Neuronal cell-cell adhesion depends on interactions of N-CAM with heparin-like molecules

Cell-cell interactions are of critical importance during neural development, particularly since the migration of neural cells and the establishment of functional interactions between growing axons and their target cells has been suggested to depend upon cell recognition processes. Neurone-neurone adhesion has been well studied in vitro, and is mediated in part by the neural cell adhesion molecule N-CAM. N-CAM-mediated cell-cell adhesion has been postulated to occur by a homophilic binding mechanism, in which N-CAM on the surface of one cell binds to N-CAM on a neighbouring cell. Studies in our laboratory have identified a cell surface glycoprotein, now known to be N-CAM, which participates in cell-substratum interactions in the developing chicken nervous system. Although this adhesion involves a homophilic binding mechanism, the binding of the cell surface proteoglycan heparan sulphate to the glycoprotein is also required. This raises the question of whether the binding of heparan sulphate to N-CAM is also required for cell-cell adhesion. Here we show that the binding of retinal probe cells to retinal cell monolayers is inhibited by heparin, a functional analogue of heparan sulphate, but not by chondroitin sulphate. Monoclonal antibodies that recognize two different domains on N-CAM, the homophilic-binding and heparin-binding domains, inhibit cell-cell adhesion. The heparin-binding domain isolated from N-CAM by selective proteolysis also inhibits cell-cell adhesion when bound to the probe cells.     

The stability of a complex social system

Analyzing communication data within a social structure represented by a wholesale agricultural market demonstrates the utility of network analysis techniques for identifying systems. A representation of these data in matrix form permits exploration of the stability properties of the system. The stability analyses lend some support for theoretical arguments pertaining to the conditions leading to system equilibrium.     

The beta-subunit of follicle-stimulating hormone is deleted in patients with aniridia and Wilms' tumour, allowing a further definition of the WAGR locus

One in 10,000 children develops Wilms' tumour, an embryonal malignancy of the kidney. Although most Wilms' tumours are sporadic, a genetic predisposition is associated with aniridia, genito-urinary malformations and mental retardation (the WAGR syndrome). Patients with this syndrome typically exhibit constitutional deletions involving band p13 of one chromosome 11 homologue. It is likely that these deletions overlap a cluster of separate but closely linked genes that control the development of the kidney, iris and urogenital tract (the WAGR complex). A discrete aniridia locus, in particular, has been defined within this chromosomal segment by a reciprocal translocation, transmitted through three generations, which interrupts 11p13. In addition, the specific loss of chromosome 11p alleles in sporadic Wilms' tumours has been demonstrated, suggesting that the WAGR complex includes a recessive oncogene, analogous to the retinoblastoma locus on chromosome 13. In WAGR patients, the inherited 11p deletion is thought to represent the first of two events required for the initiation of a Wilms' tumour, as suggested by Knudson from epidemiological data. We have now isolated the deleted chromosomes 11 from four WAGR patients in hamster-human somatic cell hybrids, and have tested genomic DNA from the hybrids with chromosome 11-specific probes. We show that 4 of 31 markers are deleted in at least one patient, but that of these markers, only the gene encoding the beta-subunit of follicle-stimulating hormone (FSHB) is deleted in all four patients. Our results demonstrate close physical linkage between FSHB and the WAGR locus, suggest a gene order for the four deleted markers and exclude other markers tested from this region. In hybrids prepared from a balanced translocation carrier with familial aniridia, the four markers segregate into proximal and distal groups. The translocation breakpoint, which identifies the position of the aniridia gene on 11p, is immediately proximal to FSHB, in the interval between FSHB and the catalase gene.     

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.     

Phorbol ester and Epstein-Barr virus dependent transformation of normal primary human skin epithelial cells

Substantial evidence has implicated Epstein-Barr virus (EBV) in the aetiology of two human neoplasms, nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma. This is supported by the presence of high antibody titres to EBV early antigen and virus capsid antigen, as well as antibody to two viral-associated enzymes, DNase and DNA polymerase. Patients with NPC, particularly the undifferentiated form, are commonly found to have EBV DNA in the tumour. Ito and others have presented strong epidemiological evidence that phorbol esters are related to the unusual geographic distribution of NPC in southeastern regions of China. There appears to be a close link between the widespread EBV infection of the Asian population and the distinct regional distribution in China of plants that produce diterpene ester. Naturally occurring phorbol esters are produced by plants of the Euphorbiaceae and Thymelaeaceae, which are used as traditional herbal medicines. Although it has been established that EBV can infect epithelial cells isolated from NPC as well as certain normal epithelial cells, there has been no in vitro evidence that EBV induces neoplastic transformation in normal human epithelial cells with or without exposure to phorbol esters. We report here evidence that transformation of normal human epithelial cells results from exposure to infectious EBV and that transformation is dependent on the presence of phorbol esters.     

Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene.

Aniridia is a semidominant disorder in which development of the iris, lens, cornea and retina is disturbed. The mouse mutation Small eye (Sey), which has been proposed as a model for aniridia, results from defects in Pax-6, a gene containing paired-box and homeobox motifs that is specifically expressed in the developing eye and brain. To test the role of PAX6 in aniridia, we isolated human cDNA clones and determined the intron-exon structure of this gene. PAX6 spans 22 kilobases and is divided into 14 exons. Analysis of DNA from 10 unrelated aniridia patients revealed intragenic mutations in three familial and one sporadic case. These findings indicate that the human aniridia and murine Small eye phenotypes arise from homologous defects in PAX6.     

Current issues in mouse genome engineering

The mouse is the foremost vertebrate experimental model because its genome can be precisely and variously engineered. Now that the mouse genome has been sequenced and annotated, the task of mutating each gene is feasible, and an international cooperation is providing mutated embryonic stem cells and mice as readily available resources. Because these resources will change biomedical research, decisions about their nature will have far-reaching effects. It is therefore timely to consider topical issues for mouse genome engineering, such as the background genotype; homologous, site-specific and transpositional recombination; conditional mutagenesis; RNA-mediated interference; and functional genomics with embryonic stem cells.