External factors involved in the regulation of an extracellular proteinase synthesis in Bacillus megaterium

Summary A mathematical model was formulated to describe the kinetics and stoichiometry of growth and proteinase production in Bacillus megaterium. Synthesis of the extracellular proteinase in a batch culture is repressed by amino acids. The specific rate of formation of the enzyme (r _E) can be described by the formula {ie373-1}, where k ₂ and k ₃ stand for the non-repressible and repressible part of enzyme synthesis respectively, k _{S 2} is a repression coefficient and S ₂ indicates the concentration of amono acids; the values of k ₂ and k _{S 2} depend on the composition of the mixture of amino acids. Even in a high concentration, a single amino acid is less effective than a mixture of amino acids. The dependence of the proteinase repression on the concentration of an external amino acid (leucine) follows the same course as its rate of incorporation into proteins, approaching saturation at concentrations higher than 50 M (half saturation approximately 10 M). However, the total uptake of leucine did not exhibit any saturation even at 500 M external concentration.Symbols X biomass concentration, g/l - E proteinase concentration, unit/l - t time, h - S ₁ concentration of glucose, g/l - S ₂ concentration of amino acids, g/l - specific growth rate, l/h - rE specific rate of enzyme production, unit/g/h - k ₁ growth kinetic constant, l/h - k ₂ product formation kinetic constant (for non-repressible part of enzyme synthesis), unit/g - k ₃ product formation kinetic constant (for repressible portion of enzyme synthesis), unit/g - k _{S 1} saturation constant, g/l - k _{S 2} repression coefficient for a certain amino acid or amino acids mixture, g/l     

Expression of HLA-DR antigens on tumour cells does not contribute to skin reactivity to autologous cholesteryl hemisuccinate (CHS)-treated tumour cells in patients with metastatic melanoma

Summary Skin tests with autologous cholesteryl hemisuccinate (CHS)-treated and untreated cells were performed in ten metastatic melanoma patients. In the majority of cases evident reaction was noted with CHS-treated cells (9/10) while the reaction with untreated cells was mostly negative (7/10). Tumour cell suspensions used for skin tests were characterized for reactivity with monoclonal antibody TAL 1B5 detecting the HLA-DR alpha chain. There were no differences between CHS-treated and untreated cells with respect to HLA-DR expression and no correlation was found between grade of skin reaction to CHS-treated cells and the proportion of HLA-DR positive cells in the injected cell sample.     

A BamHI family of highly repeated DNA sequences of Nicotiana tabacum

Summary HRS60.1, a monomer unit (184 bp) of a highly repeated nuclear DNA sequence of Nicotiana tabacum, has been cloned and sequenced. Following BamHI digestion of tobacco DNA, Southern hybridization with HRS60.1 revealed a ladder of hybridization bands corresponding to multiples of the basic monomer unit. If the tobacco DNA was digested with restriction endonucleases which have no target site in HRS60.1, the larger part of DNA homologous to HRS60.1 remained as uncleaved relic DNA. These results suggest a tandem arrangement of this DNA repeat unit. Four other clones of tobacco nuclear DNA cross-hybridized with HRS60.1, thus forming a HRS60-family. Sequencing their inserts has shown their strong mutual homology. HRS60-family comprised about 2% of the nuclear genome of N. tabacum. Computer comparisons with other tandem plant-repeated DNA sequences could not detect any other homologous sequence.     

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.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

Production of milk-clotting enzymes by basidiomycetes

BasidiomycetesPhellinus chrysoloma, Kuehneromyces mutabilis andGanoderma applanatum produce extracellular milk-clotting enzymes. The enzymes are acid proteinases stable at 40°C and within pH 3–5.5. Only the enzyme preparation fromP. chrysoloma exhibits properties comparable with animal chymosin.