Somatic cell hybrids between Friend erythroleukemia cells and mouse hepatoma cells

Conscience, J F; Ruddle, Frank H.; Skoultchi, Arthur I.; Darlington, Gretchen J.

doi:10.1007/bf01551812

Cited by 41 publications

(24 citation statements)

References 20 publications

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“…Erythroid features of the MEL parent, including globin mRNA and hemoglobin production, are extinguished. These cells also failed to produce globin mRNA upon induction with dimethyl sulfoxide (10). We confirmed the absence of P-globin transcripts in these cells by Northern (RNA) blot analysis (data not shown).…”

Section: Resultssupporting

confidence: 66%

“…While fusion of certain combinations of erythroid and nonerythroid cells leads to tr-ans-activation of globin genes in cell hybrids, other combinations lead to extinction of their expression (10,13,14). FHP2b is a hybrid between BUdRresistant MEL cells and HPRT-mouse hepatoma (Hepa la) cells.…”

Section: Resultsmentioning

confidence: 99%

“…MEL x mouse hepatoma hybrid cells (FHP2b) were grown in monolayers in Dulbecco modified Eagle medium containing 10% fetal bovine serum and hypoxanthine-aminopterin-thymidine (32) as previously described (10). Primary human fibroblast x MEL hybrid cells (Hu 11) were propagated in suspension in Dulbecco modified Eagle medium containing hypoxanthine-aminopter-in-thymidine and supplemented with 10% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

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Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication.

1989

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To investigate whether a switch in the transcriptional activity of a gene is associated with a change in the timing of replication during the S phase, we examined the replication timing of the I-globin genes in two different types of somatic cell hybrids. In mouse hepatoma (Hepa la) x mouse erythroleukemia (MEL) hybrid cells, the j8-globin gene from the MEL parent is transcriptionaily inactivated and is later replicating than in the parental MEL cell line. In human fibroblast (GM3552) x MEL hybrid cells, the human ,3-globin gene is transcriptionally activated, and all of the sequences within the human 0-globin domain (200 kilobases) we have examined appear to be earlier replicating than those in the parental fibroblast cell line. The chromatin configuration of the activated human 0-globin domain in the hybrids is relatively more sensitive to nucleases than that in the fibroblasts. Furthermore, major nuclease-hypersensitive sites that were absent in the chromatin flanking the distal 5' region of the human ,(-globin gene cluster in the parental fibroblast cell line are present in the transcriptionally activated domain in the hybrid cell line. These results suggest that timing of replication of globin genes has been altered in these hybrid cells and thus is not fixed during the process of differentiation.Genes for specialized functions usually replicate very early during the S phase in mammalian cell lines in which they are expressed (e.g., see reference 28). When they are not expressed, some of these tissue-specific genes replicate late during the S phase while others can replicate at any time during the S phase. The time at which these genes replicate in cell lines in which they are not expressed appears to depend in part on their chromosomal location (7). In erythroid cell lines, P-globin genes are expressed and replicate early in the S phase, but in lymphoid cell lines, these genes replicate late in the S phase and are inactive. Transcriptionally active immunoglobulin heavy-chain variable-region genes (such as V1 of the T15 family) and T-cell receptor p-chain constant-region genes (CTP1 and CTP2) replicate early in the S phase in B-and T-cell lines, respectively, but late in the S phase in erythroid cell lines (28). Since these represent functionally and morphologically distinct blood cell lines which arise from the same progenitor or pluripotent stem cells, our findings suggest that some tissue-specific genes undergo a change in their replication timing concomitant with the turning on of their transcription during development and differentiation.We performed studies to determine whether the time at which a gene replicates in a differentiated cell is a permanent feature or whether it can be changed when the expression of the gene is altered. Ideally, we would like to be able to turn on the expression of a gene from a completely silent level and compare the replication times of the gene in these two states. There are few genes, however, whose activities can be modulated in this manner in cultured cells. The glob...

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Section: Resultssupporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication.

1989

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“…Cell hybrids were isolated essentially according to published procedures (13) except that 50% (wt/vol) polyethylene glycol 6000 (Baker) was used to induce fusion. Air-dried chromosome preparations were made by conventional methods and stained with Atebrin (Gurr) according to Uchida and Lin (19 (12,14).…”

mentioning

confidence: 99%

Activation of human beta-globin genes from nonerythroid cells by fusion with murine erythroleukemia cells.

Pyati

Kucherlapati

Skoultchi³

1980

Proc. Natl. Acad. Sci. U.S.A.

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A human beta-globin gene derived from an established human lymphoblast cell line was introduced into murine erythroleukemia (MEL) cells by cell fusion. The globin genes in MEL cells are inducible by dimethyl sulfoxide (Me2SO); induction leads to the accumulation of mouse globin mRNA and hemoglobin. Globin mRNA was not detected in the cytoplasm of the human lymphoblast cells, even at low levels, whether or not these cells were treated with Me2SO. In cell hybrids that had retained the lymphoblast-derived beta-globin gene, human beta-globin mRNA was induced by Me2SO. Poly(A)-containing 10S human beta-globin mRNA was detected in the cytoplasm of the hybrid cells. Karyologic and isozymic analyses of a series of hybrids and subclones showed that human beta-globin gene expression occurred only in hybrids that had retained human chromosome 11. Analysis of one hybrid bearing a deletion of both the beta-globin and lactate dehydrogenase A genes indicated that the beta-globin gene is located on the short arm of human chromosome 11. No other human chromosomes are required for human beta-globin gene expression in MEL cell hybrids. We conclude that the restricted expression of a globin gene in a human nonerythroid cell can be reversed. Furthermore, all components required for the transcription, processing, and transport to the cytoplasm of a human globin mRNA appear to be present in mouse erythroleukemia cells. Thus cell fusion with MEL cells provides a way to isolate permanent cell lines with functioning human globin genes. The technique should be useful for studying the biochemical basis for abnormal function of mutant globin genes, such as those present in individuals with the thalassemia syndromes.

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“…We prepared the TK probe in a similar manner from pHSV106 by isolating the EcoRI-SacI D fragment of about 630 base pairs (18). After being washed, the filters were exposed to Kodak XR-5 film for 1 (4). Samples that were incubated without RNA exhibited 5% S1 nuclease-resistant radioactivity.…”

mentioning

confidence: 99%

Introduction and expression of a fetal human globin gene in mouse fibroblasts.

Hsiung¹,

Roginski²,

Henthorn³

et al. 1982

Mol. Cell. Biol.

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An 8.5-kilobase segment of cloned human DNA including the complete G gamma-globin gene was introduced into LMTK- cells by the calcium phosphate precipitation method in the presence or absence of carrier DNA. Transfectants containing one or more copies of intact G gamma-globin genes were obtained either by ligation of the human DNA segment to a plasmid containing the herpes simplex virus thymidine kinase gene or by nonligated cotransfer. The integrity of the integrated gamma-globin gene was established by Southern blotting experiments. Expression of the herpes simplex virus thymidine kinase and human gamma-globin genes was evaluated by Northern blotting and solution hybridization. Of 23 transfectants analyzed, 21 produced a 9S gamma-globin RNA migrating like authentic gamma-globin mRNA on denaturing agarose gels. The gamma-globin RNA is polyadenylated and present in the cytoplasm of the transfected cells; it accumulates to a level 10 times that of thymidine kinase mRNA, or about 5 to 50 molecules per transfected cell. By using plasmids in which the gamma-gene is inserted in either transcriptional orientation with respect to the thymidine kinase gene, it was possible to show that transcription occurred from the gamma-gene promoter.

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Somatic cell hybrids between Friend erythroleukemia cells and mouse hepatoma cells

Cited by 41 publications

References 20 publications

Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication.

Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication.

Activation of human beta-globin genes from nonerythroid cells by fusion with murine erythroleukemia cells.

Introduction and expression of a fetal human globin gene in mouse fibroblasts.

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