A monoclonal antibody specific for the red-absorbing form of phytochrome

Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.Abbreviations ELISA enzyme-linked immunsorbent assay - kDa kilodalton - mAb monoclonal antibody - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - (A) difference in absorbance (A ₆₆₅ ^Pr –A ₇₃₀ ^Pr )-(A ₆₆₅ ^Pfr –A ₇₃₀ ^Pfr ) - Ar/Afr spectral change ratio (SCR) - max mole fraction of Pfr following saturating red light     

Growth dynamics of a ctenophore (Mnemiopsis) in relation to variable food supply. I. Carbon biomass, feeding, egg production, growth and assimilation efficiency

This paper contains new experimental data on the growth dynamicsof a lobate coastal ctenophore, Mnenmiopsis mccradyi, whichadd significantly to our understanding of the nutritional ecologyof ctenophores and their role as opportunistic predators. Theseexperimental observations were necessary to refine the dynamiccarbon budget presented as a simulation model in another report.The ratio of carbon biomass to dry weight may vary several-folddepending on the nutritional state and size from >12% inwell-fed larvae to <1% in starved adults. Feeding effort(clearance rate) is higher for previously starved animals, fallingsharply within a few hours after re-exposure to food. Directvisual observations of feeding activity of animals confirmedthis. Assimilation efficiency can be high (72%) in these animalsbut if they continue to feed at high food concentrations, incomingfood displaces material which is only partially digested andassimilation efficiency decreases substantially. Except at verylow food concentrations, growth efficiency ranges between 20and 45%. Mnemiopsis, begins to produce eggs at a size much lessthan its maximum. Egg production is very sensitive to food supply,and somatic growth is favored over egg production at low fooddensities. Even though several thousand eggs may be producedover a few days, they represent <2% day^–1 of the carbonbiomass of the ctenophore and several-fold less than respiratorycarbon.     

Expression of a mouse metallothionein-Escherichia coli β-galactosidase fusion gene (MT-βgal) in early mouse embryos

We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli β-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolylβ- -galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. We observed staining indicative of exogenous β-galactosidase activity in 5–17% of DNA-injected embryos assayed at preimplantation stages after 16–24 h treatment with ZnSO₄. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.     

Molecular approaches to problems in biogeochemical cycling

Summary By using molecular probe techniques in combination with activity and expression measurements, it is possible to estimate bacterial populations in nature. This information can be expooited to study a number of important environmental problems. For instance, it will be possible to study ecosystem perturbation and microbial competition, by altering an ecosystem or a laboratory model of an ecosystem, and assessing corresponding changes in key activities and populations. In addition, regulation of activities in the laboratory can be compared to the response of activities and populations in situ, to develop an understanding of the key parameters that control these processes in nature. These types of approaches are important steps for determining the role of microorganisms in geochemical cycling, in both specific habitats and on a global basis.     

Nyctohemeral Rhythms of Gonadal, Thyroid and Pineal Function in the Hyperprolactinemic Male Rat

In young adult male rats bearing a donor anterior pituitary gland grafted for 3 weeks under a kidney capsule, serum prolactin (PRL) concentrations were elevated and exhibited a rhythm with the highest values in the light phase. Serum PRL in control animals did not exhibit a significant rhythm. Eutopic pituitary PRL content, manifesting a biphasic (12-hr) rhythm with crests during the day and night in controls, exhibited a similar pattern in grafted rats though an overall reduction in pituitary PRL content was seen in the grafted animals. Neither the normal biphasic serum testosterone rhythm nor the normal 24-hr rhythm (nocturnal surge) of pineal N-acetyltransferase activity and melatonin content were altered in the hyperprolactinemic rats. Serum thyroxine (T₄) and triiodothyronine (T₃) and their free indices (FT₄ I, FT₃ I) and serum thyrotropin (TSH) were highest during the day in controls and grafted rats and a 12-hr rhythmic component was detected in data for these variables. In the grafted animals, the 12-hr component was reflected in an additional peak at night detectable by testing of means. The overall serum T₄ FT₄ I, and TSH levels were lower in grafted rats though overall T₃ and FT₃I levels did not differ between grafted and controls. T₃ uptake (T₃ U) values were similar between controls and grafted rats, in both cases exhibiting a fall during the night. Changes in serum thyronines could not be explained by changes in serum binding as assessed by the T₃U₃ and thus may represent changes in thyroidal secretion of T₄. The rhythm in serum PRL of grafted rats suggests the presence of rhythmic circulating factor(s) capable of influencing ectopic lactotrophs. The reduced eutopic pituitary PRL content suggests a role for PRL in influencing eutopic lactotrophs in the pituitary-grafted hyperprolactinemic male rat model. Though circulating testosterone and pineal melatonin synthesis were not altered in this model, thyroid function appeared to be so.     

Calicotyle urolophi n. sp. (Monogenea: Monocotylidae) from stingarees,Urolophus spp. (Elasmobranchii: Urolophidae) taken in coastal waters of Southern Australia

Calicotyle urolophi n. sp. is proposed for calicotylines found in the cloaca of three stingaree species, Urolophus cruciatus, U. bucculentus and U. paucimaculatus, collected off the southeastern coast of Tasmania. Variations in the soft body parts were observed between specimens taken from U. cruciatus and U. bucculentus but were not considered sufficient for separation into two species. C. urolophi is differentiated from other Calicotyle spp. found in the South Pacific by the configuration of the tubular male copulatory organ, the structure of the intestinal caeca and the arrangement of the vaginae. Amended diagnoses for the subfamily Calicotylinae and the genus Calicotyle are provided.     

Impacts of section 404 permits requiring compensatory mitigation on wetlands in California (USA)

We analyzed data from Section 404 permits issued in California from January 1971 through November 1987 that involved impacts to wetlands and required compensatory mitigation (wetland creation, restoration, or preservation). The purpose of this study was to determine patterns and trends in permitting activity and to document cumulative effects of associated management decisions on the California wetland resource. The 324 permits examined documented that 387 compensatory wetlands (1255.9 ha) were required as mitigation for impacts to 368 wetlands (1176.3 ha). The utility of the data on wetland area was limited, however, since 38.0% of the impacted wetlands and 41.6% of the compensatory wetlands lacked acreage data. The wetland type most frequently impacted (37.8% of impacted wetlands) and used in compensation (38.2% of compensatory wetlands) was palustrine forested wetlands. Estuarine intertidal emergent wetlands had the most area impacted (52.3%) and compensated (62.5%). The majority of the wetlands were small (less than or equal to 4.0 ha in size). Wildlife habitat was the most frequently listed function of impacted wetlands (90.7% of the permits) and objective of compensatory wetlands (83.3%). Endangered species were listed as affected in 20.4% of impacted and 21.0% of compensatory projects. The number of permits requiring compensatory mitigation and the number of impacted and compensatory wetlands increased from 1971 to 1986.Documentation of the details of Section 404 permit decisions was inadequate for the permits we examined. Area information and specific locations of impacted and compensatory wetlands were lacking or of poor quality. Follow-up information was also inadequate. For example, project completion dates were specified in the permit for only 2.2% of compensatory wetlands. Furthermore, less than one-third (31.5%) of the permits required the compensatory wetland to be monitored by at least one site visit. We recommend improved documentation, regular reporting, and increased monitoring for better evaluation of the Section 404 permitting system.     

Deregulation of arginine biosynthesis in Synechococcus sp. PCC7942

Summary In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two predominant amino acids, arginine and citrulline. In-vitro activity of N-acetylglutamate kinase (NAGK), the proposed regulatory enzyme of the arginine pathway, was about 100-fold less sensitive to l-arginine inhibition in extracts from APM 31 and 40 than the enzyme in WT extracts. The enzyme from APM 1 was 20-fold less sensitive to l-arginine inhibition than WT. The most likely site of mutation in each of the APM cell lines is in the gene for NAGK, rendering the enzymes insensitive to l-arginine feedback control. These strains can be utilized for the phototrophic production of arginine. Offprint requests to: S. E. Bingham