Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21

Summary A new gene for trimethoprim resistance, dhfrV, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type I. The sequenced surroundings of dhfrV in plasmid pLMO20, were found to be almost identical with genetic areas surrounding resistance genes in transposon Tn21 and in R plasmid R388. The trimethoprim resistance genes of pLMO20 and R388 and the spectinomycin resistance gene of Tn21 could be regarded as having been inserted, by recombination, into an evolutionary older structure containg the sulfonamide resistance gene, sulI. The latter gene was sequenced and found to correspond to a peptide of 279 amino acids and with a molecular weight of 30126 daltons. The inserted genes were found to be governed by a promoter situated in the highly conserved structure and also controlling expression of sulI. The insertion points of the different resistance genes were precisely defined, and at the 3 ends of the inserted genes inverted repeats allowing the formation of stem and loop structures were found. Similar structures were found at the 3 ends of the antibiotic resistance genes in Tn7, which could indicate similar recombination mechanisms to be effective in the evolutionary construction of all these different resistance elements.     

Point mutations in the 23 S rRNA genes of four lincomycin resistant Nicotiana plumbaginifolia mutants could provide new selectable markers for chloroplast transformation

Summary Experiments designed to establish stable chloroplast transformation require selectable marker genes encoded by the chloroplast genome. The antibiotic lincomycin is a specific inhibitor of chloroplast ribosomal activity and is known to bind to the large ribosomal subunit. We have investigated a defined region of the chloroplast 23 S rRNA genes from four lincomycin resistant Nicotiana plumbaginifolia mutants and from wild-type N. plumbaginifolia. The mutants LR415, LR421 and LR446 have A to G transitions at positions equivalent to the nucleotides 2058 and 2059 in the Escherichia coli 23 S rRNA. The mutant, LR400, possesses a G to A transition at a position corresponding to nucleotide 2032 of the E. coli 23 S rRNA.     

Aestival dormancy in the cabbage moth Mamestra brassicae L. (Lepidoptera: Noctuidae)

Summary In a geographically wide distribution the life cycles of different populations of the cabbage moth Mamestra brossicae are adapted to a remarkable diversity of climatic conditions. This is undoubtedly a proof of its success in adaptation. Some populations living in regions characterized by a drought period interrupting the growth season are capable of distinguishing between one critical day length signalling the onset of the drought period and another signalling the end of the growth season. This study, therefore, is primarily concerned with the geographical patterns in the variability of the adaptional responses of populations exposed to environmental conditions requiring different strategies and tactics in, synchronizing individual, life cycles. It is also a contribution to our understanding of evolutionary mechanisms maintaining median responses to photoperiodically inductive day lengths in geographically different populations. The populations investigated originated from regions differing in predictability of the incidence, onset and duration of a drought period: Freiburg (48.0°N, Southern Germany), Avignon (44.0°N, Southern France), and Argelès (42.5°N, Southern France). Geographical variation with respect to both onset and duration of a drought period consequently results in clinal variation of the variability of innate day length thresholds triggering aestival dormancy and of innate duration of aestivation. In this paper we considered the influence of geographically changing temperatures on aestival dormancy induction. Even in southern populations of M. brassicae a temperature dependent switch off-mechanism exists which prevents aestival dormancy under certain environmental conditions. The effective temperatures vary geographically, too. What the geographical patterns in adaptive responses really are, is discussed.This research was supported by the Deutsche Forschungsgemeinschaft (Sa 259/3-1)     

The stimulating effect of a cold, dark pretreatment on the etioplast/chloroplast transformation of angiosperms. I. The stimulating effect of cold predarkening on different stages of greening under white light

Schönbohm, E., Stute, U., Thienhaus, P. and Werner, U. 1988. The stimulating effect of a cold, dark pretreatment on the etioplast/chloroplast transformation of angiosperms I. The stimulating effect of cold predarkening on different stages of greening under white light. - Physiol. Plant. 72: 541–546.
The etioplast/chloroplast transformation in angiosperms is controlled by light; most of the processes are mediated by phytochrome. We have shown that in the primary leaves of etiolated seedlings of wheat ( Triticum aestivum L. cv. Kolibri), fire-bean ( Phaseolus multiflorus L. cv. Preisgewinner) and in the cotyledons of etiolated sun flower seedlings ( Helianthus annuus L. cv. macrocarpa) the chlorophyll accumulation in the phase after the end of the lag phase can be greatly stimulated by a cold predarkening period. This effect is not necessarily coupled with a red preirradiation. Furthermore the lag phase can be dramatically shortened by the cold, dark pretreatment, whereas the amount of photoconvertible protochlorophyll(ide) in the darkness remains unaffected by the cold, dark pretreatment. The stimulating effect of a cold, predarkening period on greening is fully reversible by a warm, dark phase that is placed between the cold period and the onset of the continuous white light phase. These findings cannot be generalized: We could demonstrate that in the tropical plant Momordica charantia greening under white light was not affected by different temperature pretreatments during predarkening. The stimulating effect of a cold, predarkening period on greening is assumed to have ecological relevance.     

Local distribution of the lycaenid butterfly,Jalmenus evagoras,in response to host ants and plants

Summary The caterpillars of Jalmenus evagoras are tended by ants as they feed upon Acacia trees. In the area of Brisbane, Australia, J. evagoras require ants of the Iridomyrmex anceps species group; predation and parasitism are so intense that larvae and pupae deprived of attendant ants cannot survive (Pierce 1983). We investigated the efficiency with which J. evagoras locate and exploit the host ant resource by sampling 737 quadrats in 30 sampling grids and six study sites containing appropriate host plants; ants were collected at baits located in the center of each quadrat. J. evagoras was found in all habitats where I. anceps cooccurred with host Acacia. Nine of the ten sampling grids which had three or more I. anceps/Acacia host quadrats also had colonies of J. evagoras present (or immediately adjacent), including sites as far as 35 km apart. Of 19 sampling grids on which host quadrats were rare (i.e., less than three quadrats), none had J. evagoras (P<0.001). Within sample grids, I. anceps was distributed indepedently from Acacia trees, suggesting that they are not dependent for their survival on either Acacia or on J. evagoras. Within montane pasture habitats, I. anceps and at least one other ground-dwelling Iridomyrmex species were distributed in mutually exclusive ant mosaic territories which were stable during a one month period. I. anceps did not colonize or tend pupae of J. evagoras experimentally placed in adjacent territories of a different, nontending species of Iridomyrmex, demonstrating the integrity of territory boundaries. Sampling of ants in Acacia trees revealed that, in the absence of J. evagoras, Iridomyrmex workers are not common above ground level, and that their numbers decline in larger trees (P=0.02). In I. anceps territories, eight of nine J. evagoras pupae placed in trees over 3.0 m tall were not found after 24 h whereas all ten controls placed in low trees were found and tended (P=0.00012). This may explain why J. evagoras tends to oviposit in trees less than 2.0 m tall. An alternative hypothesis, that smaller trees have higher content of total nitrogen, and are threfore more nutritious, was not supported. We conclude that the local distribution and host tree selection by J. evagoras is dependent upon the distribution, patchiness, and foraging behavior of the host ant, I. anceps, and its spatial overlap with a number of species of host Acacia.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.Abbreviations IgG Immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TEM transmission electron micrograph     

Characterization of a delayed rectifier K+ channel in NG108-15 neuroblastomaX glioma cells: Gating kinetics and the effects of enrichment of membrane phospholipids with arachidonic acid

Summary The calcium sensitivity of exocytosis from electroper-meabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electropermeabilized cells are exposed to Ca²⁺ levels much in excess of 50 m. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca²⁺ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.Deceased     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass