The nature of the colicin K receptor of Escherichia coli Cullen.

Cell walls of E. coli strains B and Cullen contain specific receptors for colicin K and for the T2, T6, and C16 phages. The receptors for the bacteriocin and the T6 virus are located in the outer layers of the cell wall of these microorganisms and are absent in their cytoplasmic membrane. The receptors for colicin K, phage T2, and the T6 and C16 viruses differ in their stability toward enzymes and chemical reagents. Their specificity must therefore be determined by different chemical groupings. The colicin K receptor is inactivated by certain proteolytic enzymes and by reagents which combine with tryptophan. It is concluded therefore that proteins or peptides containing this amino acid are essential for biological activity of the receptor.

Methylated nucleosides in polyadenylate-containing yeast messenger ribonucleic acid.

Yeast messenger RNA was found to be methylated. A calculation of the specific methylation showed that the average yeast messenger RNA molecule contains only two methylated nucleosides which occur in one alkali stable oligonucleotide. Similar to other eukaryotic messengers, the 5' terminus of yeast messenger RNA is blocked by 7-methylguanosine, linked through a di- or triphosphate bridge to a ribosemethylated nucleoside. Contrary to the messengers of high eucaryotic organisms, no additional base methylated constituents were found.

Measurement of cytoplasmic calcium in aleurone protoplasts using indo-1 and fura-2.

Previous attempts to measure cytoplasmic Ca2+ in plant cells using the new generation of fluorescent probes, indo-1 and fura-2, have been unsuccessful. We investigated the use of indo-1 and fura-2 to measure cytoplasmic Ca2+ in barley aleurone protoplasts and found that indo-1 could be successfully used when it was loaded into protoplasts in the Ca2+-sensitive form. The acetoxymethyl esters of both dyes accumulated in aleurone protoplasts, but fura-2 was sequestered in the vacuole and indo-1 was not adequately hydrolyzed. We developed a non-disruptive method for loading the Ca2+-sensitive form of indo-1 into aleurone protoplasts in mildly acidic solutions. Using this approach, protoplasts accumulate indo-1 in a pH-dependent manner. The accumulated dye is Ca2+-sensitive, it is not sequestered in vacuoles or the endomembrane system, and it is not rapidly secreted. Fluorescence from indo-1 in individual cells was quenched by Mn2+ in the presence of digitonin. We estimate the cytoplasmic Ca2+ concentration in aleurone protoplasts to be approximately 250 nM. The Ca2+ ionophore, ionomycin does not induce changes in the fluorescence of protoplasts loaded with indo-1, but fluorescence changes could be induced by changes in extracellular Ca2+ in the presence of digitonin. We conclude that the strategy of loading indo-1 at acidic pH provides a useful means of measuring cytoplasmic Ca2+ in the barley aleurone that may also be applicable to other types of plant cells.

A simple phase-extraction assay for chloramphenicol acyltransferase activity.

A simple and convenient phase extraction assay for chloramphenicol (Cm) acetyltransferase (CAT) activity has been developed, based on the enzymatic butyrylation of radiolabelled Cm. The assay is linear over two to three orders of magnitude of enzyme concentration, is highly sensitive, and substantially less expensive than all presently available alternatives. Methods for convenient CAT assay, adapted for mammalian cells, plant protoplasts, and mammalian cell culture supernatants, are described. These methods should also simplify measurement of CAT activity in other organisms, such as yeast and bacteria. In addition, a simple pre-extraction procedure is presented for purifying radiolabelled Cm which allows a 25-fold increase in sensitivity using tritiated substrates.

Neutral selection of transfected mammalian cells using tissue plasminogen activator gene expression.

The gene that produces the proteolytic enzyme, tissue plasminogen activator (tPA), was used as a selectable marker for transfection. Cells that were successfully transfected with plasmids containing a gene for tPA (tpa) produce plaques in the caseinolysis assay. Two tpa-containing plasmids were constructed and used to transfect a variety of cell types. One plasmid contained the promoter region of simian virus 40 and the other contained the Moloney murine leukemia virus promoter. No significant difference in transfection frequencies was found for the two plasmids. However, 80% of the cell types tested produced plaques. This system allows for the identification and isolation of transfected cells under conditions that are not lethal to nontransfected cells.

Low heat resistance of Bacillus sphaericus spores correlated with high protoplast water content.

The low heat resistance (D100 = 0.554 min, z = 13.4 degrees C) of dormant lysozyme-sensitized spores of Bacillus sphaericus 9602 was correlated with a low protoplast wet density (1.305 g/ml) equivalent to a high protoplast water content (61.0%, wet weight basis). These values for these unusual spores were consistent with those correlated previously in 28 spore types of seven other species.

A general method for the detection of potyviral gene products in plant protoplasts and tissue.

A rapid immunostaining procedure for detecting potyviral antigens in individual protoplasts, isolated mesophyll cells, and epidermal strips of Nicotiana tabacum is described. Although all specific antibodies tested detected potyviral antigens in electroporated protoplasts, those against cytoplasmic inclusion (CI) protein provided the most useful results. The number of protoplasts reacting with anti-CI increased with time after inoculation, roughly in parallel with the accumulation of capsid protein, which was measured independently by enzyme-linked immunosorbent assay. Potyviral gene products were also detected in epidermal strips and mesophyll cells separated from systemically infected leaves, indicating that the immunostaining method is generally applicable and that it may prove useful for studying the movement of potyviruses from cell to cell in intact plants.

Distribution of oleanolic acid glycosides in vacuoles and cell walls isolated from protoplasts and cells of Calendula officinalis leaves.

The contents of oleanolic acid and its 3-0-glucuronide derivatives as well as of 3-0-glucoside derivatives were determined in vacuoles prepared from protoplasts and cell walls obtained from cells of Calendula officinalis leaves. In both cell compartments studied 37% of total cellular oleanolic acid were accumulated, 0.6% occurring as free oleanolic acid (only in vacuoles). Glucuronides accounted for 31.1% (20.7% in vacuoles and 10.4% in cell walls), and glucosides for 5.3% (2.6% in vacuoles and 2.7% in cell walls).

Basic techniques for DNA cloning and conditions required for streptomycetes as a host.

To develop a host-vector system in streptomycetes for DNA cloning, we examined the technical problems encountered and the conditions required for use of Streptomyces kasugaensis MB273 as the host. Basic techniques, such as plasmid DNA isolation, regeneration of mycelia from protoplasts and elimination of plasmids from cells were investigated. These techniques were found to be useful for many streptomycetes. Strain M518, a derivative of S. kasugaensis MB273, was found to have the following useful characteristics as a host. The plasmids of MB273 were easily cured by regeneration of mycelia from protoplasts. The protoplasts prepared from M518 regenerated mycelia at high frequency using an improved method and were efficiently transformed by plasmid DNA. The extra and intra cellular DNase activities were very weak, and no restriction endonuclease activity was detected. The sensitivity to various antibiotics was determined. This strain did not show any pathogenicity in mice nor suvival in the digestive organs of rats. MB273 and its derivatives died rather quickly in natural soil. M518 still forms aerial mycelial conidia. These results indicate that S. kasugaensis M518, derived from MB273, has useful characteristics as a host for DNA cloning. The techniques thus developed were found to be useful in other streptomycetes.

Biochemical characterization and visualization of plasma membrane-DNA-protein complexes from Bacillus subtilis.

Subcellular fractions containing plasma membranes with bound DNA and associated proteins were isolated from two different cultures of Bacillus subtilis 168 growing exponentially at different rates. Differences in the contents of individual between the dissociated complexes, established electrophoretically, can be explained by dynamic binding of the proteins to DNA, resulting in a control of DNA, resulting in a control of DNA replication. Electron microphotographs of isolated complexes display, in addition to unit membranes, associated filamentous structures in different arragement. Patterns obtained after treating the complexes with nucleases suggest a polydeoxyribonucleotide character of the filamentous structures.

[Protein damage after treatment with ozone of protoplasts and isolated E. coli membranes]. / Povrezhdenie belkov pri deistvii ozona na protoplasty i vydelennye membrany E. coli.

The features of ozone-induced damage of E. coli plasma membrane proteins are investigated. A conclusion is made that protein fluorescence quenching is connected with modification of amino acid residues in the vicinity of tryptophane residues. Such modification may be a consequence of reaction with either ozone itself or products of its interaction with membrane lipids and/or proteins. The suggestion of Goldstein and McDonagh that ozone has a predilection for more hydrophilical membrane domains is confirmed. The data obtained are in agreement with a supposition about the leading role of proteins in deleterious action of ozone on cells.

[Nature of the IR spectra of intact cells of Gram-negative microorganisms]. / K voposu o prirode IK-spektrov intaktnykh kletok nekotorykh gramotritsatel'nykh mikroorganizmov.

The character of changes in the infrared spectra of E. coli in the process of their mechanical disintegration has been studied. The destruction of E. coli cell structures has been shown to produce no changes in the optical density of the main analytical absorption bands in infrared spectra. This fact suggests that the infrared absorption spectra of E. coli are the sum of the spectra of all chemical components of the cell, which is confirmed by the infrared spectral study of E. coli cell-wall preparations. Similar results have been obtained in the study of the preparations of B. pertussis cell walls, protoplasts and intact cells.