A New Non-Mendelian Genetic Element of Yeast That Increases Cytopathology Produced by M1 Double-Stranded RNA in ski Strains

The Saccharomyces cerevisiae SKI (superkiller) genes are repressors of replication of M, L-A, and L-BC double-stranded (ds) RNAs; ski strains have an increased M dsRNA copy number and, as a result, are cold-sensitive for growth at 8 degrees. Growth is normal, however, at higher temperatures. We have found a new cytoplasmic genetic element [D] (for disease) that makes M1 dsRNA-containing superkiller strains grow slowly at 30 degrees, not at all at 37 degrees, and only very poorly at 20 degrees. These growth defects require three factors: a chromosomal ski mutation, the presence of M1 dsRNA, and the presence of the new cytoplasmic factor, [D]. We have isolated mutants unable to maintain [D] (mad), at least one of which is due to mutation of a single chromosomal locus. Further, [D] can be cured by growth at 37-39 degrees. We present evidence that [D] is not M, L-A, L-BC or W dsRNAs or mitochondrial DNA, 2 mu DNA, or [psi], but [D] depends on L-A for its maintenance. We also show that [D] is distinct from [B], a cytoplasmic element that allows M1 dsRNA to be stably replicated and maintained in spite of defects in certain chromosomal MAK genes that would otherwise be necessary. [D] activity is blocked by the presence of another extrachromosomal element, called [DIN] (for [D] interference). [D] and [DIN] may be different natural variants of the same molecule.     

Recalibration of the Pseudomonas aeruginosa Strain Pao Chromosome Map in Time Units Using High-Frequency-of-Recombination Donors

High-frequency-of-recombination donors of P. aeruginosa strain PAO were generated using a temperature-sensitive, replication mutant of the IncP-1 plasmid R68, loaded with the transposon Tn2521. Fourteen donors so isolated mobilized the chromosome in a polarized manner from a number of different transfer origins. The donors were used to construct a time of entry map of the entire chromosome and this was achieved by determining the time of entry of 32 randomly dispersed markers in crosses using nalidixic acid to interrupt chromosome transfer. Analysis of the time of entry data enabled the recalibration of the chromosome map to 75 min.     

Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

The impact of different fixation procedures on staining of macromolecules in a microtiter system

Summary The effects of formaldehyde, glutaraldehyde, methanol, Clarke's fixative and microwave irradiation on the quantitative staining of proteins (Naphthol Yellow S) and nucleic acids (Ethyl Green—Pyronin) in a cell culture system have been investigated. Overall, glutaraldehyde rapidly yielded the highest and most consistent levels of staining when compared to all other chemical fixatives. Although microwave irradiation was found to be uneven, 4 min exposure to 700W was found to give higher levels of protein staining than those achieveable with glutaraldehyde. Time-dependent processes were observed with all procedures. In addition, dissociations in the trends of protein and nucleic acid staining were observed. It is suggested that these results domonstrate fixation events that have not previously been resolved from the effects of reagent penetration into tissue blocks.     

Immunocytochemical study of the intracellular localization of M protein of vesicular stomatitis virus

Summary The purpose of this paper is to describe the immunocytochemical localization of M protein of vesicular stomatitis virus (VSV) in infected cells. Vero cells, MDBK cells, Swiss 3T3 cells, and BHK cells were examined at various times after infection. For immunofluorescent staining, the cells were fixed with PLP fixative and then treated with 0.05% Triton X-100 before incubation with antibodies. Three hours after infection, M protein exhibited diffuse immunostaining throughout the cytoplasm and later accumulated along the cell membrane. The localization of M protein differed from the granular localization of the nucleocapsid N protein of VSV in the cytoplasm. For electron microscopy, the cells were fixed first in a mixture of 2% paraformaldehyde and 0.05% glutaraldehyde and then with PLP fixative, this being followed by treatment with 0.05% saponin. They were then immunostained using the immunoperoxidase method. The M protein was found to be distributed throughout the cytoplasm and later under the cell membrane, especially at virus budding sites. We also used postembedding immunostaining and freeze-fracture immunostaining to avoid the translocation of M protein caused by the detergent treatment. These techniques confirmed our previous results. Our findings are consistent with the view that the M protein of VSV is synthesized on free ribosomes and is then associated with the cell membrane where viral assembly may occur.S. Ohno was a visiting fellow from the Fogarty International Center at the National Institutes of Health, USA, from September 1981 to August 1983, while some parts of this work were in progress.     

Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.     

Evolution of the Ribosomal DNA Spacers of Drosophila melanogaster : Different Patterns of Variation on X and Y Chromosomes

Length variation of the ribosomal gene spacers of Drosophila melanogaster was studied. Analysis of 47 X chromosomal and 47 Y chromosomal linked rDNA arrays collected from five continents indicates that the arrays on the two chromosomes differ qualitatively. The Y-linked arrays from around the world share little or no similarity for either their overall length or the organization of their spacers. Most of the X-linked arrays do, however, share a major length spacer of 5.1 kb. In addition, those X-linked arrays that have a major 5.1-kb band have similar spacer organization as demonstrated by genomic DNA digestions with several restriction enzymes. These data strongly support the hypothesis that spacer length patterns on only X-linked genes are maintained primarily by natural selection.     

Flagellar variation in Serratia marcescens is associated with color variation.

The pigmented enterobacterium Serratia marcescens, an opportunistic pathogen, shows a striking variation of its red color. Different strains differ greatly both in color and in the frequency with which they produce color variants. Within a strain, the variations occur at constant rates and are reversible. During an investigation of this phenomenon we observed that variation of a 39-kilodalton protein in S. marcescens 274 is closely associated with color variation. Using antibodies to this protein we identified it as being a component of the bacterial flagella. Variation of surface proteins often provides an organism with alternate offense-defense strategies for survival in a challenging environment. The pigment, in association with flagella, may provide such a function for S. marcescens.     

Physiological adaptations of anaerobic bacteria to low pH: metabolic control of proton motive force in Sarcina ventriculi.

Detailed physiological studies were done to compare the influence of environmental pH and fermentation end product formation on metabolism, growth, and proton motive force in Sarcina ventriculi. The kinetics of end product formation during glucose fermentation in unbuffered batch cultures shifted from hydrogen-acetate production to ethanol production as the medium pH dropped from 7.0 to 3.3. At a constant pH of 3.0, the production of acetate ceased when the accumulation of acetate in the medium reached 40 mmol/liter. At a constant pH of 7.0, acetate production continued throughout the entire growth time course. The in vivo hydrogenase activity was much higher in cells grown at pH 7.0 than at pH 3.0. The magnitude of the proton motive force increased in relation to a decrease of the medium pH from 7.5 to 3.0. When the organism was grown at pH 3.0, the cytoplasmic pH was 4.25 and the organism was unable to exclude acetic acid or butyric acid from the cytoplasm. Addition of acetic acid, but not hydrogen or ethanol, inhibited growth and resulted in proton motive force dissipation and the accumulation of acetic acid in the cytoplasm. The results indicate that S. ventriculi is an acidophile that can continue to produce ethanol at low cytoplasmic pH values. Both the ability to shift to ethanol production and the ability to continue to ferment glucose while cytoplasmic pH values are low adapt S. ventriculi for growth at low pH.