Construction of a hybrid bacteriophage-plasmid recombinant DNA vector.

A phage-plasmid hybrid was constructed for use as a recombinant DNA vector, allowing the propagation of cloned EcoRI restriction endonuclease fragments of about 2 X 10(6) to 11 X 10(6) daltons. The colicin E1 plasmid replicon was fused to the left arm of a lambdagt generalized transducing phage with a thermolabile repressor, yielding a genome which could be replicated either by phage lambda functions or via the colicin E1 plasmid replicon. At the nonpermissive temperature, phage functions were derepressed and phage growth occurred lytically. Alternatively, at the permissive temperature, lambda functions were repressed and the vector replicated as a covalently closed circular plasmid. The phage-plasmid hybrid vector could be maintained at a copy number determined by the colicin E1 plasmid replicon and was also sensitive to amplification after chloramphenicol treatment. An EcoRI fragment of Escherichia coli DNA encoding genes of the arabinose operon also was inserted into the central portion of the vector.     

Age-dependent changes in the hypothalamo-pituitary-ovarian axis of the laying hen.

The functional integrity of the components of the hypothalamo-pituitary-ovarian axis was examined in young and old laying hens. Ovarian function was tested by measuring the amount of progesterone released in response to an injection of LH, and pituitary function was investigated by measuring the increase in the plasma LH level after an injection of LH-RH. There were no differences between young and old birds in the response of the pituitary gland or the ovary to these stimuli. Hypothalamic function was investigated by studying the positive feedback action of a standard dose of progesterone on LH release; the positive feedback response was smaller (P less than 0.05) in old hens. It is suggested that the fall in the rate of lay in hens towards the end of their laying year is caused partly by a decrease in the response of the LH-positive feedback mechanism to progesterone.     

Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin.

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.     

Effects of cholera toxin and guanosine 5''-[betagamma-imido]triphosphate on beta-adrenergic-receptor affinity.

A comparison was made of the effects of cholera toxin and p[NH]ppG on the binding affinity of beta-adrenergic receptors in toad erythrocyte membranes. This was determined by studying the ability of isoproterenol and propranolol to compete for the receptor with (-)-[3H]dihydroalprenolol. p[NH]ppG decreased the receptor affinity for the agonist isoproterenol (i.e. a 'right' shift in the displacement-concentration curve), but was without effect on the affinity for the antagonist propranolol. Toad erythrocyte membranes after treatment with cholera toxin exhibited increased receptor affinity for isoproterenol (i.e. a 'left' shift in the displacement curve), but did not affect the affinity for propranolol. p[NH[ppG was able to exert its right shift even in cholera-toxin treated membranes. The ability of cholera toxin to alter beta-adrenergic-receptor affinity is interpreted as further evidence that the toxin affects the nucleotide-regulatory component of adenylate cyclase. The regulatory component affected may be the catecholamine-sensitive guanosine triphosphatase.     

Neuroendocrine control of reduced persistence of egg-laying in domestic hens: evidence for the development of photorefractoriness.

Egg-laying in hens exposed for more than 11 months to photostimulatory daylengths was intermittent and associated with a reduction in numbers of yellow-yolky ovarian follicles. Old laying hens (105 weeks) had lower concentrations of luteinizing hormone (LH) in the pituitary gland and plasma and reduced pituitary gland responsiveness to chicken LH-releasing-hormones (LHRH-I and II) in vivo when compared with young laying hens (28 weeks). Four weeks after transfer from 14 to 8 h light/day, egg production almost stopped in old, but not in young hens, although plasma LH concentrations decreased in all birds. After transfer from 14 to 20 h light/day, plasma LH increased in young, but not in old, hens, without a change in the rate of egg production. Reproductive function was enhanced in old hens returned to long days after induction of a moult and ovarian regression by reducing daylength and dietary restriction. Moulted hens had a greater rate of egg production, higher concentrations of plasma LH and a greater pituitary-gland responsiveness to LHRH-II in vivo than unmoulted control hens. After transfer from 14 to 8 h light/day, egg-laying decreased more rapidly in unmoulted than in moulted hens; transfer to 17 h light/day increased egg production in moulted, but not in unmoulted, birds. Induction of ovarian regression in old hens by dietary restriction alone also enhanced reproductive function after the dietary restriction was relaxed. Egg-laying was more persistent in hens brought into lay for a second year by transferring them from 3 to 11 h light/day than in hens transferred from 3 to 20 h light/day. Egg production was stimulated in hens maintained on 3 or 11 h light/day for 42 weeks, after transfer to 20 h light/day. Egg production ceased in hens maintained on 20 h light/day for 46 weeks, after transfer to 3 h light/day. These observations are consistent with the view that poor persistence of laying in hens less than 2 years old and exposed continuously to long days is caused, in part, by a reduction in hypothalamic-gonadotroph function. This reduction in neuroendocrine function may be due, in part, to the development of relative photorefractoriness.