Deletions within the mouse beta-globin locus control region preferentially reduce beta(min) globin gene expression

The mouse beta-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 beta(min) and beta(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 beta(min) expression over beta(maj). The implications of these findings for the mechanism by which the LCR controls expression of the beta(maj) and beta(min) promoters are discussed.     

The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.     

The nucleotide sequence of rabbit embryonic globin gene beta 3

The nucleotide sequence of a rabbit embryonic globin gene, beta 3, has been determined from 161 base pairs (bp) on the 5' side of the mRNA cap site to 209 base pairs beyond the 3' poly A addition site. The 5' and 3' ends of mRNA from both embryonic globin genes beta 3 and beta 4 have been determined by an S1 protection assay. Sequences that are highly conserved in the 5' flanking region of eukaryotic structural genes, AATAAAA and CCAAT, are located -25 to -31 nucleotides and -81 to -85 nucleotides, respectively, before the cap site. The CCAAT sequence is duplicated at -108 to -112 nucleotides, as it is in the human fetal gamma-globin genes. Small (124 bp) and large (817 bp) intervening sequences are located between codons 30 and 31 and between 104 and 105, respectively. The sequence AATAAA precedes the predominant poly(A) addition site by 19 nucleotides. Although rabbit globin gene beta 3 is transcribed and translated almost exclusively in embryonic erythrocytes, it shares striking homology with the human gamma-globin genes which are expressed in erythrocytes from fetal liver. The evolutionary conservation of rabbit beta 3 and human gamma correlates well with their similar chromosomal positions in the two genes families.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

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.     

Hemoglobin switching in sheep. Cloning and characterization of the beta A and beta-like embryonic globin genes from genomic DNA

Genomic DNA from a fetal sheep homozygous for the beta A gene was used to construct a library of one million cloned DNA fragments using the bacteriophage vector, Charon 4A. Screening of 150,000 plaques from this library using radioactive beta-globin gene sequences resulted in the isolation of two recombinant bacteriophage containing globin genes. One of these, S beta AG-21, contains the complete adult beta A-globin gene as demonstrated by hybridization and restriction endonuclease analysis. In common with adult globin genes from other species, the beta A gene contains small (105 base pairs) and large (900 base pairs) intervening sequences. The second recombinant bacteriophage, SG-4, contains a complete embryonic beta-like globin gene which is expressed in the sheep embryo as demonstrated by hybridization analysis with cDNA made from sheep embryonic globin mRNA. Although differing in its restriction endonuclease map from the adult beta-globin genes, SG-4 appears to contain a large intervening sequence of at least 750 base pairs in length. Finally, preliminary evidence is discussed which indicates that a Pvu II site just 5' to the Cap site may be a common feature of sheep globin genes.     

Demonstration of an enhancer activity in a fragment of DNA located at the 3' of the adult alpha globin gene in the duck

We found an enhancer element placed at the 3' side of the adult duck alpha A globin gene. The duck alpha globin gene cluster contains three genes from the 5' to 3' side: the pi embryonic gene, the alpha D minor adult gene and the alpha A adult major gene. We analyzed a 16 kb genomic domain extending from 2 kb upstream of the pi gene to 5 kb downstream of the alpha A gene. This enhancer is active in AEV transformed chicken erythroblasts. Its is inactive both in HeLa cells and in the human erythroid cells K562 which express only embryonic genes. These findings are discussed in relation to previous results concerning the duck beta globin enhancer located at the 3' side of the beta A globin gene.     

Expression of a beta thalassemia gene with abnormal splicing.

Expression of a cloned human beta thalassemia gene with a single base change at position 5 of IVS 1 has been analyzed 48 hours after transfer of the gene into HeLa cells (transient expression). Little or no normal beta globin mRNA accumulates in the presence of the abnormal beta gene in contrast to significantly more normal beta mRNA produced with other mutations at this same position. By contrast, large amounts of an abnormal beta globin mRNA are present; this is due to the use of a cryptic 5' splice site in exon 1 rather than the normal 5' splice site of IVS 1. The results indicate the variability of the effect on RNA splicing of different single base defects within IVS.