XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

Summary The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to -galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 Ile) and Val274 (Asp274 Val) substitutions over the N-terminal His4l (Arg4l His) substitution, and the intraallelic dominance of Thr45 (Arg45 Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 Leu) and Arg256 (Pro256 Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector.     

DIROM: an experimental design interactive system for directed mutagenesis and nucleic acids engineering

A computer system DIROM for oligonucleotide-directed mutagenesisand artificial gene design has been designed for better experimentalplanning and control. DIROM permits searching for optimal oligonucleotideswith respect to certain important parameters, namely sufficientenergy of oligonucleotide-target hybridization, the secondarystructure of oligonuc-tide and target DNA, the presence of alternatebinding sites in the target DNA and terminal G/C pairs. It canalso be used to plan polymerase chain reaction experiments,for optimal primer selection, in sequencing, etc. DIROM enablesone to search for both existing and potential restriction sites,to perform vector + target sequence construction. The systemconsists of a set of original algorithms that formalize theempirical knowledge of oligonucleotide action as primers.     

Species differences in the binding kinetics of 25-hydroxyvitamin D3 to vitamin D binding protein

The specific binding of 25-hydroxyvitamin D3 to its binding protein was studied in serum of the human, rhesus monkey, cow, horse, and rat. The free fraction of 25-hydroxyvitamin D3 in the rat was 0.34 +/- 0.15 pmol free/nmol total (+/- SD) and this was lower than in any of the other species (p less than 0.01). In the human, the free fraction was 1.5 +/- 0.32 pmol free/nmol total, which was higher than in any of the other species (p less than 0.001). The differences in the free fraction were mainly due to differences in dissociation constant. The relative levels of free 25-hydroxyvitamin D should be taken into account when extrapolating findings about vitamin D metabolism in animals to the human. A technical outcome of this study is that of the species tested, vitamin D binding protein from rat serum is the most suitable as a reagent component for methods used to measure total 25-hydroxyvitamin D by competitive protein binding assay.     

The Independency of Choline Transport and Acetylcholine Synthesis

The coupling of choline transport to acetylcholine synthesis has been investigated by measurement of the isotopic dilution of a pulse of [3H]choline during its incorporation into the recently synthesised acetylcholine of cerebral cortex synaptosomes. Recently synthesised acetylcholine was identified as that containing 14C-labelled precursors introduced by a preincubation before the pulse. When [14C]glucose was used to label acetyl-CoA coupling ratios (calculated as the inverse of the dilution of extracellular [3H]choline during its incorporation into [3H]acetylcholine) of about 0.05-0.2 were found at a choline concentration of 1 microM, rising to 0.5 at choline concentrations of 10-50 microM. Experiments using [14C]choline as a precursor gave similar results, and it was shown that the isotopic dilution did not occur extrasynaptosomally and was not affected by low glucose concentrations. Coupling ratios were always less than unity and rose as the choline concentration increased. It is concluded that choline transported into the nerve terminal has no privileged access to choline acetyltransferase. The results can be explained by a rate-controlling transport of choline into the terminal followed by its rapid acetylation rather than any linkage or coupling of the two processes.     

Physiological and biochemical contributions to the taxonomy of the genera Ankistrodesmus and Scenedesmus

The 17 Ankistrodesmus strains studied do not produce extracellular amylolytic enzymes. 20 out of 28 Scenedesmus strains, however, are able to hydrolyse starch.The groups of strains previously characterized physiologically and biochemically were assigned to established species of the genera Ankistrodesmus (including Monoraphidium) and Scenedesmus.Abbreviation GC Guanine + cytosine     

Na+-dependent,potential-sensitive l-ascorbate transport across brush border membrane vesicles from kidney cortex

l-Ascorbate is taken up into brush border vesicles from kidney cortex of rat, rabbit and guinea pig by an efficient, Na⁺-dependent and potential-sensitive transport process. This uptake shows saturation ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>:0.1–0.3\; mM</mtext>}}$) and is strongly stimulated by low concentrations of N₃^?. Erythorbate (d-isoascorbate) seems to be another, but poorer, substrate of the same transporter.     

Neonatal thyroxine treatment enhances classical conditioning in the infant rat

Neonatal rats injected with either thyroxine (T₄) or vehicle (NaOH) on postnatal Days 1, 2, and 3 were given classical-conditioning pairings of an odor with footshock when 7, 9, or 11 days of age. In accord with the conventional acceleration of other indices of maturation following the T₄ treatment, 24-hr retention of the conditioned odor aversion was substantially enhanced among the 11 day-old rats given the earlier T₄ treatment. This effect was marginally significant among 9-day olds but not among 7-day olds.     

Intracellular appearance of nitrite and nitrate in nitrogen-starved cells of Ankistrodesmus braunii

Summary The occurrence of heterotrophic nitrification in nitrogen-starved cells of Ankistrodesmus braunii was confirmed. The levels of nitrate and nitrite were measured over a period of four weeks. The validity of quantitative determinations in the presence of highly active nitrate and nitrite reductases is discussed. Whereas free hydroxylamine as an intermediate could not be detected, increased hydroxylamine oxidase activity was found in nitrogen-starved cultures. Nitrite reductase and hydroxylamine oxidase can be assigned to particles by sucrose density gradient centrifugation. The possible involvement of microbodies, which were found to be present in Ankistrodesmus, in metabolic processes during nitrogen starvation is discussed.Abbreviations NR nitrate reductase - NiR nitrite reductase - NNEDA N-(1-naphthyl)ethylenediaminedihydrochloride - DCPIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - TCA trichloroacetic acid - DAB 3,3-diaminobenzidine - AT 3-amino-1H-1,2,4-triazole - AMP 2-amino-2-methyl-1,3-propanediol