Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli

The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA.     

3-Hydroxypropyl amide formation from 1-aminocyclopropane-1-carboxylic acid by a cell-free ethylene-forming system from olive leaves

The low ethylene yield in a cell-free ethylene-forming system from olive tree leaves ( Olea europaea L. cv. Picual) was investigated. During the incubation, 1-aminocyclopropane-1-carboxylic acid (ACC) was extensively transformed into 3-hydroxypropyl amide (HPA). Enzyme extract, Mn²⁺ and oxygen are responsible for this reaction. Horseradish peroxidase (EC 1.11.1.7) can substitute for the enzyme extract in this reaction. HPA formation could be one reason for the poor in vitro conversion efficiency of ACC to ethylene.     

Metabolism of ethylene in olive leaves

Olive tree tissues are able to metabolize ethylene. This metabolism is inhibited by heat killing and carbon disulfide. Rubber stoppers usually employed to close the incubation vials release carbon disulfide, thereby modifying the values obtained for ethylene. The ethylene consumption rate has been found to be 9 nl/h.g, an intermediate value among the different plant tissues so far examined.     

Effects of the Phosphatase Inhibitors Calyculin A and Okadaic Acid on Acetylcholine Synthesis and Content of Rat Hippocampal Formation

Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 n M ) and okadaic acid (50 n M ) reduced significantly ( p < 0.01) the synthesis of [³H]ACh from [³H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner ( p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly ( p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner ( p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly ( p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [³H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced V _max values for high- and low-affinity choline uptake ( p < 0.01) with no significant change in K _m values ( p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.     

Expression of mutations and protein release by yeast conditional autolytic mutants in batch and continuous cultures

Thermosensitive mutant strains of Saccharomyces cerevisiae that fail to generate an osmotically stable cell wall when grown at a non-permissive temperature release their cell contents upon expression of the mutation. Therefore, they may represent an alternative for the production of homologous or heterologous protein preparations. In order to analyse the expression of two of these mutations, lyt2 and slt2, we grew the corresponding strains under precisely defined conditions in batch and continuous fermentors. A switch in the temperature of batch cultures from 24° C to 37° C determined lysis of the cells with a significant release of intracellular enzymes. These include alkaline phosphatase and periplasmic proteins such as glucan-degrading enzymes, the pattern of cell lysis and protein release being maintained for about 6 h. One-stage continuous cultures of a lyt2 mutant were maintained for long periods at 37° C; a fraction of the population lysed and released the indicated proteins, but eventually a revertant of the lytic phenotype was selected. To avoid this, a two-stage continuous culture system was developed by connecting two fermentors in series, the effluent from the first one at 24°C being fed to the second one adjusted to 37° C. A steady state of cell lysis and protein liberation was reached in the second-stage fermentor without any evidence of selection of revertants. This system can be very useful for developing conditions for the use of yeast strains to produce protein preparations. Correspondence to: C. Nombela     

Partial deletion of 4p16 band in a ring chromosome and wolf syndrome

Summary A new case of ring chromosome 4 in a 2-day-old female child with multiple malformations is described. By means of the GTG-banding technique, a karyotype 46,XX,r(4), (p16q35) was determined. The characteristics of the child's karyotype and the relationship with the structure of the chromosome, especially the location of the deletion that produces the syndrome, are compared with previous reports.     

Biodegradation of Crude Oil by Thermophilic Bacteria Isolated from a Volcano Island

One-hundred and fifty different thermophilic bacteria isolated from a volcanic island were screened for detection of an alkane hydroxylase gene using degenerated primers developed to amplify genes related to the Pseudomonas putida and Pseudomonas oleovorans alkane hydroxylases. Ten isolates carrying the alkJ gene were further characterized by 16s rDNA gene sequencing. Nine out of ten isolates were phylogenetically affiliated with Geobacillus species and one isolate with Bacillus species. These isolates were able to grow in liquid cultures with crude oil as the sole carbon source and were found to degrade long chain crude oil alkanes in a range between 46.64% and 87.68%. Results indicated that indigenous thermophilic hydrocarbon degraders of Bacillus and Geobacillus species are of special significance as they could be efficiently used for bioremediation of oil-polluted soil and composting processes.