Preventing aneuploidy: the contribution of mitotic checkpoint proteins

Aneuploidy, an abnormal number of chromosomes, is a trait shared by most solid tumors. Chromosomal instability (CIN) manifested as aneuploidy might promote tumorigenesis and cause increased resistance to anti-cancer therapies. The mitotic checkpoint or spindle assembly checkpoint is a major signaling pathway involved in the prevention of CIN. We review current knowledge on the contribution of misregulation of mitotic checkpoint proteins to tumor formation and will address to what extent this contribution is due to chromosome segregation errors directly. We propose that both checkpoint and non-checkpoint functions of these proteins contribute to the wide array of oncogenic phenotypes seen upon their misregulation.     

Coamplification of Eukaryotic DNA with 16S rRNA Gene-Based PCR Primers: Possible Consequences for Population Fingerprinting of Complex Microbial Communities

The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V₃, V₃-V₅, and V₆-V₈ hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V₃ and V₃-V₅ primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.     

Purification, isolation and search for possible functions of a phase-related 6080-Da peptide from the haemolymph of the desert locust, Schistocerca gregaria

Abstract. A novel 6-kDa peptide has been isolated from the haemolymph of the desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae) , and fully identified. Its concentration is higher in crowd-reared (gregarious) animals than in isolated-reared (solitarious) ones. Its concentration decreases progressively from generation to generation with solitarization of gregarious locusts. The peptide is also present in freshly laid eggs. The concentration in eggs is higher in those from crowd-reared locusts. It is likely that the peptide is transferred from the female's haemolymph into the eggs because injection of the peptide into females before oviposition increases the amount of the 6-kDa peptide in the eggs. A two-step HPLC purification procedure for this peptide is described. It allowed several bioassays to test for a possible function. Although the peptide's concentration in the haemolymph is high (0.1 m m ), which suggests some physiological function, we have as yet not been able to identify a function. We hypothesize that the 6-kDa peptide may somehow play a role as a maternal factor in the determination of the phase-state of the offspring.     

Novel phenotypes identified by plasma biochemical screening in the mouse

We used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for both gene function studies and use as new animal models of human disease (Nolan et al. 2000b). One focus of the program was the development of a blood biochemistry screen. At 8-12 weeks of age, approximately 300 ml of blood was collected from F1 offspring of ENU mutagenized male mice. This yielded approximately 125 ml of plasma, used to perform a profile of 17 standard biochemical tests on an Olympus analyzer. Cohorts of F1 mice were also aged and then retested to detect late onset phenotypes. In total, 1,961 F1s were screened. Outliers were identified by running means and standard deviations. Of 70 mice showing consistent abnormalities in plasma biochemistry, 29 were entered into inheritance testing. Of these, 9 phenotypes were confirmed as inherited, 10 found not to be inherited, and 10 are still being tested. Inherited mutant phenotypes include abnormal lipid profiles (low total and HDL cholesterol, high triglycerides); abnormalities in bone and liver metabolism (low ALP, high ALP, high ALT, and AST); abnormal plasma electrolyte levels (high sodium and chloride); as well as phenotypes of interest for the study of diabetes (high glucose). The gene loci bearing the mutations are currently being mapped and further characterized. Our results have validated our biochemical screen, which is applicable to other mutagenesis projects, and we have produced a new set of mutants with defined metabolic phenotypes.     

Invertebrates in field margins: taxonomic group diversity and functional group abundance in relation to age

Sown, temporary field margins are a common agri-environment scheme (AES) in the Netherlands. Despite their wide application, though, there has been scarcely any long-term monitoring of the succession of invertebrates. In the field margins of 40 farms, invertebrate diversity and the abundance of three functional groups were assessed in relation to age. The diversity in terms of number of species groups was found to increase with the age of the margins. The abundance of herbivores and detritivores also showed a positive correlation with the age of the margins. However, the abundance of predators decreased with increasing age. Older margins showed a higher total vegetation cover and fewer plant species, also resulting in lower plant species evenness. We suggest several changes to the current AES regulations. For the conservation of invertebrate diversity, longer-lasting field margins are desirable. In addition, old margins are favoured by detritivores, a group that has particular difficulty finding suitable habitats in agricultural landscapes. However, such margins are less favourable from an agricultural perspective, as they appear unsuitable for high abundances of potentially useful predators and the high vegetation cover attracts many potentially harmful herbivores. To circumvent this, the AES might be extended by incorporating hay-making, which would reduce standing biomass and might lead to more predators and fewer herbivores.     

Purple non-sulphur bacteria and plant production: benefits for fertilization,stress resistance and the environment

Purple non-sulphur bacteria (PNSB) are phototrophic microorganisms, which increasingly gain attention in plant production due to their ability to produce and accumulate high-value compounds that are beneficial for plant growth. Remarkable features of PNSB include the accumulation of polyphosphate, the production of pigments and vitamins and the production of plant growth-promoting substances (PGPSs). Scattered case studies on the application of PNSB for plant cultivation have been reported for decades, yet a comprehensive overview is lacking. This review highlights the potential of using PNSB in plant production, with emphasis on three key performance indicators (KPIs): fertilization, resistance to stress (biotic and abiotic) and environmental benefits. PNSB have the potential to enhance plant growth performance, increase the yield and quality of edible plant biomass, boost the resistance to environmental stresses, bioremediate heavy metals and mitigate greenhouse gas emissions. Here, the mechanisms responsible for these attributes are discussed. A distinction is made between the use of living and dead PNSB cells, where critical interpretation of existing literature revealed the better performance of living cells. Finally, this review presents research gaps that remain yet to be elucidated and proposes a roadmap for future research and implementation paving the way for a more sustainable crop production.     

Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. ‘Grande Naine’ (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.