Two molecular mechanisms of T cell-mediated cytotoxicity, one perforin-based, the other Fas-based, have been demonstrated. To determine the extent of their contribution to T cell-mediated cytotoxicity, a range of effector cells from normal control or perforin-deficient mice were tested against a panel of target cells with various levels of Fas expression. All cytotoxicity observed was due to either of these mechanisms, and no third mechanism was detected. Thus, the perforin- and Fas-based mechanisms may account for all T cell-mediated cytotoxicity in short-term in vitro assays.

We present here the first fully integrated, comprehensive map of the canine genome, incorporating detailed cytogenetic, radiation hybrid (RH), and meiotic information. We have mapped a collection of 266 chromosome-specific cosmid clones, each containing a microsatellite marker, to all 38 canine autosomes by fluorescence in situ hybridization (FISH). A 1500-marker RH map, comprising 1078 microsatellites, 320 dog gene markers, and 102 chromosome-specific markers, has been constructed using the RHDF5000-2 whole-genome radiation hybrid panel. Meiotic linkage analysis was performed, with at least one microsatellite marker from each dog autosome on a panel of reference families, allowing one meiotic linkage group to be anchored to all 38 dog autosomes. We present a karyotype in which each chromosome is identified by one meiotic linkage group and one or more RH groups. This updated integrated map, containing a total of 1800 markers, covers >90% of the dog genome. Positional selection of anchor clones enabled us, for the first time, to orientate nearly all of the integrated groups on each chromosome and to evaluate the extent of individual chromosome coverage in the integrated genome map. Finally, the inclusion of 320 dog genes into this integrated map enhances existing comparative mapping data between human and dog, and the 1000 mapped microsatellite markers constitute an invaluable tool with which to perform genome scanning studies on pedigrees of interest.

The purebred dog population consists of >300 partially inbred genetic isolates or breeds. Restriction of gene flow between breeds, together with strong selection for traits, has led to the establishment of a unique resource for dissecting the genetic basis of simple and complex mammalian traits. Toward this end, we present a comprehensive radiation hybrid map of the canine genome composed of 3,270 markers including 1,596 microsatellite-based markers, 900 cloned gene sequences and ESTs, 668 canine-specific bacterial artificial chromosome (BAC) ends, and 106 sequence-tagged sites. The map was constructed by using the RHDF5000-2 whole-genome radiation hybrid panel and computed by using MULTIMAP and TSP͞CONCORDE. The 3,270 markers map to 3,021 unique positions and define an average intermarker distance corresponding to 1 Mb. We also define a minimal screening set of 325 highly informative well spaced markers, to be used in the initiation of genome-wide scans. The well defined synteny between the dog and human genomes, established in part as a function of this work by the identification of 85 conserved fragments, will allow follow-up of initial findings of linkage by selection of candidate genes from the human genome sequence. This work continues to define the canine system as the method of choice in the pursuit of the genes causing mammalian variation and disease.dog ͉ microsatellites ͉ ESTs ͉ bacterial artificial chromosome ends

We have investigated the molecular basis of biological differences observed among cell line-adapted isolates of the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and the simian immunodeficiency virus (SIV) in response to receptor binding by using a soluble form of CD4 (sCD4) as a receptor mimic. We find that sCD4 binds to the envelope glycoproteins of all of the HIV-1 isolates tested with affinities within a threefold range, whereas those of the HIV-2 and SIV isolates have relative affinities for sCD4 two- to eightfold lower than those of HIV-1. Treatment of infected cells with sCD4 induced the dissociation of gp120 from gp41 and increased the exposure of a cryptic gp41 epitope on all of the HIV-1 isolates. By contrast, neither dissociation of the outer envelope glycoprotein nor increased exposure of the transmembrane glycoprotein was observed when sCD4 bound to HIV-2- or SIV-infected cells. Moreover, immunoprecipitation with sCD4 resulted in the coprecipitation of the surface and transmembrane glycoproteins from virions of the HIV-2 and SIV isolates, whereas the surface envelope glycoprotein alone was precipitated from HIV-1. However, treatment of HIV-1-, HIV-2-, and SIV-infected cells with sCD4 did result in an increase in exposure of their V2 and V3 loops, as detected by enhanced antibody reactivity. This demonstrates that receptor binding to the outer envelope glycoprotein induces certain conformational changes which are common to all of these viruses and others which are restricted to cell line-passaged isolates of HIV-1.

SummsryWe studied the coupling of the TCK/CD3 complex to a T cell effector function, namely Fasbased T cell-mediated cytotoxicity. Encounter or re-encounter with antigen was mimicked by treating 5 d mixed lymphocyte culture cells or T cell hybridomas with anti-CD3 antibody. This TCR/CD3 engagement induced swift expression of Fas-based cytotoxicity in these cells. Induction of Fas-based cytotoxicity was Ca2+-dependent, while its execution was not; induction was sensitive to macromolecular synthesis inhibitors, in line with a demonstrable increase of the Fas ligand (Fas-L) message. We also used T cell hybridomas transfected with various constructs to dissect the involvement of distinct components of the TCR/CD3 complex. The cytoplasmic domain of the CD3 ~" chain was able to transduce by itself a signal leading to Fas-L expression, unless there were mutations in its activation receptor homology sequence 1 (ARH-1) motifs. On the one hand, these findings are relevant to signal transduction pathways coupled to the TCR/CD3, and on the other hand, to the involvement of Fas-based T cell-mediated cytotoxicity in various physiological and possibly pathophysiological situations.

Purebred dogs are a unique resource for dissecting the molecular basis of simple and complex genetic diseases and traits. As a result of strong selection for physical and behavioral characteristics among the 300 established breeds, modern dogs are characterized by high levels of interbreed variation, complemented by significant intrabreed homogeneity. A high-resolution map of the canine genome is necessary to exploit the mapping power of this unusual resource. We describe here the integration of an expanded canine radiation hybrid map, comprised of 600 markers, with the latest linkage map of 341 markers, to generate a map of 724 markers-the densest map of the canine genome described to date. Through the inclusion of 217 markers on both the linkage and RH maps, the 77 RH groups are reduced to 44 syntenic groups, thus providing comprehensive coverage of most of the canine genome.

No abstract

We have probed the structure of the C4 and V3 domains of human immunodeficiency virus type 1 gpl20 by immunochemical techniques. Monoclonal antibodies (MAbs) recognizing an exposed gpl20 sequence, (E/ K)VGKAAMYAPP, in C4 were differentially sensitive to denaturation of gpl20, implying a conformational component to some of the epitopes. The MAbs recognizing conformation-sensitive C4 structures failed to bind to a gpl20 mutant with an alteration in the sequence of the V3 loop, and their binding to gpl20 was inhibited by both V3 and C4 MAbs. This implies an interaction between the V3 and C4 regions of gpl20, which is supported by the observation that the binding of some MAbs to the V3 loop was often enhanced by amino acid changes in and around the C4 region.