Expression Of Human Beta-globin Gene Research Articles

Expression of chimeric human beta- and delta-globin genes during erythroid differentiation.

To determine whether sequences contained within the small intervening sequence (IVS 1) or large intervening sequence (IVS 2) are involved in the regulated expression of the human beta-globin gene, chimeric genes containing portions of the human beta- and delta-globin genes were stably transfected into mouse erythroleukemia (MEL) cells. Since MEL cells can be induced to differentiate in culture, the expression of the chimeric genes was compared to the expression of beta and delta both before and after the induction of erythroid differentiation. The expression of beta delta 1, a beta-globin gene containing delta IVS 1 in place of beta IVS 1, was comparable to the expression of a beta-globin gene both before and after erythroid differentiation. However, the base-line expression of human beta-globin genes containing delta IVS 2 in place of beta IVS 2 was dramatically decreased. Furthermore, the substitution of delta IVS 2 for beta IVS 2 prevented the regulated increase in expression of the beta-globin gene upon induction. The results also indicate that sequences present in beta IVS 2 are not sufficient for this induced increase in expression since the substitution of beta IVS 2 for delta IVS 2 in a delta gene does not increase the regulated expression of delta during differentiation. These experiments suggest that either the presence of delta IVS 2 in a beta gene interrupts sequences required for the induced expression of beta-globin or that sequences in beta IVS 2 act in concert with other beta globin sequences not present in the delta-globin gene to permit optimal expression.

Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element.

Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element.

Erythroid-specific expression of human beta-globin genes in transgenic mice.

Transgenic mice carrying human beta-globin genes were produced by microinjecting linear DNA molecules containing cloned beta-globin genes with up to 4300 bp of 5'-flanking sequence and 1700 bp of 3'-flanking sequence. Most (15 of 20) of these transgenic mice expressed the human beta-globin genes in blood cells and the level of expression in some mice was comparable with that obtained from endogenous beta-globin genes. Human beta-globin gene expression appeared to be restricted to cells of the erythroid lineage and was first detected between 11 and 14 days of development, in parallel with mouse beta-globin. Constructs with as little as 48 bp of 5'-flanking sequence also appeared to be expressed appropriately. The mRNA transcripts had correct 5' ends and directed human beta-globin synthesis in reticulocyte lysates. Human beta-globin protein was detectable in mature erythrocytes from progeny of one of these mice. The frequency and extent of expression was severely depressed when the procaryotic vector DNA was not removed prior to microinjection.

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

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.