Ultrastructural localization of interferon receptors on the surfaces of cultured cells and erythrocytes.

The bindings sites for interferon (IFN) on the limiting cell membranes of human and mouse fibroblasts and erythrocytes were revealed by an indirect immunoferritin technique. Mouse IFN-beta and human IFN-beta of high specific activity were used with the corresponding purified antibodies. Species-specific IFN binding was demonstrated by ferritin deposition on human erythrocytes and fibroblast membranes treated with human IFN and on mouse erythrocytes and fibroblast membranes treated with mouse IFN, but not on human erythrocytes or fibroblast membranes treated with mouse IFN. IFN binding sites on fibroblasts were located on regions of membranes between microvilli, whereas diphtheria toxin receptors were demonstrated mainly on microvilli. IFN binding altered the diphtheria toxin after IFN treatment. This reduced toxicity correlated with a decrease in the quantity of receptors for diphtheria toxin on the cell membrane. Thus, the species-specific binding of IFN appears to depend on membrane receptors in discrete regions of the limiting membrane which are present not only on functionally responsive fibroblasts but also on erythrocytes.

Plasmid transformation in Haemophilus influenzae.

Purified 34-megadalton-plasmid deoxyribonucleic acid from antibiotic-resistant strains of Haemophilus influenzae transforms competent strains of H. influenzae more efficiently if the recipient strains contain certain other 30-megadalton plasmids.

Physical size of the donor locus and transmission of Haemophilus influenzae ampicillin resistance genes by deoxyribonucleic acid-mediated transformation.

The properties of donor deoxyribonucleic acid (DNA) from three clinical isolates and its ability to mediate the transformation of competent Rd strains to ampicillin resistance were examined. A quantitative technique for determining the resistance of individual Haemophilus influenzae cells to ampicillin was developed. When this technique was used, sensitive cells failed to tolerate levels of ampicillin greater than 0.1 to 0.2 mug/ml, whereas three resistant type b beta-lactamase-producing strains could form from the colonies in 1- to 3-mug/ml levels of the antibiotic. DNA extracted from the resistant strains elicited transformation of the auxotrophic genes in a multiply auxotrophic Rd strain. For two of the donors, transformation to ampicillin resistance occurred after the uptake of a single DNA molecule approximately 104-fold less frequently than transformation of auxotrophic loci and was not observed to occur at all with the third. The frequency of transformation to ampicillin resistance was two- to fivefold higher in strain BC200 (Okinaka and Barnhart, 1974), which was cured of a defective prophage. All three clinical ampicillin-resistant strains were poor recipients, but the presence of the ampicillin resistant genes in strain BC200 did not reduce its competence. Sucrose gradients of DNA from ampicillin-resistant transformants of BC200 and from the original ampicillin-resistant strains showed that: (i) all the DNA preparations had high molecular weights; (ii) donor activity for ampicillin resistance sedimented heterogeneously and in parallel with genome DNA up to the highest molecular weights observed (100 x 106 to 200 x 106); and (iii) genetic transformation of ampicillin resistance from strain BC200-amp90383 required the physical integrity of a linearly integrated segment of DNA having a molecular weight of about 25 x 106 to 30 x 106.

Prophage S2 mutants in Haemophilus influenzae: a technique for their production and isolation.

A procedure utilizing nitrosoguanidine has been developed to produce defective and temperature-sensitive mutants of prophage (S2) in lysogenic Haemophilus influenzae. The system should be generally applicable to all temperate phage systems. At saturating concentrations of phage DNA, more than 25 percent of recipient mutant lysogenic bacteria can be transformed to the wild type.