rose appeared to be less affected by the poor aeration. Increasing air content improved rooting but best results were obtained at moisture tensions of 4 to 8 cm. Rooting seems to be better correlated with oxygen diffusion rate (ODR) than with air content.For poinsettia cuttings the optimum temperature for rooting was 24 to 28°C. At low temperatures rooting was delayed while at higher temperatures it was almost completely inhibited. Callus formation increased with temperature but decreased with increasing moisture tension. Conditions which induced large callus formation inhibited root formation.High light intensity during rooting reduced overall rooting performance and the inhibition was most pronounced in conjunction with high moisture tensions.Report No. 255.     

High recombination between the breakpoint of a reciprocal translocation in rye (Secale cereale L.) and an interstitially located gene

Summary The recombination fraction between the interstitially located gene an and interchange 303 of rye was found to be 0.244±0.038 in a test cross using the translocation as the male parent. In first metaphase translocation configurations in pollen mother cells of the same plant, the chiasma frequency between an and the translocation breakpoint was found to be significantly more than twice the recombination fraction. Recombination was concluded to be masked by a difference in the alternate frequency between configurations without interstitial chiasmata and configurations with interstitial chiasmata, the effect of the first type being of major importance. Random centromere orientation of translocation multivalents with interstitial chiasmata was concluded to be a realistic assumption. The exceptionally high recombination between an and translocation 303 is discussed. Consideration is also given to the use of interchanges in the establishment of a marker's chromosomal position, and to the use of translocation chromosomes in balanced systems for hybrid breeding purposes.