Biomedical subjects
Publications and source records attributed to G Banting.
The X-linked gene, MIC5, encodes a human cell-surface antigen, R1. We have assigned MIC5 to the region between HPRT and G6PD on the long arm of the X chromosome. Regional localization was based on the pattern of reactivity of the R1 monoclonal antibody with human-rodent somatic cell hybrids which contained different fragments of the human X chromosome.
MIC2, which encodes the 12E7 antigen, is the only well-defined pseudoautosomal gene in man. We have isolated cDNA and genomic sequences corresponding to MIC2 and have produced monoclonal antibodies reacting with the 12E7 antigen. These molecular tools have been used to investigate the genetics and biochemistry of the MIC2 system. Recent results suggest that MIC2 is the most proximal of the currently defined pseudoautosomal markers and that the escape of MIC2 from X-inactivation may be intrinsic to an associated HTF island found at the 5' end of the gene. Investigation of the inter-relationship between MIC2 and the XG locus has led us to postulate the existence of a second pseudoautosomal gene in man.
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Two monoclonal antibodies (TRA-1-60 and TRA-1-81) recognizing distinct cell surface antigens on human embryonal carcinoma (EC) cells were produced and characterized. These antibodies reacted strongly with undifferentiated human EC cells in indirect radioimmunoassays (RIA) and immunofluorescence (IF) assays, but only weakly or not at all with cells derived from pluripotent EC cells differentiating in vitro or in xenograft tumors, nor with other germ cell tumor cell lines that did not also express the typical features of human EC cells. They did not react with murine teratocarcinoma cell lines. A survey of other human tumor cell lines and normal human tissues disclosed that molecules recognized by these antibodies are not confined to human EC cells but that cross-reacting epitopes appear on several neoplastic and normal tissues, although in a different anatomical pattern for each antibody. Both antibodies immunoprecipitated a major polypeptide (apparent molecular weight approximately 240,000) and a minor polypeptide (apparent molecular weight approximately 415,000) from lysates of 125I surface-labeled human EC cells, in this respect resembling another monoclonal antibody, 8-7D, previously described by Blaineau et al. (1,2) However, sequential immunoprecipitation revealed that each of the three antibodies reacted with different molecules of slightly different molecular weights. The epitopes defined by the present antibodies differ from those recognized by the other human EC cell-specific monoclonal antibodies that have been described and provide new markers for studying the differentiation of pluripotent human EC cells.
We have mapped two new genes to chromosome 11 which control the cell-surface expression of two distinct antigens defined by monoclonal antibodies. One of the antigens has a general tissue distribution and is associated with a molecular complex of two polypeptides of 80,000 dalton and 40,000 dalton molecular weight. The second antigen has a restricted tissue distribution and is carried on a polypeptide of 100,000 daltons. We have used a combination of genetic and biochemical techniques to demonstrate that these new markers are distinct from the antigens defined by the monoclonal antibodies F10.44.2 and W6/34 which are also encoded by genes on chromosome 11. It is concluded that human chromosome 11 carries at least four distinct genes controlling cell-surface antigen expression.
The monoclonal antibody, GERO, agglutinated all samples of red cells with the exception of Gerbich-negative cells of both Ge and Yus types. Using the antiglobulin test, Gerbich-negative cells reacted as strongly with GERO as did Gerbich-positive cells.
The monoclonal antibody, F10.44.2, is directed against a human antigenic determinant of restricted tissue distribution. In this report it is shown that this determinant is coded for by a gene, MIC4, which is on human chromosome 11, as shown by its reactivity with a panel of somatic cell hybrids. Synteny with an antigen recognized by another monoclonal antibody, W6/34, is demonstrated by hybrid and fluorescence-activated cell sorter analysis, although it is clear from absorption analysis that the determinants involved are on different molecules.
The monoclonal antibody OKT-9 has been shown to recognize the human transferrin receptor. We have exploited the species specificity of OKT-9 to map a gene controlling human transferrin receptor expression to chromosome 3, using human-mouse somatic cell hybrids. The gene for the human transferrin receptor and the gene controlling transferrin expression may be linked in humans.
We have developed an approach to human developmental biology which exploits somatic cell genetics. With this system we have examined the production of the HLA-A,B,C antigens, A human-mouse somatic cell hybrid was constructed which contained a human X-7 chromosome translocation carrying the HLA region; this hybrid was used as a donor of the X-6 translocation in the technique of microcell transfer. The X-6 chromosome recipient was the mouse embryonal carcinoma cell line PCC4. The microcell hybrid MCP-6 retained the embryonal carcinoma phenotype as judged by shape and absence of H-2 expression. Nonetheless, the expression of the HLA-A,B,C genes was not extinguished. HLA-A,B,C antigen production of the cell surface, however, was not detected because this hybrid apparently could not make beta 2-microglobulin.
We have constructed hybrids between human thymocytes and the mouse thymoma BW5147. These hybrids, and others, have been used to show that the expression of a thymocyte antigen is controlled by an X-lined gene.
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The mammalian sex chromosomes are thought to be related to each other by sharing a common origin. That is, the X and Y chromosomes originally evolved from a pair of chromosomes that only differed at the locus determining sexual differentiation. For example, this evolutionary relationship is reflected during meiosis in chromosomal pairing between the tip of the human X chromosome short arm and the Y chromosome which presumably implies sequence homology. However, compelling genetic evidence for functional homology between the mammalian X and Y chromosome is lacking. We describe here the localization of a gene to the tip of the short arm of the human X chromosome and evidence for a related gene on the Y chromosome.
The monoclonal antibody R1 defines the product of the MIC5 locus which has been localized to the long arm of the human X chromosome in the same region as the fragile site FRAXA. DNA transfectants, selected by R1 on the fluorescence-activated cell sorter (FACS), have been used to demonstrate that a previously described polyclonal antiserum, S10 (Buck & Bodmer, 1976), recognizes the same cell surface molecule as R1. Immunoprecipitation from metabolically and surface-labelled cells has shown that this molecule is a Mr 200 K phosphoprotein which is synthesized as a Mr 180 K precursor and subsequently modified to a Mr 195 K extracellular form. A Mr 150 K molecule is coprecipitated with the Mr 200 K phosphoprotein, but is only detected in lysates of surface-labelled cells, raising the possibility that the 200 K molecule is a cell surface receptor and the 150 K molecule a ligand.
TGN38 is a heavily glycosylated, type I integral membrane protein which is predominantly localized to the trans Golgi network (TGN), but which constitutively traffics between the TGN and the cell surface. The trafficking of TGN38 has been extensively studied in non-polarized cells, and a short, tyrosine-based, peptide motif within the cytosolic domain of the protein has been shown to be necessary and sufficient for its rapid internalization from the cell surface and efficient delivery to the TGN. Such tyrosine-based motifs have also been shown to act as basolateral targeting signals, whilst N-linked glycans (as occur on the extracytosolic domain of TGN38) can act as apical targeting signals. TGN38 has previously been shown to be sorted to the basolateral surface of polarized canine MDCK cells; a polarized cell line in which biosynthetic sorting decisions concerning the eventual destination of apical or basolateral targeted plasma membrane proteins are made at the TGN. We now show that TGN38 is targeted exclusively to the basolateral domain of polarized human Caco-2 cells, a cell line in which newly synthesized membrane proteins destined for either the apical or basolateral plasma membrane may be sorted for delivery to their final destination either at the TGN or at the cell surface. These data also demonstrate that the heavily glycosylated, extracytosolic domain of TGN38 does not contain a dominant apical targeting signal.