Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Über Bastarde zwischen Fisole (Phaseolus vulgaris L.) und Feuerbohne (Phaseolus multiflorus Lam.) und ihre eventuelle praktische Verwertbarkeit

Ohne Zusammenfassung     

Pregnancy diagnosis in zoo animals by estrogen determination in feces

Estrogen concentration in feces was investigated in five different herbivorous species of zoo animals. Using a nonspecific estrogen radioimmunoassay, in four species (red buffalo, yak, Grevy's zebra, and Nubian ibex) pregnancy was revealed by measuring estrogen concentration in feces. In hippopotamus, the levels of fecal estrogens were not different between pregnant and nonpregnant animals.     

Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication

The kilB locus (which is unclonable in the absence of korB) of broad-host-range plasmid RK2 (60 kb) lies between the trfA operon (co-ordinates 16.4 to 18.2 kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co-ordinates 20.0 to 27.0 kb). Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter. The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2. We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis. Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBI) of the region analysed results in a Tra- phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From the kilBI DNA sequence KilBI is predicted to be 34995 Da, in line with M(r) = 36,000 observed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and contains a type I ATP-binding motif. The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tumefaciens Ti plasmid DNA from bacteria to plant cells. The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI may be involved in assembly of the surface-associated Tra functions. The data presented in this paper provide the first demonstration of coregulation of genes required for vegetative replication and conjugative transfer on a bacterial plasmid.     

Phenotypic heterogeneity of Gross virus-induced thymic lymphomas in the rat: cellular origins and migratory properties

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross leukemia virus (RAGV) were analyzed for a variety of differentiation markers. The neoplasms from individual rats all expressed the antigenic phenotype MP+, W3/13+, Thy-1+, RT-1+, RT-7+, W3/25-. However, approximately two-thirds of the neoplasms were positive for the OX 8 antigen, and one-third were negative. The OX 8- neoplasms only involved the thymus, whereas approximately 40% of the OX 8+ neoplasms involved the spleen as well as the thymus. Virtually all OX 8+ and OX 8- neoplastic cells contained terminal deoxynucleotidyl transferase (TdT), and both OX 8+ and OX 8- lymphomas expressed the lactate dehydrogenase (LDH)-5' isozyme and the primary, but not the secondary, ADA isozyme. This enzymatic phenotype is characteristic of thymocyte precursors, but not thymocytes. Our results therefore indicate that RAGV-induced lymphomas arise from transformed prethymic TdT+ cells which contain the LDH-5' and the primary ADA isozymes. These preleukemic cells presumably migrate to the thymus where they express the RT-7 pan-T-cell antigen and, in some instances, the OX 8 antigen during the development of overt leukemia. The OX 8+ neoplasms, being more differentiated than their OX 8- counterparts, then migrate to peripheral lymphoid tissues.     

Upper limits of temperature for growth inChlorella (Chlorophyceae)

The upper limit of temperature for growth is a species-specific character in the genusChlorella. The limits of 14Chlorella species range from 26–30°C (C. saccharophila) to 38–42°C (C. sorokiniana), withC. fusca var.vacuolata (34°C) andC. kessleri (34–36°C) assuming an intermediate position. Thus, there is no wide gap in the temperature limits between the normal (“low-temperature”) species ofChlorella and the “high-temperature” species,C. sorokiniana.