Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification

To successfully treat cancer we will likely need a much more detailed understanding of the genes and pathways meaningfully altered in individual cancer cases. One method for achieving this goal is to derive cancers in model organisms using unbiased forward genetic screens that allow cancer gene candidate discovery. We have developed a method using a “cut-and-paste” DNA transposon system called Sleeping Beauty (SB) to perform forward genetic screens for cancer genes in mice. Although the approach is conceptually similar to the use of replication competent retroviruses for cancer gene identification, the SB system promises to allow such screens in tissues previously not amenable to forward genetic screens such as the gastrointestinal tract, brain, and liver. This article describes the strains useful for SB-based screens for cancer genes in mice and how they are deployed in an experiment.     

Prevalence and distribution of airborne and waterborne fungi and actinomycetes in the Nile river

The objective of this research was to investigate the prevalence and distribution of airborne and waterborne fungi and actinomycetes along the main stream of the Nile river during April to July, 2005. Air and water samples were collected at eight sites within a ~50 km stretch of the river. The distribution and prevalence of air and water microorganisms varied with location. The highest counts of airborne fungi (516 CFU/p/h) and actinomycetes (222 CFU/p/h) were detected at suburban sites near cultivated areas. However, the highest counts of waterborne fungi (56.4 CFU/ml) and actinomycetes (15.4 CFU/ml) were detected at Al-Galaa (city centre) and Kafr-El-elwe (south Cairo), respectively. A total of 1,816 fungal colonies (943 isolates from air and 873 from water samples) belonging to 27 genera were identified. Aspergillus, Alternaria, Cladosporium, and yeasts were the predominant fungal types in both air and water environments. Dreschlera, Emericella, Nigrospora, Spicaria, Stachybotrys, and Verticillium were only detected in the air, and Epicoccum, Philaphora, Phoma and Ulocladium were only detected in the water. Mycotoxin-producing fungi represented by Aspergillus flavus, Aspergillus parasiticus, Penicillium, Fusarium, and Trichoderma were found in the air and water environments. Significant differences (P ≤ 0.05) were found between fungal populations in air and water at different sampling sites. No significant differences (P ≥ 0.05) were found between waterborne actinomycetes. Sampling location, human activity, and pollution load are the main factors affecting the variability and biodiversity of microorganisms in different microenvironments.     

Analyses of spontaneous pink mutant events in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating growth chamber

In order to obtain more fundamental data on Tradescantia clone BNL 4430, one of the most suitable testers for environmental mutagens, the occurrences of spontaneous somatic pink mutations in the stamen hairs were scored for 52 weeks from 12 December 1998 to 10 December 1999, cultivating the young inflorescence-bearing shoots with roots in a nutrient solution circulating (NSC) growth chamber. The environmental conditions in the chamber were 22.0±0.5°C during the 16 h day with the light intensity of 7.5 klx from white fluorescent tubes, and 20.0±0.5°C at night. During the scoring period, 697,443 stamen hairs with an average cell number of 25.36 were observed and 2642 pink mutant events (PMEs) were detected. The overall spontaneous mutation frequency was 1.56±0.03 PMEs per 10⁴ hair-cell divisions, and the frequency was significantly lower in May, July and August and significantly higher in November and December. By analyzing the sectoring patterns of 1856 PMEs (70.25% of PMEs detected), the most of 172 cases of multiple (two to five) pink sectors observed in the same hairs (scored as 232 PMEs for calculating mutation frequency) were found to be the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells. This finding clearly shows the significance of somatic recombinations in producing such multiple sectors (382 sectors in total) which occupied 19.0% of the 2006 pink sectors in total analyzed. Somatic recombinations were considered to be playing a significant role also in producing single PMEs in the stamen hairs.     

Should congruence between intra- and interspecific ecomorphological relationships be expected? A case study with the great tit,Parus major

We studied the relationship between leg morphology and posture while feeding in a population of great tits (Parus major) under controlled conditions to investigate to what extent morphology and ecology are linked at the individual level. From predictions generated at the interspecific level within the genus Parus (Moreno and Carrascal 1993), we tested whether intra- and interspecific ecomorphological relationships are consistent. Within our population, neither leg bone lengths nor leg muscle morphology were related to the feeding posture of individuals. However, differences in body weight were correlated with inter-individual differences in time spent hanging. These results demonstrate that the association between intra- and interspecific ecomorphological relationships is not uniform. We argue that, at the intraspecific level, body weight overrides the significance of other traits that have a functional meaning at the interspecific level (i.e. leg segment lengths, muscular morphology), due to isometric variation of morphological traits (muscular and skeletal) with body mass. Thus, the discrepancy between the ecomorphological associations at intra- and interspecific levels is the result of a problem of scale (morphological changes in evolutionary time and isometric variation of morphological traits with body mass in ecological time).     

Bioreactor studies on the effect of dissolved oxygen concentrations on growth and differentiation of carrot (Daucus carota L.) cell cultures

A bioreactor control system was used to investigate the effects of two dissolved oxygen concentrations (10% and 100%) on the growth and differentiation of Daucus carota L. cell cultures. The strategy used allowed the dissolved oxygen concentration to be controlled without the need for changing either the agitator speed or the total gas flow rate. During the proliferation phase, reducing oxygen resulted in a lower growth rate and in a delay in sugar uptake kinetics. Nonetheless, varying levels of oxygen were observed to have no effect on the final dry biomass. The higher alcohol dehydrogenase activity obtained under reduced oxygen conditions suggests that proliferating cultures adapted to the hypoxic environment by inducing alcoholic fermentation. Cell differentiation was highly sensitive to reduced oxygen since under this condition, the somatic embryo production was inhibited by about 75%. Sugar uptake and embryo formation were also delayed.Abbreviations ADH alcohol dehydrogenase - 2,4-D 2,4-dichlorophenoxyacetic acid - DO2 dissolved oxygen - SE somatic embryos - Tris tris(hydroxymethyl)-aminoethane     

Analysis of the mitochondrial DNA of the somatic hybrids of Solanum brevidens and S. tuberosum using non-radioactive digoxigenin-labelled DNA probes

Summary Mitochondrial (mt) DNAs of somatic hybrids obtained by electrical and chemical fusion of mesophyll protoplasts of S. brevidens and a dihaploid line of S. tuberosum PDH 40 were analysed by Southern hybridization using the digoxigenin-labelled mtDNA sequences nad5 or orf25. In the Southern analysis of the hybrid mtDNA probed with nad5, most of the 19 hybrids analyzed had an RFLP pattern similar, but not identical, to one of the parents, S. tuberosum, PDH40. Nineteen percent of the hybrids had most of the S. brevidens fragments. Five of the hybrids had an identical RFLP pattern to either one of the parents while another two hybrids had novel RFLP patterns. Similar results were obtained by Southern analysis with orf25. These results clearly show that mtDNA rearrangements had occurred at a high frequency in the somatic hybrids. There were no differences in the frequencies of rearrangements observed between the hybrids regenerated from chemical and electrical fusions.     

Detecting significant change in stream benthic macroinvertebrate communities in wilderness areas

A major challenge in the biological monitoring of stream ecosystems in protected wilderness areas is discerning whether temporal changes in community structure are significantly outside of a reference condition that represents natural or acceptable annual variation in population cycles. Otherwise sites could erroneously be classified as impaired. Long-term datasets are essential for understanding these trends, to ascertain whether any changes in community structure significantly beyond the reference condition are permanent shifts or with time move back to within previous limits. To this end, we searched for long-term (>8 years) quantitative data sets of macroinvertebrate communities in wadeable rivers collected by similar methods and time of year in protected wilderness areas with minimal anthropogenic disturbance. Four geographic areas with datasets that met these criteria in the USA were identified, namely: McLaughlin Nature Reserve in California (1 stream), Great Smoky Mountains National Park in Tennesse-North Carolina (14 streams), Wind River Wilderness Areas in Wyoming (3 streams) and Denali National Park and Preserve in Alaska (6 streams).Two statistical approaches were applied: Taxonomic Distinctness (TD) to describe changes in diversity over time and non-metric multidimensional scaling (MDS) to describe changes over time in community persistence (Jaccards Index) and community stability (Bray–Curtis Index). Control charts were used to determine if years in MDS plots were significantly outside a reference condition. For Hunting Creek, TD showed three years outside natural variation which could be attributed to severe hydrological events but years outside the natural-variation funnel at sites in other geographical areas were inconsistent and could not be explained by environmental variables. TD identified simulated severe pollutant events which caused the removal of entire invertebrate assemblages but not simulated water temperature shifts.Within a region, both MDS analyses typically identified similar years as exceeding reference condition variation, illustrating the utility of the approach for identifying wider spatial scale effects that influence more than one stream. MDS responded to both simulated water temperature stress and a pollutant event, and generally outlying years on MDS plots could be explained by environmental variables, particularly higher precipitation. Multivariate control charts successfully identified whether shifts in community structure identified by MDS were significant and whether the shift represented a press disturbance (long-term change) or a pulse disturbance. We consider a combination of TD and MDS with control charts to be a potentially powerful tool for determining years significantly outside of a reference condition variation.     

The enigmatic molar from Gondolin, South Africa: Implications for Paranthropus paleobiology

The specific attribution of the large hominin M₂ (GDA-2) from Gondolin has significant implications for the paleobiology of Paranthropus. If it is a specimen of Paranthropus robustus it impacts that species' size range, and if it belongs to Paranthropus boisei it has important biogeographic implications. We evaluate crown size, cusp proportions and the likelihood of encountering a large-bodied mammal species in both East and South Africa in the Early Pleistocene. The tooth falls well outside the P. robustus sample range, and comfortably within that for penecontemporaneous P. boisei. Analyses of sample range, distribution and variability suggest that it is possible, albeit unlikely to find a M₂ of this size in the current P. robustus sample. However, taphonomic agents - carnivore (particularly leopard) feeding behaviors - have likely skewed the size distribution of the Swartkrans and Drimolen P. robustus assemblage. In particular, assemblages of large-bodied mammals accumulated by leopards typically display high proportions of juveniles and smaller adults. The skew in the P. robustus sample is consistent with this type of assemblage. Morphological evidence in the form of cusp proportions is congruent with GDA-2 representing P. robustus rather than P. boisei. The comparatively small number of large-bodied mammal species common to both South and East Africa in the Early Pleistocene suggests a low probability of encountering an herbivorous australopith in both. Our results are most consistent with the interpretation of the Gondolin molar as a very large specimen of P. robustus. This, in turn, suggests that large, presumptive male, specimens are rare, and that the levels of size variation (sexual dimorphism) previously ascribed to this species are likely to be gross underestimates.