A rapid method for the determination of the base composition of bacterial DNA

A rapid method to evaluate the (G+C)-content of bacterial DNA is described. About 2 × 10⁸ cells are lysed by the combined action of detergents and enzymes and put into a CsCl-gradient without any purification. Up to five samples may be run at once. The high background absorption of cell-derived material other than DNA is overcome by novel collimating optics.     

Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

Enhanced Rate of Expression and Biosynthesis of Neuropeptide Y After Kainic Acid-Induced Seizures

Recent studies have shown marked increases in brain content of neuropeptide Y (NPY) after seizures induced by intraperitoneal injection of kainic acid and after pentylenetetrazole kindling in the rat. We have now investigated possible changes in the rate of biosynthesis of NPY after kainic acid treatment, by using pulse-labeling of the peptide and by determining prepro-NPY mRNA concentrations. For pulse labeling experiments, [3H]tyrosine was injected into the frontal cortex, and the incorporation of the amino acid into NPY was determined after purifying the peptide by gel filtration chromatography, antibody affinity chromatography, and reversed-phase HPLC. At 2 and 30 days after kainic acid treatment, the rate of tyrosine incorporation was enhanced by approximately 380% in the cortex. In addition, concentrations of pre-pro-NPY mRNA were determined in four different brain areas by hybridization of Northern blots with a complementary 32P-labeled RNA probe 2, 10, 30, and 60 days after kainic acid treatment. Marked increases were observed in the frontal cortex (by up to 350% of controls), in the dorsal hippocampus (by 750%), and in the amygdala/pyriform cortex (by 280%) at all intervals investigated. In the striatum only a small, transient increase was observed. The data demonstrate increased expression of prepro-NPY mRNA and an enhanced rate of in vivo synthesis of NPY as a result of seizures induced by the neurotoxin kainic acid.     

Eight newRubus species described from Czech Republic

Eight newRubus species are described from the area of Czech Republic (western Czechoslovakia), four of them belonging to ser.Discolores, and one by one to subsect.Rubus, ser.Rhamnifolii, ser.Micantes and ser.Hystrices. *** DIRECT SUPPORT *** AAY02002 00005     

Comparison of 4-hydroxynzoate decarboxylase and phenol carboxylase activities in cell-free extracts of a defined, 4-hydroxybenzoate and phenol-degrading anaerobic consortium

Summary Two 4-hydroxybenzoate decarboxylase activities and a phenol carboxylase activity were found in cell-free extracts of a defined, 4-hydroxybenzoate- or phenol-grown consortium. Both decarboxylase activities were loosely membrane-associated and required K⁺ but a different pH and ion strength. Loss of activity of both decarboxylases by EDTA could be compensated by Zn²⁺ ions. The K _m values for 4-hydroxybenzoate and K⁺ of the decarboxylase activities with pH optima at 6.4 or 7.8 were 0.02 and 2.5 or 0.004 and 0.5 mm, respectively. 3,4-Dihydroxybenzoate, 3,4,5-tridydroxybenzoate, 3,5-dimethoxy-4-hydroxybenzoate and 3-chloro-4-hydroxybenzoate were also decarboxylated by both enzyme activities. The phenol carboxylase was a soluble enzyme with its pH optimum at 6.5. It required K⁺, Rb⁺ or NH _inf4 ^sup+ as monovalent, Zn²⁺, Mg²⁺, Mn²⁺ or Ni²⁺ as divalent cations and catalysed the carboxylation of phenol if 2,4-,2,3,4- or 2,4,6-hydroxybezoates were absent. The three enzyme activities were not influenced by Avidin and thus were probably not biotin-dependent enzymes. Offprint requests to: J. Winter     

Control of the Lysine Biosynthesis Sequence in Corynebacterium glutamicum as Analyzed by Overexpression of the Individual Corresponding Genes

The gene cluster that codes for feedback-resistant aspartate kinase (lysCα and lysCβ) and aspartate semialdehyde dehydrogenase (asd) was cloned from a mutant strain of Corynebacterium glutamicum. Its functional analysis by subcloning, enzyme assays, and type of aspartate kinase regulation enabled the isolation of a fragment for separate expression of the feedback-resistant kinase without aspartate semialdehyde dehydrogenase expression. This was used together with other clones constructed (J. Cremer, L. Eggeling, and H. Sahm, Mol. Gen. Genet. 220:478-480, 1990) to overexpress individually each of the six genes that convert aspartate to lysine. Analysis of lysine formation revealed that overexpression of the feedback-resistant kinase alone suffices to achieve lysine formation (38 mM). Also, sole overexpression of wild-type dihydrodipicolinate synthase resulted in lysine formation but in a lower amount (11 mM). The other four enzymes had no effect on lysine secretion. With a plasmid overexpressing both relevant enzymes together, a further increase in lysine yield was obtained. This shows that of the six enzymes that convert aspartate to lysine the kinase and the synthase are responsible for flow control in the wild-type background and can be useful for construction of lysine-producing strains.     

Picomole assay for N-methylhistidine by gas chromatography—Mass spectrometry

A simple, sensitive method for measuring N^τ-methylhistidine in biological samples using a deuterated internal standard and methane chemical ionization gas chromatography-mass spectrometry is described. After sample preparation, a single analysis can be completed in 3 min; analysis in duplicate, including sample preparation for 40 samples, can be completed at a rate of 15 min per sample. Nanomole amounts of N^τ-mmethylhistidine in urine or plasma samples are determined with a precision of 0.5%. Picomole amounts, released during in vitro rat epitrochlaris muscle incubations, are measured with a precision of 10%.