Through the use of man-rodent somatic cell hybrids we intend to map the structural genes for human heavy and light chain immunoglobulins (Ig), for the IgA secretory piece and for the J chain of polymeric Ig. For the purposes of mapping, man-rodent hybrids of defined human chromosomal constitution will be examined to determine whether or not they produce human Ig. To determine the chromosomal constitution of hybrids, karyotypic analyses and enzyme electrophoresis will be carried out. Ig production will be examined using the following technics: surface immunofluorescence, radio-immunoelectrophoresis, affinity chromatography, radio-immunoassay. Information on the role of glycosyl transferases on the intra-cellular transport and secretion of Ig will be obtained by determining whether Ig which accumulates on the cell surface and that which is secreted into the medium is glycosylated in the same manner as the corresponding Ig produced by human and mouse cells. Glycosylation will be studied by cultivating Ig producing clones in the presence of a number of different radio-labelled sugars then isolating the Ig and determining the content of labelled sugar. The data will then be scrutinized to determine whether additional human chromosomes besides those thought to be carrying the structural genes for Ig production, are necessary for Ig glycosylation. In this way it may be possible to determine the chromosomal localization of genes coding for the glycosyl transferases which act on Ig. Some aspects of the role of nuclear-cytoplasmic interaction in the control of Ig synthesis will be studied through the use of Cytochalasin B which allows dissociation of cells into cytoplasts and karyoplasts. Hybrid cells will then be reconstituted using Sendai virus. Experiments will be carried out using a number of different parental cells, e.g. Ig producing cells and non-Ig producing cells and both inter-specific and intra-specific hybrids will be produced.
NIH ExPorter
NIH ExPorter