Plant regeneration of grapevine (Vitis sp.) protoplasts isolated from embryogenic tissue

Summary Protoplasts with high embryogenic competence could be isolated from leaf-disk-derived embryos and embryoids of Vitis sp. cv. Seyval blanc. After a 4-week induction treatment in NN-69 medium supplemented with 4.0mg/l naphthoxyacetic acid (NOA) and 0.9mg/l thidiazuron (TDZ) and subsequent subcultivation in hormone-free medium, 38.5% of the developed microcalluses showed somatic embryogenesis. In contrast, only few formed somatic embryos after induction in CPW-13 medium with either 1.0mg/l 2,4-dichlorophenoxyacetic acid and 0.5mg/l benzylaminopurine treatment (13.8%) or NOA/TDZ treatment (1.4%). Up to 30% of these embryos germinated and about half of them regenerated into typical in vitro grapevines when transferred onto LS-medium in culture tubes.Abbreviations BAP benzylaminopurine - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-[N-morpholino]-ethanesulfonic acid - NOA naphthoxyacetic acid - PCR polymerase chain reaction - PVP polyvinylpyrrolidone - TDZ thidiazuron     

Restriction map of Chinese hamster mitochondrial DNA containing replication coordinates: comparison with Syrian hamster mitochondrial genome

A precise physical map, containing the structurally and operationally defined D-loop origin, terminal region, and direction of heavy-strand replication, has been constructed for mitochondrial DNA (mtDNA) from ovary (CHO-KI) and lung cells of Chinese hamster (Cricetulus griseus 2 N = 22), and compared with our previously established genome coordinates for mtDNA from Syrian hamster ( Mesocricetus auratus 2 N = 44). All four HpaI sites in Cricetulus are conserved in Mesocricetus (8 sites). Extensive variation exists for hexanucleotides cleaved by EcoRI HindIII PstI. KpnI and BamHI. Sequence divergence between Chinese and Syrian hamster mtDNAs, as reflected from analysis of the mapped recognition sites for these six endonucleases, is estimated as 5–9% base substitutions. mtDNAs from both hamster and several other mammalian species contain a commonly conserved HpaI site in the region of light strand initiation.     

Molecular Characterization of the S-Layer Gene, sbpA, of Bacillus sphaericus CCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5′ end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA_31-1068). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA_31-1068. Labeling of the square S-layer lattice formed by recrystallization of rSbpA_31-1068/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.     

Pigs’ preferences for rooting materials measured in a three-choice maze-test

The aim of this experiment was to investigate pigs’ preferences for rooting materials. Eighteen materials were allocated to six categories each of which consisted of three similar materials based on characteristics such as structure, size of particles, complexity, destructibility and digestibility. Twelve pairs of pigs chose among the three materials of each of the six categories in a balanced design. Within each category each pair was given four instantaneous choices among the three materials in a three-armed maze. ‘No choice’ was scored if the pigs did not enter one of the maze-arms within 90 s. Thus there were four options in each choice situation. The results were analysed using a random utility model incorporating random intercepts to account for the repeated testing of the same animals. The pigs expressed clear preferences within the category EARTH, where compost and peat were preferred to wood-shavings and no choice. In the category CHIP the most probable rank-order was spruce chip, willow chip, fir chip and no choice, while in the category ROUGH the most probable rank-order was maize-silage, grass silage, sugar beets and no choice. However, in these two categories none of the probabilities were sufficiently large to signify a preference for any of the three materials although the probabilities of the ‘no choice’ option were low. The pigs expressed no preferences among any of the four options including ‘no choice’ in the categories TOY (sisal robe, Bite-Rite, wooden beam), HAY (alfalfa hay mixed with straw, seed grass hay, barley straw with under-seed), and STRAW (long straw, chopped straw and straw pellets).     

TraM of plasmid R1 regulates its own expression

Regulation of the traM gene, which encodes a factor essential for conjugation of resistance plasmid R1, was studied in vivo using translational gene fusions. tram″lacZ fusion constructs were transferred to the chromosome via the recombinant phage λRZ5. The level of β-galactosidase expressed by the lysogens indicates that the traM promoters are very active. Expression of traM was diminished five- to sixfold when the single-copy plasmids R1 or R1-19 were present in trans. When recombinant plasmids carrying traM were present at higher copy numbers, traM expression was reduced as much as 45-fold. The negative effect of R1 plasmids on traM expression in trans, which we interpret as autoregulation, was observed regardless of whether the plasmids were conjugatjvely repressed or derepressed. Site-specific mutagenesis of the region encoding the N-terminus of the TraM protein eliminated the autoregulative effect indicating that the N-terminal amino acids of the protein are important to its DNA-binding function. The autoregulatory behaviour of TraM is allele specific. R1- or P307-encoded TraM molecules were found to recognize only the cognate DNA.     

Plasma-membrane-Bound mcromoleculas are dynamically aggregated to form non-random codistribution patterns of selected functional elements. Do pattern recognition processes govern antigen presentation and intercellular interactions?

Molecular recognition processes between cell surface elements are discussed with special reference to cell surface pattern formation of membrane-bound integral proteins. The existence, as detected by flow cytometric resonance energy transfer (Appendix), and significance of cell surface patterns involving the interleukin-2 receptor, the T-cell receptor–CD3 system, the intercellular adhesion molecule ICAM-1, and the major histocompatilibilty complex class I and II molecules in the plasma membrane of lymphocytes are described. The modulation of antigen presentation by transmembrane potential changes is discussed, and a general role of transmembrane potential changes, and therefore of icon channel activities, adduced as one of the major regulatory mechanisms of cell–cell communications. A general role in the mediation and regulation of intercellular interactions is suggested for cell-surface macromolecular patterns. The dynamic pattern of protein and lipid molecules in the plasma membrane is generated by the genetic code, but has a remarkable flexibility and may be one of the major instruments of accomodation and recognition processes at the cellular level.