Cytogenetic study of F1 hybrids betweenCrepis dinarica andCrepis froelichiana (Asteraceae)

Crepis dinarica andC. froelichiana are two closely related species of theC. praemorsa complex. Even though they exhibit the same chromosome number (2n = 8) and similar idiogram shape, they differ widely in quantity and distribution of heterochromatin bands. The hybrids between these two species comprise three morphological types. Parental genomes were distinguished in hybrids by Giemsa differential staining (C-banding). Although meiosis presents only a few abnormalities (about 2.4%), the percentage of aborted pollen grains is very high (90%).     

Factors influencing the landing of maleEpiphyas postvittana (Walker) exhibiting pheromone-mediated flight (Lepidoptera: Tortricidae)

The effects of changes in various visual and olfactory properties of a white card surface on the landing position of male Epiphyas postvittanaexhibiting pheromone-mediated flight were studied in a wind tunnel. Males landed predominantly at the most downwind position of a surface in line with the pheromone source, regardless of the strength of the source. The position on the surface that males landed was strongly influenced by visual factors. The landing position of males appeared to be influenced by visual cues along all three axes of the surface. Decreases in either the dimension horizontally perpendicular to the wind direction or the vertical dimension resulted in greater numbers of males landing farther upwind on the surface than the downwind edge. Visual changes in the axis along the wind direction also affected the position at which males landed. For example, when presented with two white card surfaces with a 4- cm gap between them, males tended to land on the downwind edge of the upwind surface (on which the source was located). When the gap was bridged with clear Mylar, the landing pattern was significantly different, with the greater proportion of males landing on the downwind surface. However, when Mylar was placed on the plexiglass floor of the tunnel (in addition to bridging the gap), the landing pattern on the surface was not significantly different from that on the two surfaces without the Mylar bridge. It is suggested that during the prelanding and landing phases of pheromone-mediated flight, male moths orient to visual features of the surface containing the pheromone source rather than to visual features of the source (conspecific female moth) itself.     

The membrane dialysis bioreactor with integrated radial-flow fixed bed —a new approach for continuous cultivation of animal cells

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran^® carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l^?1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran^® carriers with 3–5 mm diameter showed an advantage compared to those with 1–2 mm diameter. For the 3–5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s^?1 and 0.75 mm s^?1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.     

Determination of the chromosomal site for the human radiosensitive ataxia telangiectasia gene by chromosome transfer

The chromosomal localization of the gene which complements radiation hypersensitivity of AT cells was studied by microcell-mediated chromosome transfer. A 6-thioguanine-resistant derivative of an immortalized AT cell line, AT2KYSVTG, was used as a recipient for microcell-mediated chromosome transfer from 4 strains of mouse A9 cells, 3 of which carried a human X/11 recombinant chromosome containing various regions of chromosome 11, while the other carried an intact X chromosome. HAT-resistant microcell hybrids were isolated and examined for their radiosensitivity and chromosome constitution. The microcell hybrid clones obtained from the transfer of an intact X chromosome or an X/11 chromosome bearing the pter → q13 region of chromosome 11 did not show a difference in radiosensitivity from parental AT cells, while those obtained from the transfer of X/11 chromosomes bearing either the p11 → qter or the pter → q23 region of chromosome 11 exhibited a marked radioresistance which was comparable to normal human fibroblasts. A HAT-resistant but radiosensitive variant was further obtained from the microcell fusion with an A9 cell strain carrying an X/11 chromosome bearing the 11p11 → qter region, in which a deletion at the 11q23 region was found. The results indicate that the gene which complements a radiosensitive phenotype of AT is located at the q23 region of chromosome 11.     

Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

Morphogenesis of the antenna of the male silkmoth. Antheraea polyphemus, III. Development of olfactory sensilla and the properties of hair-forming cells

During adult development of the male silkmoth Antheraea polyphemus, the anlagen of olfactory sensilla arise within the first 2 days post-apolysis in the antennal epidermis (stage 1-3). Approximately on the second day, the primary dendrites as well as the axons grow out from the sensory neurons (stage 4). The trichogen cells start to grow apical processes approximately on the third day, and these hair-forming 'sprouts' reach their definite length around the ninth day (stages 5-6). Then the secretion of cuticle begins, the cuticulin layer having formed on day 10 (stage 7a). The primary dendrites are shed, the inner dendritic segments as well as the thecogen cells retract from the prospective hair bases, and the inner tormogen cells degenerate around days 10/11 (stage 7b). The hair shafts of the basiconic sensilla are completed around days 12/13 (stage 7c), and those of the trichoid sensilla around days 14/15 (stage 7d). The trichogen sprouts retract from the hairs after having finished cuticle formation, and the outer dendritic segments grow out into the hairs: in the basiconic sensilla directly through, and in the trichoid sensilla alongside, the sprouts. The trichogen sprouts contain numerous parallel-running microtubules. Besides their cytoskeletal function, these are most probably involved in the transport of membrane vesicles. During the phase of cuticle deposition, large numbers of vesicles are transported anterogradely from the cell bodies into the sprouts, where they fuse with the apical cell membrane and release their electron-dense contents (most probably cuticle precursors) to the outside. As the cuticle grows in thickness, the surface area of the sprouts is reduced by endocytosis of coated vesicles. When finally the sprouts retract from the completed hairs, the number of endocytotic vesicles is further increased and numerous membrane cisterns seem to be transported retrogradely along the microtubules to the cell bodies. Here the membrane material will most probably be used again in the formation of the sensillum lymph cavities. Thus, the trichogen cells are characterized by an intensive membrane recycling. The sensillum lymph cavities develop between days 16-20 (stage 8), mainly via apical invaginations of the trichogen cells. The imago emerges on day 21.     

Sex determination and differentiation in organisms other than higher plants

The understanding of sex determination in general, but in particular in mammals, has been a subject of scientific speculation for a long time. It has been shown that in many vertebrate and invertebrate species, the sex of an individual is determined by the individual's chromosomal constitution. Initial studies of classical genetic searching for sex-transforming mutations and the scrupulous analyses of modified phenotypes have shed light on the mechanism(s) of sex-determination. They paved the road to successful studies at molecular level. After a brief review on sex determination in chosen model species, the “Drosophila system” is presented to exemplify a possible general principle for sex determinism.     

Sex determination in cucumber (Cucumis sativus) as a model system for molecular biology

Floral biology and sex determination are reviewed in cucumber, one of the best studied monoecious plant systems. Sexual differentiation is controlled by genotypic and environmental factors. Sex conversion has been achieved by a variety of chemical treatments, some of which being extensively used for commercial purposes. Sex expression can be shifted in either direction: femaleness is promoted by ethylene, auxines and ethylene releasing compounds, while maleness is induced by gibberellins and chemicals counteracting ethylene action. Agrobacterium transformation affects, albeit rather nonspecifically, sex expression. An important collection of sex and floral mutants has been developed. The expression of sex genes has been shown to be under the control of modifier genes or the environment. Cloning strategies can take profit of the fact that sex conversion can be modulated alone or in combination by genetical, chemical and/or environmental parameters.     

DNA probes for the Y-chromosome ofSilene latifolia,a dioecious angiosperm

Summary In order to obtain markers for the Y chromosome ofSilene latifolia, we pooled equal weights of leaf tissue from 18 female siblings into one sample and repeated the process with 18 male siblings. Pooling was intended to provide a common genetic background for each sample, leaving the absence or presence of the Y chromosome as the primary difference between the two samples. DNA was extracted from each sample and subjected to polymerase chain reaction (PCR) amplification with arbitrary 10 bp primers. Four of 60 primers used gave an amplification with the male DNA not found among those from the female DNA. Each of these was subsequently shown to provide a reliable marker for the Y chromosome.     

Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity.

An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS)