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T Papayannopoulou

Publications and source records attributed to T Papayannopoulou.

At least 19 recordsLinked to original sources
PubMed1998

Targeted expansion of genetically modified bone marrow cells.

The ability to specifically target a mitogenic signal to a population of genetically modified primary cells would have potential applications both for gene and cell therapy. Toward this end, a gene encoding a fusion protein containing the FK506-binding protein FKBP12, fused to the intracellular portion of the receptor for thrombopoietin (mpl), was introduced into primary murine bone marrow cells. Dimerization of this fusion protein through the addition of a dimeric form of the drug FK506, called FK1012, resulted in a marked proliferative expansion of marrow cells that was restricted to the genetically modified population. FK1012's proliferative effect was sustained and reversible. An apparent preference for differentiation along the megakaryocytic lineage was observed. This approach allows for the specific delivery of a mitogenic signal to a population of genetically modified primary cells and may have applications for studies in hematopoiesis and receptor biology, and for gene and cell therapy.

Animals↗
PubMed1998

Anti-VLA4/VCAM-1-induced mobilization requires cooperative signaling through the kit/mkit ligand pathway.

Although a large body of data on mobilization have yielded valuable clues, the mechanism(s) dictating egress of stem/progenitor cells during baseline hematopoiesis and after their mobilization are poorly understood. We have previously provided functional in vivo evidence that cytoadhesion molecules, specifically the beta1 integrins, are involved in mobilization; however, the mechanism by which this was achieved was unclear. To provide further insights into the anti-very late antigen 4 (VLA4)/anti-vascular cell adhesion molecule 1 (VCAM-1)-induced mobilization, we used these antibodies to treat mutant mice with compromised growth factor receptor function. We found that mobilization by anti-VLA4 does not depend on a functional granulocyte colony-stimulating factor, interleukin-7 (IL-7), or IL-3alpha receptor. By contrast, the functional kit receptor is required, because W/Wv mice responded minimally, whereas Steel-Dickie (Sl/Sld) responded normally. Both Wv and Sl/Sld mice did not respond to anti-VCAM-1 treatment, in contrast to their +/+ littermates and despite normal levels of VCAM-1 expression in bone marrow cells. The defective response to anti-VCAM-1 in W/Wv mice was corrected after their transplantation with +/+ cells. mev/mev mice showed increased numbers of circulating progenitors before treatment and a heightened response after anti-VLA4 or anti-VCAM-1 treatment. Downmodulation of kit expression was detected in normal bone marrow cells after anti-VLA4 treatment. On the strength of the above findings we conclude that (1) anti-VLA4/VCAM-1-induced mobilization likely requires signaling for stimulation of cell migration; (2) this cooperative signaling involves the kit/kit ligand pathway, and provides a novel example of integrin/cytokine crosstalk; and (3) migration mediated through the kit/kit ligand pathway may be a common contributor to different mobilization stimuli. Dissection of the exact molecular pathways that lead to mobilization remains a future challenge.

Animals↗
PubMed1997

Antibodies to VLA4 integrin mobilize long-term repopulating cells and augment cytokine-induced mobilization in primates and mice.

Although the use of cytokine-mobilized peripheral blood stem cells has gained a significant momentum in clinical transplantation, the mobilization schemes practiced are guided by a great deal of empiricism. The mechanism(s) by which cytokines or chemokines, alone or in combination, bring about redistribution of stem/progenitor cells from bone marrow to peripheral blood are poorly understood. Likewise the fate of mobilized stem/progenitor cells and their biological properties are incompletely defined. One of the leading hypotheses to explain the mechanism of cytokine-induced mobilization encompasses the view that cytokines disrupt, directly or indirectly, cytoadhesive interactions of stem/progenitor cells with their bone marrow stroma. Compatible with this view are changes in the expression and/or function of several cytoadhesion molecules, especially integrins, postmobilization, and extensive in vitro experimentation supporting the concept of cytokine/integrin interactions. To provide a further insight on the cytokine/integrin interplay in vivo, we have combined cytokine treatments with anti-integrin treatments for mobilization in primates and mice. We found that anti-VLA4 treatment combined with either granulocyte colony-stimulating factor (G-CSF ) treatment or kit ligand treatment leads to significant enhancement of mobilization efficiency (fivefold to eightfold) well above the levels produced by either cytokine alone or anti-VLA4 treatment alone. Similar enhancement was seen when combinations of cytokines, ie, G-CSF plus kit ligand or G-CSF plus Flt3-ligand were used with anti-VLA4 in primates and mice. Furthermore, when anti-VLA4 was given in 5-Fluorouracil-treated primates, significant numbers of progenitor cells were circulating for several days during the recovery period only in the anti-VLA4 treated animals. These data suggest that (1) the effect of anti-VLA4 on mobilization, when used alone, is unlikely to be mediated by secondary cytokine elaboration in vivo; (2) three different cytokines and their combinations do not appear to influence the in vivo responsiveness to anti-VLA4 in coadministration schemes; (3) even if cytokine treatments on their own exert downmodulation of VLA4 function, the target progenitor cells influenced by anti-VLA4 or by cytokines may not necessarily overlap; and (4) augmentation of mobilization in cytokine/anti-VLA4 treatments is most likely caused by an amplification of the pool of target cells on which anti-VLA4 exerts its effects. Because cytokines or anti-VLA4 are each capable of mobilizing long-term repopulating cells and because we show with the present studies that anti-VLA4 in an autologous bone marrow cell transplantation setting does not cause any delay in engraftment, the combination of cytokine/anti-integrin treatment enhancing mobilization may have a clinical use.

Animals↗
PubMed1997

In vivo effects of Flt3/Flk2 ligand on mobilization of hematopoietic progenitors in primates and potent synergistic enhancement with granulocyte colony-stimulating factor.

The Flt3 receptor is expressed in primitive hematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To expand on the functional properties of Flt3 ligand (FL) in vivo we treated nonhuman primates with FL and tested its ability to mobilize stem/progenitor cells when given alone or in combination with granulocyte colony-stimulating factor (G-CSF) treatment. FL alone (200 microg/kg/day) mobilizes progenitors with slow kinetics and with a peak effect at the end of 2 weeks of treatment. The spectrum of mobilized progenitors includes myeloid, lymphoid, megakaryocytic, and osteoclastogenic but a low proportion of burst-forming unit (BFU)e. Bone marrow (BM) studies before and during the treatment suggested that proliferative effects in BM may have preceded effects on peripheral blood mobilization. To assess the synergy of FL with G-CSF in mobilization of progenitors we used two schemes: one in which G-CSF was used for the last 5 days of a 12-day treatment with FL; the other in which both cytokines were given concurrently for 5 days only (FL, 200 microg/kg; G-CSF, 100 microg/kg). Both schemes yielded much higher progenitor mobilization levels (peak levels of colony-forming cells [CFSs] 41,000 to 95,000/mL blood) than observed with either FL (CFC 4,600 to 7,300/mL) or G-CSF (8,405 +/- 3,024/ mL) used alone at the same doses. Furthermore, there was a progressive and significant expansion of progenitors in vitro during 2 weeks in suspension cultures of mononuclear cells or of CD34+ cells only in the animal with the combined treatment. Likewise, substantial mobilization of osteoclastogenic progenitors was documented only with the combined treatment. Given the functional properties of FL, its synergistic mobilization with G-CSF, and its anticipated good tolerance (because of the absence of an effect on mast cell activation), a clinical use is projected for this cytokine in peripheral blood transplantation settings, as well as in experiments with ex vivo gene transfer.

Animals↗
PubMed1997

Human platelets display high-affinity receptors for thrombopoietin.

Thrombopoietin (Tpo) is a major regulator of megakaryopoiesis both in vivo and in vitro. Tpo initiates its biologic effects by binding to the Mpl receptor, which is a member of the hematopoietin receptor family. To define the Tpo binding characteristics of the Mpl receptor, we iodinated purified 70-kD recombinant human Tpo using the Bolton-Hunter reagent. Autoradiographic analysis of (125)I-Tpo binding to normal human marrow mononuclear cells showed many grains specifically associated with megakaryocytes; there were no grains specifically associated with myeloblasts or erythroblasts. Equilibrium binding experiments with (125)I-Tpo and normal human platelets showed a single class of high-affinity receptors (kd, 190 pmol/L) with approximately 30 Mpl receptors per platelet. Affinity cross-linking with (125)I-Tpo showed that the Mpl receptor on platelets is of molecular weight approximately 98 kD. Despite their sequence similarity, erythropoietin and Tpo did not cross-compete for binding to BaF3 cells engineered to coexpress Mpl receptor and erythropoietin receptor. Progeny of normal human burst-forming units-erythroid (BFU-E) contained Mpl receptor mRNA, and flow cytometric analysis showed the presence of Mpl receptor protein on the surface of these cells. These data indicate that display of the Mpl receptor is not limited to the megakaryocytic lineage, but also includes progeny of BFU-E. Like receptors for other hematopoietic cytokines, the binding affinity of the Mpl receptor for Tpo is high, with relatively few receptors displayed per cell. These results suggest that the effects of Tpo to speed red blood cell recovery after myelosuppressive therapy in vivo and to enhance colony-forming unit-erythroid generation in vitro may be mediated by direct interaction of Tpo and erythroid progenitor cells.

Adult↗
PubMed1997

Cytokine prestimulation as a gene therapy strategy: implications for using the MDR1 gene as a dominant selectable marker.

A major obstacle to stem cell gene therapy is the extremely low efficiency of stem cell transduction. In vivo selection is a strategy for enriching a minor population of genetically modified bone marrow cells through the introduction of a drug resistance gene, followed by subsequent administration of the corresponding cytotoxic drug in vivo. Achieving persistent effects from in vivo selection is expected to require selection at the level of stem cells or, minimally, selection at the level of progenitors. Major limitations to in vivo selection are the nonhematologic toxicities of the cytotoxic drugs used and the resistance of stem cells and progenitors to killing by most cytotoxic agents. Experiments were performed in mice to evaluate whether the drugs used for selection in combination with multiple drug resistance gene 1 (MDR1) could have an enhanced effect on clonogenic progenitors if preceded by administration of the cytokine, stem cell factor (SCF). Single doses of taxol, navelbine, or vinblastine produced 10-fold reductions in the total number of mononuclear cells per femur, indicating a significant depletion of nonclonogenic precursor cells. However, for each of these agents, clonogenic progenitors, assayed as colony-forming unit cells and day-12 spleen colony-forming units, were relatively spared. Administration of SCF before taxol, navelbine, or vinblastine completely abrogated the progenitor-sparing phenomenon, because clonogenic progenitors were depleted as effectively as nonclonogenic precursor cells. Furthermore, the administration of SCF before drug administration allowed the dosages of taxol and vinblastine to be reduced by more than half, while retaining reductions in progenitor numbers that were unachievable using very high doses of the cytotoxic drug alone. Doxorubicin administration resulted in a 30- to 40-fold depletion in progenitors that was not significantly altered by preceding SCF administration. These results suggest that previous observations of in vivo selection using MDR1 gene transfer followed by taxol administration may have resulted from selection at the level of relatively mature, nonclonogenic precursor cells. Furthermore, these data suggest that cytokine prestimulation may be a useful strategy for improving the selection of drug-resistant clonogenic progenitors and, possibly, stem cells in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Homing and trafficking of hemopoietic progenitor cells.

Investigations of the homing of transplanted hemopoietic cells into preconditioned recipients have in most studies been referable to parameters that determine engraftment. The principles, however, that govern their early traffic into the host's blood and tissues have remained much less explored. Early studies and experiments from our own laboratory suggest that intravenously administered hemopoietic cells, including progenitors, are not selectively taken up by bone marrow, as they are distributed widely in several tissues (liver, lung, kidney, spleen, bone marrow). As the hemopoietic cells are later on found almost exclusively in the bone marrow (and spleen in the mouse), the data argue for either preferential retention in these tissues or just only preferential survival and proliferation. Our recent studies showing modulation of cells lodged to the bone marrow, before proliferation ensues (i.e. 3 h after infusion), would favor preferential retainment and/or survival of these cells within the bone marrow. Furthermore we established that the VLA4/VCAM-1 adhesion pathway plays a significant role in this process, thus defining VCAM-1 as the dominant bone marrow endothelial addressin in hemopoietic cell homing. Since homing likely represents a cascade of adhesive interactions between hemopoietic cells and bone marrow stroma and/or its extracellular matrix, other adhesion pathways are likely to be involved and remain to be defined. Finally, our data on mobilization of hemopoietic progenitors from normal individuals, induced by blocking the VLA4/VCAM-1 adhesion pathway, suggest that the molecular pathways involved in homing are also of importance in governing hemopoietic progenitor cell trafficking in and out of the bone marrow.

Animals↗
PubMed1996

The hematological effects of folate analogs: implications for using the dihydrofolate reductase gene for in vivo selection.

Previous studies have shown that dihydrofolate reductase (DHFR) gene transfer protects marrow from methotrexate (MTX)-mediated toxicity; however, MTX treatment in vivo has not convincingly been shown to enrich DHFR-transduced progenitors or stem cells. Experiments were performed to better characterize the hematological effects of MTX, and maneuvers were tested with the aim of improving the utility of MTX as an agent for in vivo selection. Progenitors were assayed as colony forming unit cells in culture (CFU-C) and in the spleens of irradiated mice (day 11 CFU-S). A single injection of MTX at doses up to 250 mg/kg (more than three times the LD10) failed to reduce CFU-C numbers significantly in the femur or spleen assayed 1-3 days later. However, consistent declines in the number of mononuclear cells per femur reflected a significant depletion of nonclonogenic precursor cells. Preceding administration of pegylated stem cell factor (SCF), 100 micrograms/kg per day, increased CFU-C killing by a single dose of 5-fluorouracil (5-FU) 15- to 65-fold in the femur, and 5- to 15-fold in the spleen, consistent with previous reports. In contrast, despite preceding SCF administration there was no significant progenitor killing by MTX. Similar results were obtained using a second folate analog, trimetrexate. These results suggest that the mechanism by which folate analogs exert their hematological toxicity is through the depletion of relatively mature, nonclonogenic precursor cells, and not by killing progenitors. This information is relevant to the use of DHFR in gene therapy protocols, and suggests that folate analogs are poorly suited agents for selection at the level of clonogenic progenitor cells in vivo.

Animals↗
PubMed1996

Globin gene switching. In vivo protein-DNA interactions of the human beta-globin locus in erythroid cells expressing the fetal or the adult globin gene program.

To characterize the protein-DNA interactions important for the developmental control of the human beta-globin locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult globin developmental program. In the locus control region (LCR) of the beta-globin locus, in vivo footprints on NF-E2 (or AP-1) and GATA-1 motifs remained the same regardless of whether the fetal or the adult globin genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult globin program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult globin expressing cells displayed more extensive footprints than the fetal globin expressing cells in the 3' regulatory sequences of both the Agamma- and the beta-globin genes, suggesting a role of these 3' elements in beta-globin gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of globin genes takes place in the gamma- and beta-globin gene promoters, and that GT motifs of the beta-globin locus LCR may play a role in the developmental regulation of human beta-globin gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult globin gene.

Adult↗
PubMed1996

Identification of new genes expressed in a human erythroleukemia cell line.

We cloned novel cDNAs from MB02 human erythroleukemia cells using PCR based approaches: a) Differential display by means of RT-PCR using one 5' primer CTTGATTGCC and four different 3' primers (T12AA, T12CA, T12GA, and T12AT). Ninety-three percent of the differential clones which were reamplified and sequenced were cDNAs of previously unidentified genes. b) Cloning using degenerate TFIIIA-like zinc finger domain specific oligonucleotide. Of the 54 clones sequenced, 20 contained two or more zinc finger sequences. Ten of these clones were new zinc finger cDNAs and one belonged to a known zinc finger gene (ZFP7). c) Cloning using degenerate tyrosine kinase(TK) domain-specific oligonucleotides corresponding to the highly conserved amino acid sequences IHRDLAA and DVWSFG. Of the 28 cDNA clones sequenced, 7 were JAK1 TK, one was atk TK, one was tec TK. The remaining sequences belonged to new ESTs or to ribosomal genes. d) Cloning using degenerate POU domain-specific oligonucleotides corresponding to sequence FK(QV)RRIK of the POU-specific domain and to sequence VWFCN(RQ)R of the POU-homeodomain. Sixteen clones were sequenced and all but one were identical with the Oct-1 transcriptional factor. Differential display RT-PCR and PCR-based cDNA cloning using degenerate primers for zinc finger motifs yielded the largest number of new genes expressed in MBO2 cells.

Amino Acid Sequence↗
PubMed1996

Biologic effects of thrombopoietin, the Mpl ligand, and its therapeutic potential.

In vitro experiments and data from studies in Mpl-null and thrombopoietin (TPO)-null mice have shown that TPO is the major regulator of megakaryocytopoiesis. In addition to its effect on megakaryocyte production and maturation, TPO has a proliferative effect on other progenitors, particularly erythroid progenitors, when used in combination with other cytokines (erythropoietin, interleukin 3, and kit ligand). The precise effect of TPO on platelet release is uncertain and warrants further study. In myeloablated animal models (mice and primates), TPO shortened the period of critical neutrocytopenia and accelerated the recovery of platelet counts, thereby showing great promise for use in patients postchemotherapy and posttransplantation.

Animals↗
PubMed1996

Human CD34+ cell EST database: single-pass sequencing of 402 clones from a directional cDNA library.

A directional cDNA library was constructed from human CD34+ cells, and single-pass DNA sequence analysis was used to sequence 402 randomly picked cDNA clones. Sequence homology searches against the GenBank/EMBL databases revealed that 144 clones (35.8%) represented new genes, whereas 39 clones (9.7%) matched the partial sequences of cDNA clones (expressed sequence tags [ESTs]) from cDNA libraries from other tissues or cells. One hundred three (25.6%) of the clones were highly homologous or identical to genes of known function. 18.6% of the clones were ribosomal and 6 % were mitochondrial sequences. Seventeen (4.2%) of the clones were coding for Alu or L1 repetitive sequences. Our results suggest that random single-pass sequencing of cDNA clones provides a rapid way to obtain a profile of genes expressed in CD34+ cells and to identify new and potentially interesting genes.

Antigens, CD34↗
PubMed1996

Insights into the cellular mechanisms of erythropoietin-thrombopoietin synergy.

Using suspension cultures of purified bone marrow CD34+ cells, we have analyzed the effects of the combination of erythropoietin (Epo) and thrombopoietin (Tpo) on the in vitro differentiation toward erythropoiesis and thrombopoiesis. The number of CD41+ cells that accumulated over 2 weeks of culture, as well as the number of globin+ cells in the same cultures, was found to be significantly higher with the Epo+Tpo combination compared to either cytokine alone. No evidence was found that Tpo affected the differentiative action of Epo. Instead, there was a significant expansion of erythroid progenitors, both erythroid colony-forming and burst-forming units (CRU-E and BFU-E), by 7 days in culture, suggesting a proliferative effect of Tpo on erythroid cells in vitro. To determine the phenotypic features of erythroid progenitor cells which were targets of Tpo's action, and specifically to inquire whether the effect was directed mainly toward bipotent erythroid/megakaryocytic (E+Mk) progenitors, we isolated subsets enriched for both erythroid and megakaryocytic progenitors from CD34+ cells. We found that 1) BFU-E and CFU-Mk co-segregate in the subset of CF34+ cells that is negative for the phosphatase isoform CD45RA; 2) the presence of CD41 on this subset appears to segregate late erythroid and late CFU-Mk from early erythroid and early CFU-Mk, which are CD41-negative; 3) bipotent erythroid/Mk progenitors, studied by single-cell culture assays, were found mainly in the CD41+ and rarely in the CD41- subsets which included more multipotent progenitors; 4) by comparing the frequencies of pure erythroid or pure megakaryocytic progenitors to that of bipotent E+Mk progenitors, we conclude that the erythroid-enhancing effect of Tpo is directed mainly toward pure erythroid progenitors expressing CD41 and Mpl, as suggested by independent experiments employing anti-Mpl antibody, rather than only on bipotent E+Mk progenitors.

Antigens, CD34↗
PubMed1995

Stimulation of fetal hemoglobin production by short chain fatty acids.

Butyrate, a four-carbon fatty acid, and its two-carbon metabolic product, acetate, are inducers of gamma-globin synthesis. To test whether other short-chain fatty acids share this property, we first examined whether propionic acid, a three-carbon fatty acid that is not catabolized to acetate, induces gamma-globin expression. Sodium propionate increased the frequency of fetal hemoglobin containing erythroblasts and the gamma/gamma + beta mRNA ratios in adult erythroid cell cultures and F reticulocyte production in a nonanemic juvenile baboon. Short-chain fatty acids containing five (pentanoic), six (hexanoic), seven (heptanoic), eight (octanoic), and nine (nonanoic) carbons induced gamma-globin expression (as measured by increase in gamma-positive erythroblasts and gamma/gamma + beta mRNA ratios) in adult erythroid burst-forming unit cultures. There was a clear-cut relationship between the concentration of fatty acids in culture and the degree of induction of gamma-globin expression. Three-, four-, and five-carbon fatty acids were better inducers of gamma globin in culture as compared with six- to nine-carbon fatty acids. These results suggest that all short-chain fatty acids share the property of gamma-globin gene inducibility. The fact that valproic acid, a derivative of pentanoic acid, also induces gamma-globin expression suggests that short-chain fatty acid derivatives that are already approved for human use may possess the property of gamma-globin inducibility and may be of therapeutic relevance to the beta-chain hemoglobinopathies.

Adult↗
PubMed1995

The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen.

Selective lodgement or homing of transplanted hemopoietic stem cells in the recipient's bone marrow (BM) is a critical step in the establishment of long-term hemopoiesis after BM transplantation. However, despite its biologic and clinical significance, little is understood about the process of homing. In the present study, we have concentrated on the initial stages of homing and explored the functional role in vivo of some of the adhesion pathways previously found to mediate in vitro adhesion of hemopoietic cells to cultured BM stroma. We have found that homing of murine hemopoietic progenitors of the BM of lethally irradiated recipients at 3 h after transplant was significantly reduced after pretreatment of the donor cells with an antibody to the integrin very late antigen 4 (VLA4). This inhibition of marrow homing was accompanied by an increase in hemopoietic progenitors circulating in the blood and an increased uptake of these progenitors by the spleen. Similar results were obtained by treatment of the recipients with an antibody to vascular cell adhesion molecule 1 (VCAM-1), a ligand for VLA4. Furthermore, we showed that administration of the same antibodies (anti-VLA4 or anti-VCAM-1) to normal animals causes mobilization of hemopoietic progenitors into blood. These data suggest that hemopoietic cell lodgement in the BM is a regulatable process and can be influenced by VLA4/VCAM-1 adhesion pathway. Although additional molecular pathways are not excluded and may be likely, our data establish VCAM-1 as a BM endothelial addressin, analogous to the role that mucosal addressin cell adhesion molecule (MAdCAM) plays in lymphocyte homing. Whether splenic uptake of hemopoietic progenitors is passive or controlled through different mechanisms remains to be clarified. In addition, we provide experimental evidence that homing and mobilization are related phenomena involving, at least partly, similar molecular pathways.

Animals↗
PubMed1995

Transcriptional activation of human adult alpha-globin genes by hypersensitive site-40 enhancer: function of nuclear factor-binding motifs occupied in erythroid cells.

The developmental stage- and erythroid lineage-specific activation of the human embryonic zeta- and fetal/adult alpha-globin genes is controlled by an upstream regulatory element [hypersensitive site (HS)-40] with locus control region properties, a process mediated by multiple nuclear factor-DNA complexes. In vitro DNase I protection experiments of the two G+C-rich, adult alpha-globin promoters have revealed a number of binding sites for nuclear factors that are common to HeLa and K-562 extracts. However, genomic footprinting analysis has demonstrated that only a subset of these sites, clustered between -130 and +1, is occupied in an erythroid tissue-specific manner. The function of these in vivo-occupied motifs of the alpha-globin promoters, as well as those previously mapped in the HS-40 region, is assayed by site-directed mutagenesis and transient expression in embryonic/fetal erythroid K-562 cells. These studies, together with our expression data on the human embryonic zeta-globin promoter, provide a comprehensive view of the functional roles of individual nuclear factor-DNA complexes in the final stages of transcriptional activation of the human alpha-like globin promoters by the HS-40 element.

Adult↗
PubMed1995

Coexpression of epsilon, G gamma and A gamma globin mRNA in embryonic red blood cells from a single copy beta-YAC transgenic mouse.

We utilized reverse transcription polymerase chain reaction (RT-PCR) to amplify epsilon, G gamma and A gamma globin cDNAs from single red blood cells isolated from a day-10 transgenic fetus harboring a single copy of the human beta-YAC. A detailed structural analysis of the beta-YAC showed a single copy of each beta-like globin gene is present and linked to the locus control region (LCR). RNase protection analysis of RNA isolated from erythroid tissues from day-8 to day-16 of development and the adult stage showed proper developmental switching of the beta-like globin gene expression. Using epsilon / gamma and G gamma / A gamma primer sets in separate RT-PCR reactions on RNA from single day-10 red blood cells we observed an intercellular variation in the epsilon and gamma RT-PCR products that may be indicative of a change in the LCR preference from the epsilon gene promoter to the gamma gene promoter during switching. We also found that the majority of the red blood cells examined contain all three globin mRNA species. These observations suggest that either the LCR is capable of interacting simultaneously with more than one globin gene promoter or alternatively, the LCR may interact with only one promoter at any given time, but its interaction oscillates between promoters (flip-flop mechanism) resulting in expression of more than one gene from a single beta-globin locus.

Animals↗