Subunit structure of chromosomes in mitotic nuclei of Physarum polycephalum

We have investigated the subunit structure of mitotic chromosomes of the acellular slime mould Physarum polycephalum, using the nuclease susceptibility of isolated mitotic nuclei as a probe. A characteristic pattern of DNA digestion products is obtained, containing approximately integral multiples of a basic 140 base pair DNA segment that resembles very closely the pattern in G₂ phase nuclei of Physarum and of calf lymphocyte nuclei. These results demonstrate that during the process of chromosome condensation there is no alteration at the primary level of chromatin structure that is responsible for the characteristic DNA digestion pattern.     

Photocontrol of anthocyanin formation in turnip seedlings

Summary As measured by in vivo spectrophotometry the phytochrome content in etiolated turnip seedlings was higher in cotyledons than in hypocotyls; in the latter, it is confined to the apical part. During early growth in darkness the amount increased in both tissues to a maximum, reached about 40 hours after sowing; the levels then gradually declined. Separation of seedlings into hypocotyl and cotyledons increased the rate of phytochrome loss in the former, but not in the latter.Following 5 minutes of red light P _frdecayed very rapidly in darkness; after 1.5 hours all of the phytochrome was present as P _r, which was presumably not converted initially. In continuous red light the total phytochrome was reduced to below the detection level within 3 hours. Seedling age markedly affected the loss of phytochrome following red light; more was destroyed in older than in younger hypocotyls and apparent new synthesis occurred only in young seedlings. The capacity to synthesise phytochrome differed in cotyledons and hypocotyl. In cotyledons, synthesis occurred following shots of red light varying from 10 seconds, to 6×I minute, but the amount of newly formed phytochrome was not related to the amount destroyed: after 5 hours of continuous red light no new synthesis occurred. In hypocotyls, the amount of phytochrome synthesised was related to the amount previously destroyed, and the phytochrome content after 24 hours of darkness was similar following all red light treatments of 1 minute or longer: new synthesis occurred following 5 hours of continuous red light.In far-red light phytochrome decayed very slowly, approaching the limit of detection after 48 hours. In cotyledons some loss was already observed after 5 hours of far-red and, in hypocotyls, after about 10 hours.These results are discussed in relation to the possible role of phytochrome as the pigment mediating anthocyanin synthesis in prolonged far-red light.     

Zur Biologie und Ökologie des Landeinsiedlerkrebses Coenobita scaevola Forskål am Roten Meer

Zusammenfassung Coenobita scaevola lebt an der Küste des Roten Meeres in sehr großer Individuenzahl. Sie ist an die Küstenlinie gebunden, da sie regelmäßig Wasser in ihre Kiemenhöhle und ihr Schneckenhaus aufnehmen muß, da sie nur hier Nahrung findet (Strandanwurf) und da sie nur hier Schneckenhäuser zum Schutz des weichen Hinterleibes gegen Strahlung, Austrocknung und mechanische Beschädigung findet. Die Tiere sind in Gebieten mit sehr wenig Strandanwurf nicht standorttreu, in Gebieten mit guten Lebensbedingungen dagegen bleiben sie lange Zeit. In verschiedenen, oft sehr nahe beieinander liegenden Gebieten sind die Einsiedler zu verschiedenen Tageszeiten rege, ohne daß eine Beziehung zu Temperatur, Luftfeuchte, Gezeiten und Feinden erkennbar wäre. Es wird erstmalig ein aktives Vermeiden von Bodenfallen durch Coenobita beschrieben. Coenobita scaevola lebt bei günstiger Temperatur (16–20°C) und günstiger Salinität (35–40) lange Zeit (über 20 Tage) unter Wasser.Die Osmoregulation der Landeinsiedler erfolgt in prinzipiell anderer Weise als bei den bisher bekannten Typen, sie ist mit den Ausdrücken Poikilosmotie oder Homoiosmotie nicht zu umschreiben. Vielmehr pendelt sich der Innendruck bei hohen Außendrucken auf höhere Werte ein als bei niedrigen Außendrucken; mittlere Blutwerte bleiben nur bei gleichzeitiger Gabe von Süß- und Seewasser erhalten.Die statischen Probleme, die sich für ein phylogenetisch junges Lufttier am Lande ergeben, meistert Coenobita durch eine regelmäßige Folge der Beinbewegung und durch anatomische Umkonstruktionen in den Beinen.Als phylogenetisch junges Landtier ist Coenobita von einer Fauna mariner und terrestrischer Herkunft besiedelt.

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Biology and ecology of the terrestrial hermit crab coenobita scaevola forskål of the Red Sea

Summary The terrestrial hermit crab Coenobita scaevola is very common on the coast of the Red Sea. The species depends on the sea for its source of food (wrack-fauna), source of drinking-water and water for moistening gills and abdomen. Only in the supra-litoral zone they find gastropod shells to protect their abdomen against insolation, desiccation and mechanical damage. Coenobita scaevola stays in one place for a long time if good living conditions are available. The time of activity of the juveniles differs from one place to another. Some are diurnal, others are nocturnal. There is no evident relation to the ecological factors. Most of the adults are nocturnal. No Coenobita could be collected in Barber traps. The avoidance of such traps by arthropodes has never been observed before. Coenobita scaevola can live for quite a long time under water of sufficient temperature and salinity. The osmotic regulation of the land-hermit crab differs from that of other shore animals. Coenobita can tolerate a wide range of blood concentrations (25–70). It controls the concentration of its blood by selecting water of the appropriate salinity.The static problems of Coenobita are solved by regular movement of the legs and special articulation of the legs.As Coenobita scaevola is a phylogenetically young land animal it carries many inhabitants of marine and terrestrial origin.

     

Antibody humanization by molecular dynamics simulations—in‐silico guided selection of critical backmutations

Monoclonal antibodies represent the fastest growing class of biotherapeutic proteins. However, as they are often initially derived from rodent organisms, there is a severe risk of immunogenic reactions, hampering their applicability. The humanization of these antibodies remains a challenging task in the context of rational drug design. “Superhumanization” describes the direct transfer of the complementarity determining regions to a human germline framework, but this humanization approach often results in loss of binding affinity. In this study, we present a new approach for predicting promising backmutation sites using molecular dynamics simulations of the model antibody Ab2/3H6. The simulation method was developed in close conjunction with novel specificity experiments. Binding properties of mAb variants were evaluated directly from crude supernatants and confirmed using established binding affinity assays for purified antibodies. Our approach provides access to the dynamical features of the actual binding sites of an antibody, based solely on the antibody sequence. Thus we do not need structural data on the antibody–antigen complex and circumvent cumbersome methods to assess binding affinities. © 2016 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.     

Evolutionary conservation of kinetochore protein sequences in plants

The evolutionary conservation of structural/functional kinetochore proteins has been studied on isolated nuclei and pro-/metaphase chromosomes of mono- and dicot plants. The cross-reactivities of antibodies against human CENPC, CENPE and CENPF, and against maize CENPCa with the centromeric regions of mitotic chromosomes of Vicia faba and/or Hordeum vulgare are shown. Putative homologs of the kinetochore protein SKP1 (suppressor of kinetochore protein 1p of yeast) were found in both species and of CBF5p (centromere binding factor 5 of yeast) in barley. Antibodies against synthetic peptides derived from partial sequences encoding these proteins were produced and recognized the centromeric regions on mitotic chromosomes as detected by indirect immunofluorescence.     

Probing the unfolding region of ribonuclease A by site-directed mutagenesis.

Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent beta-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in DeltaG can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule.     

Colocalization of a partially dominant gene for yellow seed colour with a major QTL influencing acid detergent fibre (ADF) content in different crosses of oilseed rape (Brassica napus).

Quantitative trait loci (QTLs) contributing to yellow seed colour and acid detergent fibre (ADF) were localized and compared in 3 mapping populations developed from 2 crosses (designated 'YE1' and 'YE2') between 2 distinct sources of true-breeding yellow-seeded oilseed rape (Brassica napus) and 2 different black-seeded genotypes. A clear correlation was observed between seed colour and ADF content in both crosses. In all 3 populations, a major QTL, with a large effect on both seed colour and ADF in multiple environments, was detected at the same position on chromosome N18. In YE1, a second minor QTL, with a small effect on seed colour but not on ADF content, was localized on chromosome N1. In YE2, no QTL was observed on N1; however, 2 minor seed-colour loci were localized to N15 and N5. A second major QTL for ADF was localized in YE1 on N13; in YE2, no other QTLs for ADF were detected. Combined QTL and segregation data for seed colour and ADF content in the different populations suggest that a partially dominant B. napus gene for seed colour on N18 contributes to a reduction in fibre content in different yellow-seeded B. napus genotypes. The other QTLs that were identified appear to represent different genes in the 2 yellow-seeded rapeseed sources, which, in each case, affect only fibre content or seed colour, respectively. Potential candidate genes and implications for marker-assisted breeding of oilseed rape with reduced seed dietary fibre content are discussed.