N-terminal Gag domain required for foamy virus particle assembly and export

Among the Retroviridae, foamy viruses (FVs) exhibit an unusual way of particle assembly and a highly specific incorporation of envelope protein into progeny virions. We have analyzed deletions and point mutants of the prototypic FV gag gene for capsid assembly and egress, envelope protein incorporation, infectivity, and ultrastructure. Deletions introduced at the 3' end of gag revealed the first 297 amino acids (aa) to be sufficient for specific Env incorporation and export of particulate material. Deletions introduced at the 5' end showed the region between aa 6 and 200 to be dispensable for virus capsid assembly but required for the incorporation of Env and particle egress. Point mutations were introduced in the 5' region of gag to target residues conserved among FVs from different species. Alanine substitutions of residues in a region between aa 40 and 60 resulted in severe alterations in particle morphology. Furthermore, at position 50, this region harbors the conserved arginine that is presumably at the center of a signal sequence directing FV Gag proteins to a cytoplasmic assembly site.     

Emerin expression in early development of Xenopus laevis

Emerin is an integral protein of the inner nuclear membrane in the majority of differentiated vertebrate cells. In humans, deficiency of emerin causes a progressive muscular dystrophy of the Emery-Dreifuss type. The physiological role of emerin is poorly understood. By screening and sequencing of EST clones we have identified two emerin homologues in Xenopus laevis, Xemerin1 and Xemerin2. Xemerins share with mammalian emerins the N-terminal LEM domain and a single transmembrane domain at the C-terminus. As shown by immunoblot analysis with Xemerin-specific antibodies, both proteins have an apparent molecular mass of 24 kDa but differ in their isoelectric points. Xemerin1 and Xemerin2 proteins are not detectable in oocytes nor during early embryogenesis. Protein expression is first found at stage 43 and persists in somatic cells. However, RT-PCR and Northern blot analysis show Xemerin mRNAs of approximately 4.0 kb to be present in oocytes and throughout embryogenesis. During embryogenesis the level of Xemerin mRNAs increases at stage 22 and is particularly abundant in mesodermal and neuro-ectodermal regions of the embryo. These data provide the necessary background to further investigate the role of emerin in nuclear envelope assembly, gene expression and organ development of X. laevis as a model organism.     

Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells.

The three capsid proteins VP1, VP2, and VP3 of the adeno-associated virus type 2 (AAV-2) are encoded by overlapping sequences of the same open reading frame. Separate expression of these proteins by recombinant baculoviruses in insect cells was achieved by mutation of the internal translation initiation codons. Coexpression of VP1 and VP2, VP2 and VP3, and all three capsid proteins and the expression of VP2 alone in Sf9 cells resulted in the production of viruslike particles resembling empty capsids generated during infection of HeLa cells with AAV-2 and adenovirus. These results suggest a requirement for VP2 in the formation of empty capsids. Individual expression of the AAV capsid proteins in HeLa cells showed that VP1 and VP2 accumulate in the cell nucleus and VP3 is distributed between nucleus and cytoplasm. Coexpression of VP3 with the other structural proteins also led to nuclear localization of VP3, indicating that the formation of a complex with VP1 or VP2 is required for accumulation of VP3 in the nucleus.     

Bench-scale calorimetry – application in biotechnology

Two calorimetric techniques are described allowing us to monitor and control microbial processes at relatively high cell densities: dynamic and continuous calorimetry. These techniques have been applied to study microbial systems, e.g. the dependence of thermodynamic efficiency and heat yield on specific growth rate, the heat yield of mesophilic and thermophilic microorganisms, the oxy-caloric coefficient during aerobic microbial growth, as well as the energetics of the yeast-cell cycle. Such studies are of importance for optimizing biotechnical processes.     

Isolation and characterization of thermophilic bacteria from natural and artificial habitats

Several thermophilic Bacillus Strains were isolated from natural as well as artificial habitats. They grow optimally on a carbohydrate-containing medium at a temperature of 65 to 68°C and a pH value of 6 to 7 under aerobic conditions. They utilize glucose, sucrose and sodium acetate as carbon and energy sources. They can be differentiated by acid formation and composition of intracellular fatty acid fraction as well as growth on xylose, lactose, starch, cellobiose, ribose and galactose.     

Conversion of bacteriophage fd into an efficient single-stranded DNA vector system

Summary Single-stranded DNA vectors were constructed in vitro by insertion of various DNA fragments into the Intergenic Region of the single-stranded DNA phage fd. These inserts introduce into the phage genome unique cleavage sites for restriction nucleases which are suited for sticky joining in cloning experiments. Since these sites are usually located within genes coding for antibiotic resistance, inactivation of a resistance gene by insertion can be used as a marker for the successful cloning of a DNA fragment. Resistance genes also allow to select for recombinant DNA phages and to minimize the loss of DNA inserts which otherwise becomes significant above an insert size of about one kb. Cloning of several DNA fragments is described and strand separation of double-stranded DNA fragments by means of cloning into fd DNA is given as an example for application of single-stranded DNA vectors.Abbreviations Ap ampicillin - Cm chloramphenicol - Km kanamycin - Sm streptomycin - kb, kbp a unit length equivalent to 1000 bases, respectively 1000 base pairs - wt wild type     

Origin of replication in episomal bovine papilloma virus type 1 DNA isolated from transformed cells.

The origin of replication of bovine papilloma virus type 1 (BPV-1) has been determined by isolating replicative intermediates (RI) of BPV-transformed hamster embryo fibroblasts (HEF-BPV). These RI were treated with single cut restriction enzymes to determine the start-position (origin) of the extending replication eyes using electron microscopic techniques. 'Cairns'-type RI molecules were shown to contain one replication eye in monomeric as well as in dimeric molecules. The position of this eye was localized at 6940 +/- 5% bp in the physical map. In a second set of experiments BPV-1 DNA fragments cloned in pBR322 were tested for transient episomal replication. Transfected cells were harvested after increasing periods of time and screened for replication with isoschizomeric restriction enzymes to differentiate between input and replicated DNA. The part of the BPV genome harboring the replication origin spans the BPV ClaI-C restriction fragment corresponding to the non-coding region of the BPV genome and coincides with the DNase I-hypersensitive control region in the chromatin, isolated from transformed cells.     

Single-gene heterozygotes derived from the polygenic pappus part system ofMicroseris hybrid C34 (Asteraceae—Lactuceae)

Microseris strain C34 is a hybrid between the Chilean speciesM. pygmaea (10 pappus parts) and the CalifornianM. bigelovii (5 pappus parts). The plants are propagated by selfing from the original hybrid specimen. Each plant has from 5 to 10 pappus parts per achene with an average value that is additively determined by four unlinked quantitatively acting genes. Single-gene heterozygote sublines have been obtained for two of these,pp-1 (shown to be linked to a modifier of acid phosphatase-1) andpp-4. Sublines homozygous for all four additive genes show residual genetic variation influencing pappus part number. At least one additional gene can be demonstrated by its linkage with leucine aminopeptidase-1. Lines for the further characterization of these hypostatic genes are selected.