Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.     

Genetics of salt tolerance in higher plants: theoretical and practical considerations

Summary An interdisciplinary approach to breeding for stress tolerance in plants has gained considerable recognition in the past few years. Accordingly, this article presents a synthesis of the genetic, physiological, and ecological aspects of salt tolerance in plants. An understanding of these aspects and the interrelationships between them is essential for an efficient breeding program.A significant part of the presentation concentrates on the basic problems associated with the genetics of tolerance to stresses and of quantitative characters in general, since many of the unsolved problems relevant to the genetics of salt tolerance are still general. Significant progress in the breeding of quantitative as well as qualitative traits in multicellular organisms depends on an understanding of the genetic and epigenetic dimensions of gene action. The discussion therefore includes an overview of (1) the limited existing knowledge on the genetic control of salt tolerance and (2) the physiological mechanisms and molecular targets central to the control of salt resistance as expressed by the amount and stability of yield.An additional subject emphasized here concerns the main strategies of adaptation of wild species to their natural habitats. An understanding of them is essential to (1) enable distinction between traits that can increase agricultural yield and traits that are favorable only for survival under natural conditions (such a distinction is essential, especially when wild species are used as a gene source), and (2) predict the best combinations of characters for efficient agricultural production in stressful environments.     

Purification, biochemistry and molecular cloning of an insect glycosylasparaginase from Spodoptera frugiperda

Glycosylasparaginase (EC 3.5.1.26 [EC] ) from Sf9 cells (Spodopterafrugiperda) was purified to homogeneity with a specific activityof 2.1 unit/mg. The enzyme is composed of two non-identical     

The SER primer and climate change

The SER Primer on Ecological Restoration provides a succinct introduction to, and overview of, the rapidly growing field of ecological restoration. The Primer was issued initially in 2002 by the Society for Ecological Restoration (SER) and reissued verbatim 2 years later in a more attractive format ( http://www.ser.org/resources;resources-detail-view/ser-international-primer-on-ecological-restoration ). A SER committee recently began deliberations to update the Primer, and much discussion is underway. As two of the Primer's principal authors, we were invited to share our views on how the Primer can be advantageously revised in the light of any changes or new insights since 2002. In particular, we were asked how the Primer might be modified to reflect the ways that ecological restoration address conservation issues raised by climate change and other rapid environmental shifts and global changes. We also touch on questions relating to the benefits of ecological restoration to human society, as this is an area where the Primer needs sharper focus. We have structured the following in a ‘Frequently Asked Questions’ format to highlight issues raised in the recent literature and to focus attention on other issues that merit consideration in the Primer revision process.     

A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice

A -lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3 region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3 end, with BLG sequences of exon 6/intron 6/exon 7 and the 3 region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3 flanking sequences. The BLG 5 regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.     

The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.