Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases

High-throughput methods to produce a large number of soluble recombinant protein variants are particularly important in the process of determining the three-dimensional structure of proteins and their complexes. Here, we describe a collection of protein expression vectors for ligation-independent cloning, which allow co-expression strategies by implementing different affinity tags and antibiotic resistances. Since the same PCR product can be inserted in all but one of the vectors, this allows efficiency in versatility while screening for optimal expression strategies. We first demonstrate the use of these vectors for protein expression in Escherichia coli, on a set of proteins belonging to the ubiquitin specific protease (USP) Family. We have selected 35 USPs, created 145 different expression constructs into the pETNKI-His-3C-LIC-kan vector, and obtained 38 soluble recombinant proteins for 21 different USPs. Finally, we exemplify the use of our vectors for bacterial co-expression and for expression in insect cells, with USP4 and USP7 respectively. We conclude that our ligation-independent cloning strategy allows for high-throughput screening for the expression of soluble proteins in a variety of vectors in E. coli and in insect cells. In addition, the same vectors can be used for co-expression studies, at least for simple binary complexes. Application in the family of ubiquitin specific proteases led to a number of soluble USPs that are used for functional and crystallization studies.     

A unique coral reef formation discovered on the Great Astrolabe Reef, Fiji

 A spectacular mound-like reef formation (126 m in circumference, 10 m high) dominated by highly arched and record-size colonies of the unattached mushroom coral Halomitra pileus, along with 17 other species of the family Fungiidae, occurs in 31 m of water on the sedimentary lagoon floor of the Great Astrolabe Reef, Fiji. Core samples show radiocarbon dates which indicate that the formation hypothetically began building ∼4500 y ago, with a calculated mean accretion rate of 2.2 mm ⋅ y^-1. The majority of fossil and living material is contributed by H. pileus colonies between 40–70 cm mean diameter, with some individuals up to 1.5 m in diameter. The size, fungiid diversity, and geological history of the bioherm is unprecedented and represents the first example of a coral reef constructed almost entirely by Fungiidae. Accepted: 29 July 1996     

HETEROMORPHIC LIFE-HISTORY STRATEGIES IN THE BROWN ALGA SCYTOSIPHON LOMENTARIA (LYNGB.) LINK1

The adaptive significance of a life-history strategy, expressed as divergent morphological forms, was examined for the heteromorphic alga Scytosiphon lomentaria. Successional studies were performed by physically clearing mature, temporally-constant intertidal communities on San Clemente I., California. At week six, after clearing, complanate thalli dominated the successional plots (mean cover = 23.5%) and began to decline as the cylindrical form became abundant. The latter attained its peak cover (82.3%) at week 13, whereupon it too began a precipitous reduction. The crustose ralfsioid form appeared surprisingly early (4–13 wks) in trace amounts but did not achieve its greatest cover (85.0%) until week 43. The ranking from high to low primary productivity (cylindrical form = 8.1 mg C · g dry. wt^-1·h^-1, complanate form = 6.5 mg, crustose form = 0.5 mg) corresponded closely with the data for photosynthetic vs. structural components (cylindrical = 92.3% pigmented, complanate = 65.3%, crustose = 32.0%). This finding indicates that selection in the crust form, which is more readily accessible to epilithic grazers, has tended to increase allocation of materials to nonpigmented structural tissue at the expense of photosynthetic tissue and reduced production rates. The results for thallus losses to urchin grazing over 48 h were complanate form = 82.7% lost, cylindrical = 81.4% and crustose = 16.2%, which correlates with the calorific contents of the three forms (i.e. complanate = 4.97 kcal · ash-free g dry wt.^-1, cylindrical = 4.46 kcal and crustose = 3.55 kcal). The crustose form had tougher thalli (26 g · mm^-2 to penetrate thallus) than either the complanate form (12 g) or the cylindrical form (15 g). It is likely that opposing selective factors have resulted in the evolutionary divergences observed in algae with heteromorphic life histories. Previous work may have overemphasized the selective role of grazing because the crustose form is also adapted to withstand physical forces (sand-scouring, burial and wave-shearing) or as an overwintering stage under physiologically stressful conditions.     

Comparisons of N- and P-limited productivity between high granitic islands versus low carbonate atolls in the Seychelles Archipelago: a test of the relative-dominance paradigm

This exploratory study suggests that different geological systems (carbonate vs. granitic) in tropical waters have contrasting patterns of photosynthetic nutrient limitation correlated with inorganic nitrogen (N) and phosphorus (P) availability. Physiological assays for 21 predominant macrophyte species show that inorganic N and P are much less limiting to photosynthesis on granitic islands than is the case on carbonate islands and that, of the two, P is more likely to limit production in carbonate-rich tropical waters. Patterns of nutrient limitation in turn are reflected by differences in the relative dominance of functional groups of sessile, epilithic, photosynthetic organisms. Surveys at 33 sites on 10 islands revealed that nearshore waters on high granitic islands tend to be characterized by large and species-rich standing stocks of frondose macroalgae, often dominated by Sargassum spp., whereas waters around low carbonate islands tend to be dominated by hermatypic corals. Macrophyte tissue and seawater analyses also indicate a possible trend toward higher levels of N and P in granitic vs. carbonate islands. Pagode Island, a low carbonate island influenced by guano from seabird colonies, is an exception, with few corals, relatively high levels of tissue and seawater N and P, and a predominance of macroalgae (mostly Dictyosphaeria cavernosa).     

Algal resistance to herbivory on a Caribbean barrier reef

Field and laboratory research at Carrie Bow Cay, Belize showed that macroalgae, grouped in functional-form units resisted fish and urchin herbivory in the following order (from high to low resistance): Crustose-Group, Jointed Calcareous-Group, Thick Leathery-Group, Coarsely Branched-Group, Filamentous-Group and Sheet-Group; thereby supporting the hypothesis that crustose, calcareous and thick algae have evolved antipredator defenses and should show the greatest resistance to herbivory with a gradation of increasing palatability towards filaments and sheets. Of the 21 species examined, several (e.g.,Dictyota cervicornis on grids,Laurencia obtusa andStypopodium zonale) had exceptionally low losses to fish grazing, probably due to chemical defences. The sea urchin,Diadema antillarum, was more inclined to feed on algae with known toxic secondary metabolites than were herbivorous fishes; hypothetically related to the differences in mobility and concomitant modes of feeding. Tough leathery forms such asSargassum polyceratium andTurbinaria turbinata resisted grazing by bottom feeding parrotfishes (Scaridae) and surgeonfishes (Acanthuridae) but were susceptible when suspended midway in the water column, possibly due to the presence of rudderfishes (Kyphosidae) which readily consume drift Sargassaceae. The overall tendencies support our predicted relationship between grazer-resistance and algal morphology. In conjunction with our previously reported findings concerning primary productivity, toughness and calorimetry for many of the same species, these results lend credence to generalizations relating form with function in marine macroalgae.     

Productivity and nutrient relationships in psammophytic versus epilithic forms of Bryopsidales (Chlorophyta): comparisons based on a short-term physiological assay

Members of the green algal order Bryopsidales (= Caulerpales) are important calcifying agents of tropical reefs and comprise two fundamentally different life-form groups: (1) epilithic species with limited attachment structures and (2) psammophytic forms that have extensive subterranean rhizoidal systems. Because the shallow-water habitats of the former have relatively low nitrogen (N) to phosphorus (P_i) ratios compared to the pore waters of the sedimentary carbonate-rich substrata in which the latter are anchored, we hypothesized that epilithic forms should tend to be relatively more limited by N, while psammophytic species should tend to show P_i limitation. In partial support of the hypothesis, light-saturated net photosynthesis (P_max) in the epilithic forms, Halimeda opuntia, H. lacrimosa and H. copiosa, tended to be enhanced by N, while P_i was inhibitory or had no effect. In contrast, the psammophytic forms, Udotea sp., U. conglutinata, H. monde, H. tuna and H. simulans, tended to be stimulated more by P_i, whereas N had little effect. The utility of a bioassay to assess macroalgal nutrient limitation, based on a physiological response (net P_max) to short-term nutrient pulses, is demonstrated.     

Complex interactions in the control of coral zonation on a Caribbean reef flat

Summary This study uses short-term assays and long-term transplant experiments to document the potential importance of fish predation and herbivory to the distribution and abundance of reef-building corals in a Caribbean back-reef system. Experimental manipulations of fish access reveal that the zonal patterns of the two reef-building corals Porites astreoides and P. porites f. furcata, dominant on shallow back-reef habitats, are strongly associated with the feeding intensity of parrotfishes. Differential palatability of the two corals to parrotfishes, the proximity of protective cover for large grazers and the availability of small refugia to harbor a cryptic grazer fauna are suggested as major features contributing to the observed patterns. A model predicting the interactions of various algivore/corallivore guilds on the relative dominance of Porites and algal populations is presented.     

The characterization of the EBV alkaline deoxyribonuclease cloned and expressed in E. coli.

Studies of nucleic acid homology suggest the BGLF5 open reading frame of Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase) sharing some homology with that of herpes simplex virus. We report here the expression of the BGLF5 open reading frame in E. coli and the expression of high levels of a novel alkaline DNase activity in induced cells. This alkaline DNase has been purified to apparent homogeneity as a single protein species. This is the first report of the expression of a herpesvirus coded DNase in a prokaryotic system and of the purification of the EBV DNase to demonstrable purity. It has the biochemical characteristics of a typical herpesvirus alkaline exonuclease showing a high pH optimum, an absolute requirement for Mg2+ for activity and sensitivity to high salt concentrations and polyamines. The enzyme activity was neutralized by sera from patients with nasopharyngeal carcinoma and was reactive with these sera in Western blot analysis. Thus the prokaryotic expression system described here provides an economical and efficient source of the EBV DNase for biochemical and seroepidemiological analysis.