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PubMed1981

Dispensable sequences and packaging constraints of DNA from the Streptomyces temperate phage phi C31.

Deletion mutants of the temperate Streptomyces phage phi C31 were selected by two methods: resistance to the chelating agent sodium pyrophosphate, and plating of a phi C31::pBR322 hybrid phage on Streptomyces albus G to obtain large plaque mutants. The deletions defined a 7.7 kilobase (kb) segment of the phi C31 genome which is inessential for plaque formation, in addition to a shorter segment including the repressor gene. Analysis of deletions and insertions suggested that the minimum size of the phi C31 genome allowing plaque formation is 37.5 kb (91% of the wild-type length of 41.2 kb), and the maximum is at least 42.4 kb (103%). These results indicate that it should be possible to introduce up to 10 kb of foreign DNA into a suitably developed phi C31 vector.

Bacteriophages↗
PubMed1984

Thermo-inducible gene expression in Bacillus subtilis using transcriptional regulatory elements from temperate phage phi 105.

A Bacillus subtilis/Escherichia coli shuttle plasmid vector containing a transcriptionally silent chloramphenicol acetyl transferase gene (cat-86) was constructed by ligation of pPL603 (Williams et al., 1981a) and pUC8 (Vieira and Messing, 1982) at their unique EcoRI sites. Using this "promoter probe" vector we have obtained, by direct Cm resistance selection, a collection of cloned Sau3A fragments from the temperate phage phi 105 genome exhibiting promoter activity in B. subtilis. 18 promoter plasmids were subsequently transferred to an acceptor cell containing a functional repressor gene of phage phi 105 inserted into the temperature-sensitive replicon pE194. A repressor-controlled promoter was identified on the basis of its ability to confer thermo-inducible Cm resistance. The promoter is located on a 650-bp Sau3A fragment, mapping within the 3.2-kb EcoRI-F fragment, which also contains the phi 105 repressor gene. By assaying cloned subfragments of EcoRI-F for expression of immunity against phi 105 infection, the repressor gene could be assigned to a 1.1-kb EcoRI-HindIII fragment, which partially overlaps the promoter fragment. Taken together, these results suggest that, like the cI-coded repressor in coliphage lambda, the phi 105 repressor interacts with an operator sequence mapping very close to its own gene.

Acetyltransferases↗
PubMed1985

Isolation and characterization of the Streptomyces cattleya temperate phage TG1.

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.

Attachment Sites, Microbiological↗
PubMed1992

Gene expression in the Streptomyces temperate phage phi C31.

The repressor gene, c, of the temperate Streptomyces phage, phi C31 was previously cloned and sequenced, and predicted to encode a 74-kDa protein. The c gene actually produces three in-frame, N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa. The repressor proteins are translated from a corresponding nest of transcripts. Genetic and biochemical evidence suggests that the transcription of the c locus is autoregulated possibly by the 42-kDa protein binding to a highly conserved 16-bp perfect inverted repeat. The 16-bp sequence is present at at least twelve loci throughout the phi C31 genome. Transcription of the 'early' region is complex, possibly involving phage-specific promoters. The phi C31 terminators display sequence conservation and may be regulated. The phi C31 gene 'k' may encode a nucleotide kinase-encoding gene.

Bacteriophages↗
PubMed1993

Genetic organization and sequence of the region encoding integrative functions from Lactobacillus gasseri temperate bacteriophage phi adh.

A 2.0-kb fragment from the Lactobacillus gasseri temperate bacteriophage phi adh contained the essential genetic determinants for site-specific integration. The nucleotide sequence of this fragment was determined. An open reading frame (intG), which adjoined the phage attachment site (attP), encoded a deduced protein related to the integrase family. The organization of this region was comparable to other phage site-specific recombination systems.

Amino Acid Sequence↗
PubMed1994

Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development.

The temperate phage phi C31 is the most studied bacteriophage infecting Streptomyces spp., and has been used to develop an extensive and widely used series of cloning vectors. The sequence of 10 kb of phi C31 DNA containing most or all of the essential early genes was determined. Among the ORFs, 14 (perhaps 15) appear to be protein-coding, and these have been designated ORF1 to ORF14 and ORFX. Previously mapped transcripts appear to initiate upstream from ORFs 1, 8, 11 and 12, and within ORF3 and ORF12, in each case close to one example of the unusual ('21-mer') sequences that appear to serve as a recognition site for RNA polymerase early in the phi C31 lytic cycle [Ingham et al., Mol. Microbiol. 9 (1993) 1267-1274]. Further copies of the 21-mer are upstream from ORF2 and ORF13. There are four recognisable examples of a conserved inverted repeat sequence motif (CIR) thought to bind phi C31 repressor [Smith and Owen, Mol. Microbiol. 5 (1991) 2833-2844]. Only one CIR is closely associated with a 21-mer sequence, though three are located between known transcription units. Of all 14 ORFs, only one (ORF11) would encode a protein unmistakably resembling other known proteins; its product appears to be a DNA polymerase. Strikingly, two codons, TTA (Leu) and AGG (Arg), are absent from the 14 ORFs.

Amino Acid Sequence↗
PubMed2005

The effects of UVB radiation on temperate southern hemisphere forests.

The temperate forests of the southern hemisphere are the most likely forests to be affected by increased levels of ultraviolet-B (UVB) radiation resulting from reduced ozone. The review describes these forests and then discusses the morphological changes, physiological effects, and protection mechanisms, particularly UV absorbing compounds that result from present day and increasing UVB radiation. Possible avenues for future research are explored.

Africa, Southern↗
PubMed2005

A comparative assessment of heavy metal accumulation in soft parts and byssus of mussels from subarctic, temperate, subtropical and tropical marine environments.

Existing data on metal concentrations in mussels from subarctic, temperate, subtropical and tropical waters were analyzed using multivariate statistics in order to assess regional variations in metal contamination. Potential errors were reduced by only analyzing data from surveys that employed the same protocols, analytical methodologies and analysts. Factor analysis demonstrated that mussels inhabiting extremely contaminated areas (e.g. from Japanese and Swedish metallurgy sources) could be separated from mussels from other contaminated areas, and that metals such as Cd, Pb, Cu and Zn could be used to identify heavily contaminated samples while Co, Fe, Cr and Ni concentrations were good markers for exposure to inputs from different industrial sources. Furthermore byssus, like soft tissue, selectively and sensitively reflects variations of certain metal concentrations in ambient waters and thus serves as a reliable biomonitor for these contaminants in a variety of coastal and estuarine areas.

Animals↗
PubMed2006

Factors affecting the ozone sensitivity of temperate European grasslands: an overview.

This overview of experimentally induced effects of ozone aims to identify physiological and ecological principles, which can be used to classify the sensitivity to ozone of temperate grassland communities in Europe. The analysis of data from experiments with single plants, binary mixtures and multi-species communities illustrates the difficulties to relate individual responses to communities, and thus to identify grassland communities most at risk. Although there is increasing evidence that communities can be separated into broad classes of ozone sensitivity, the database from experiments under realistic conditions with representative systems is too small to draw firm conclusions. But it appears that risk assessments, based on results from individuals or immature mixtures exposed in chambers, are only applicable to intensively managed, productive grasslands, and that the risk of ozone damage for most of perennial grasslands with lower productivity tends to be less than previously expected.

Biodiversity↗
PubMed2004

New method for rapid and sensitive quantification of sulphide-producing bacteria in fish from arctic and temperate waters.

The offensive, fishy, rotten H2S-off-odours in spoiled, aerobically and cold stored fish from arctic and temperate waters are generally caused by sulphide-producing bacteria (SPB), mainly Shewanella putrefaciens. In the present work, a new, rapid, simple and accurate method for estimation of the SPB content in fish from these areas is described. The quantification is based on the formation rate of iron sulphide during growth of SPBs incubated at 30 degrees C in a liquid growth medium containing cysteine, sodium thiosulphate and iron(III)citrate as specific substrates for iron sulphide formation. The iron sulphide turns the medium grey and masks the background fluorescence in the medium when the SPB content in the assay is approximately 10(9) cfu/ml. The fluorescence change could be detected instrumentally and the colour change visually. The method was developed and evaluated in tests with S. putrefaciens CCUG 13452 DT as well as naturally occurring SPBs in cod, salmon, wolf fish and coal fish. A linear correlation between the SPB count and detection time was obtained over the entire range from 1 to 10(9) cfu SPB/g, corresponding to detection times 17 and 1 h, respectively. The correlation is described by the equation: log cfu/g fish= -0.59(+/- 0.17) x DT+ 9.65(+/- 0.09), where DT is the detection time in hours. The model was valid for all the tested fish species and all tested naturally occurring SPBs in these species. The regression coefficients (R2) for cod, coal fish, wolf fish and salmon were 0.99, 0.92, 0.97 and 0.97, respectively. The detection level of the method is 1 SPB per sample tube, corresponding to 16 cfu/g fish. The method could be used to predict the remaining shelf life of the fish for different markets, even when the time-temperature history during storage of the fish is unknown.

Animals↗
PubMed2003

EMERGY-based environmental systems assessment of a multi-purpose temperate mixed-forest watershed of the southern Appalachian Mountains, USA.

Emergy (with an 'm') synthesis was used to assess the balance between nature and humanity and the equity among forest outcomes of a US Forest Service ecosystem management demonstration project on the Wine Spring Creek watershed, a high-elevation (1600 m), temperate forest located in the southern Appalachian mountains of North Carolina, USA. EM embraces a holistic perspective, accounting for the multiple temporal and spatial scales of forest processes and public interactions, to balance the ecological, economic, and social demands placed on land resources. Emergy synthesis is a modeling tool that allows the structure and function of forest ecosystems to be quantified in common units (solar emergy-joules, sej) for easy and meaningful comparison, determining 'system-value' for forcing factors, components, and processes based on the amount of resources required to develop and sustain them, whether they are money, material, energy, or information. The Environmental Loading Ratio (ELR), the units of solar emergy imported into the watershed via human control per unit of indigenous, natural solar emergy, was determined to be 0.42, indicating that the load on the natural environment was not ecologically damaging and that excess ecological capacity existed for increasing non-ecological activities (e.g. timbering, recreation) to achieve an ELR of 1.0 (perfect ecological-economic balance). Three forest outcomes selected to represent the three categories of desired sustainability (ecological, economic, and social) were evaluated in terms of their solar emergy flow to measure outcome equity. Direct economic contribution was an order of magnitude less (224 x 10(12)solar emergy-joules (sej) ha(-1)) than the ecological and social contributions, which were provided at annual rates of 3083 and 2102 x 10(12)sejha(-1), respectively. Emergy synthesis was demonstrated to holistically integrate and quantify the interconnections of a coupled nature-human system allowing the goals of ecological balance and outcome equity to be measured quantitatively.

Appalachian Region↗
PubMed2006

Soil organic carbon decomposition and carbon pools in temperate and sub-tropical forests in China.

Decomposition of soil organic carbon (SOC) is a critical component of the global carbon cycle, and accurate estimates of SOC decomposition are important for forest carbon modeling and ultimately for decision making relative to carbon sequestration and mitigation of global climate change. We determined the major pools of SOC in four sites representing major forest types in China: temperate forests at Changbai Mountain (CBM) and Qilian Mountain (QLM), and sub-tropical forests at Yujiang (YJ) and Liping (LP) counties. A 90-day laboratory incubation was conducted to measure CO(2) evolution from forest soils from each site, and data from the incubation study were fitted to a three-pool first-order model that separated mineralizable soil organic carbon into active (C(a)), slow (C(s)) and resistant (C(r)) carbon pools. Results indicate that: (1) the rate of SOC decomposition in the sub-tropical zone was faster than that in the temperature zone, (2) The C(a) pool comprised approximately 1-3% of SOC with an average mean residence time (MRT) of 219 days. The C(s) pool comprised approximately 25-65% with an average MRT of 78 yr. The C(r) pool accounted for approximately 35-80% of SOC, (3) The YJ site in the sub-tropical zone had the greatest C(a) pool and the lowest MRT, while the QLM in the temperature zone had the greatest MRT for both the C(a) and C(s) pools. The results suggest a higher capacity for long-term C sequestration as SOC in temperature forests than in sub-tropical forests.

Carbon↗
PubMed2007

Chlorine-39 in rainfall at a temperate latitude (40 degrees N).

Rainwater samples were collected and measured for the radioactivity to confirm the presence of Chlorine-39 (T1/2=56.2 min) formed by the interaction of cosmic-ray slow negative muons, mu- with Argon-40 in the lower atmosphere. The presence of 39Cl was confirmed by estimating the half-life of a radionuclide present in the rainwater ranging from 58 to 63 min and averaging 60.4 min. The disintegration rate of this radionuclide varied from 2.1 to 2.7 Bq L(-1) (125-160 dpm/L) of rainwater and averaged 2.3 Bq L(-1) (140 dpm/L) of rainwater in Thessaloniki (40 degrees 38'N, 22 degrees 58'E), Northern Greece. This concentration is indicative of mid or temperate latitudes. This is clearly less than that reported in the literature, 1.7 Bq L(-1) (100 dpm/L) of rainwater, for a southern latitude 31 degrees 54'N, 34 degrees 49'E (Rehovot, Israel), due to the latitudinal effect of cosmic radiation.

Air Pollutants, Radioactive↗
PubMed2004

Computational analysis of ligand binding dynamics at the intermolecular hot spots with the aid of simulated tempering and binding free energy calculations.

Equilibrium binding dynamics is studied for a panel of benzimidazole-containing compounds at the remodeled interface between human growth hormone (hGH) and the extracellular domain of its receptor (hGHbp), engineered by mutating to glycine hot spot residues T175 from the hormone and W104 from the receptor. Binding energetics is predicted in a good agreement with the experimental data for a panel of these small molecules that complement the engineered defect and restore the binding affinity of the wild-type hGH-hGHbp complex. The results of simulated tempering ligand dynamics at the protein-protein interface reveals a diversity of ligand binding modes that is consistent with the structural orientation of the benzimidazole ring which closely mimics the position of the mutated W104 hot spot residue in the wild-type hGH-hGHbp complex. This structural positioning of the benzimidazole core motif is shown to be a critical feature of the low-energy ligand conformations binding in the engineered cavity. The binding free energy analysis provides a plausible rationale behind the experimental dissociation constants and suggests a key role of ligand-protein van der Waals interactions in restoring binding affinity.

Computational Biology↗
PubMed2004

Desertification of Mediterranean rocky reefs caused by date-mussel, Lithophaga lithophaga (Mollusca: Bivalvia), fishery: effects on adult and juvenile abundance of a temperate fish.

Macroalgal beds in temperate rocky habitats provide shelter and food for many adult reef fishes and their juvenile stages. In the Mediterranean Sea, the fishery of the endolithic date-mussel Lithophaga lithophaga (which involves dismantling of rocky substrates inhabited by these bivalves) may cause formation of barrens in shallow rocky reefs. Preliminary data collected in SW Apulia (SE Italy) show that rocky reefs impacted by this destructive fishery display different distribution patterns of adult Coris julis (a common labrid fish in the Mediterranean basin), and lower abundance of juveniles. The ecological implications of date-mussel fishery for dynamics of fish populations and rocky-reef ecosystem functioning (e.g., nursery role) are discussed.

Analysis of Variance↗
PubMed2005

Cloning, expression, purification, and characterisation of the dUTPase encoded by the integrated Bacillus subtilis temperate bacteriophage SPbeta.

The cDNA encoding dUTPase, an enzyme catalysing the hydrolysis of dUTP to dUMP and pyrophosphate, from the integrated Bacillus subtilis temperate bacteriophage SPbeta has been cloned and over-expressed at high levels in Escherichia coli. The resulting recombinant dUTPase was purified to homogeneity in one step by phosphocellulose chromatography with a final yield of 700 mg pure crystallisable protein per litre of bacterial culture. The molecular mass of the 142 amino acid polypeptide was 16 kDa as judged by electrophoretic analysis and gel filtration chromatography revealed the enzyme to exist as a homotrimer in solution. Isoelectric focusing indicated the isoelectric point to be 7. Functionality of the purified recombinant dUTPase was proven by demonstrating catalytic activity towards the substrate dUTP. The optimal activity of SPbeta dUTPase proved to be dependent on the presence of divalent metal ions, with Mg(2+) conferring the highest activity.

Amino Acid Sequence↗