Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.     

Variation in Photosynthesis and Enzyme Activity in Cenchrus ciliaris L.

Populations of Cenchrus ciliaris differ significantly in relativegrowth-rate (R), net assimilation rate (E), and leaf-area ratio(F). Differences in R were related to differences in E ratherthan in F. The populations also differed in the rate of apparentphotosynthesis of individual leaves as measured by infra-redgas analysis. Warburg and ^l4CO₂ techniques. Temperature andlight-response measurements revealed optimum temperature forphotosynthesis of 35 °C and the rate in most populationscontinued to increase up to the highest levels of irradianceemployed (200 W m²). The relative order of the populations wassimilar in all methods of analysis, but there was no relationshipevident between leaf photosynthesis and E derived from growthanalysis. The activity of the enzyme PEP carboxylase, expressedin terms of unit leaf area, was correlated with photosyntheticrate of the leaf. Activities of PEP carboxylase and of proteinsynthesis in vitro expressed on a basis of soluble protein contentwere correlated with E, but no such relationship was found forthe enzymes acid phosphatase and alanine--ketoglutarate aminotransferase.     

Life budgets for a population of Tipula sacra (Diptera; Tipulidae)

Abstract.

• 1 A population of Tipula sacra was studied intensively from 1969 to 1974 in a series of abandoned beaver ponds. Beavers recolonized the ponds from 1974 to 1976 and again in 1978.
• 2 The basic life-cycle is of 2 years duration, but cohort-splitting occurs in the autumn as a result of some larvae growing faster and completing the life-cycle in 1 year.
• 3 Some periods are more favourable for growth than others and the size of the fast-growing cohort varies from year to year. This insight allows some anomalies in the estimates of population size to be explained and life budgets have been produced for three year-classes.
• 4 The short-fall in realization of egg potential is massive, apparently due to heavy female mortality by predation and the prevention of oviposition by inclement weather.
• 5 Mortality rate is relatively low and constant from stage II larvae until pupation, but increases to adult emergence. There is a correlation between larval density and mortality rate.
• 6 Certain aspects of the life-history (e.g. high fecundity, variability in growth rate) appear to fit the species to life in a temporally unstable habitat.

     

The Conservation of Sea Turtles: Practices and Problems

The various techniques in common use for conservation and restorationof depleted sea turtle populations are reviewed, namely: banninginternational commerce; operating artificial hatcheries, bothin the natural beach environment and in styrofoam and othertypes of incubators; "head-starting" of hatchlings in captivity;protection of nesting females by means of beach patrols; andtranslocation of eggs or hatchlings to distant areas from whichturtles have been extirpated or to which it is desired to introducenew colonies. The difficulties of monitoring the results ofall of these techniques are discussed, and potential dangersor disadvantages of each approach are reviewed. It is concludedthat, until unequivocal data become available, turtle conservationistsshould continue to pursue common sense or logically sound restorationprograms, but should constantly re-evaluate their actions inthe light of the latest available knowledge and modify or desistfrom current approaches as necessary.     

Characterization of tetranucleotide microsatellites for Rio Grande cutthroat trout and rainbow trout,and their cross‐amplification in other cutthroat trout subspecies

We describe the isolation and characterization of 12 tetranucleotide microsatellites for Rio Grande cutthroat trout (Oncorhynchus clarkii virginalis) and rainbow trout (Oncorhynchus mykiss), and subsequently investigate their performance in Colorado River cutthroat trout (Oncorhynchus clarkii pleuriticus), greenback cutthroat trout (Oncorhynchus clarkii stomias) and Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri). All 12 loci are polymorphic in all subspecies of O. clarkii examined.     

Diversity of carbon use strategies in a kelp forest community: implications for a high CO2 ocean

Mechanisms for inorganic carbon acquisition in macroalgal assemblages today could indicate how coastal ecosystems will respond to predicted changes in ocean chemistry due to elevated carbon dioxide (CO₂). We identified the proportion of noncalcifying macroalgae with particular carbon use strategies using the natural abundance of carbon isotopes and pH drift experiments in a kelp forest. We also identified all calcifying macroalgae in this system; these were the dominant component of the benthos (by % cover) at all depths and seasons while cover of noncalcareous macroalgae increased at shallower depths and during summer. All large canopy‐forming macroalgae had attributes suggestive of active uptake of inorganic carbon and the presence of a CO₂ concentration mechanism (CCM). CCM species covered, on average, 15–45% of the benthos and were most common at shallow depths and during summer. There was a high level of variability in carbon isotope discrimination within CCM species, probably a result of energetic constraints on active carbon uptake in a low light environment. Over 50% of red noncalcifying species exhibited values below ?30‰ suggesting a reliance on diffusive CO₂ uptake and no functional CCM. Non‐CCM macroalgae covered on average 0–8.9% of rock surfaces and were most common in deep, low light habitats. Elevated CO₂ has the potential to influence competition between dominant coralline species (that will be negatively affected by increased CO₂) and noncalcareous CCM macroalgae (neutral or positive effects) and relatively rare (on a % cover basis) non‐CCM species (positive effects). Responses of macroalgae to elevated CO₂ will be strongly modified by light and any responses are likely to be different at times or locations where energy constrains photosynthesis. Increased growth and competitive ability of noncalcareous macroalgae alongside negative impacts of acidification on calcifying species could have major implications for the functioning of coastal reef systems at elevated CO₂ concentrations.     

Spatial and temporal nature of reactive oxygen species production and programmed cell death in elm (Ulmus pumila L.) seeds during controlled deterioration

Seed deterioration is poorly understood and remains an active area for research. Seeds of elm (Ulmus pumila L.) were aged at 37 °C above water [controlled deterioration treatment (CDT)] for various lengths of time to assess programmed cell death (PCD) and reactive oxygen species (ROS) product in embryonic tissues during a 5 d period. The hallmarks of PCD were identified in the elm seeds during CDT including TUNEL experiments, DNA laddering, cytochrome c (cyt c) leakage and enzymatic activities. These analyses indicated that PCD occurred systematically and progressively in deteriorated elm seeds. Cyt c release and increase in caspase‐3‐like/DEVDase activity occurred during CDT, which could be suppressed by ascorbic acid (AsA) and caspase‐3 inhibitor Ac‐DEVD‐CHO, respectively. In situ localization of ROS production indicated that the distinct spatial‐temporal signature of ROS during CDT coincided with the changes in PCD hallmark features. Multiple antioxidant elements were activated during the first few days of CDT, but were subsequently depleted as PCD progressed. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during elm seeds CDT and suggest an important role for PCD in seeds deterioration.