Transformation of a new Gram-positive methylotroph,Brevibacterium methylicum,by plasmid DNA

Summary Brevibacterium methylicum is a newly isolated Gram-positive facultatively methylotrophic bacterium that uses the NAD⁺-dependent methanol dehydrogenase for methanol oxidation and assimilates its carbon via the ribulose monophosphate cycle. Protoplasts prepared by lysozyme treatment of B. methylicum cells grown in the presence of glycine were transformed by plasmid shuttle vectors pCEM500 (16.5 kb; Sm^r/Sp^r, Km^r/Gm^r) and pEC71 (7.1 kb; Km^r/Nm^r) constructed on the basis of B. lactofermentum plasmid pAM330 and replicating in Escherichia coli and in amino-acid-producing coryneform bacteria. The resistance markers were found to be expressed in B. methylicum and autonomous plasmid DNAs of various size were isolated from the transformants. The presence of the pAM330 replicon in these plasmids was demonstrated by DNA-DNA hybridization experiments. Offprint requests to: J. Nevera     

A new vector for cloning of genes and replicons in yeasts

A novel Escherichia coli-Saccharomyces cerevisiae shuttle vector lambda MAN78 has been constructed. The vector contains phage lambda 47.1 DNA, Sacch. cerevisiae chromosomal segment with TRP1 gene and the yeast ARS1 replicator. This vector may be propagated as a phage and, similar to parental lambda 47.1, allows direct selection of large DNA inserts (15-24 kbp) in E. coli. lambda MAN78 can efficiently transform LiCl-treated yeast cells (3-5 X 10(3) transformants per 1 microgram DNA). Replication of hybrid molecules in E. coli cells does not influence the ability of the molecules to transform yeast cells and replicate in the cells.     

Glutamicin CBII,a bacteriocin-like substance produced by

Corynebacterium glutamicum CBII, in the stationary phase of growth, was found to produce spontaneously a substance resembling bacteriocins by its bactericidal properties. This substance designated glutamicin CBII was observed to exhibit bactericidal activity against coryneform bacteria (12 species tested) but not against unrelated gram-positive (3) and gram-negative (3) bacteria, while its action on bacteria with no quite known relatedness to the coryneform group (14) was found to be variable. Glutamicin CBII was partially purified by precipitation with ammonium sulphate (70% saturation), selective heat precipitation and gel chromatography on Sepadex G-50. The antibacterial substance diffused through cellophane membrane with an approximate cut-off of 10000 dalton and its sedimentation coefficient was determined to be 1.1. S by ultracentrifugation. Heating at 100°C for 30 min had no effect on its activity. Glutamicin CBII was proved to be resistant to chloroform, trypsin, chymotrypsin, pronase, and subtilisin. According to its staining behaviour and ¹H NMR spectra it probably represents a glycoprotein containing only a minor protein component.

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Optimization of the exposure of normal and transformed cell populations to phase-specific agents

Dynamic behavior of stem cells population of the "critical" tissue (normal population) and tumor cell population under periodic treatment with a phase-specific cytotoxic agent was considered. The results were used for optimization of anticancer chemotherapy. The schedules of treatment were found which provide a maximum rate of tumor-cell elimination for any given rate of the normal population size decrease. If the mean generation times of normal and tumor populations differ (which was stated for many tumors), usage of the optimal period markedly increases the selectivity of therapy, while application of other periods can result in selective elimination of the normal population. Problems concerned with practical realization of the proposed regimes are discussed.     

Bacteriocin production inAgrobacterium tumefaciens

Agrobacterium tumefaciens C58 forms “plaques” during layer cultivation. The “plaques” were shown not to be caused by the presence of a temperate bacteriophage or by random contamination. The “plaques” and their central microcolonies were used to repeatedly isolate cultures producing an antibiotic substance against the original strainA. tumefaciens C58, other nopaline strains, some octopine strains ofA. tumefaciens and some strains of the related genusRhizobium. The substance is thus a bacteriocin; in analogy to agrocins 84 and D286 it was named agrocin C58. The agrocin is not inactivated by trypsin. Its production by strain C58 was found only on cultivation on solid but not liquid media. The producing isolate ofA. tumefaciens C58 (strain C58i2) contains neither plasmid pTiC58 nor the plasmid analogous to pAgK84 which controls the production of agrocin 84 inA. radiobacter K84.     

Tritium labeling of tRNA by the tritium thermal activation method

Tritium labelled E. coli total tRNA and tRNAPhe are prepared by action of thermally activated tritium atoms. The preparations, having the molar radioactivity up to 3.6 Ci/mmol, are useful for functional investigations.     

Responses of cell populations of three chlorococcal algae to the action of streptomycin

Time doses of a single concentration of streptomycin and its concentration doses acting for the same time period in a liquid medium had different effects on three strains of chlorococcal algae. This concerned both the physiological responses and permanent changes in the characteristics of cell colonies growing from treated cells. Significant differences were recorded in: the number of autospores produced during the first division of the treated cells on the surface of a solid medium, the length of the lag phase, the growth rate of the diameter of cell colonies, and the survival of the treated cells. The permanent changes in the characteristics of the growing colonies were very different in the individual algal strains in quality and frequency. Physiological and the mutation effects were compared and discussed.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

Nuclear function during early development of remote fish hybrids

The participation of paternal genome was studied in the development of remote hybrids obtained as a result of artificial insemination of the loach (Misgurnus fossilis) eggs by the sperm of aquarial Cyprinids (Brachydanio rerio, Danio malabaricus, Barbus tetrazona, Razbora heteromorpha, Carassius auratus) and Cobitids (Acanthophtalmus kuhlii). The hybrids obtained differed at the stage of hatching both from each other and from the loach by some morphological features. To study the function of heterologous nuclei, haploid nucleocytoplasmic hybrids were obtained by means of chromosome inactivation in the loach eggs by heavy doses of X-rays. The participation of paternal genome in development was estimated by comparison of the curves of viability of diploid and haploid hybrids with those of diploid, haploid and "anuclear" loach embryos. Patterns of mortality of embryos and larvae in each hybrid combination (percentage, stage) suggest the functioning of paternal genome already at the early stages of development. The activity of hybrid and heterologous nuclei was also estimated by the onset and the intensity of morphogenetic function which was determined by the time of embryonic death following nuclear inactivation at different stages. The onset of nuclear function in all hybrids coinsides with that in the loach, except B. rerio in which it occurs somewhat earlier. The data obtained prove the participation of paternal genes in development and maintenance of viability of embryos at all developmental stages beginning from the early ones (blastula).     

The mechanism of photodynamic inactivation ofStaphylococcus aureus by deuteroporphyrin

The mechanism ofStaphylococcus aureus inactivation by deuteroporphyrin (DT) and light was studied with singlet oxygen quenchers or hydroxyl radical scavengers. The light-activated DT (10 /ml) reduced the viability of the culture to less than 1%, whereas methionine, tryptophan, and 1,4-diazabicyclo-2,2,2-octane (DBCO) used as singlet oxygen quenchers provided almost 60% protection. Propylgallate, which is a hydroxyl free radical scavenger, also provided 60% protection. The presence of a singlet oxygen quencher and propylgallate provided almost complete protection from inactivation (96%). Photoinactivation in the absence of culture media (in saline) increased the killing rate and decreased the ability of the singlet oxygen quenchers to protect. In the same conditions damage from hydroxl free radicals was well protected by propyl gallate. The present results indicate thatS. aureus photoinactivation by DT and light is mediated by both singlet oxygen and hydroxyl free radicals.