Cloning and expression of the 3' portion of the T4 denV gene as a lacZ fusion gene

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage lambda gt11 T4 dC DNA library was screened for phage which produced a beta-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25% of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within lambda gt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native beta-galactosidase.     

Effects of lung congestion and oleic acid injury on the Hering-Breuer reflex

Breathing and the Hering-Breuer (HB) reflex may be stimulated by congestion and by acute lung injury, but there is disagreement about the effects of both stimuli. This study evaluated these effects using greater stimulus isolation and control of secondary interactions than have previously been employed. Pressurization of lung vessels and left heart and oleic acid injury were individually imposed on anesthetized open-chest dogs perfused with an external pump and gas exchanger. Lungs were inflated in steps before and during those stimuli. The HB reflex was evaluated from graphs of breathing frequency (fr) vs. airway pressure. Congestion itself had no significant sustained effect on fr, but it slightly depressed the HB reflex. Oleic acid tachypnea that was depressed to pretreatment fr by inflation, implying enhancement of the HB response. Capsaicin and oleic acid had similar effects. Vagal cooling to 8 degrees C slightly depressed the effects of oleic acid and capsaicin, had no effect on the sustained fr response to congestion, and reversed the inhibitory effect of inflation. A stimulation of breathing or an enhancement of the HB reflex by congestion was not confirmed, but oleic acid increased fr and the HB reflex.     

Interaction of reduced glutathione with bovine brain tubulin

Incubation of phosphocellulose-purified tubulin with GSH at 30 degrees C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30 degrees C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.     

High-frequency spontaneous deletion of DNA sequences flanked by direct DNA repeats which also contain an internal palindrome

The DNA sequences associated with a very high-frequency, spontaneous deletion event have been determined to be two 11-base direct repeats which also contain an internal 6-base palindrome. A parental M13 replicative form (RF) DNA harboring DNA fragments of the T4 denV gene contained these direct repeats and could only be maintained at 5% of the total RF DNA within an infected cell. The remaining RF DNA was deleted for all intervening sequences between the direct repeats (2.2-kb), but one copy of the direct repeat was retained after the deletion had occurred. This site-specific deletion was highly reproducible in that if parental-sized M13 RF DNA was gel purified and transformed back into cells, the deletion occurred at precisely the same sequence as before. Electron microscopic analyses of DNA extracted from cells transformed with parental-sized DNA revealed the presence of excised 2.2-kb double-stranded circular DNA molecules. This observation thus rules out a copy choice replication/deletion mechanism to account for this high-frequency deletion event.     

Effects of male partners upon proceptivity in ovariectomized estradiol-treated marmosets (Callithrix jacchus)

Effects of male partners upon the expression of female proceptivity were examined in two experiments using 16 ovariectomized marmosets. Experiment 1 showed that the female's proceptive tongue-flicking display (PTF) is triggered specifically by eye contact with the male. Stimulation of PTF by administration of estradiol-17 beta (E2) to ovariectomized females depends in part upon the male's responsiveness to female displays, such as "staring" and "freezing," which may serve to attract his attention and to establish eye contact. Experiment 2 provided evidence that females' proceptivity decreases if their male partners are lesioned in the preoptic area/anterior hypothalamus. Such males are sexually hypoactive and less responsive to the females' visual displays. However, E2 still activates PTF if females succeed in initiating eye contact with males. Results indicate that variability in effects of E2 upon proceptivity in marmosets may be influenced by subtle aspects of facial communication between the sexes as well as by individual differences in hormonal sensitivity. Copulatory activity in males is not essential for E2 to exert its stimulatory action upon proceptivity in female marmosets.     

Glyphosate tolerant flax plants from Agrobacterium mediated gene transfer

Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector containing a chimeric NPT-II gene and a glyphosate resistance plant-derived 5-enolpyruvylshikimate-3-phosphate synthase gene was used to transform flax hypocotyl tissues. Transformed shoots could be regenerated from the inoculated tissue and were proven to be transgenic by the combination of leaf callus assays, nopaline assays and progeny tests. Co-segregation was observed in the progeny for kanamycin and glyphosate resistance.