Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

Oviposition deterring pheromone in Rhagoletis cerasi: Behavioral laboratory test to measure pheromone activity

Current investigations concerning the identification of the chemical nature of the oviposition deterring pheromone of the European cherry fruit fly, Rhagoletis cerasi L., are conducted in an interdisciplinary research program by combining chemistry with behavioral evaluation. The methods used to collect and evaluate the pheromone by an improved semi-quantitative behavior test are described.

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Zusammenfassung Nach einer einleitenden Übersicht über den heutigen Stand der Erkenntnisse auf dem Gebiet der eiablage-hemmenden Pheromone (ODP) bei Fruchtfliegen und insbesondere der Kirschenfliege wird der verbesserte Verhaltenstest beschrieben, welcher für den halb-quantitativen Nachweis von ODP-Aktivität entwickelt worden ist. Im laufenden interdisziplinären Forschungsprogramm an der Eidg. Forschungsanstalt Wädenswil wird versucht, durch enge Zusammenarbeit zwischen Biologe, Chemiker und Elektrophysiologe das ODP der Kirschenfliege möglichst mit geringem Anteil an Verunreinigung einzusammeln, zu reinigen und einer chemischen Identifikation der ODP Komponenten zugänglich zu machen.Die Möglichkeiten und Grenzen des Verhaltenstests werden diskutiert und die Empfindlichkeit des Verfahrens mittels einer Konzentrationsreihe von Pheromonextrakt aufgezeigt.

     

60-Hz electric fields: detection by female rats

Female rats were trained to detect a vertical, 60-Hz electric field using the same apparatus and procedure we used previously to study behavioral detection of the field by male rats. Each rat was trained individually to press a lever in the presence of the field and not to press in its absence. Correct detections occasionally produced a food pellet. The probability of detecting the field increased as field strength increased. The threshold of detection--ie, the field strength required for detections at a probability of 0.5 after correction for errors--varied among rats between 3 and 10 kV/m. Behavioral detection by female rats was indistinguishable from that by male rats.     

Effects of steroid hormones on the neuropil of the hypothalamic paraventricular nucleus of male chickens

Summary Testosterone and corticosterone, administered in doses of 0.5 mg/day for two weeks to three-day-old male chickens, induced alterations in the distributional pattern and in the number of synapses in the rostral neuropil of the hypothalamic paraventricular nucleus. This avian nucleus is a target area for both above-mentioned hormones and also one of the most important centers involved in the regulation of behavioral patterns related to reproduction. Testosterone increased the number of synapses in the rostral paraventricular nucleus, while corticosterone altered their distributional pattern causing an increase in type-B terminals; according to morphological criteria the latter are regarded to represent aminergic endings. Similar results were induced by simultaneous administration of both testosterone and corticosterone. Precocious sexual behavior was also provoked by double treatment.Preliminary results have been presented on the occasion of the 5th ENA meeting (Liège, Belgique, Sept. 1981) and the 1st Italian Meeting of Cell Biology (Rimini, Italy, April 1982)This study was supported by CNR bilateral grants (82.00215.04 & 83.00492.04), MPI 40% and European Training Program in Brain and Behaviour Researchs (twinning grant)     

Management of a harem breeding colony of rhesus monkeys to reduce trauma-related morbidity and mortality

A management procedure was developed for a harem breeding colony of rhesus monkeys to reduce trauma-related injuries and deaths resulting from the periodic removal of pregnant monkeys for research and their subsequent return to the population. Lower morbidity and mortality rates, a reduced mean conception interval, and a higher mean conception rate occurred when monkeys were maintained in permanent harems to which returning females were reintroduced compared to new social groups formed from aggregates of unfamiliar animals.     

Termination of parental care due to small clutch size in the temperate damselfish,Chromis notata

Synopsis Reproductive behavior of the temperate damselfish, Chromis notata, was investigated on the island of Mukaishima, Japan, almost daily during the breeding season in 1982. Both males and females repeated reproductive cycles many times during the breeding season. Females had a strong tendency to spawn a whole clutch on one nest during a few hours. The average number of eggs which a male gained per reproductive cycle was estimated at 38560 (480–131100 eggs). Males ordinarily cared for eggs until just prior to hatching, but abandoned more than half of the nests with the eggs numbering less than 11568. Contribution 207 from the Mukaishima Marine Biological Station.     

Transient breakdown in the selective permeability of the plasma membrane ofChlorella emersonii in response to hyperosmotic shock: Implications for cell water relations and osmotic adjustment

Summary In osmotic experiments involving cells of the euryhaline unicellular green algaChlorella emersonii exposed to hyperosmotic stress by immersion in a range of low molecular weight organic and inorganic solutes, a temporary breakdown in the selective permeability of the plasma membrane was observed during the initial phase of transfer to media of high osmotic strength (up to 2000 mosmol kg^–1). Thus, although the cells appeared to obey the Boyle-van't Hoff relationship in all cases, showing approximately linear changes in volume (at high salinity) as a function of the reciprocal of the external osmotic pressure, the extent of change was least for the triitols, propylene glycol and glycerol, intermediate for glucose, sorbitol, NaCl and KCl, with greatest changes in media containing the disaccharides sucrose and maltose. In NaCl-treated cells, uptake of external solute and loss of internal ions was observed in response to hyperosmotic treatment while sucrose-treated cells showed no significant uptake of external solute, although loss of intracellular K⁺ was observed. These observations suggest that the widely used technique of estimating cellular turgor, and osmotic/nonosmotic volume by means of the changes in volume that occur upon transfer to media containing increasing amounts of either a low molecular weight organic solute or an inorganic salt may be subject to error. The assumption that all algal cells behave as ideal osmometers, with outer membranes that are permeable to water but not to solutes, during the course of such experiments is therefore incorrect, and the data need to be adjusted to take account of hyperosmotically induced external solute penetration and/or loss of intracellular osmotica before meaningful estimates of cell turgor and osmotic volume can be obtained.     

Social stimulation and the resumption of copulation in Rhesus (Macaca mulatto) and stumptail (Macaca arctoides) Macaques

Copulatory data derived from observations of social groups of rhesus and stumptail macaques were analyzed to test the hypothesis that pairs of animals would resume copulation significantly sooner if a second male copulated with the female shortly after the first male’s ejaculation. Data from both groups supported the hypothesis. These results, extending previous studies in Macaca nemestrina,suggest that the shortening of copulatory intervals by social stimuli occurs in several species, both in social groups and in experimentally created triads. These findings also are consistent with the hypothesis that socially mediated resumption of mating is related to intrasexual competition among males.     

Behavior of Japanese monkey (Macaca fuscata) mothers and neonates at parturition

Parturitional behavior in 12 caged Macaca fuscatawas analyzed. Wild-caught mothers showed adequate maternal behaviors immediately following the neonate’s expulsion. Parity differences existed in the behaviors; primiparae were more idiosyncratic than were multiparae. Among multiparae, those with two or more offspring were uniformly adequate, but those with a single birth experience varied in the adequacy of the maternal care they provided at parturition. Mothers embraced and licked their neonates and had ventroventral contact with them frequently immediately after parturition but decreased these behaviors after expulsion of the placenta. In contrast, mothers showed allogrooming after consuming the placenta. Placentophagy was correlated with the level of orality represented by maternal licking behaviors. An isolation-reared primipara reacted to her newborn in a basically negative manner, although she showed little actual aggression. She showed a rapid shift in her negative behavior during the immediate postpartum period. This mother’s newborn sought contact with her, indicating the neonate’s active role in establishing a stable mother-neonate bond.     

Depth distribution of Campostoma grazing scars in an Ozark stream

Synopsis Campostoma spp., widespread and abundant herbivorous minnows of eastern North America, produce distinctive grazing scars when feeding on algae attached to natural substrates in streams. These scars are particularly prominent upon the low growth forms of blue-green algae that dominate the attached algal flora of many upland streams. In one stream pool in the Ozark uplands of Oklahoma, numbers and sizes of grazing scars coincided with numbers and sizes of individual Campostoma that occurred across a depth gradient, demonstrating that the information contained in the scars can provide quantification of microhabitat use and grazing intensity of these important herbivores. The results also support the hypothesis that in environments free of aquatic predators, larger fish use deeper parts of available stream habitats, particularly if threats from terrestrial or avian predators exist.