Linkage studies with chromosome 17 DNA markers in 45 neurofibromatosis 1 families

A locus for von Recklinghausen neurofibromatosis (NF1) has recently been mapped near the chromosome 17 centromere. We have extended these linkage studies by genotyping 45 NF1 families with three DNA probes known to be linked to the chromosome 17 centromeric region. Of 34 families informative for NF1 and at least one of the three probes, 28 families show no recombinants with the disease gene. These data provide additional support for genetic homogeneity of NF1 and for a primary NF1 locus linked to the chromosome 17 centromere. Among the informative families were 7 families with apparent new NF1 mutations. Our data suggest that these mutations are probably at the chromosome 17 NF1 locus.     

Identification of an octapeptide involved in homophilic interaction of the cell adhesion molecule gp80 of dictyostelium discoideum

During development of Dictyostelium discoideum, a surface glycoprotein of Mr 80,000 (gp80) is known to mediate EDTA-resistant cell-cell adhesion via homophilic interaction. Antibodies directed against a 13 amino acid sequence (13-mer) near the NH2 terminus of the protein were found to inhibit cell reassociation. This 13-mer also inhibited gp80-cell interaction and gp80-gp80 interaction. The cell binding site was mapped to the octapeptide sequence YKLNVNDS by using shorter peptide sequences to inhibit gp80 interaction. High salt concentrations inhibited homophilic interactions of both the 13-mer and gp80, suggesting that ionic interactions are involved in the forward binding reaction. Since disruption of homophilic interactions between the bound molecules required the presence of Triton X-100, hydrophobic interactions may occur after the initial ionic binding.     

Purification and characterization of a murine cellular retinoic acid-binding protein

A cellular retinoic acid-binding protein from 1-day-old mouse pups has been purified to homogeneity. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE cellulose, and chromatofocusing on PBE9-4 ion exchange resin. The chromatofocusing step was most useful in removing the major contaminants, which were otherwise difficult to remove. The binding protein was finally subjected to two cycles of high performance liquid chromatography on a DEAE-5PW column to achieve homogeneity. The protein has an isoelectric point of 4.75 and consists of a single polypeptide, migrating with an apparent Mr of 14,600 in SDS--polyacrylamide gel electrophoresis. Amino-terminal sequence analysis showed that the mouse cellular retinoic acid-binding protein has a high percentage of amino acid identity with other retinoid-binding proteins. However, it is immunologically distinct from the cellular retinol-binding protein.     

Tumor dormancy. I. Regression of BCL1 tumor and induction of a dormant tumor state in mice chimeric at the major histocompatibility complex

The growth of the BCL1 tumor in murine H-2 chimeras was studied. Lethally x-irradiated BALB/c mice were reconstituted with C57BL/6 bone marrow that had been depleted of T cells. When chimerism was established 90 to 120 days later, large doses of BCL1 cells were injected. The tumor grew progressively, reaching a peak level of as many as 10(9) tumor cells per animal by 40 days after inoculation. After that time, the tumor regressed in all the chimeric animals, and by 100 days after inoculation, virtually all the animals appeared disease free as judged by an absence of BCL1-idiotype-positive cells in the spleen and peripheral blood, a normal spleen size, and absence of an elevated white blood cell count. Such animals were followed for as long as 8 mo after tumor inoculation and remained disease free. However, transfer of graded numbers of splenocytes from these animals into normal BALB/c recipients resulted in development of tumor in recipients receiving 100 or more spleen cells. These results indicate a large tumor burden in the spleen of each donor, namely, 10(6) to 10(7) BCL1 cells. The present model should facilitate characterization of the mechanisms underlying tumor dormancy.     

Temperature-sensitive high affinity [3H]serotonin binding: Characterization and effects of antidepressant treatment

Characterization of temperature-sensitive [³H]serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S₁ and S₂ receptors. $\text{In vivo}$ pretreatment (48 h before) with mianserin did not alter Bmax or Kd for the 1 nM Kd [³H]5-HT site, although [³H]ketanserin (S₂) densities were decreased by 50%. This suggested that possible S₂ components of [³H]5-HT binding must be negligeable, even though ketanserin competed with high affinity (IC₅₀ = 3 nM) for a portion of the 1 nM Kd [³H]5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd [³H]5-HT site in a non-competitive manner, as shown by a decrease in Bmax with no change in Kd after $\text{in vitro}$ incubation. The complex inhibition data may therefore represent indirect interactions through another site.     

Cell-cell adhesion and morphogenesis in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire EDTA-resistant cell-cell adhesion at the aggregation stage. The EDTA-resistant cell binding activity is associated with a cell surface glycoprotein of Mr 80,000 (gp80), which mediates cell-cell binding via homophilic interaction. Analysis of the structure of gp80 deduced from cDNA sequence reveals the presence of three internally homologous segments in the NH2-terminal domain, which also contains regions with homology to the neural cell adhesion molecule. Secondary structure predictions show an abundance of beta-structures and very few alpha-helices. This is confirmed by circular dichroism measurements. It is likely that the homologous segments are organized into globular structures, extended from the cell surface by a Pro-rich stalk domain. The cell binding activity of gp80 resides within the first globular repeat of the NH2-terminal domain and has been mapped to a 51 amino acid region between Val123 and Leu173. Synthetic oligopeptides corresponding to sequences within this region have been prepared and assayed for their ability to bind to cell surface gp80. Results lead to identification of the homophilic binding site to an octapeptide sequence within this region. Synthetic peptides containing this octapeptide sequence and univalent antibodies directed against this site block the formation of organized cell streams during aggregation. Although cell aggregates are eventually formed, most fail to undergo further development to give rise to slugs and fruiting bodies, indicating that cell-cell adhesion involving gp80 is an important step in normal morphogenesis.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Rapid accumulation and disappearance of plasma membrane proteins during development of wild-type and mutant strains of Dictyostelium discoideum

Plasma membranes were purified from the cellular slime mold Dictyostelium discoideum at different developmental stages and the protein compositions compared. The protein components of the plasma membrane of vegetative cells are largely conserved during development. Specific morphogenetic events are accompanied by synthesis and accumulation of several new proteins which are subsequently lost as development progresses. Proteins with apparent molecular weights of 38,000, 36,500 and 10,000 to 12,000 rapidly accumulate during the first six hours of development and then disappear from the plasma membrane after 12 hours. Later in development, several new high molecular weight proteins are synthesized and appear in the membrane. The pattern of accumulation of membrane proteins in wild-type and mutant strains suggests that appearance of membrane proteins is linked to a dependent sequence of events.