The growth rate variability ofChlorella vulgaris beijer. Clones grown after mutagen treatment under autotrophic or heterotrophic conditions on the surface of a solid medium

Populations of theChloretta vulgaris Beijeb. strain ?esnokov “V” clones (progenies of single cells) grown up from cells treated by chemical mutagens were cultivated on the surface of solid media. The differentiation of growth types according to their growth rates in the populations originating from the same sample of cells but growing under different conditions was compared. The strain studied exhibited much greater growth rate variability on the complete medium than on the minimal one. Short time changes of light regime during the cultivation for the purpose of inducing reparation processes in the mutagen damaged cells did not manifest themselves significantly in the composition of resulting clonal populations. The possibility of selection of growth types under conditions studied was considered.     

Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus.

The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.     

Expression of the threonine operon fromEscherichia coli inBrevibacterium flavum andCorynebacterium glutamicum

Summary The threonine operon fromEscherichia coli was cloned in plasmid pBR322, subcloned into the shuttle vector pCEM300 and the resulting recombinant plasmid was transferred intoBrevibacterium flavum andCorynebacterium glutamicum. The expression ofE. coli threonine genes in these coryneform bacteria was demonstrated by complementing thethrA andthrB mutations and by assaying homoserine dehydrogenase activity.     

Plasmid cloning vectors replicating in Escherichia coli,amino acid-producing coryneform bacteria and Methylobacillus sp.

Summary Four hybrid plasmids were constructed from the cryptic plasmid pAM330 (from Brevibacterium lactofermentum; 4.5 kb) and the broadhost-range plasmid pGV1106 (9.0 kb; Km^r Sm^r) isolated from Escherichia coli. All of them were mobilized from E. coli into the Gram-negative methylotrophic bacterium Methylobacillus sp. and two of these constructs (pCEM300 and pCEM400) were transferred by transformation into B. flavum and Corynebacterium glutamicum. Their kanamycin-resistance determinant coming from Gram-negative hosts was expressed in these Gram-positive bacteria. Both pCEM300 and pCEM400 are very stably maintained in B. flavum and represent suitable vectors for gene cloning in coryneform producers of amino acids.     

Cytophotometric and biochemical research on the amount of DNA in the nuclei of loach embryos injected with low-molecular RNA

The cell cycle structure in the cells of loach embryos at the early blastula stage (5 h of development at 21 degrees) is markedly altered under the influence of injection of homologous low molecular weight nuclear RNA and, as a result, the number of cells in G2-phase. The DNA amount in the embryo increases by 20%. At the midblastula stage (7 h) no increase in the number of cells in G2-phase was found.     

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.