Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis

The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA⁺ strain but more than pKM101 in a uvrA⁻ strain. muc⁻ point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Export of species to islands from sources of differing maturity

Export of species from sources (epicenters) of differing ages and complexities was examined using laboratory microcosms. Polyurethane foam (PF) artificial substrates were colonized by protozoans for different time periods in a small pond. Substrates were returned to the laboratory and used as epicenters for protozoan colonization of barren PF islands in initially sterile microcosms. Islands were exposed to epicenters for either 24 h or continuously for 28 d. Islands from pairs of microcosms exposed to epicenters of identical ages were sampled on 1, 3, 7, 14, 21, 28 and 46 d after initial epicenter exposure. Colonization parameters were estimated by fitting numbers of colonizing species to the MacArthur-Wilson equilibrium model. Islands exposed continuously to epicenters were colonized by significantly more species than those exposed for only 24 h. Islands exposed to immature, species poor epicenters were colonized by a greater proportion of the source community than those exposed to more mature, species rich epicenters. All islands were depauperate compared to epicenters except those exposed to the most immature (1 d old) epicenter. Colonization continued at a reduced rate in spite of the absence of the epicenter. Results from communities with rapid species turnover and rapidly reproducing species suggest that the continuous presence of a species source is less important for colonization of a new habitat. Dispersal of potential colonists occurs rapidly in these communities. Less mature communities dominated by pioneer forms are more effective at producing colonists than more mature communities.     

Proof that the endogenous, heat-stable glucocorticoid receptor-activating factor is thioredoxin

Extraction of rat liver cytosol with charcoal inactivates glucocorticoid-binding capacity and receptors can be reactivated to the steroid-binding state by an endogenous reducing system utilizing NADPH and a Mr = 12,000, heat-stable, endogenous, cytosolic protein (Grippo, J. F., Tienrungroj, W., Dahmer, M. K., Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 13658-13664). In this paper we show that NADPH-dependent conversion of the rat liver glucocorticoid receptor from a nonbinding to a steroid-binding form is blocked in an immune-specific manner by antisera raised against purified rat liver thioredoxin reductase or thioredoxin. The inhibition produced by thioredoxin reductase antiserum may be circumvented by dithiothreitol or overcome by addition of purified thioredoxin reductase. These observations prove that the endogenous glucocorticoid receptor-activating factor is thioredoxin and that the enzyme required for generating the steroid-binding conformation of the glucocorticoid receptor by the endogenous receptor-activating system is thioredoxin reductase.     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

Long term regeneration by somatic embryogenesis in barley (Hordeum vulgare L.) tissue cultures derived from apical meristem explants

Callus cultures were initiated from apical meristem explants of one to four-week-old aseptically-grown barley (Hordeum vulgare L. cv. Atlas 57) plants. Embryogenic callus and plants were produced in three separate experiments; the cultures have retained regenerative capacity for three years after initiation. Our results demonstrate that explants other than immature embryos are embryogenically competent in barley and that regeneration occurs by both somatic embryogenesis and organogenesis.     

Behavioral and biochemical assessment of time-related changes in globus pallidus and striatal dopamine induced by intranigrally administered neurotensin

Microinjection of neurotensin (NT; 2 and 5 μg) into the substantia nigra zona compacta caused an increase in dopamine (DA) and DA metabolites in the rodent globus pallidus and striatum which persisted for at least 20 hours after peptide administration. Similar NT treatments given unilaterally into the nigra caused circling away from the injected side in amphetamine-pretreated rats, but were without effect when microinjected into saline-pretreated animals. Circling also occurred when the animals were given amphetamine 20 hours after intranigral NT administration. Contralateral rotation was observed with unilateral intranigral injections of gamma-hydroxybutyric acid (GHB; 400 μg) or with lower intranigral GHB doses (250 μg) in amphetamine-pretreated animals. The effects of GHB and NT differed in the manner in which the animals rotated as well as in the profile of DA and DA metabolite changes induced by these drugs. These studies indicated that: (1) dopaminergic functions of the globus pallidus are influenced, like the striatum, by manipulations of the substantia nigra; (2) NT and GHB likely act via different mechanisms to effect nigral dopamine-containing cells; and (3) NT was capable of inducing changes in dopamine neurons which had long term consequences.     

The effect of nisin-sodium chloride interactions on the outgrowth of Bacillus licheniformis spores

The effect of salt (NaCl) on the efficacy of nisin in preventing outgrowth of Bacillus licheniformis spores was determined in Plate Count Agar (PCA). An equivalent liquid medium was used for heat activation. Nisin and salt were added to the heat-activation medium, the PCA, or both. The spores were extremely sensitive to nisin; outgrowth were completely inhibited in salt-free media when 10 iu/ml of nisin was present in both the heat-activation and the growth media or when 100 iu/ml nisin was present in either the heat-activation and the growth medium. In media supplemented with 1% salt, outgrowth occurred from 1% of spores exposed to 100 iu/ml nisin in either the heat-activation or the growth medium. A 3% salt supplement was necessary before detectable outgrowth occurred when both the heat-activation and the growth media contained 100 iu/ml nisin. Salt appears to antagonize the sporicidal action of nisin by interfering with nisin adsorption onto the spore.     

Rapid measurement of creatine kinase activity in a coronary care unit using a portable benchtop reflectance photometer.

Rapid measurements of plasma creatine kinase activity using an inexpensive benchtop reflectance photometer (Ames Seralyzer) and disposable reagent strips were evaluated in the laboratory and coronary care unit. The system proved simple to use and capable of yielding rapid (four minutes per analysis), precise (coefficient of variation less than 9%), and accurate results (correlation with routine method 0.995) when used by medical staff. Creatine kinase values were available 6.5-102 hours earlier than routine laboratory data, depending on the time and day of sampling, thereby facilitating appropriate and economic patient management. This instrument might be used to supplement the routine enzyme service for selected admissions, resulting in greatly improved availability of results and hence contributing to the early discharge of patients from intensive care facilities.     

Conserved sequences in both coding and 5'' flanking regions of mammalian opal suppressor tRNA genes.

The rabbit genome encodes an opal suppressor tRNA gene. The coding region is strictly conserved between the rabbit gene and the corresponding gene in the human genome. The rabbit opal suppressor gene contains the consensus sequence in the 3' internal control region but like the human and chicken genes, the rabbit 5' internal control region contains two additional nucleotides. The 5' flanking sequences of the rabbit and the human opal suppressor genes contain extensive regions of homology. A subset of these homologies is also present 5' to the chicken opal suppressor gene. Both the rabbit and the human genomes also encode a pseudogene. That of the rabbit lacks the 3' half of the coding region. Neither pseudogene has homologous regions to the 5' flanking regions of the genes. The presence of 5' homologies flanking only the transcribed genes and not the pseudogenes suggests that these regions may be regulatory control elements specifically involved in the expression of the eukaryotic opal suppressor gene. Moreover the strict conservation of coding sequences indicates functional importance for the opal suppressor tRNA genes.