Chromosome-mediated transfer of murine alleles for hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and ouabain resistance into human cell lines

Genetic drug-resistance markers were transferred via purified metaphase chromosomes from mouse L cells into the human fibrosarcoma line HT1080 and HeLa S3 cells. Interspecific chromosome-mediated transfer of hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) from mouse L cells into HGPRT^– HT1080 cells occurred at a frequency of approximately 1×10^–7. The presence of the mouse allele for HGPRT in transferent isolates was confirmed by isoelectric focusing. Transfer of ouabain resistance from mouse L cells to HT1080 and HeLa S3 cells occurred at an average frequency of approximately 4×10^–7. Expression of the mouse trait in transferent isolates was confirmed by their ability to withstand doses of ouabain which would be lethal to spontaneous ouabain-resistant mutants of the human cells but not to mouse L cells. Ouabain-resistant transferents of human cells showed 10⁴- to >10⁵-fold enhanced drug resistance, characteristic of either wild-type or mutant alleles, respectively, from ouabain-resistant donor L cells. Unstable expression of the transferred phenotypes in the absence of selection was seen in some isolates, but expression was lost at slow rates.This work was supported by National Institutes of Health Grant GM30383/21665 to RMB, Core Grants CA14051 to S. E. Luria and CA24538 to E. Mihich, and institutional predoctoral Training Grant GM07287.     

Selection studies on anthelmintic resistant and susceptible populations of Trichostrongylus colubriformis of sheep

Waller P. J., Dobson R. J., Donald A. D., Griffiths D. A. and Smith E.F. 1985. Selection studies on anthelmintic resistant and susceptible populations of Trichostrongylus colubriformis of sheep. International Journal for Parasitology15: 669–676. A T. colubriformis population (BCK), formerly resistant to benzimidazole anthelmintics, but now highly resistant to levamisole after 6 years exposure to this drug alone in the field, was passed through 12 generations in the laboratory in three separate lines exposed either to selection with thiabendazole or levamisole, or to no selection. Another population (McM) not previously exposed to these anthelmintics was treated similarly in two lines, selected with thiabendazole or not selected.Selection with thiabendazole resulted in a return of benzimidazole resistance in the BCK line which occurred faster than in the McM line, but a similar level of resistance was reached in each by the twelfth generation. Resistance ratios in both selected lines compared with the unselected McM line were less than 20: 1, and only 1.5 times the recommended dose rate of thiabendazole was required to remove more than half of the resistant population. This suggests that a polygenic vigour tolerance rather than a specific resistance had been selected.In the case of levamisole resistance, the BCK population was found to contain two distinct subpopulations, one susceptible and the other highly resistant. Resistance ratios for the highly resistant subpopulation were greater than 4000: 1, implying a specific resistance controlled by a major gene. During the 12 generations of levamisole selection, the proportion of resistant phenotypes fluctuated about an average level of 70%, suggesting that susceptibility alleles were being maintained in the population through superior heterozygote fitness. This conclusion is supported by a significant decline in levamisole resistance in the absence of levamisole selection. Moreover, thiabendazole selection hastened the reversion to levamisole suceptibility.The results provide support for the reintroduction of a benzimidazole anthelmintic to control this helminth population, and for a slow rotation in the use of drugs with different modes of action.     

Interdependence of growth, water relations and abscisic acid level in Phaseolus vulgaris during waterlogging

The interdependence between changes in growth and water relations after waterlogging was investigated by recording simultaneously growth, transpiration, water potential, turgor, leaf diffusion resistance and abscisic acid content in Phaseolus vulgaris L. cv. bruine Noord-Hollandse. Growth was inhibited immediately after flooding, whereas transpiration decreased gradually to a low level in about three days. The first two days after flooding a small increase in abscisic acid content in the leaves was observed which was accompanied by an increase in diffusion resistance. The increase in abscisic acid content could result from an inhibited export from the leaves. After the first two days a decrease in water potential and turgor was accompanied by a drastic increase in both abscisic acid content and diffusion resistance. This large increase in abscisic acid content occurred before the turgor had reached its minimum value. The change in diffusion resistance kept showing a lag of about one day with the change in abscisic acid content. The possibility is discussed that besides abscisic acid also its metabolite phaseic acid is involved in stomatal closure. After the formation of adventitious roots on the hypocotyl, abscisic acid level, diffusion resistance, water potential and turgor returned to the control values. Transpiration showed a slow recovery from the sixth day after flooding, whereas growth was inhibited for at least nine days. A remarkable similarity exists between our observations on the responses of bean plants to flooding and the well known responses to drought.     

Isolation and cryopreservation of O-methylthreonine-resistant Rosa cell lines altered in the feedback sensitivity of l-threonine deaminase

O-Methylthreonine (OMT) inhibits the growth of plated Rosa cells (ID₅₀6·10^-6M). Isoleucine is able to reverse efficiently and specifically this OMT toxicity. From OMT-resistant colonies occurring at a frequency of 1.58·10^-7 variants per cell plated at 10^-4M OMT, the variant strains OMT^R-1 and OMT^R-2 were isolated, cloned via protoplasts and characterized. Both variants were ten times more resistant to OMT than the wildtype and were cross-resistant to another isoleucine analog, dl-4-thiaisoleucine. The resistant variants retained their resistance after storage for three years in liquid nitrogen. Both resistant strains were stable for several months when subcultured in the absence of OMT although it was shown in a reconstitution experiment that wildtype cells overgrow OMT^R-2 variant cells if co-cultivated for many passages in drug-free medium. One case of instability was observed upon long-term subculturing in drug-free medium: the strain OMT^R-1D^* partially lost phenotypic properties. Resistance to OMT was followed qualitatively by a new method based on inhibition-zone formation in cell suspensions plated in agar medium. The OMT-resistant variants showed a reduction in sensitivity of the enzyme l-threonine deaminase to feedback inhibition by isoleucine, a decreased stability of l-threonine deaminase when stored at-18°C or incubated at +55°C and a two- to threefold increase of the free isoleucine pool within the cells. The genetical events and the biochemical mechanisms which might lead to the observed stable and biochemically defined character are discussed with particular reference to the high ploidy level of the Rosa cell line.Abbreviations OMT l-O-methylthreonine - TD l-threonine deaminase     

Influence of aerial dispersal on persistence and spread of pesticide-resistant Metaseiulus occidentalis in California almond orchards

Aerial dispersal of the phytoseiid Metaseiulus occidentalis (Nesbitt) was evaluated as a component in managing pesticide-resistant populations established in California almond orchards. Peak dispersal occurred in late July and early August during 1982 and 1983. Most predators (and spider mites) left the orchards on the prevailing winds from the northwest. Within the orchard, the prevailing winds had less influence, and dispersal was usually random. Both spider mites and predators dispersed randomly with regard to height from the almond trees, but data obtained during one 24-h interval suggest they do not disperse randomly throughout the day. Most aerial movements occurred between 16–22 h when relative humidity and wind speeds increased and temperatures decreased. Spider mites and predators were trapped on panels located 200 m from the orchard. A survey of carbaryl resistance levels in M. occidentalis collected from almond orchards surrounding the release sites indicates that carbaryl-resistant M. occidentalis dispersed at least 800 m between 1981–83. However, growers wishing to use the resistant strains should release them in their orchards as natural dispersal appears to be too slow. Migration of native M. occidentalis into the release sites appeared to be sufficiently rare that dilution of carbaryl-resistant populations was minimal during a 2–4 year period.

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Résumé La dispersion aérienne du phytoseïdae, M. occidentalis (Nesbitt), a été estimée comme élément de la lutte contre les populations résistantes aux insecticides établies dans les vergers de Californie. La dispersion maximale s'est produite fin juillet et début a oût en 1982 et 1983. La plupart des prédateurs (et des acariens) quittent les vergers avec les vents dominants du nordouest. Dans le verger, les vents dominants sont moins importants et la dispersion est généralement au hasard. Tant les acariens que les prédateurs se dispersaient au hasard par rapport à la taille des amandiers, mais les relevés sur 24 heures laissent supposer qu'il n'y a pas une distribution aléatoire pendant la journée. La plupart des mouvements aériens se produisirent entre 16 et 22 heures quand HR et vitesse du vent augmentaient et température diminuait. Les acariens et prédateurs ont été piégés sur des panneaux à 200 m du verger.

     

Selection and Inbreeding of Heterodera glycines on Glycine max

Few soybean cyst nematodes (SCN), Heterodera glycines, of a diverse gene pool developed into females on soybeans PI 89772 or PI 209332. Nematodes surviving the selection pressure were then inbred for nine generations by single cyst transfers on the same selecting soybean line. These nematodes appeared to tolerate concurrent selection and inbreeding. Effects of selection-inbreeding, selection only, and secondary selection were evaluated by relative ability to produce cysts on 11 soybean lines. The genetic differences of PI 89772 (also Peking and Pickett 71) and PI 209332 were reaffirmed. The random effects of inbreeding indicated that Ilsoy and Williams may have genes for resistance different from those in PI 89772 or PI 209332. Egg inoculum obtained from soil resulted in very few cysts in some tests. Fresh egg inoculum (from cysts on 27-30-day-old plants) generally resulted in more cysts and more consistent results. Concurrent with the change in inoculum, there was a large increase in relative numbers of cysts on several soybean lines but no change on other lines; the true cause of this large interaction is unknown. Secondary selection of two inbreds was effective and suppressed cyst numbers on the line on which one inbred was selected initially. These results are consistent with the allelism linkage of some SCN genes reported previously.     

Large scale selection of aluminum-resistant mutants from plant cell culture: expression and inheritance in seedlings

Summary A large number of aluminum-resistant variants, selected from non-mutagenized homozygous diploid cell cultures of Nicotiana plumbaginifolia Viv., are characterized. Of 115 variants cloned and reselected from single cells, 67 retained Al resistance in callus cultures after 6–9 months of growth in the absence of Al. There was no association between Al resistance and callus growth in the absence of Al, suggesting that the Al-resistant phenotype is not detrimental in the absence of Al challenge and that Al resistance is not the result of increased vigor. Plants regenerated from initially resistant callus lines that subsequently lost their resistance failed, with one exception, to transmit resistance to their seedling progeny. Fertile plants were regenerated from 40 of the 67 variants that retained stable Al resistance in callus culture. All 40 transmitted Al resistance to their seedling progeny (selfed and backcrossed) in segregation ratios expected for a single dominant mutation. The selfed progeny of many variants also segregated for recessive lethal mutations which were attributed to independent mutations that occurred during cell culture.     

Stomatal response to air humidity and its relation to stomatal density in a wide range of warm climate species

The gas exchange of 19 widely different warm climate species was observed at different leaf to air vapour pressure deficits (VPD). In all species stomata tended to close as VPD increased resulting in a decrease in net photosynthesis. The absolute reduction in leaf conductance per unit increase in VPD was greatest in those species which had a large leaf conductance at low VPDs. This would be expected even if stomata of all species were equally sensitive. However the percentage reduction in net photosynthesis (used as a measure of the relative sensitivity of stomata of the different species) was also closely related to the maximal conductance at low VPD. Similarily the relative sensitivity of stomata to changes in VPD was closely related to the weighted stomatal density or crowding index.The hypothesis is presented that stomatal closure at different VPDs is related to peristomatal evaporation coupled with a high resistance between the epidermis and the mesophyll and low resistance between the stomatal apparatus and the epidermal cells. This hypothesis is consistent with the greater relative sensitivity of stomata on leaves with a high crowding index.The results and the hypothesis are discussed in the light of selection, for optimal productivity under differing conditions of relative humidity and soil water availablility, by observation of stomatal density and distribution on the two sides of the leaf.Visiting scientist, plant physiologist and research assitant of the Cassava Program