Β-globin cDNA Research Articles

CD34+ cells from dental pulp stem cells with a ZFN-mediated and homology-driven repair-mediated locus-specific knock-in of an artificial β-globin gene.

In humans, mutations in the β-globin gene (HBB) have two important clinical manifestations: β-thalassemia and sickle cell disease. The progress in genome editing and stem cell research may be relevant to the treatment of β-globin-related diseases. In this work, we employed zinc-finger nuclease (ZFN)-mediated gene integration of synthetic β-globin cDNA into HBB loci, thus correcting almost all β-globin mutations. The integration was achieved in both HEK 293 cells and isolated dental pulp stem cell (DPSCs). We also showed that DPSCs with an artificial gene knock-in were capable of generating stable six-cell clones and were expandable at least 108-fold; therefore, they may serve as a personalized stem cell factory. In addition, transfection with non-integrated pCAG-hOct4 and culturing in a conditioned medium converted the genome-edited DPSCs to CD34+ HSC-like cells. We believe that this approach may be useful for the treatment of β-globin-related diseases, especially the severe form of β-thalassemia.

Molecular Cloning and Expression of α-Globin and β-Globin Genes from Crocodile (Crocodylus siamensis)

The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co(2+)-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein.

Evidence that a consensus element found in naturally intronless mRNAs promotes mRNA export

We previously showed that mRNAs synthesized from three genes that naturally lack introns contain a portion of their coding sequence, known as a cytoplasmic accumulation region (CAR), which is essential for stable accumulation of the intronless mRNAs in the cytoplasm. The CAR in each mRNA is unexpectedly large, ranging in size from ∼160 to 285 nt. Here, we identified one or more copies of a 10-nt consensus sequence in each CAR. To determine whether this element (designated CAR-E) functions in cytoplasmic accumulation of intronless mRNA, we multimerized the most conserved CAR-E and inserted it upstream of β-globin cDNA, which is normally retained/degraded in the nucleus. Significantly, the tandem CAR-E, but not its antisense counterpart, rescued cytoplasmic accumulation of β-globin cDNA transcripts. Moreover, dinucleotide mutations in the CAR-E abolished this rescue. We show that the CAR-E, but not the mutant CAR-E, associates with components of the TREX mRNA export machinery, the Prp19 complex and U2AF2. Moreover, knockdown of these factors results in nuclear retention of the intronless mRNAs. Together, these data suggest that the CAR-E promotes export of intronless mRNA by sequence-dependent recruitment of the mRNA export machinery.

Epstein-Barr virus protein EB2 stimulates cytoplasmic mRNA accumulation by counteracting the deleterious effects of SRp20 on viral mRNAs.

The Epstein–Barr Virus (EBV) protein EB2 (also called Mta, SM and BMLF1), is an essential nuclear protein produced during the replicative cycle of EBV. EB2 is required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes. EB2 is an RNA-binding protein whose expression has been shown to influence RNA stability, splicing, nuclear export and translation. Using a yeast two-hybrid screen, we have identified three SR proteins, SF2/ASF, 9G8 and SRp20, as cellular partners of EB2. Then, by using siRNA to deplete cells of specific SR proteins, we found that SRp20 plays an essential role in the processing of several model mRNAs: the Renilla luciferase reporter mRNA, the human β-globin cDNA transcript and two EBV late mRNAs. These four mRNAs were previously found to be highly dependent on EB2 for their efficient cytoplasmic accumulation. Here, we show that SRp20 depletion results in an increase in the accumulation of these mRNAs, which correlates with an absence of additive effect of EB2, suggesting that EB2 functions by antagonizing SRp20. Moreover, by using RNA-immunoprecipitation assays we found that EB2 enhances the association of SRp20 with the β-globin transcript suggesting that EB2 acts by stabilizing SRp20’s labile interactions with the RNA.

Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

Letter to the editor] Application of linear polyacrylamide coprecipitation of denatured templates for PCR amplification of ultra-rapidly reannealing DNA

Letter to the editor] Application of linear polyacrylamide coprecipitation of denatured templates for PCR amplification of ultra-rapidly reannealing DNA

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

Erythroid-Specific Expression of β-Globin by the Sleeping Beauty Transposon for Sickle Cell Disease

Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type beta- or gamma-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type beta-globin expression cassette into the human genome for sustained expression of beta-globin. We initially constructed a beta-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken beta-actin promoter (CAGGS) and full-length beta-globin cDNA, as well as truncated forms lacking either the 3' or 3' and 5' untranslated regions (UTRs), to optimize expression of beta-globin. Beta-globin with its 5' UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-beta-globin plasmids using a minimal beta-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, ankyrin-1 promoter), IHbetap (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, beta-globin promoter), or HS3betap (HS3 core element from human betaLCR, beta-globin promoter) to establish erythroid-specific expression of beta-globin. Stable genomic insertion of the minimal gene and expression of the beta-globin transgene for >5 months at a level comparable to that of the endogenous gamma-globin gene were achieved using a SB-Tn beta-globin cis construct. Interestingly, erythroid-specific expression of beta-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of beta-globin.

Assembly of Recently Translated Human γ-Globin Chains with a Pool of α-Globin Chains To Form Hb F in a Cell-Free System.

A pool of free α chains as well as hemin appears compatible with cellular homeostasis and important for ensuring efficient assembly with non-α globin chains to form functional hemoglobin tetramers in erythroid cells. Furthermore, globin-chain assembly with partner chains and efficient assembly of subunits to form functional hemoglobin is critical to prevent accumulation of unstable single globin chains which could lead to cell destruction. Efficient assembly also is critical for optimizing gene therapy for the treatment of hemoglobinopathies, since one therapeutic scenario involves expression of excess β-chain variants or γ-globin chains. We studied determinants for efficient assembly of αγ hetero-dimers followed by tetrameric Hb F formation. Indeed, γ-globin chains in Hb F are negatively charged; and, therefore, they would be expected to assemble efficiently with positively charged α chains. However, Hb F formation in vitro using purified γ- and α-globin chains is very slow compared to Hb A formation from purified β- and α-globin chains. This slow Hb F formation in vitro is to some extent due to stable γ2-dimer formation. Differences in amino acids at α1β1 and α1γ1 interaction sites and differences in surface charge also lead to different in vitro assembly rates for Hb A and Hb F. In contrast, expressed radiolabeled γ- and β-globin chains made in a wheat germ, coupled cell-free transcription/translation system using γ- and β-globin cDNA expression vectors with excess unlabeled α chains added prior to translation initiation showed similar formation rates for radiolabeled Hb A and Hb F. These results suggested in the absence of free α chains the newly translated monomeric γ chains form stable homo-dimers in erythroid cells, which leads to lower levels of αγ dimers. Therefore, we assessed formation of Hb A and Hb F after expression of radiolabeled β- or γ-globin chains in a wheat germ, coupled cell-free transcription/translation system. Results indicate that assembly of Hb F, but not Hb A, is enhanced by early addition of excess free α chains that act by inhibiting the formation of stable γ2 homo-dimers. Mutational analysis of truncated γ-globin chains showed that removal of more than 11 but not 2 amino acids from the carboxy terminus of γ chains inhibits assembly with α chains. Polysome profile studies showed that all carboxy-terminal deleted γ-chain mutants retained on polysomes due to removal of the translation termination codon in the cDNA expression vector do not assemble with unlabeled holo α chains. However, the two amino acid-deleted γ chains coded from vectors lacking a stop codon only assembled with α chains after puromycin-induced polysome release. In addition, small amounts of radiolabeled α γ dimers were detected on polysomes in reactions using wild type γ-chain templates only when excess unlabeled α chains were present. Together, our results suggest that Hb F formation is enhanced by a stable pool of free α chains that interact with newly synthesized γ chains, either before or soon after their release from the ribosome and prior to formation of stable γ2 homo-dimers.

3′-Untranslated Region of Phosphoenolpyruvate Carboxykinase mRNA Contains Multiple Instability Elements That Bind AUF1

Phosphoenolpyruvate carboxykinase (PEPCK) is regulated solely by alterations in gene expression that involve changes in rates of PEPCK mRNA transcription and degradation. A tetracycline-responsive promoter system was used to quantify the half-life of various chimeric beta-globin-PEPCK (betaG-PCK) mRNAs in LLC-PK -F(+) cells. The control betaG mRNA was extremely stable (t(1/2) = 5 days). However, betaG-PCK-1 mRNA, which contains the entire 3'-UTR of the PEPCK mRNA, was degraded with a half-life of 1.2 h. RNase H treatment indicated that rapid deadenylation occurred concomitant with degradation of the betaG-PCK-1 mRNA. Previous studies indicate that PCK-7, a 50-nucleotide segment at the 3'-end of the 3'-UTR, binds an unidentified protein that may contribute to the rapid decay of the PEPCK mRNA. However, the chimeric betaG-PCK-7 mRNA has a half-life of 17 h. Inclusion of the adjacent PCK-6 segment, a 23-bp AU-rich region, produced the betaG-PCK-6/7 mRNA, which has a half-life of 3.6 h. The betaG-PCK-3 mRNA that contains the 3'-half of 3'-UTR was degraded with the same half-life. Surprisingly, the betaG-PCK-2 mRNA, containing the 5'-end of the 3'-UTR, was also degraded rapidly (t((1/2)) = 5.4 h). RNA gel shift analyses established that AUF1 (hnRNP D) binds to the PCK-7, PCK-6, and PCK-2 segments with high affinity and specificity. Mutational analysis indicated that AUF1 binds to a UUAUUUUAU sequence within PCK-6 and the stem-loop structure and adjacent CU-region of PCK-7. Thus, AUF1 binds to multiple destabilizing elements within the 3'-UTR that participate in the rapid turnover of the PEPCK mRNA.

Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement.

As an adhesion receptor, the beta2 integrin lymphocyte function-associated antigen-1 (LFA-1) contributes a strong adhesive force to promote T lymphocyte recirculation and interaction with antigen-presenting cells. As a signaling molecule, LFA-1-mediates transmembrane signaling, which leads to the generation of second messengers and costimulation resulting in T cell activation. We recently have demonstrated that, in costimulatory fashion, LFA-1 activation promotes the induction of T cell membrane urokinase plasminogen activator receptor (uPAR) and that this induced uPAR is functional. To investigate the mechanism(s) of this induction, we used the RNA polymerase II inhibitor 5, 6-dichloro-1-beta-D-ribobenzimidazole and determined that uPAR mRNA degradation is delayed by LFA-1 activation. Cloning of the wild-type, deleted and mutated 3'-untranslated region of the uPAR cDNA into a serum-inducible rabbit beta-globin cDNA reporter construct revealed that the AU-rich elements and, in particular the nonameric UUAUUUAUU sequence, are crucial cis-acting elements in uPAR mRNA degradation. Experiments in which Jurkat T cells were transfected with reporter constructs demonstrated that LFA-1 engagement was able to stabilize the unstable reporter mRNA containing the uPAR 3'-untranslated region. Our study reveals a consequence of adhesion receptor-mediated signaling in T cells, which is potentially important in the regulation of T cell activation, including production of cytokines and expression of proto-oncogenes, many of which are controlled through 3' AU-rich elements.

Cloning and sequencing of cDNAs of the β-globin gene family in carp.

A total blood cell cDNA library was constructed using a 3-year-old carp Cyprinus carpio. A β-globin cDNA (CβG1) was identified from the library by the polymerase chain reaction using a β-globin-specific primer deduced from the carp β-globin-A amino acid sequence. Also, five additional types of β-globin cDNAs (CβG2∼6) were isolated by colony hybridization using CβG1 as a probe. Sequence analysis revealed that these CβGs encoded 147 amino acids, and the deduced amino acid sequences showed high identity (89·1-95·2%) to previously reported carp β-globin amino acid sequences. The nucleotide sequences of the CβGs were very similar (identity 96·0-99·6%) and the expression levels of CβG1∼6 were 28·6, 28·6, 21·4, 14·3, 3·6 and 3·6% of the total number of cloned CβGs, respectively. Although the complete amino acid sequence identities between the CβGs and the β-globin of higher vertebrates were low, functionally important regions such as the α-βcontact region and haem contact region were well conserved. These data showed that, as in higher vertebrates, the adult carp has a multiple β-globin gene family (at least six members). However, transcripts encoding four types of peptides (CβG1 type, CβG2 and 3 type, CβG5 type, and CβG4 and 6 type) were expressed at relatively high levels, this being a unique character of the carp haemoglobin system.

Do the hemoglobinless icefishes have globin genes?

Among piscine taxa, the Antarctic icefishes (family Channichthyidae) prosper in the absence of erythrocytes and hemoglobin, a unique condition among adult vertebrates. The genomes of three icefish species and four red-blooded notothenioid species were probed using α- and β-globin cDNA from the red-blooded Antarctic fish Notothenia coriiceps. High-stringency hybridization signals with the α-globin probe, but none with the β-globin probe, on genomic DNAs of both hemoglobinless and red-blooded fishes suggest that icefishes retain remnants of α-globin genes in their genomes but have lost the gene that encodes for β-globin, either through deletion or through rapid mutation. Northern blot analysis of major hematopoietic and non-hematopoietic tissues shows that the α-globin-related sequences of icefishes are nonexpressed derivatives of the α-globin genes of their red-blooded relatives. Mechanisms leading to the hemoglobinless phenotype are discussed in relation with the expression of myoglobin in the Channichthyidae family.

The mouse histone H2a gene contains a small element that facilitates cytoplasmic accumulation of intronless gene transcripts and of unspliced HIV-1-related mRNAs.

Histone mRNAs are naturally intronless and accumulate efficiently in the cytoplasm. To learn whether there are cis-acting sequences within histone genes that allow efficient cytoplasmic accumulation of RNAs, we made recombinant constructs in which sequences from the mouse H2a gene were cloned into a human beta-globin cDNA. By using transient transfection and RNase protection analysis, we demonstrate here that a 100-bp sequence within the H2a coding region permits efficient cytoplasmic accumulation of the globin cDNA transcripts. We also show that this sequence appears to suppress splicing and can functionally replace Rev and the Rev-responsive element in the cytoplasmic accumulation of unspliced HIV-1-related mRNAs. Like the Rev-responsive element, this sequence acts in an orientation-dependent manner. We thus propose that the sequence identified here may be a member of the cis-acting elements that facilitate the cytoplasmic accumulation of naturally intronless gene transcripts.

Characterization of Adult α- and β-Globin Genes in the Zebrafish

AbstractDevelopmental switching of hemoglobins (Hbs) occurs in most vertebrates, yet the cellular and molecular basis for this process remains elusive. The zebrafish is a new genetic and developmental system that can be used to study embryogenesis, and mutants with a variety of defects in hematopoiesis have recently been derived. To initiate our studies on Hb switching in this organism, we have characterized the globins expressed in the adult. Reversed-phase high performance liquid chromatography and mass spectrometric analyses of adult peripheral blood hemolysates showed that there are three major α globins and two β globins in circulating erythroid cells. In addition, we have isolated and characterized zebrafish adult α- and β-globin cDNA clones that encode some of these globins. High levels of α- and β-globin gene expression were detected in adult erythroid cells, whereas embryonic erythroid cells expressed little, if any, of these RNAs. We have also shown that the α- and β-globin genes are tightly linked on the same chromosome and are arrayed in a 3′-5′ to 5′-3′ configuration, respectively. The characterization of these genes and regulatory elements in this globin locus will provide insight into the process of globin gene transcription. With these reagents, future studies of Hb switching in zebrafish mutants with defective hematopoiesis will be possible.

Synthesis of Human Globin Polypeptides Mediated by Recombinant Adeno-Associated Virus Vectors

Adeno-associated virus, serotype 2 (AAV2)-based chimeric plasmids that harbored a near-full-length human α- or β-globin cDNA were constructed. The cDNAs were spliced into an AAV plasmid, pAAV∆K, downstream from the viral P40 promoter, substituting the capsid gene region. The correctness of the insertion with regard to the transcriptional polarity was ascertained by both restriction enzyme analysis and DNA sequencing. One of the constructs, pAAVcHBBLCR, contained the erythroid-specific enhancer elements, the locus control region, HS1 and HS2, to ensure an efficient and tissue-specific gene expression. Use of a defective complementing helper, pAVXB (Dixit, M.; et al. Gene1991, 104, 253-257.) and adenovirus 2 made it possible to prepare recombinant AAVs (rAAVs). Infection of human 293 cells (embryonal kidney cell line) with the resultant rAAV (AAVcHBB) and cotransfection of mouse erythroleukemia (MEL) cells with the β-globin construct (pAAVcHBBLCR) and an α-globin construct (pAAVcHAB) triggered efficient synthesis of human globin polypeptides in the cells, as analyzed by biochemical and immunohistochemical means. The LCR made the construct respond to an inducer, N,N'-hexamethylenebisacetamide, the amount of expressed human β-globin reaching a similar level as the endogenous mouse β-globin in MEL cells. Electrotransfection of mouse bone marrow hematopoietic stem/progenitor cells with the constructs dramatically increased the number of benzidine-positive cells in liquid suspension culture, indicating expression and synthesis of a human hemoglobin in these cells. Thus, the rAAV constructs may be useful for gene therapy of hemoglobinopathies.

An Episomal Expression Vector System for Monitoring Sequence-Specific Effects on mRNA Stability in Human Cell Lines

A plasmid expression system has been developed which allows sequence-specific effects on mRNA degradation rates to be determined. This system uses stable, nonintegrating vectors that provide consistent levels of mRNA expression without the position effects common to integrating vectors. cDNAs encoding putative instability elements may be subcloned into the 5′ untranslated region (5′UTR), the coding region, or the proximal 3′UTR of a β-globin cDNA reporter. The effects of these sequences on mRNA stability may then be determined by actinomycin time course analyses of the fusion mRNAs and recombinant β-globin mRNA in human cell lines. To demonstrate the utility of the vector system we fused an 820-bp fragment of the cDNA encoding the proximal 3′UTR of human 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase to the 3′UTR of the β-globin reporter and introduced the vector into the human hepatocarcinoma cell line, HepG2. The fusion mRNA was degraded at a rate 2- to 2.5-fold greater than that of β-globin alone, at a rate similar to that reported for HMG CoA reductase mRNA in normal rat liver. Similar to a number of other relatively unstable mRNAs, the rate of fusion mRNA degradation was greatly decreased by treatment with cycloheximide.

Construction and replication of an adeno-associated virus expression vector that contains human β-globin cDNA

With the goal of using adeno-associated viruses (AAV) as the gene-transfer vector for gene therapy of hemoglobinopathies, human β-globin cDNA was ligateddownstream from the P40 promoter of the AAV type-2 (AAV2) genome. To circumvent difficulties of cloning DNA containing palindromic sequences, two of which exist in the termini of AAV genome, a step-wise approach handling one palindrome at a time was devised to construct the chimeric expression vector. Electroporation of the construct into human 293 cells (embryonal kidney cell line) resulted in expression of the cloned human β-globin cDNA, as evidenced by the synthesis of transcripts hybridizable to human β-globin cDNA probe. Addition of the 3′-end region of AAV DNA that contains both the transcription termination signal and origin of DNA replication for AAV to the construct permitted the recombinant AAV genome to be rescued and replicate in the cell.

A cell type-specific enhancer drives expression of the chick muscle acetylcholine receptor α-subunit gene

The regulation of acetylcholine receptor α-subunit gene expression was analyzed by transient expression assays. Using rabbit β-globin cDNA as a reporter gene, we have confirmed that the 5′-flanking sequence of the chicken acetylcholine receptor a-subunit gene directs specific expression in differentiated C2C12 cells, a mouse muscle cell line, but not in undifferentiated C2C12 cells and mouse 3T3 fibroblasts. Testing chimeric plasmids containing Bal31 deletion mutants of the a-subunit gene upstream sequence, we found the - 116 to -81 region of the α-subunit to be responsible for tissue- and stage-specific expression. This 36 by fragment stimulates the activity of both α-subunit and SV40 promoters in a distance- and orientation-independent manner, thus fulfilling the criteria of an enhancer.

The β-major and β-minor globin genes in murine erythroleukemia cells replicate during the same early interval of the S phase

The time of replication in S phase was determined for the 7.3 kb EcoRI segment containing the β-major globin gene and the 14 kb EcoRI segment containing the β-minor globin gene in a murine Friend erythroleukemia virus transformed (MEL) cell line. Cells were obtained from different intervals of S phase by centrifugal elutriation to avoid artifacts of chemical synchronization. Newly synthesized DNA from different parts of S phase were obtained by isolation of 5-bromouracil (BU) labelled DNA from these cells. The BU-DNA synthesized during four different intervals of S was transferred to diazobenzyloxymethyl (DBM) paper and hybridized with a β-globin cDNA probe. Quantitation showed that both β-globin segments were replicated in the first quarter of the S phase with no significant difference in their time of replication in this MEL cell line.