Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).     

A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins

A temperature-sensitive E. coli mutant with a mutation in the spc ribosomal protein operon was found to have a conditional defect in the processing of precursor proteins destined for the periplasmic space or the outer membrane. At high temperatures, significant amounts of precursor proteins having unprocessed signal sequences are detected in the mutant cell by pulse-labeling. The precursors are processed at very slow rates during a subsequent chase. Genetic analysis indicates that the mutation impairs the function of a gene, termed secY, located at the promoter-distal part of the spc operon. The secY gene is distinct from those genes previously known to specify ribosomal proteins, yet it is within the spc operon. It is suggested that the product of the secY gene is a component of the cellular apparatus that is essential for protein secretion across the cytoplasmic membrane. The gene secY is probably identical with prlA, previously identified as a suppressor of signal sequence mutations.     

Standardization of the Feulgen-Schiff technique

Summary Four fuchsin analogues (Pararosaniline, Rosaniline, Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches.Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 m.Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects: APPLICATIONS TO BISPECIFIC MOLECULES AND MONOVALENT IgG

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.     

The Azure Dyes: Their Purification and Physicochemical Properties. I. Purification of Azure A

A method is described for the separation of azure A from commezcial samples of polychrome methylene blue. Up to 300 mg of the pure dye may be isolated in this way. The method is based on chromatography using columns 90 cm high, 7 cm in diameter, loaded with 3 g of polychrome methylene blue. The absorbent is silica gel, the eluent a mixture of acetic and formic acid.

Poor solubility of the dye acetate in water necessitates dissociation of the acetate by alkalinization and subsequent conversion of the dye to the chloride with diluted Ha. Demethylation that occasionally occurs during this procedure is negligible. Pure azure A does not spontaneously demethylate under ordinary conditions.     

Vitalfärbung mit Derivaten des Phenothiazins

Zusammenfassung Zellkulturen wurden vergleichend mit Handelsfarbstoffen und gereinigten Farbstoffen aus der Phenothiazingruppe (Thionin und seine methylierten Homologe — Azurfarbstoffe — bis zum Methylenblau vitalgefärbt.Granuläre Farbstoffspeicherung wird erst dann beobachtet, wenn im zweifach aminosubstituierten Phenothiazin (Thionin) mindestens zwei Methylgruppen gegen Protonen an den Aminogruppen ausgetauscht worden sind, wie dies bei Azur A der Fall ist.Dieser Befund läßt sich nur bei Verwendung gereinigter Farbstoffe erheben. Die hier verwendeten Handelsfarbstoffe Azur C sind in solchem Ausmaß mit höher methylierten Homologen vermischt, daß deren Effekt eine Eignung des Azur C zur granulären Farbstoffspeicherung vortäuscht. Gereinigtes Azur C ist dazu jedoch ungeeignet.Phasenoptisch erscheint die mit Azur A, Azur B und Methylenblau erreichte Farbstoffspeicherung mehr vakuolär als granulär. Die vom jeweils verwendeten Farbstoff induzierten vakuolären Einschlüsse zeigen morphologische Eigenheiten.Die Befunde werden diskutiert, unter besonderer Berücksichtigung der mit den kationischen Farbstoffen vermutlich reagierenden anionischen zellulären Bestandteilen.

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Vital staining with phenothiazine derivativesComparative application of commercial and purified dyes

Summary In a comparative study cells grown in monolayer have been vitally stained with commercial dye samples and dyes purified by thin layer chromatography and column chromatography. The dyes used were derivatives of amino-substituted phenothiazine (thionine to methylene blue).Granular storage of dyes has been observed only after the protons of the amino groups had at least been substituted by two methyl groups, as represented by the compound azure A.This observation is based on the use of purified dyes. Commercial samples of azure C are mixed with fractions of the higher methylated homologues of thionine to a degree that these impurities imply suitability of azure C for intracytoplasmic granular storage. Purified azure C, however, does not induce formation of cytoplasmic vacuoles.In the phase contrast microscope, inclusions formed under the influence of azure A, azure B and methylene blue exhibit a more vacuolar than granular appearance. There are morphologic differences, between vacuoles formed under the influence of the respective dyes.The observations are discussed with special regard to the anionic cellular constituents supposed to react with the cationic dyes.

Die Arbeit wurde mit Unterstützung der F. Hoffmann-La Roche und Co. AG., Basel, durchgeführt.