Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus

Summary A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.Abbreviations GUS ß-glucuronidase - PPV Plum Pox Virus - BA 6-benzylaminopurine - NPTII neomycin phosphotransferase II - CP coat protein - CaMV Cauliflower Mosaic Virus - P35S 35S promoter - MS Murashige and Skoog - PCR polymerase chain reaction - P/C/I phenol/chloroform/isoamylalcohol - RNase ribonuclease - dNTP deoxyribonucleosidetriphosphate - DMSO dimethyl sulfoxide     

Simple sequences

Simple sequences (or microsatellites) are stretches of monotonous repetitions of short (1–5 bp) nucleotide motifs that are distributed across the whole genome in eukaryotes. They are probably generated by slippage during replication and their primary mutation rate seems to be controlled predominantly by the efficiency of the mismatch repair system. Although most mutations in simple sequence loci appear to be neutral, some mutations in particular stretches have been implicated as having a role in human genetic diseases.     

Asynchronous mitotic domains during blastoderm formation inMusca domestica L. (Diptera)

Summary We describe the mitotic cleavage patterns during blastoderm stage of the house flyMusca domestica L. Nuclear divisions up to mitotic stage 11 are apparently synchronous. Beginning with stage 12, nuclear divisions in the posterior third of the embryo lag behind, resulting first in a parasynchronous and finally in an asynchronous cleavage pattern. Thus a stage exists where all nuclei in the anterior region have completed 14 nuclear division cycles, while those in the posterior region have completed only 13 cycles. The border region between these nuclei is well defined and lies at 35% EL (egg length), the expression border of a gap gene. This border region is about 4–5 nuclei wide and shows a specialized mitotic behaviour.     

Identification of a Rapidly Labelled 350K Histidine-Rich Protein in Neonatal Mouse Epidermis

Abstract. The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6–9 h lag period. A number of putative precursor HRPs in the 70–300 K molecular weight range were initially identified using short pulse labelling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [³H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100°C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.     

Veränderungen des Gibberellingehaltes von Salatsamen nach Belichtung

Zusammenfassung Samen der obligat lichtkeimenden Salatsorte Tenax wurden auf ihren Gibberellingehalt untersucht. Sie enthalten bei Quellung im Dunkeln nur wenig gibberellinähnliche Substanzen. Nach Belichtung mit hellrotem Licht nimmt der Gibberellingehalt innerhalb 1 Std sehr stark zu. Es wird gefolgert, daß Lichtkeimer einen zu niedrigen Gibberellingehalt besitzen, um im Dunkeln keimen zu können, und daß unter dem Einfluß des Lichtes die zur Keimungsauslösung notwendige Gibberellinmenge entsteht.

---

Changes in gibberellin-like substances of lettuce seeds after light exposure

Summary The contents of gibberellin-like substances in lettuce seeds were determined. In darkness the seeds contain only small amounts of gibberellin-like substances, but upon illumination with near red light there is a sharp increase in the gibberellin content within one hour. It is concluded that lettuce seeds in darkness do not contain the amounts of gibberellin necessary for germination and that under the influence of light these amounts are provided.

     

The Degree and Length of O-Glycosylation of Recombinant Proteins Produced in Pichia pastoris Depends on the Nature of the Protein and the Process Type

The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N-linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O-glycosylation. O-glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O-mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O-glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, P_GAP and P_AOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI-MS. In the samples expressed with P_GAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with P_AOX1. The results indicate that the O-glycosylation level is markedly higher when the protein is produced in a methanol-based expression system.     

Downscaling screening cultures in a multifunctional bioreactor array-on-a-chip for speeding up optimization of yeast-based lactic acid bioproduction

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (V_t = 15 µl) in a 26 mm × 76 mm area with in-line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab-on-a-chip was used in a proof-of-principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4- to 6-fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.     

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.     

<Emphasis Type="Italic">her11</Emphasis> is involved in the somitogenesis clock in zebrafish

Somitogenesis requires an intricate process of pre-patterning, which is driven by an oscillator mechanism consisting of the Delta-Notch pathway and hairy- (h) and Enhancer of split- [E(spl)] related genes. With the aim of unravelling the complex mechanism of somite pre-patterning, we have conducted an extensive search for h/E(spl)-related genes in the third release of the Danio rerio genomic sequence. We identified 14 new h/E(spl) genes and analysed them by in situ hybridisation for their potential role in the somitogenesis process. We describe here the functional analysis of one of these genes, which we have named her11. her11 is a paralogue of her1 and, similar to her1, is arranged in a head to head fashion with another her gene, namely the previously described her5. It shares an expression in the midbrain-hindbrain boundary with her5, but is in addition cyclically expressed in patterns overlapping those of her1 and her7 and complementary to those of hey1. Furthermore it is expressed in the anterior half of the most caudally formed somites. We show that Delta-Notch pathway genes and fused somites (fss) are necessary for the control of her11 expression. However, some aspects of the her11 regulation suggest that at least one additional as yet unknown gene of the Delta-Notch cascade is required to explain its expression. Morpholino-oligonucleotide-mediated knockdown of her11 shows that it is involved in the zebrafish somitogenesis clock via an interaction with her1 and her7. We have also studied the role of hey1 by morpholino injection, but could not find a direct function for this gene, suggesting that it reflects the output of the clock rather than being a core component of the mechanism.Edited by B. Herrmann