Biomedical subjects
T V Gopalakrishnan
Publications and source records attributed to T V Gopalakrishnan.
Epigenetic activation of phenylalanine hydroxylase in mouse erythroleukemia cells by the cytoplast of rat hepatoma cells.
Friend mouse erythroleukemia cells do not synthesize detectable levels of phenylalanine hydroxylase [phenylalanine 4-monooxygenase; L-phenylalanine, tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] and hence are unable to grow in medium totally lacking tyrosine. These cells were fused with the cytoplasts of rat hepatoma cells that synthesize phenylalanine hydroxylase constitutively. Cytoplasmic hybrids [cybrids, Bunn, C. L., Douglas, C. W. & Eisenstadt, J. M. (1974) Proc. Natl. Acad. Sci. USA 71, 1681--1685] were selecte in medium without tyrosine. Cybrid clones expressed phenylalanine hydroxylase enzyme, which was of mouse type as determined by immunotitration and isoelectric focusing. This phenotype has been mainta ined even in the absence of any selective pressure. In contrast, in whole cell hybrids derived between the same parents, the expression of the phenylalanine hydroxylase gene was totally extinguished. One interpretation of these results is that the cytoplasm of rat hepatoma cells contain a positively acting factor(s) for the phenylalanine hydroxylase gene that brings about the activation of this gene in erythroleukemia cells.
Maintenance of hemoglobin inducibility in somatic cell hybrids of tetraploid (2S) mouse erythroleukemia cells with mouse or human fibroblasts.
It has previously been reported that Friend mouse erythroleukemia (MEL) cells synthesize hemoglobin when exposed to 2% dimethylsulfoxide, and that hybrids between MEL cells and fibroblasts (or other nonerythroid cells) do not synthesize hemoglobin. We have been successful in obtaining hybrids (3/15) between MEL cells and mouse L-cell fibroblasts that maintain hemoglobin inducibility by preserving nonadherent cells after fusion. The proportion of hemoglobin inducible hybrids can be increased (8/11) by using a stable 2S (pseudotetraploid) MEL parent in addition to preserving nonadherent cells after fusion. All hybrids which were nonadherent were hemoglobin inducible, and all hybrids which were adherent were not. Five nonadherent hybrid clones were analyzed from fusions between a stable 2S MEL parent and a human fibroblast (WI-38, VA-2). All these clones were inducible for hemoglobin. It is concluded that gene dosage is effective in increasing the proportion of hemoglobin inducible hybrids, but hybrid morphology is the phenotype characteristic that correlates most closely with expression of hemoglobin inducibility.
Active conformation of the globin genes in uninduced and induced mouse erythroleukemia cells.
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Cellular and molecular studies on globin gene expression.
Globin gene expression has been studied with the use of a combination of cell and molecular biology techniques. With a somatic cell hybrid between mouse erythroleukemia (MEL) and human erythroid cells, human and mouse globin genes can be coexpressed in the same hybrid cell. Somatic cell hybridization between MEL cells and nonerythroid cells (e.g., human fibroblasts) results in hybrid cells that cannot be induced to produce hemoglobin of any type. Molecular hybridization of cellular DNA and RNA with globin cDNA indicates that the globin genes are present but that no globin mRNA is in the nonexpressing hybrid cell. This finding demonstrates that the loss of globin gene expression in the hybrid cell occurs at the level of transcription or mRNA processing. The nucleus of the nonerythroid cell is not necessary for the extinction of globin gene expression, since cybrids formed from MEL cells with enucleated nonerythroid cells also result in cells which cannot be induced to synthesize hemoglobin even after 40 or more generations. These data suggest that the cytoplasm of cells contains diffusible regulatory molecules which influence globin gene expression. In our attempt to develop an intracellular assay to test for regulatory molecules, we studied the procedure microinjection with red blood cell (RBC) ghosts. Rabbit globin mRNA (with hemin) was loaded into RBC's and then, by Sendai virus-mediated fusion, into Chinese hamster ovary (CHO) cells growing in culture; these microinjected CHO cells synthesized hemoglobin. Since this microinjection procedure appears to be effective in inserting both proteins and nucleic acids into intact cells, it should be possible to develop modifications for inserting aliquots of fractionated cytoplasm into growing cells. The procedure might then be useful as an assay for putative genetic regulatory molecules.
Effect of concanavalin A on tyrosine aminotransferase in rat hepatoma tissue culture cells. Rapid reversible inactivation of soluble enzyme.
Concanavalin A added to intact cells at 37 degrees caused rapid and reversible inactivation of a soluble enzyme, tyrosine aminotransferase, in two lines of rat hepatoma tissue culture cells grown in monolayer culture. This temperature-dependent process was independent of de novo protein and RNA synthesis and independent of increased uptake of Ca2+ and Mg2+ or glucose. The inactivation could be reversed by adding alpha-methyl-D-mannopyranoside a competing sugar for concanavalin A binding. Other lectins known to bind to different sugars did not bring about the inactivation of tyrosine aminotransferase. Addition of concanavalin A did not result in the inactivation of another soluble enzyme, lactic dehydrogenase. The maintenance of tyrosine aminotransferase in an inactive form after the binding of concanavalin A to the cells required the continued presence of concanavalin A. This effect of concanavalin A could not be mimicked either by dibutyryl cyclic adenosine or guanosine monophosphoric acid. Incubation of cell extracts with concanavalin A did not result in inactivation nor did mixing of extracts from concanavalin A-treated cells with extracts from untreated cells. On the basis of these results we conclude that the following are the essential requirements for concanavalin A to bring about the inactivation of tyrosine aminotransferase: (a) the binding of native concanavalin A to the cells; (b) integrity of certain structural elements of the cells.
The stability of tyrosine aminotransferase and other proteins in enucleated rat hepatoma tissue culture cells.
The role of the nucleus in bringing about the induction of tyrosine aminotransferase (TAT) by glucocorticosteroid hormone and its deinduction upon steroid removal has been studied in enucleated rat hepatoma tissue culture cells (FU5-5). Both processes require the presence of the nucleus. However, cytoplasts from preinduced cells show an initial rapid decline in enzyme activity immediately after enucleation followed by maintenance of a constant level of activity. This initial decline in enzyme activity can be partially prevented by trypan blue, an inhibitor of lysosomal activity. This suggests that the early fall in enzyme activity could be due to an increase in the level of lysosomal activity immediately after enucleation. The subsequent constant level of activity seems due to maintenance rather than synthesis and degradation since it is not affected by cycloheximide. The absence of degradation applies to other kinds of proteins in enucleated FU5-5 cells and enucleated mouse fibroblast L cells. These experiments suggest that some kind of labile RNA or protein dependent on the presence of the nucleus is required for the degradation of all classes of proteins in different kinds of cells.
Extinction of hemoglobin inducibility in Friend erythroleukemia cells by fusion with cytoplasm of enucleated mouse neuroblastoma or fibroblast cells.
Friend mouse erythroleukemia cells (T3c1-2 and its subline 5000) can be induced to synthesize hemoglobin after treatment with 1.5% (vol/vol) dimethylsulfoxide. When these cells are fused with nonerythroid cells (namely, mouse neuroblastoma or L cells) hemoglobin induction is extinguished. In order to determine if the nucleus of the nonerythroid cell is necessary for this extinction, fusions were performed between mouse erythroleukemia cells and enucleated neuroblastoma or L cells. Hemoglobin induction was reduced or eliminated in clones of these hybrids even after 6 months of continuous culture. These results suggest that the cytoplasm of nonerythroid cells contains factor(s) that extinguish hemoglobin inducibility in erythroleukemic cells and that this new phenotype can be inherited.
A method for enucleating cultured mammalian cells.
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