Genetic studies on the yeast Saccharomycopsis lipolytica. Inactivation and mutagenesis

Spontaneous mutants of Saccharomycopsis lipolytica were selected and partially characterized. Several antibiotics and antimetabolites were used for selection of spontaneous resistant mutants from Saccharomycopsis lipolytica. The frequencies of such mutants were mainly arranged between 1 X 10(-7) and 5 X 10(-6) mutants per cell. But one class of glucosamine resistant mutants (GAMRA) occurred more frequently. Among the resistant mutants different types of dominant and recessive resistant mutants could be observed. UV light was used for inactivation of cells and induction of mutants from S. lipolytica. Comparing four haploid strains only small differences were detected in sensitivity to UV light. UV light at a dosage of 135 J/m2 was applied to increase the mutant frequencies in three haploid strains. Besides auxotrophic, temperature sensitive and colony morphology mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, allylalcohol, glucosamine, 2-deoxyglucose or nystatin resistant mutants, hitherto not described for S. lipolytica, were isolated and partially characterized.     

The lectin neuraminidase inhibition test: a new method for the detection of antibodies to neuraminidase

Two methods for the detection of neuraminidase antibodies were compared. The lectin neuraminidase inhibition test (LNI-test) gave results comparable with those provided by the conventional neuraminidase inhibition test (NI-test). Reproducibility and repeatability were better with the LNI-test which used smaller amounts of materials, was less time consuming than the NI-test and was more sensitive.     

Genetic identification of an endoproteinase encoded by the adenovirus genome

The temperature-sensitive adenovirus type 2 mutant H2ts1 is defective for polypeptide processing at the non-permissive temperature. We have in the present study mapped the mutation by marker rescue and DNA sequencing techniques: the mutation is a C/T transition located at map co-ordinate 61.1. Previous sequencing studies have identified an uninterrupted translational reading frame in this part of the adenovirus genome, encoding a hypothetical 23 X 10(3) Mr polypeptide. The mutation leads to a proline/leucine substitution in the 23 X 10(3) Mr polypeptide.     

Amino acid sequence data on glial fibrillary acidic protein (GFA); implications for the subdivision of intermediate filaments into epithelial and non-epithelial members.

Determination of 50% of the sequence of the astrocyte-specific intermediate filament (IF) protein documents the hypervariable regions as well as parts of the coiled-coil array of glial fibrillary acidic protein (GFA). The results show that the four non-epithelial IF proteins (myogenic desmin, mesenchymal vimentin, GFA and neurofilament 68 K protein) known to form homopolymers are much more closely related than the epithelial keratins, which seem to form heteropolymers only. Of the four non-epithelial proteins, desmin and vimentin are the most closely related, since GFA has a shorter non-alpha-helical array at the amino terminus. We discuss the possibility that the non-alpha-helical terminal arrays, because of their sequence and length variability, are responsible for differences of distinct IF with respect to physical-chemical properties such as the low ionic strength-induced depolymerization into protofilaments.     

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts.

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Formation of genes coding for hybrid proteins by recombination between related, cloned genes in E. coli.

We describe a method for the formation of hybrid genes by in vivo recombination between two genes with partial sequence homology. DNA structures consisting of plasmid vector sequences, flanked by the alpha 2 interferon gene on the one side and a portion of the alpha 1 interferon gene (homology about 80%) on the other, were transfected into E. coli SK1592. Appropriate resistance markers allowed the isolation of colonies containing circular plasmids which arose by in vivo recombination between the partly homologous interferon gene sequences. Eleven different recombinant genes were identified, six of which encoded new hybrid interferons not easily accessible by recombinant DNA techniques.     

Uridine transport and RNA synthesis in growing and in density-inhibited animal cells

Both chick embryo fibroblasts and mouse 3T3 cells reduce the rate at which they incorporate H³ uridine into RNA as their growth becomes inhibited at high cell density. This reduction occurs as a function of the cell population density, and with chick embryo cells (in contrast to 3T3 cells) it is not accompanied by significant medium alterations. This indicates the importance of the cell population density in the control of cellular metabolism. The decline in H³ uridine incorporation is paralleled by a decline in the rate of uptake of the isotope into the acid-soluble pool, suggesting that decreased entry of H³ uridine into the cell, rather than a decreased rate of RNA synthesis, is responsible for the reduced rate of incorporation into RNA of density-inhibited cells. This suggestion was confirmed by finding that when the restriction on uridine uptake was overcome by increasing the concentration of uridine in the medium, the density-dependent inhibition of uridine incorporation was largely reversed. We conclude that, even though the rate of H³ uridine incorporation into RNA is reduced three- to five-fold in density-inhibited cells, the rate of synthesis of pulse-labeled RNA continues at 70 to 85% of the rapidly-growing rate.     

Molecular weights of Cucurbita sieve tube proteins

Summary By means of SDS-polyacrylamide-gel electrophoresis, molecular weights of 15000, 28000, 59000, 116000 and 220000 were determined for the main sieve tube proteins from Cucurbita maxima.