A molecular model for illegitimate recombination in Bacillus subtilis.

The recombinant DNA junctions at which pUB110 and Bacillus subtilis chromosomal DNA were joined to form the plasmid pKBT1 were cloned and sequenced. From the sequencing data we conclude that the pUB110 sequence is intact in the pair of cloned pKBT1 fragments and pTL12 sequences are not present. A molecular model for the formation of pKBT1 based on structural motifs characteristic of the joint sites is presented.

Illegitimate recombination in Bacillus subtilis: a site-specific mechanism in the formation of plasmid pKBT1.

The Bacillus subtilis plasmid pKBT1, the product of in vivo recE4-independent recombinal events, contains segments derived from pUB110 and the B. subtilis chromosome. To determine whether the pUB110 sequence is intact in PKBT1, two 1 kb fragments, each containing a site at which chromosomal and pUB110 sequences are joined, were cloned and sequenced. Sequencing data revealed that: 1). An intact copy of pUB110 is present in pKBT1; 2) The apparent recombination sites were adjacent to the Bam HI-generated ends of pUB110 sequences; 3) pTL12-derived sequences from the original transforming DNA were limited to no more than 1 bp outside the Bgl II recognition sequence; 4) Recombination sites at both ends of pUB110 contain a 19 bp inverted repeat with 15 homologous nucleotides. These findings suggest a site-specific mechanism acting during in vivo formation of pKBT1.

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1.

The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kanr/Neor) and Bg/II digests of pTL12 (Tmpr, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid pKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.

An analysis of altered attention in monkeys exposed to delta-9-tetrahydrocannabinol during development.

Monkey infants born to mothers treated chronically with 2.4 mg/kg delta-9-tetrahydrocannabinol (THC) showed an alteration in visual attention characterized by a failure to limit the duration of attention when looking at novel stimuli. Five off-spring of treated mothers were compared to nine controls in a standardized situation in which they could view slides through a small peephole. THC offspring looked longer at slides early in a session and during the first session in which a given slide was seen. The time of appearance of the peak duration in a series of attention episodes also differed between groups. These alterations in visual attention could have an influence on perceptual and cognitive processes.

Regulation of visual attention in offspring of female monkeys treated chronically with delta 9-tetrahydrocannabinol.

Visual attention was studied in a group of rhesus monkey infants whose mothers received daily oral treatment with low levels of delta 9-tetrahydrocannabinol (THC, 2,4 mg/kg/day) prior to and during pregnancy and throughout lactation (3.5 postnatal months). Attention was measured at 1 and 2 years of age in a standardized test situation in which animals looked at projected slides. In comparison with controls (offspring of untreated mothers), THC offspring directed more attention at slides on the 1st trial of a session. The THC offspring also engaged in relatively longer individual periods of attention on the 1st vs 2nd trials of the session and during the 1st vs 2nd session in which a given slide was presented. Further experiments varying novelty and complexity of visual stimuli suggested that changes in visual attention of THC offspring can be characterized as a failure to limit the response to novel stimuli.

Mother-infant interaction in rhesus monkeys treated clinically with delta-9-tetrahydrocannabinol.

Behavior was studied in 15 rhesus monkeys mother-infant pairs, 5 of whom were exposed to delta-9-tetrahydrocannabinol continuously throughout gestation and the nursing period via drug treatment of mother (2.4 mg/kg/day). The drug-exposed mother-infant pairs were similar to nontreated controls in the amount and types displayed at 10 and 90 days of age, and adequate maternal care was demonstrated by all mothers. Drug-exposed pairs, however, showed no increase in interaction patterns that signal the onset of mother-infant independence at about 3 months of age. This finding suggests that mother-infant attachment may be a behavioral system that is particularly sensitive to modification by perinatal exposure to psychoactive drugs.

Mechanisms of the flare reaction in human skin.

The diffuse area of arteriolar vasodilation surrounding a region of recently injured human skin (axon reflex flare) is dependent upon the integrity of nerve fibers with cell bodies located in dorsal root ganglia. Evidence is presented to indicate that a vasodilator peptide similar to a kinin, neurotensin, or substance P, is implicated in the chain of biochemical events responsible for the transient shift in vascular tonus observable as the flare reaction.

Primate social behavior as a method of analysis of drug action: studies with THC in monkeys.

The use of primate social behavior tests systems to characterize alteration of central nervous system (CNS) function by lesioning, telestimulation, and psychoactive drugs is discussed. Current observations of effects of acute and long-term chronic administration of delta9-tetrahydrocannabinol in group-caged rhesus monkeys are cited to demonstrate the sensitivity and specificity of primate social behavior test systems in characterization of CNS drugs. The time course of drug action proceeds from the acute intoxication stage (sedation, stimulation, decreased social interaction) through behavioral tolerance to the emergence of irritable aggressiveness (which is demonstrated in four different social contexts). The influence of social enviornmental factors and individual differences in social roles on the manifestation of drug effects are discussed. Possible mechanisms of drug action (on biogenic amines and synaptic receptors) compatible with the behavioral data are suggested.

Anacystis nidulans mutants resistant to aromatic amino acid analogues.

Three classes of mutants of Anacystis nidulans were selected on the basis of resistance to fluorophenylalanine and 2-amino-3-phenylbutanoic acid. The most frequent type exhibited DAHP synthetase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate-D-erythrose-4-phosphate-lyase [pyruvate phosphorylating], EC 4.1.2.15) activity identical to that of the parental strain. The second type was characterized by extremely low levels of the activity. The third type had a DAHP synthetase showing decreased sensitivity to inhibition by L-tyrosine. The enzyme was purified 140-fold from wild-type and feedback-insensitive strains, and the kinetics of the reaction was examined. The activity of the wild-type enzyme was inhibited 75% in the presence of 2.0 X 10-3 M tyrosine, and the altered enzyme was inhibited 10%. The following apparent constants were obtained from kinetic studies with partially purified wild-type enzyme: S0.5 for D-erythrose-4-phophate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 1.4 X 10-4 M. Inhibition by tyrosine was mixed with respect to binding of both D-erythrose-4-phosphate and phosphoenolpyruvate. In addition, tyrosine promoted cooperative interactions in the binding of phosphoenolpyruvate. For the altered enzyme the following apparent constants were obtained: S0.5 for D-erythrose-4-phosphate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 2.9 X 10-4 M. Inhibition by tyrosine was mixed with respect to D-erythrose-4-phosphate and competitive with respect to phosphoenolpyruvate. Tyrosine did not promote cooperative effects in the binding of phosphoenolpyruvate to the altered enzyme.

Properties of some norvaline-resistant mutants of Bacillus subtilis.

DL-Norvaline inhibits growth of wild-type Bacillus subtilis. A number of mutants resistant to growth inhibition by this analogue were isolated and studied. Cross-feeding experiments and paper chromatography of culture supernatants indicated that the mutants excreted leucine and possibly valine and glutamate. Enzymic analysis indicated that the mutants were derepressed for acetohydroxy-acid synthetase and alpha-isopropylmalate synthetase; however, no derepression of threonine deaminase, dihydroxyacid dehydrase or transaminase B was observed.

Degradative acetolactate synthase of Bacillus subtilis: purification and properties.

A degradative acetolactate synthase (acetolactate pyruvate-lyase [carboxylating], EC 4.1.3.18) from Bacillus subtilis has been partially purified and characterized. The synthesis of the enzyme was induced by growth of cells in minimal medium plus isobutyrate or acetate. The enzyme was partially purified by ammonium sulfate fractionation, gel filtration, and hydroxyapatite chromatography. The pH optimum of the purified enzyme was 7.0 in phosphate buffer. When assayed in phosphate buffer (pH 7.0), activity was stimulated by acetate and inhibited by sulfate. When assayed in acetate buffer (pH 5.8), activity was inhibited both by sulfate and phosphate. Michaelis-Menten kinetics was observed when the enzyme was assayed in phosphate buffer (pH 6.0 or 7.0), and inhibition by sulfate was competitive and activation by acetate was noncompetitive. When assayed in acetate buffer (pH 5.8), nonlinear Lineweaver-Burk plots were obtained; inhibition by phosphate appeared to be competitive and that by sulfate was of the mixed type. The approximate molecular weight of the purified enzyme was 250,000 as determined by gel filtration.