Chemical mutagenesis of the mouse genome: an overview

The careful comparison of the phenotypic variations generated by different alleles at a given locus, including of course, those alleles with a deleterious effect, is often an important source of information for the understanding of gene functions. In fact, every time it is possible to match a specific alteration observed at the genomic level with a particular pathology, it is possible to establish a relationship between a gene and its function. When considered from this point of view, the production of new mutations by experimental mutagenesis appears as an alternative to the strategy of in vitro gene invalidation by homologous recombination in embryonic stem (ES) cells, with the advantage that experimental mutagenesis does not require any previous knowledge of the gene structure at the molecular level. Homologous recombination in ES cells is a gene driven approach, in which mutant alleles are produced for those genes that we already know. Experimental mutagenesis, on the contrary, is a phenotype driven approach, in which unknown genes are identified based on phenotypic changes. Also, while homologous recombination in ES cells requires a rather sophisticated technology, mutagenesis is simple to achieve but relies greatly on the efficiency of the mutagenic treatment as well as on the use of an accurate protocol for phenotyping. In this review, we will address a few comments about the different techniques that can be used for the induction of point mutations in the mouse germ line with special emphasis on chemical mutagenesis. We will also discuss the limitations of experimental mutagenesis and the necessity to look for alternative ways for the discovery of new genes and gene functions in the mouse.     

Chemical diversity of several Betulaceae species: comparison of phenolics and terpenoids in northern birch stems

 Phenolics and terpenoids characteristic for silver birch (Betula pendula Roth) were screened in current-growth twigs of seedlings (1-growing-season-old individuals) and current-growth twigs of saplings (3 to 5-year-old individuals) of several birch species. Seedlings were grown in a greenhouse for 1 growing season. Saplings were field cultivated or wild-stand individuals. Chemicals were methanol-extracted, purified and HPLC-diode array detector-, gas chromatograph-flame ionizaton detector- and gas chromatograph-mass selective-analyzed. Species-specific qualitative and quantitative variation of secondary chemicals in birch stems were considerable and dependent on the age of the individual. The chemical diversity and amount were lower in seedlings than in saplings. In saplings, (+)-catechin and its derivatives were found in nearly all species, while arylbutanoids (±rhododendrol glucosides), an arylheptanoid (platyphylloside) and dammaraneterpenoids (papyriferic acid, deacetylpapyriferic acid and pendulic acid) showed a more restricted distribution. The chemicals analyzed in birch stems are a useful tool for recognition of species. Received: 18 September 1995 / Accepted: 19 December 1995     

Metabolism of plastid terpenoids: in vitro inhibition of phytoene synthesis by phenethyl pyrophosphate derivatives

The inhibition of partially purified phytoene synthetase activity from Capsicum annuum chromoplasts was investigated using aminophenethyl pyrophosphate and azidophenethyl pyrophosphate. These compounds were effective inhibitors of phytoene synthesis and kinetic analysis showed that they were competitive with respect to the substrate isopentenyl pyrophosphate. These data were strengthened by the ability of azidophenethyl pyrophosphate to photoinactivate irreversibly the activity of the enzyme complex. These results suggest that the primary targets of these analogs are at the level of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthetase.     

Adaptive management: a synthesis of current understanding and effective application

Summary Adaptive management (AM) remains a commonly cited, yet frequently misunderstood, management approach. The aim of AM is to improve environmental management through ‘learning by doing’ and understand the impact of incomplete knowledge, but AM more commonly consists of ad hoc changes in managing environmental resources in the absence of adequate planning and monitoring. Here, we trace and review the development of AM, the central roles of consultation, collaboration and of monitoring, and of quantitative models and simulations. We identify a series of formalized, structured steps included in one AM cycle and review how current AM programs build upon such cycles. We conclude that the best AM outcomes require rigorous and formalized approaches to planning, collaboration, modelling and evaluation. Finally, simulating potential outcomes of an AM cycle in the presence of existing uncertainty can help to identify management strategies that are most likely to succeed in relation to clearly articulated goals.     

"Rheumatoid Arthritis": A balanced overview of current research

This is a brief review of the third volume of the series edited by AN Theofilopoulos, "Current Directions in Autoimmunity". This hard-cover volume comprises 282 pages, 46 figures and 14 tables. The book has a cover price of 196 Swiss Francs, or 255 Deutschmarks, or 170.50 US dollars.     

Genome browsing with Ensembl: a practical overview.

A wealth of gene information is accruing in public databases. Genome browsers such as Ensembl are needed to organize and depict this information in the context of the genome. Ensembl provides an open source gene set based on experimental evidence for over 30 species, the majority of which are vertebrates. Genes and annotation are accessible through the Ensembl browser (http://www.ensembl.org), and through direct queries of its databases using the Perl API (Application Programme Interface), MySQL or BioMart.     

Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.     

Chemical synthesis of a P(k)-antigenic globotriose analogue with a functionalized aglycon

6-Aminohexyl alpha-D-galactopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside (2), a globotriose analogue with a functionalized aglycon, was synthesized, using alpha-D-galactopyranosyluronic acid-(1-->4)-D-galactopyranosyluronic acid [di-GalA (3)] as the starting material, which is commercially available or can be readily prepared from pectin.     

Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models

The dystrophin-glycoprotein complex (DGC) is a multisubunit complex that connects the cytoskeleton of a muscle fiber to its surrounding extracellular matrix. Mutations in the DGC disrupt the complex and lead to muscular dystrophy. There are a few naturally occurring animal models of DGC-associated muscular dystrophy (e.g. the dystrophin-deficient mdx mouse, dystrophic golden retriever dog, HFMD cat and the delta-sarcoglycan-deficient BIO 14.6 cardiomyopathic hamster) that share common genetic protein abnormalities similar to those of the human disease. However, the naturally occurring animal models only partially resemble human disease. In addition, no naturally occurring mouse models associated with loss of other DGC components are available. This has encouraged the generation of genetically engineered mouse models for DGC-linked muscular dystrophy. Not only have analyses of these mice led to a significant improvement in our understanding of the pathogenetic mechanisms for the development of muscular dystrophy, but they will also be immensely valuable tools for the development of novel therapeutic approaches for these incapacitating diseases.     

Chemical synthesis of a gene for somatomedin C.

A synthetic gene for somatomedin C, a human growth factor, has been assembled by a single ligation of 23 oligodeoxyribonucleotides, which were chemically synthesized by an improved solid phase phosphotriester method.     

Chemical synthesis of a 7 kDa insect gonadotropic neurohormone

An original insect neurohormone of 65 residues was synthesized by the solid-phase methodology using t-Boc strategy and Boc-Val-PAM–resin. The purification, conducted by several steps of liquid chromatography having mass, polarity or charge as separative criteria, yielded the product with the correct molecular weight of 6922 Da determined by mass spectrometry. The synthetic peptide had both the same affinity for the antinative neurohormone serum and the same biological activity as the native neurohormone.     

Shoal bass life history and threats: a synthesis of current knowledge of a Micropterus species

The shoal bass (Micropterus cataractae) is a black bass species native to the Apalachicola-Chattahoochee-Flint Basin of the southeastern U.S. Damming in the basin has created extensive habitat loss; consequently, shoal bass have been extirpated from several areas of their native range. Early shoal bass research focused on age and growth, spawning habits, and distribution. The formal recognition of the species in 1999 increased interest in research and restoration. Recent research has described critical habitat, movements, and systematic information about shoal bass. As researchers continue to investigate the life history of the species, several threats have become apparent including habitat modification, interactions with non-native black basses, and the effects of angling. Currently, management needs include basic population assessments and investigation of factors causing population declines. Despite increased interest in the species, the outlook for the long-term conservation of the shoal bass is uncertain.     

Counter-current chromatography for the separation of terpenoids: a comprehensive review with respect to the solvent systems employed

Natural products extracts are commonly highly complex mixtures of active compounds and consequently their purification becomes a particularly challenging task. The development of a purification protocol to extract a single active component from the many hundreds that are often present in the mixture is something that can take months or even years to achieve, thus it is important for the natural product chemist to have, at their disposal, a broad range of diverse purification techniques. Counter-current chromatography (CCC) is one such separation technique utilising two immiscible phases, one as the stationary phase (retained in a spinning coil by centrifugal forces) and the second as the mobile phase. The method benefits from a number of advantages when compared with the more traditional liquid–solid separation methods, such as no irreversible adsorption, total recovery of the injected sample, minimal tailing of peaks, low risk of sample denaturation, the ability to accept particulates, and a low solvent consumption. The selection of an appropriate two-phase solvent system is critical to the running of CCC since this is both the mobile and the stationary phase of the system. However, this is also by far the most time consuming aspect of the technique and the one that most inhibits its general take-up. In recent years, numerous natural product purifications have been published using CCC from almost every country across the globe. Many of these papers are devoted to terpenoids—one of the most diverse groups. Naturally occurring terpenoids provide opportunities to discover new drugs but many of them are available at very low levels in nature and a huge number of them still remain unexplored. The collective knowledge on performing successful CCC separations of terpenoids has been gathered and reviewed by the authors, in order to create a comprehensive document that will be of great assistance in performing future purifications.     

Chemical synthesis of sulfated oligosaccharides with a beta-D-Gal-(1-->3)

The syntheses of two sulfated pentasaccharides: beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc-(1-->6)]-alpha-D-GalNAc-->OMe (1) and beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc6SO3Na-(1-->6)]-alpha-D-GalNAc-->OMe (2) by using Lewisx trisaccharides 12 and 16 as glycosyl donors are described. Sulfated oligosaccharides 1-2 and intermediate compounds are fully characterized by 2D 1H-1H DQF-COSY and 2D ROESY experiments.     

Chemical synthesis of UDP-4-O-methyl-GlcNAc, a potential chain terminator of chitin synthesis

Chitin synthase converts uridine diphosphoryl-N-acetylglucosamine (UDP-GlcNAc) to chitin (poly-beta-(1-->4)-GlcNAc). During polymerization, elongation occurs at the 4-OH (nonreducing) terminus of the growing chitin chain. Blockage of the 4-OH via incorporation of UDP-N-acetyl-4-O-methylglucosamine (UDP-4-OMe-GlcNAc, 3) can potentially terminate chitin polymerization, and represents a novel strategy for chitin synthase inhibition. The chemical synthesis of 3 and preliminary evaluation of its possible incorporation by chitin synthase are reported herein.