Qa alloantigen expression on functional T lymphocytes from spleen and thymus

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (>40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2^–, Ly-1^hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.     

Development of an improved medium for the isolation of 'Haemophilus somnus'

A medium containing lincomycin (3 μg/ml), cycloheximide (100 μg/ml) and chloral hydrate (0–1 %) was superior to all others examined for the isolation of 'Haemophilus somnus' from material contaminated with Proteus species.     

Interspecific crosses inHordeum (Poaceae)

A crossing programme including 30 species and 40 cytotypes within the genusHordeum was undertaken. Viable hybrids were obtained in 302 combinations, 15 of which were intraspecific. Differences in seed set and in germination were observed in crosses between different groups of species. Obtaining crosses between different taxonomic groups was generally more difficult when diploid material was used. Some species, e.g.,H. lechleri, H. jubatum, andH. brachyantherum showed a higher crossability than others. The chromosome numbers of the hybrids were usually those expected from the parental numbers but aneuploid series around the expected numbers were rather frequent. Three cases of unreduced gametes were found. Selective chromosome elimination was restricted to combinations including eitherH. vulgare orH. bulbosum.—Despite a very diverse morphology, all South American diploid species together with the two North American diploidsH. intercedens andH. pusillum appear to be closely related. The hexaploid American speciesH. procerum, H. lechleri, andH. arizonicum are also related. The two North American tetraploid speciesH. jubatum andH. brachyantherum sometimes form semifertile hybrids. The Asiatic speciesH. roshevitzii appears to be related to both North and South American taxa.     

Physiology of F-Pilin Synthesis and Utilization

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to study the synthesis and turnover of F-pilin in membrane preparations of Escherichia coli K-12 under conditions which have been reported to affect the production of F-pili. Incorporation of [³⁵S]methionine into membrane F-pilin by cells in log phase was barely detectable at 25°C, but increased with temperature. The labeled pilin band was prominent in membranes from 37°C cultures and even more prominent if the growth temperature was raised to 42°C. The appearance of other tra products in the membranes was similarly temperature dependent. In cultures grown in glucose minimal medium at 37°C, the relative amount of membrane pilin and traT product synthesis remained unchanged from early log phase through early stationary phase; provision of glycerol or arabinose as a substitute carbon source had no obvious effect. Turnover of traT product and membrane F-pilin, as assessed in an Flac tra mutant strain which is incapable of elaborating pili, was not rapid. Both traT product and pilin subunits labeled in mid-log phase cells were still apparent in the membranes after growth of the cells to stationary phase. The relative amount of labeled pilin decreased with prolonged incubation in stationary phase, but the relative amount of traT product did not decrease even after the culture was incubated for 24 h. When wild-type Flac piliated cells were used, a similar result was obtained, but in this case, loss of F-pilin from the preparations could be acclerated by blending the cells. Although intermittent blending during culture growth caused a slow depletion of the labeled pilin pool, continuous blending resulted in the rapid disappearance of this pool from our preparations. Loss of other membrane polypeptides was not accelerated by our blending procedure, and blending did not affect the turnover of the pilin pool of the Flac tra mutant. Our data are consistent with a model in which pilin subunits are assembled transiently into pili, conserved by retraction, and made available for subsequent reassembly. Growth in 0.01% sodium dodecyl sulfate did not accelerate loss of pilin from the Flac strain compared with the Flac tra strain, and we suggest that in the presence of sodium dodecyl sulfate at this concentration, F-pili are not elaborated from cell surfaces.     

DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli

Summary Two active enzymes of the galactose operon of Escherichia coli, uridyl transferase and galactokinase have been synthesized with high yields in a DNA dependent system for protein synthesis. The unspecific blank values amount to less than two percent of the rate obtained under optimal conditions and permit the accurate determination of even a small fraction of the maximum synthesis rate. Therefore this system provides a sensitive assay for the biological activity of DNA that contains the intact galactose operon of Escherichia coli.The synthesis of these galactose enzymes is to a high extent dependent on the presence of cyclic adenosine-3:5-monophosphate.D-fucose, known as an inducer of the galactose operon in vivo, stimulates the synthesis of galactokinase, indicating that the repressor of the galactose operon in active under these conditions. This stimulation is not observed, if the bacterial extract is prepared from a strain defective for the galactose repressor or if the DNA carries an operator constitutive mutation in the galactose operon. Therefore the stimulation by D-fucose is true derepression.     

Phycobilisomes in Blue-Green Algae

Fifteen species of freshwater blue-green algae, including unicellular, filamentous, and colonial forms, were subjected to a variety of fixatives, fixation conditions, and stains for comparison of the preservation of phycobilisomes. Absorption spectra of the corresponding in vivo and released photosynthetic pigments, in 10 of the species that were maintained in culture, demonstrated the presence of phycocyanin in all 10 species and phycoerythrin in only 2 of them. Spectroscope and electron microscope evidence was obtained for localization of phycobiliproteins in phycobilisomes of Nostoc muscorum. Phycobilisomes were observed in all species examined in situ, strenghening the hypothesis that phycobilisomes are common to all phycobiliprotein-containing photosynthetic blue-green algae.     

The reaction of alkylating agents with bacteriophage R17. Biological effects of phosphotriester formation

The extent of biological inactivation and of the degradation of the RNA after reaction of bacteriophage R17 with ethyl methanesulphonate, isopropyl methanesulphonate and N-ethyl-N-nitrosourea was studied. Formation of breaks in the RNA chain probably results from hydrolysis of phosphotriesters formed in the alkylation reactions. Near neutral pH the ethyl and isopropyl phosphotriesters are sufficiently stable for the kinetics of the hydrolysis reaction to be followed. Results indicate that the rate of hydrolysis increases rapidly as the pH is raised. The evidence shows that a phosphotriester group does not itself constitute a lethal lesion. The extent of phosphotriester formation by the different agents is discussed in terms of reaction mechanism.