Extinction of globin gene expression in human fibroblast x mouse erythroleukemia cell hybrids.

We have chosen human fibroblast x mouse erythroleukemia hybrid cells as a model system to examine regulation of unique genes. The globin genes were studied as a marker of erythroid differentiation. Three separate hybrid cell lines were incubated in 2% dimethylsulfoxide, an agent which induces erythroid differentiation of the parental erythroleukemia cells. Neither human nor mouse globin mRNA sequences could be detected by a sensitive molecular hybridization assay which utilized globin complementary D N A. However, td n a from one of the cell lines was shown to contain both the mouse and humand globin genes. Thus, loss of the genes by chromosomal segregation did not account for their failure to be expressed. Cocultivation of the mouse erythroleukemia cells with excess human fibroblasts did not prevent erythroid differentiation of the erythroleukemia cells in the presence of dimethylsulfoxide. Similarly globin gene expression was preserved in tetraploid cells generated by fusion of two erythroleukemia lines. Thus, extinction of globin geneated by fusion of two erythroleukemia lines. Thus, extinction of blobin gene expression in the human fibroblast x erythroleukemia hybrids occurred at the level of mRNA production and appeared to be due to the presence of the fibroblast genome within the hybrial cell.     

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.     

Regulated expression of human A gamma-, beta-, and hybrid gamma beta-globin genes in transgenic mice: manipulation of the developmental expression patterns

We have introduced the human fetal gamma- and adult beta-globin genes into the germ line of mice. Analysis of the resulting transgenic mice shows that the human gamma-globin gene is expressed like an embryonic mouse globin gene; the human beta-globin gene is expressed (as previously shown) like an adult mouse globin gene. These results imply that the regulatory signals for tissue- and developmental stage-specific expression of the globin genes have been conserved between man and mouse but that the timing of the signals has changed. Because the two genes are expressed differently, we introduced a hybrid gamma beta-globin gene construct. The combination of the regulatory sequences resulted in the expression of the hybrid gene at all stages in all the murine erythroid tissues.     

Differential expression of mouse beta/goat beta c, mouse beta/goat beta F, and mouse beta/goat epsilon II hybrid globin genes in murine erythroleukemia cells.

We assembled three hybrid beta-globin genes by fusing the mouse beta-major promoter and initial transcribed region to one of three goat beta-like globin gene bodies: beta c (preadult), beta F (fetal), or epsilon II (embryonic). Thymidine kinase (tk)-deficient murine erythroleukemia (MEL) cells were cotransformed with one of these constructs and a separate plasmid bearing the tk gene. Half of the 24 cell lines containing either the mouse beta/goat beta c or mouse beta/goat beta F genes expressed the transferred genes at significant levels; in many cases the hybrid genes were, like the endogenous beta-globin genes, inducible with dimethyl sulfoxide. We obtained 13 cell lines containing the mouse beta/goat epsilon II hybrid gene, 6 of which were cotransfected with a mouse beta/human beta fusion gene known to function in MEL cells. In contrast to the results with the other fusion genes, the mouse beta/goat epsilon II hybrid was very poorly expressed: in two separate experiments, 0 of 13 and 2 of 13 lines showed significant mouse beta/goat epsilon II RNA levels after induction. In all these lines the endogenous mouse beta and cotransfected mouse beta/human beta genes were expressed. As an initial test of possible reasons for the inactivity of the mouse beta/goat epsilon II hybrid, we recloned this fusion gene into a tk-bearing plasmid, adjacent to the tk gene. Of 12 cell lines transformed with this plasmid, 11 produced mouse beta/goat epsilon II RNA; in 6 cases the expression was both strong and dimethyl sulfoxide inducible.     

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.     

Fibroblast interferon in man is coded by two loci on separate chromosomes.

We have examined viral and poly(rl):poly(rC) induction of interferon synthesis in several human, mouse and Chinese hamster cell lines, and in hybrids derived from the fusion of such cells. We observed species and cell-type differences in inducer effectiveness and in the kinetics of interferon production. In some cases, parental characteristics are preserved in somatic cell hybrids, and in other cases, the expression of the donor phenotype is modulated by the epigenetic state of the recipient cell. Mapping studies in human/mouse and human/Chinese hamster hybrids indicate that there are at least two structural genes for human fibroblast interferon. Chromosomes 2 and 5 each contain genetic information for the synthesis of fibroblast interferon. Gene dosage experiments indicate that one gene is on the long arm of chromosome 2 and another is on the short arm of chromosome 5. Leukocyte interferon genes could not be mapped to these chromosomes, but this negative result could be influenced by the epigenetic state of the hybrid cells.     

Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells.

The structural gene encoding liver-specific tyrosine aminotransferase (TAT; EC 2.6.1.5) was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of murine Tat-1 gene sequences by genomic Southern blotting. This assignment demonstrated that the Tat-1 structural gene was not syntenic with Tse-1, a chromosome 11-linked locus that negatively regulates TAT expression in trans (A. M. Killary and R. E. K. Fournier, Cell 38:523-534, 1984). We also showed that the fibroblast Tat-1 gene was systematically activated in hepatoma X fibroblast hybrids retaining fibroblast chromosomes 8 in the absence of chromosome 11 but was extinguished in cells retaining both fibroblast chromosomes. Thus, the TAT structural genes of both parental cell types were coordinately regulated in the intertypic hybrids, and the TAT phenotype of the cells was determined by the presence or absence of fibroblast Tse-1.