Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human ^Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin ^Aγ-globin (βsp/^Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/^Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the ^Aγ-globin gene promoter showed developmental switching and expressed ^Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.     

Deletions within the Mouse β-Globin Locus Control Region Preferentially Reduce β Globin Gene Expression

The mouse β-globin gene cluster is regulated, at least in part, by a locus control region (LCR) composed of several developmentally stable DNase I hypersensitive sites located upstream of the genes. In this report, we examine the level of expression of the β^min and β^maj genes in adult mice in which HS2, HS3, or HS5,6 has been either deleted or replaced by a selectable marker via homologous recombination in ES cells. Primer extension analysis of RNA extracted from circulating reticulocytes and HPLC analysis of globin chains from peripheral red blood cells revealed that all mutations that reduce the overall output of the locus preferentially decrease β^min expression over β^maj. The implications of these findings for the mechanism by which the LCR controls expression of the β^maj and β^min promoters are discussed.     

Characterization of the 5′-to-5′linked adult α- and β-globin genes from three sciaenid fish species (Pseudosciaena crocea, Sciaenops ocellatus, Nibea miichthioides)

Recently, we cloned the adult α-globin genes from large yellow croaker Pseudosciaena crocea, cuneate drum Nibea miichthioides and red drum Sciaenops ocellatus. All these α-globins have a unique Gly insertion at the 47th residue. In this paper, the three sciaenid globin complexes were identified and compared in detail. Linkage analysis indicated that the sciaenid α- and β-globin genes were oriented head-to-head relative to each other. The sciaenid intergenic regions between the linked α- and β-globin genes were the smallest in reported fish globin gene complexes to date. Classical promoter elements were condensed and the CCAAT box unstable duplication was found in these regions. The promoter function of the intergenic region from large yellow croaker was tested by transient expression of EGFP in Vero cells. We also described a method for studying luciferase reporter gene transient expression in primary fish erythrocytes. We used the method to assess the promoter strength of the three intergenic regions between the sciaenid α- and β-globin genes.     

Characterization of a Widely Expressed Gene (LUC7-LIKE; LUC7L) Defining the Centromeric Boundary of the Human α-Globin Domain

We have identified the first gene lying on the centromeric side of the α-globin gene cluster on human 16p13.3. The gene, called 16pHQG;16 (HGMW-approved symbol LUC7L), is widely transcribed and lies in the opposite orientation with respect to the α-globin genes. This gene may represent a mammalian heterochromatic gene, encoding a putative RNA-binding protein similar to the yeast Luc7p subunit of the U1 snRNP splicing complex that is normally required for 5′ splice site selection. To examine the role of the 16pHQG;16 gene in delimiting the extent of the α-globin regulatory domain, we mapped its mouse orthologue, which we found to lie on mouse chromosome 17, separated from the mouse α-cluster on chromosome 11. Establishing the full extent of the human 16pHQG;16 gene has allowed us to define the centromeric limit of the region of conserved synteny around the human α-globin cluster to within an 8-kb segment of chromosome 16.     

The tkNeo gene, but not the pgkPuro gene, can influence the ability of the beta-globin LCR to enhance and confer position-independent expression onto the beta-globin gene

Whether drug-selectable genes can influence expression of the beta-globin gene linked to its LCR was assessed here. With the tkNeo gene placed in cis and used to select transfected cells, the beta-globin gene was expressed fourfold lower when it was positioned upstream of the LCR rather than downstream. This difference did not occur when the pgkPuro gene replaced tkNeo. Moreover, the beta-globin gene situated upstream of the LCR was transcribed without position effects when it was cotransfected with a pgkPuro-containing plasmid, whereas cotransfection with a tkNeo plasmid gave measurable position effects. Previous results from transfected cells selected via a linked tkNeo gene suggested that the 3' end of the beta-globin gene has no impact on LCR-enhanced expression. Here, removal of the 3' end of the beta-globin gene resulted in lower and much more variable expression in both transgenic mice and cells cotransfected with pgkPuro. Together, the results suggest that tkNeo, but not pgkPuro, can strongly influence expression of the beta-globin gene linked to its LCR. The findings could partly explain why data on beta-globin gene regulation obtained from transfected cells have often not agreed with those obtained using transgenic mice. Hence, one must be careful in choosing a drug-selectable gene for cell transfection studies.     

The sequence and phylogenesis of the α-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion)

In order to investigate the polymorphism of α-globin chain of hemoglobin amongst caprines, the linked ^Iα and ^IIα globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5′ and 3′ untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, had almost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different α genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the ^Iα-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.     

Conservation of Position and Sequence of a Novel, Widely Expressed Gene Containing the Major Human α-Globin Regulatory Element

We have determined the cDNA and genomic structure of a gene (−14 gene) that lies adjacent to the human α-globin cluster. Although it is expressed in a wide range of cell lines and tissues, a previously described erythroid-specific regulatory element that controls expression of the α-globin genes lies within intron 5 of this gene. Analysis of the −14 gene promoter shows that it is GC rich and associated with a constitutively expressed DNase 1 hypersensitive site; unlike the α-globin promoter, it does not contain a TATA or CCAAT box. These and other differences in promoter structure may explain why the erythroid regulatory element interacts specifically with the α-globin promoters and not the −14 gene promoter, which lies between the α promoters and their regulatory element. Interspecies comparisons demonstrate that the sequence and location of the −14 gene adjacent to the α cluster have been maintained since the bird/mammal divergence, 270 million years ago.     

Activation of β-Globin Promoter by Erythroid Krüppel-Like Factor

Erythroid Krüppel-like factor (EKLF), an erythroid tissue-specific Krüppel-type zinc finger protein, binds to the β-globin gene CACCC box and is essential for β-globin gene expression. EKLF does not activate the γ gene, the CACCC sequence of which differs from that of the β gene. To test whether the CACCC box sequence difference is the primary determinant of the selective activation of the β gene by EKLF, the CACCC boxes of β and γ genes were swapped and the resulting promoter activities were assayed by transient transfections in CV-1 cells. EKLF activated the β promoter carrying a γ CACCC box at a level comparable to that at which it activated the wild-type β promoter, whereas EKLF failed to activate a γ promoter carrying the β CACCC box, despite the presence of the optimal EKLF binding site. Similar results were obtained in K562 cells. The possibility that overexpressed EKLF superactivated the β promoter carrying the γ CACCC box, or that EKLF activated the mutated β promoter through the intact distal CACCC box, was excluded. To test whether the position of the CACCC box in the β or γ promoter determined EKLF specificity, the proximal β CACCC box sequence was created at the position of the β promoter (−140) which corresponds to the position of the CACCC box on the γ promoter. Similarly, the β CACCC box was created in the position of the γ promoter (−90) corresponding to the position of the CACCC box in the β promoter. EKLF retained weak activation potential on the β^−140CAC promoter, whereas EKLF failed to activate the γ^−90βCAC promoter even though that promoter contained an optimal EKLF binding site at the optimal position. Taken together, our findings indicate that the specificity of the activation of the β promoter by EKLF is determined by the overall structure of the β promoter rather than solely by the sequence of the β gene CACCC box.     

Cloning and sequence analysis of a cDNA for the α-globin mRNA of carp, Cyprinus carpio

A cDNA for α-globin mRNA of the carp, Cyprinus carpio, was cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2 (1982) 161–170) and its complete nucleotide sequence was determined. The 5′ non-coding region contained 23 nucleotides. Following this region, there was an open reading frame encoded with an α-globin polypeptide consisting of 142 amino acids. The 3′ non-coding region was 88 nucleotides in length, including two copies of the hexanucleotide AATAAA and a poly(A) site of the GC dinucleotide. There were 16 discrepancies between the reported amino acid sequence of the carp α-globin chain and the amino acid sequence predicted from the DNA sequence of the clone. The possible explanations for these differences in amino acid sequence are discussed.     

Derivatives of β- -fructofuranosyl α- -galactopyranoside

De-etherification of 6,6′-di-O-tritylsucrose hexa-acetate (2) with boiling, aqueous acetic acid caused 4→6 acetyl migration and gave a syrupy hexa-acetate 14, characterised as the 4,6′-dimethanesulphonate 15. Reaction of 2,3,3′4′,6-penta-O-acetylsucrose (5) with trityl chloride in pyridine gave a mixture containing the 1′,6′-diether 6 the 6′-ether 9, confirming the lower reactivity of HO-1′ to tritylation. Subsequent mesylation, detritylation, acetylation afforded the corresponding 4-methanesulphonate 8 1′,4-dimethanesulphonate 11. Reaction of these sulphonates with benzoate, azide, bromide, and chloride anions afforded derivatives of β- -fructofuranosyl α- -galactopyranoside (29) by inversion of configuration at C-4. Treatment of the 4,6′-diol 14 the 1,′4,6′-triol 5, the 4-hydroxy 1′,6′-diether 6 with sulphuryl chloride effected replacement of the free hydroxyl groups and gave the corresponding, crystalline chlorodeoxy derivatives. The same 4-chloro-4-deoxy derivative was isolated when the 4-hydroxy-1′,6′-diether 6 was treated with mesyl chloride in N,N-dimethylformamide.     

1′,2′-Dideacetylboronolide, an α-pyrone from Iboza riparia

The structural elucidation of 1′,2′-dideacetylboronolide, 5,6-dihydro-6-(3′-acetoxy-1′,2′-dihydroxyheptyl)2-pyrone, a new α-pyrone isolated from the leaves of Iboza riparia has been performed. Additionally, three sterols, sitosterol, stigmasterol and campesterol, have been identified in this species.     

Organization of the V Gene Segments in Mouse T-Cell Antigen Receptor α/δ Locus

The mouse T-cell receptor (TCR) α/δ locus was mapped using 17 Vα and 4 Vδ subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The α/δ locus spans about 1 Mb. The distance between the 3′-most V gene segment (Vδ1) and the δ constant gene (Cδ) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual Vα gene segment subfamilies are dispersed throughout the locus. In contrast, the Vδ gene segments Vδ1 to 5 are clustered at the 3′ end of the V gene segment cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the α/δ locus and on organizational features that might influence the expression of specific V gene segments in γδ cells.