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PIGMENT GENE EXPRESSION IN FIBROBLAST X MELANOMA FUSIONS: I. EXTINCTION OF DOPA OXIDASE IN HETEROKARYONS. II. MAINTENANCE OF EXPRESSION OF DOPA OXIDASE IN NONDIVIDING CYBRIDS
Abstract
Techniques of single cell analysis were developed to study differentiated gene expression in fibroblast x melanocyte fusion products. Chicken embryo fibroblasts containing normal genes for pigment production were radioactively labeled with ('3)H-thymidine and fused with mitomycin C-treated pigmented B-16 mouse melanoma cells. The resulting heterokaryons, incapable of division, contained one radioactively labeled nucleus, a second unlabeled nucleus and pigment granules. These cells were identified using the light microscope and assayed for pigment gene expression using the transmission electron microscope. In a separate set of experiments the chicken embryo fibroblasts were radioactively labeled with ('3)H-leucine and enucleated by a combination of cytochalasin B treatment and centrifugation. Cytoplasts (the enucleated cytoplasms) were then fused with the mitomycin C-treated pigmented B-16 mouse melanoma cells. The resulting nondividing cybrids, cytoplasmic hybrids incapable of division, contained cellular radioactive label, one mouse nucleus and pigment granules. These cells were also identified using the light microscope and assayed for pigment gene expression using the transmission electron microscope. Pigment gene expression was assayed using a cytochemical test for dihydroxyphenylalanine (dopa) oxidase, the enzyme responsible for the conversion of tyrosine to dopa and dopa to melanin. Cells were scored according to the extent of golgi-related electron dense end product with a score of 0 representing no reaction product; 1 representing scant, vesicular reaction product; and 2 representing vesicular and cisternal reaction product. The fibroblast x melanoma heterokaryons showed significantly less dopa reaction product than mitomycin C-treated whole melanoma cells, while the nondividing cybrids showed no significant change in enzyme activity compared with their control populations. Extinction of pigment production in heterokaryons agrees with the results found in hybrids using biochemical analyses and suggests that extinction is not due to chromosome loss in hybrids but is the result of gene control interactions between the two parent cells, in this case from different species. The presence of the intact nuclear membranes of the fibroblast and melanoma nuclei did not restrict the interactions resulting in altered gene expression. The maintenance of dopa oxidase activity in the nondividing cybrids shows that the fibroblast cytoplasm alone is incapable of suppressing pigment gene expression. Thus, the presence of the fibroblast nucleus is necessary to extinguish the expression of luxury functions in the differentiated nucleus.
Subject Area
Genetics
Recommended Citation
SCHWARTZ, MARLENE SUE, "PIGMENT GENE EXPRESSION IN FIBROBLAST X MELANOMA FUSIONS: I. EXTINCTION OF DOPA OXIDASE IN HETEROKARYONS. II. MAINTENANCE OF EXPRESSION OF DOPA OXIDASE IN NONDIVIDING CYBRIDS" (1981). ETD collection for University of Nebraska-Lincoln. AAI8127164.
https://digitalcommons.unl.edu/dissertations/AAI8127164