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.     

Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.     

Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination

GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the beta-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the beta- and delta-globin genes also allowed the simultaneous generation of a delta-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the gamma-globin genes and the termination codon of the beta-globin gene enabled the generation of reporter constructs for both (A)gamma- and (G)gamma-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the epsilon- and beta-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.     

Isolation of the chicken beta-globin gene and a linked embryonic beta-like globin gene from a chicken DNA recombinant library.

A library of random chicken DNA fragments, 15-22 kb long, has been prepared in the vector lambda Charon 4A. This library was screened with combined adult and embryonic globin cDNA, and several independent globin gene-containing recombinants were isolated. One of these recombinants, lambda Chicken beta-globin 1 (lambda C beta G1), contains the adult chicken beta-globin gene and a closely linked embryonic beta-like globin gene. Both genes are transcribed in the same direction with the adult gene located 5' to the embryonic gene. Electron microscopic visualization of R loop structures generated by hybridization of globin RNA to lambda C beta G1 demonstrates that both globin genes contain major intervening sequences about 800 bp long, similar to those present in mammalian beta-globin genes. The adult beta-globin gene also contains a minor (approximately 100 bp long) intervening sequence analogous to the one observed in mammalian beta-globin genes. Restriction enzyme analysis of the adult beta-globin gene on lambda C beta G1 is consistent with the hypothesis that its two intervening sequences occur in the same positions with respect to the beta-globin amino acid sequence as do the corresponding mammalian intervening sequences.     

Restriction endonuclease mapping of gamma-delta-beta-globin region in G gamma (beta)+ HPFH and a Chinese A gamma HPFH variant.

Restriction endonuclease mapping of the beta-globin genomic region was used for studying the molecular basis of two variants of hereditary persistence of fetal hemoglobin (HPFH): an African G gamma (beta)+ HPFH and a Chinese HPFH variant with predominant synthesis of A gamma chains. HPFH and control DNA samples were digested with a battery of restriction enzymes, and the fragments were identified by hybridization to a family of discrete probes. DNA fragments from the A gamma HPFH (Chinese) and the G gamma (beta)+ HPFH individuals were identical with those of the normal controls. These findings suggest that the two mutants are the result of small structural anomalies of DNA sequences that play a role in the regulation of the expression of gamma-globin genes.     

Regulated expression of human A gamma-, beta-, and hybrid gamma beta-globin genes in transgenic mice: manipulation of the developmental expression patterns

We have introduced the human fetal gamma- and adult beta-globin genes into the germ line of mice. Analysis of the resulting transgenic mice shows that the human gamma-globin gene is expressed like an embryonic mouse globin gene; the human beta-globin gene is expressed (as previously shown) like an adult mouse globin gene. These results imply that the regulatory signals for tissue- and developmental stage-specific expression of the globin genes have been conserved between man and mouse but that the timing of the signals has changed. Because the two genes are expressed differently, we introduced a hybrid gamma beta-globin gene construct. The combination of the regulatory sequences resulted in the expression of the hybrid gene at all stages in all the murine erythroid tissues.     

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.     

Cleavage map of the simian-virus-40 genome by the restriction endonuclease III of Haemopholus aegyptius.

Enzymic digestion of Simian virus 40 (SV40) DNA with Haemophilus aegyptius restriction endonuclease Hae III results in 10 major and eight minor fragments. These were resolved by electrophoresis on graduated polyacrylamide slab gels. All fragments have been characterized with respect to the size relative to the Haemophilus influenzae Rd fragments (Hind). They were ordered on the SV40 DNA map by means of overlap analysis of the double cleavage products derived from sequential digestion of Hind fragments with Hae III endonuclease and Hae fragments with Hind II + III enzyme, as well as by other reciprocal cleavage experiments, including those involving Haemophilus para-influenzae fragments. In this way the 18 Hae III cleavage sites and the 13 Hind sites have been localized on the circular SV40 DNA map.     

Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel type of secondary modification of two CCGG residues in the human gamma delta beta-globin gene locus

The majority of the CCGG residues in the human gamma delta beta-globin gene locus are cleaved by Msp I, irrespective of the tissue of origin of the DNA, although these sites show differential sensitivity to Hap II as a result of methylation of the internal C residue of the cleavage site (ref 6). Two CCGG sites, at homologous positions 54 nucleotides in front of the G gamma- and A gamma-globin genes respectively, are not cleaved by Msp I in DNA from several human tissues, although DNA from placenta, foetal liver and from some established cell lines is cut at these sites. We have cloned the A gamma-globin gene from foetal blood DNA where the relevant CCGG site is not cut by Msp I. After cloning, the CCGG site can be cut by Msp I. The failure to cleave at this CCGG site in foetal blood DNA therefore, is not the result of a change in the DNA sequence of the cleavage site. Most likely the external C residue and perhaps both C residues are blocked by methylation at these two specific sites.     

Amplification and characterization of a beta-globin gene synthesized in vitro.

Full-length, double-stranded globin DNA was synthesized in vitro starting from rabbit globin mRNA. Several restriction endonuclease cleavage sites with known recognition sequences were mapped on this DNA as a means of assessing the accuracy of in vitro synthesis. By comparing this map with the nucleotide sequences known or predicted from the amino acid sequences of alpha-and beta-chain rabbit hemoglobin, it was possible to show that the synthetic globin DNA is a faithful copy of beta-globin mRNA. Amplification of the synthetic globin DNA was achieved by inserting the molecule into the plasmid PMB9 using the poly(dA)-(dT) joining procedure, and transforming E. coli with the hybrid DNA. Transformants carrying beta-globin DNA were identified by colony hybridization using purified 125I-beta-mRNA probe. Comparison of the restriction maps of the synthetic and inserted globin DNAs showed that the entire synthetic globin DNA molecule was amplified without sequence rearrangements. Both the synthetic and the cloned DNA include the entire coding sequence of the beta-globin gene plus a substantial portion of the untranslated regions flanking the structural gene.     

Identification of the Hind III polymorphic site in the PAI-1 gene: analysis of the PAI-1 Hind III polymorphism by PCR

The identification of the Hind III polymorphic site in the 3' end of the plasminogen activator inhibitor 1 (PAI-1) gene and a simple method to identify the Hind III polymorphism rapidly in the PAI-1 gene using PCR is described. The Hind III restriction site was identified by restriction site mapping and sequence analysis from a cosmid DNA clone. Genomic DNA was isolated from individual human umbilical cords and a 754-bp fragment of the human PAI-1 gene was amplified by PCR. Aliquots of the PCR products were digested with Hind III and analyzed by agarose gel electrophoresis. The presence of two fragments, 754 and 567 bp, was identified, and they were designated as 1/1 (750-bp band), 1/2 (754- and 567-bp bands), and 2/2 (567-bp band). The PCR method is considerably less time consuming than the conventional DNA genotyping using Southern blot analysis. To ensure that this new method identified the same PAI-1 genotypes as previously identified by Hind III restriction fragment length polymorphism (RFLP), samples were simultaneously genotyped by PCR and Southern blot analysis. Both methods identified the same Hind III genotypes in all the samples, confirming the reliability of this new PCR method for the rapid identification of the Hind III polymorphism in the human PAI-1 gene.     

A complete cleavage map of Neurospora crassa mtDNA obtained with endonucleases Eco RI and Bam HI.

A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases. The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III. The sequence of the Eco RI and Bam HI fragments has been established by analysis of partial fragments. By digestion of the Eco RI fragments with Bam HI, a complete overlapping map has been constructed. The position of the largest Hind III fragment on this map has also been determined. The map is circular and the added molecular weight of the fragments is 40 - 10(6), which is in good agreement with earlier measurements on intact DNA, using the electron microscope.     

Heterogeneity of the gamma-globin gene sequences in Japanese individuals: implication of gene conversion in generation of polymorphisms

The linked fetal globin genes (the G gamma- and A gamma-globin genes) were cloned from Japanese individuals with three different haplotypes of the HindIII polymorphisms within the gamma-globin genes. Determination of nucleotide sequences of the segment spanning from IVS2 to the 3' flanking region of each gamma-globin gene revealed that nucleotide differences are located at 43 positions and a stretch of simple GT or GC sequences. Almost half of the nucleotide changes could be accounted for by gene conversion between the G gamma- and A gamma-globin genes. We found that gene conversion had created the SacI polymorphic site just downstream of the A gamma-globin coding region. Association of the SacI polymorphic site with the HindIII polymorphic site suggests that the region containing these two sites was derived from that of the linked G gamma-globin gene through a gene conversion event. The nucleotide sequences obtained here are identical to those of the Caucasoid fetal globin genes of the same haplotypes, with the exception of some sequence changes in the hot spots of mutations. These results indicate that the sequence heterogeneity of the gamma-globin genes can be classified into three major categories according to HindIII haplotypes. The possible mechanisms of generation of the heterogeneity of the gamma-globin gene sequences are discussed.     

The ovalbumin gene: alleles created by mutations in the intervening sequences of the natural gene.

Two allelic forms of the natural chicken ovalbumin gene have been independently cloned. These alleles differ from each other by an Eco RI restriction cleavage site in one of the seven intervening sequences within the natural ovalbumin gene. Restriction endonuclease mapping and sequence analyses of these cloned genotypic alleles have shown identical sequence organization and molecular structures of the interspersed structural and intervening sequences except for the particular Eco RI cleavage site. Sequencing data of the cloned DNA suggest that this Eco RI site may be created or eliminated by a single base mutation in the intervening sequence of the ovalbumin gene. The occurrence of apparent homozygous and heterozygous allelic forms of the ovalbumin gene in individual hens and roosters within the same breed has been observed. 10 and 40% of the chickens examined are homozygous for the ovalbumin gene with and without the extra Eco RI site, respectively, while 50% of them are heterozygous. Further analysis of individual chicken DNA cleaved by restriction endonuclease Hae III has revealed that there may be a series of such mutational variations within the ovalbumin gene. We have identified two Hae III cleavage sites that do not occur in all of the chickens, thus giving rise to several additional allelic variations of the ovalbumin gene. At least one of these Hae III sites is situated in the intervening sequence of the ovalbumin gene, and its lcoation has been mapped. Such allelic variations must be taken into consideration when determining eucaryotic gene structure by restriction mapping of the genomic DNA. Furthermore, this type of mutation within the intervening sequences of an eucaryotic gene has no known phenotypic manifestation. It represents an extrastructural silent mutation that must be taken account of in studies to estimate the rates of eucaryotic gene sequence divergence during evolution.     

Abnormal arrangements in the alpha- and gamma-globin gene clusters in a relatively large group of Japanese newborns.

Data were obtained on blood samples from a relatively large group (264) of healthy Japanese newborns, collected at hospitals in Tokyo, Kurashiki, and Ube. The studies included an evaluation of anomalies in alpha-globin gene and gamma-globin gene arrangements using gene mapping and gamma-chain composition analyses. The results confirmed the rarity of alpha-thalassemia among Japanese; only a few babies had alpha-thalassemia-2 trait (the -3.7-kilobase [kb] deletion), while others had alpha-globin gene triplications (both the alpha alpha alpha anti-3.7 and the alpha alpha alpha anti-4.2 types). Among the gamma-globin gene anomalies that were observed, a few babies had the -A gamma-A gamma- globin gene arrangement or the -G gamma A gamma- type of deletion. The gamma-chain triplication (-G gamma-A gamma G gamma-A gamma-) occurred in 10 out of 256 newborns, and its frequency exceeded that of its corresponding -G gamma A gamma- deletion by a factor of 5. The restriction endonuclease XmnI was a useful tool, in addition to the enzymes Bg1II and BclI, to evaluate and confirm the gamma-globin gene deletion and triplication. The A gamma T variant, which is the product of a mutant A gamma-globin gene, occurred at a frequency of 0.156. The chromosome carrying this mutant A gamma gene had a characteristic haplotype that was originally seen in black and Mediterranean patients with Hemoglobin (Hb) S or with beta-thalassemia.     

CG sites and their nucleotide environment in the human gamma-delta-beta-globin gene cluster: distribution, frequency and possible frame for differential methylation

Available restriction endonucleases including CG dinucleotides in their target sequences (most of them being unable to cut the DNA when the cytosine of the CG sequence is methylated) have been used to map cloned DNA covering the human gamma-delta-beta globin gene cluster. Since the human DNA fragments were cloned in Escherichia coli, only the internal cytosine in the sequence CCAT GG could be methylated. Thus, any recognized "CG enzyme" site can be detected since they are unmethylated. Results show that frequencies of "CG enzyme" sites regularly decrease from the gamma-globin region to the beta-globin region, the latter being very poor in "CG enzyme"' sites. The array of enzymes used here detects 4 times more CG sites than the classical MspI/HpaII system. Examination of previously sequenced parts of the gamma-delta-beta globin gene cluster shows that CG dinucleotides correspond to an average frequency of 1 out of 104 nucleotides in the gamma-globin region and 1 out of 138 nucleotides in the beta-globin region. In the gamma-globin region, 1 CG out of 4 or 5 may be detected by the enzymes used; the detected frequency is less than 1 out of 10 CG in the beta-region. Analysis of nucleotide environment around CG dinucleotides shows occurrence of local differences, the main sequences being CGG in the 5' side flanking the gamma genes and ACG in the corresponding area of the beta gene. The results presented introduce some new considerations about analysis of cytosine methylation which has been previously proposed as playing a role in the control of the activity of gamma, delta and beta genes respectively.