Structure and expression of human globin genes introduced into mouse fibroblasts

The human delta- and beta-globin genes, contained in a recombinant bacteriophage (lambda H beta G1), were introduced into mouse fibroblasts by cotransformation with a plasmid (chi 1) containing the herpes simplex thymidine kinase gene using the calcium phosphate precipitation technique. A molar ratio of lambda H eta G1 to chi 1 DNA of 3:1 was used. Four of the eleven stable transformants obtained contained intact delta- and beta-globin genes as determined by Southern blot analysis. To assess methylation in the segment of human DNA introduced into mouse cells, digestion with Hpa II or Msp I alone or with a second restriction enzyme was performed. The sites examined near the human delta- and beta-globin genes in transformed cells were not methylated. RNA extracted from the transformed cells was analyzed by RNA-cDNA hybridization; no more than 100 copies of human beta-globin mRNA/cell were found. Although hypomethylation of sites surrounding expressed globin genes in erythroid cells has been described, this property is not sufficient to ensure a high level of expression in fibroblasts.     

Transfer of human and murine globin-gene sequences into transgenic mice.

We have studied the transfer of human and murine globin gene sequences into fertilized mouse oocytes by microinjection. Germline transmission was demonstrated for the human delta- and beta-globin genes contained in the bacteriophage lambda H beta G1. Expression of these human globin-gene sequences was not detectable in either erythroid or nonerythroid tissues. A recombinant plasmid containing the murine beta maj promoter region coupled to the prokaryotic coding sequence for galactokinase was also successfully transferred to two mice, and stable germline transmission of integrated DNA was demonstrated for at least 3 generations. Despite the presence of a murine globin-promoter sequence, expression of the mouse beta maj galactokinase fusion gene was not observed in primary or secondary animals in erythroid or nonerythroid tissues. Analysis of primary and secondary animals from both series of injections revealed extensive de novo methylation in the integrated microinjected DNA. Administration of 5-azacytidine to mice containing the mouse beta maj-promoted galactokinase gene resulted in partial hypomethylation was associated with an apparent two- to threefold increase in galactokinase (gal K) gene expression.     

Functional dissection of structural domains in the receptor for colony-stimulating factor-1.

Receptor tyrosine kinases (RTKs) transduce external signals to the interior of the cell via a cytoplasmic kinase domain. We demonstrated previously that ligand-induced kinase activation of the colony-stimulating factor-1 receptor (CSF-1R) occurs via receptor oligomerization without propagation of conformational changes through the transmembrane (TM) domain (Lee, A. W., and Nienhuis, A. W. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7270-7274). We have now examined the role of the different subdomains in the metabolic and signaling properties of CSF-1R. Two types of chimeric receptors have been utilized, Glyfms A, with the extracellular and TM domains of glycophorin A (GpA) and the cytoplasmic domain of CSF-1R, and Glyfms B, where only the extracellular domain originates from GpA. Glyfms A was found to exhibit a higher basal level of in vitro kinase activity, an increased associated phosphatidylinositol (PtdIns) 3-kinase activity and to support enhanced cellular mitogenesis, compared with wild-type CSF-1R or to Glyfms B. The constitutive activation of Glyfms A is consistent with the hypothesis that the TM domain may play a role in receptor oligomerization. Cross-linking with anti-GpA antibodies activated the kinase function of Glyfms B leading to an increase in PtdIns 3-kinase association and to the transmission of a mitogenic signal. Our results indicate that an activated kinase domain contains the major determinant for coupling with PtdIns 3-kinase, independent of extracellular and TM sequences and of ligand binding. Both chimeric receptors underwent internalization in the presence of anti-GpA antibodies but were not degraded, including the tyrosine-phosphorylated and kinase-active population. These results suggest that structural determinants in the extracellular domain must be important for targeting internalized receptors for lysosomal degradation.     

The human homolog of the Moloney leukemia virus integration 2 locus (MLV12) maps to band p14 of chromosome 5

The Moloney leukemia virus integration 2 (MLV12) locus represents a common region for proviral integration and a putative oncogene involved in the induction of thymic lymphomas in rodents. The human homolog of the MLV12 locus has been cloned and studies have been initiated to determine its possible role in the induction and progression of human neoplasms. In this study we used a panel of human X rodent somatic cell hybrids and in situ hybridization to metaphase chromosomes to map MLV12 to the short arm of the human chromosome 5, band p14.     

Variance for water-use efficiency among ecotypes and recombinant inbred lines of Arabidopsis thaliana (Brassicaceae)

Knowledge of natural variation among ecotypes and recombinant inbred lines of Arabidopsis thaliana L. Heynh for season-long water-use efficiency (WUE, moles of carbon accumulated per mole of water used) is useful in the design of experiments to understand the genetic control of this phenomenon. Water-use efficiency among 31 container-grown Arabidopsis ecotypes ranged from a high of 2.40 to a low of 1.86 mg/g (grams of dry aerial biomass per gram of water used). Genetic variance for WUE was observed among 80 F₅ recombinant inbred lines (RILs) derived from a cross between the highest (Lip-0) by the lowest (Edi-0) ecotype. The heritability of WUE was calculated as 0.18 ± 0.07. The mean WUE for the ten highest and ten lowest RILs was 2.42 and 1.05 mg/g, respectively. A significant difference was observed in the composition of stable isotopes of plant carbon (δ¹³C) between the mean of the ten highest (-31.23±) and the mean of the ten lowest (-31.96±) RILs based on WUE. Characterization of the 80 RILs provides a structured population for further genetic and physiologic study of WUE.     

The Oosterschelde estuary: an evaluation of changes at the ecosystem level induced by civil-engineering works

The interest in storm-flood protection has recently gained momentum, owing to the wide international discussion on the impact of sea-level rise on society. The Oosterschelde project is technically and scientifically unique. The storm-surge barrier represents an important breakthrough in marine civil engineering. The project also offered ample opportunities to perform integrated physical, chemical, geological and biological research. Integration of the knowledge gained, raised the entire project to the level of a case study of a changing estuarine ecosystem, and demonstrated the effects of human interference in a non-polluted estuary. Notwithstanding considerable changes in the environment, the Oosterschelde has retained most of its favourable abiotic factors, labeling the estuary as a high quality marine system. The water quality in the post-barrier period more closely resembles that of the North Sea than in the period before. Significant changes in erosion and sedimentation and the consequent redistribution of fine sediments, are continuing. The ecosystem has shown responses to various factors. Effects of severe winters and impact of mussel- and cockle fisheries could be distinguished from other factors. The physical response of the ecosystem to the civil-engineering project could be quantified in terms of changes in habitat availability, maintenance of biological productivity, and restricted maintenance of the carrying capacity as an internationally recognized wetland and fisheries area.     

Mechanism of autocrine stimulation in hematopoietic cells producing interleukin-3 after retrovirus-mediated gene transfer.

Endogenous expression of the interleukin-3 (IL3) gene introduced with a retrovirus vector renders hematopoietic cells autonomous of exogenous growth factor. To investigate the mechanism of autocrine stimulation, 25 clones were isolated after retrovirus transduction of IL3 into 32D-cl23 or FDC-P1 cells. Medium conditioned by these autonomous IL3-producing clones supported the growth of factor-dependent 32D cells. Although there was a severalfold variation in the amount of IL3 secreted (some clones secreted barely detectable levels), the doubling time of each clone in the absence of added IL3 was identical to that of the parental cell line maximally stimulated by exogenous IL3. Concentrated monoclonal and polyclonal antibodies, both highly effective in neutralizing exogenous IL3, were assayed for ability to inhibit autocrine growth. Minimal inhibitory effects were observed only on washed autocrine clones secreting low levels of IL3. To test the activity of cytoplasmically synthesized IL3, the nucleotides encoding the signal sequence of IL3 were deleted and replaced with an in-frame ATG in the context of a consensus translation initiation sequence. Ten 32D clones expressing this restructured IL3 genome were obtained. Despite the presence of biologically active IL3 in cell lysates, all clones remained dependent on exogenous IL3, with the same dose-response as that found for 32D cells. Our data are most compatible with a mechanism whereby endogenously produced IL3 interacts with its receptor prior to surface display.     

Elevated CO2 affects shell dissolution rate but not calcification rate in a marine snail

As CO₂ levels increase in the atmosphere, so too do they in the sea. Although direct effects of moderately elevated CO₂ in sea water may be of little consequence, indirect effects may be profound. For example, lowered pH and calcium carbonate saturation states may influence both deposition and dissolution rates of mineralized skeletons in many marine organisms. The relative impact of elevated CO₂ on deposition and dissolution rates are not known for many large-bodied organisms. We therefore tested the effects of increased CO₂ levels—those forecast to occur in roughly 100 and 200 years—on both shell deposition rate and shell dissolution rate in a rocky intertidal snail, Nucella lamellosa. Shell weight gain per day in live snails decreased linearly with increasing CO₂ levels. However, this trend was paralleled by shell weight loss per day in empty shells, suggesting that these declines in shell weight gain observed in live snails were due to increased dissolution of existing shell material, rather than reduced production of new shell material. Ocean acidification may therefore have a greater effect on shell dissolution than on shell deposition, at least in temperate marine molluscs.     

Fish zonations and guilds as the basis for assessment of ecological integrity of large rivers

Longitudinal zonation concepts describe the downstream changes in chemico-physical and biological properties of rivers. Including information on ecological fish guilds can enhance the usefulness of fish zonation concepts, in a way that they can be used as tools for assessment and management of the ecological integrity of large rivers. We present an ecological characterization of fish zones and fish communities in near-natural and in regulated large rivers in Europe (the River Doubs in France and the Rivers Rhine and Meuse in the Netherlands), using guild classifications of several life-history traits of fish and national Red Lists of threatened species. The Doubs data set was also analyzed using indices of the sensitivity of fish species to environmental degradation and indices for eurytopy. In these rivers, the number of ecological guilds per zone increases downstream, and there are clear shifts in the structure of the guilds. Flow preference and reproduction ecology of river fish are closely linked. The proportion of rheophilic species in the fish community decreases downstream, and the proportions of limnophilic and eurytopic species increase. Lithophilic and psammophilic spawners are dominant in the upper zones, whereas the lower zones are dominated by phytophilic and phytolithophilic spawners. The proportion of zoobenthivorous and periphytivorous species decreases downstream, and the proportion of zooplanktivorous and phytivorous species increases. However, because the European fish fauna mainly consists of feeding generalists, the discriminative abilities of simplistic feeding guild classifications are not very high. Guilds of sensitive, stenoecious species that share life history strategies that are highly adapted to specific riverine conditions (rheophils and limnophils) have declined far more than generalist species that can survive in a wide range of habitats that are not characteristic of natural river ecosystems. Because of the subsequent over-abundance of the eurytopic species the original longitudinal fish zonations are hardly recognizable anymore in heavily impacted large rivers such as the River Rhine. Hence these rivers do not meet the criteria for ecological integrity. Within a specific fish region, a suitable way of analyzing and monitoring the impact of human disturbance on the structure of the fish community is by comparing the guild structure of the present state of a fish zone with that of the reference situation.