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.     

A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli

Abstract The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 3D gene from a serotype Cl virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells.     

Parthenogenesis in Tubificidae

The ability of tubificids to reproduce parthenogenetically following or in place of bisexual reproduction has been proved. During parthenogenesis the spermatogenesis ceases at an early stage of sexual cell development, which, together with some peculiarities of the structure of the sexual system, rules out the possibility of self-fertilization in the worms. For two years of cultivation the life cycles of 3 parthenogenetic generations in Tubifex tubifex and 2 in Limnodrilus hoffmeisteri were traced.     

Inhibition of CI857-controlled recombinant gene expression in Escherichia coli at very low concentrations of glucose

Summary The effects of glucose on biomass and temperature-mediated recombinant gene expression has been studied in Escherichia coli batch cultures. Glucose, above 2 g/L, has inhibitory effects on aerobic cell growth and specially on gene expression. Although lower concentrations have no influences on biomass, a tenuous, but still remarkable concentration-dependent reduction of gene expression levels was observed even at about 0.1 g/L.