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.     

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.     

Towards an integrated linkage map of common bean. 4. Development of a core linkage map and alignment of RFLP maps

 Three RFLP maps, as well as several RAPD maps have been developed in common bean (Phaseolus vulgaris L.). In order to align these maps, a core linkage map was established in the recombinant inbred population BAT93×Jalo EEP558 (BJ). This map has a total length of 1226 cM and comprises 563 markers, including some 120 RFLP and 430 RAPD markers, in addition to a few isozyme and phenotypic marker loci. Among the RFLPs mapped were markers from the University of California, Davis (established in the F₂ of the BJ cross), University of Paris-Orsay, and University of Florida maps. These shared markers allowed us to establish a correspondence between the linkage groups of these three RFLP linkage maps. In total, the general map location (i.e., the linkage group membership and approximate location within linkage groups) has been determined for some 1070 markers. Approaches to align this core map with other current or future maps are discussed. Received: 10 March 1998 / Accepted: 22 April 1998     

Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder.

A high-titer, recombinant retroviral vector produced in psi 2 packaging cells has been used to introduce the murine interleukin-3 (IL-3) gene into mouse hematopoietic cells. Integration and expression of the IL-3 gene was observed in spleen foci from which could be derived factor-independent, continuously proliferating cell lines. Irradiated or genetically anemic W/Wv recipients of infected hematopoietic cells developed a myeloproliferative syndrome characterized by a marked elevation in leukocyte count, bone marrow hyperplasia, and enlargement of the liver and spleen. The syndrome reflected proliferation of one or more stem cell clones, the progeny of which were capable of repopulating secondary recipients. One animal developed the syndrome primarily by a paracrine mechanism. Endogenous IL-3 production caused amplification of hematopoietic cells but did not appear to alter the maturational or self-renewal potential of these cells.     

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.     

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.     

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.     

A family of long reiterated DNA sequences, one copy of which is next to the human beta globin gene.

An unusually long repeated DNA sequence was identified in cloned DNA, three kb 3' to the human beta-globin gene. Other members of this repeated sequence family were isolated from a human genomic DNA library and characterized by Southern blotting techniques, electron microscopy, and solution hybridization. The copy located next to the beta-globin gene was found to be 6.4 +/- 0.2 kb long and continuous over that length. This repeated sequence family comprises about 1% of the human genome and contains 3000-4800 copies of moderate sequence divergence which are interspersed with other less-highly repeated DNA. The 6.4 kb repeated unit does not appear to be composed of any smaller tandemly repeated subunits, nor is it expressed at a high level in bone marrow cell RNA.