A silent deletion in the beta-globin gene cluster.

A survey of the gamma-globin gene region of over 1000 normal individuals revealed a novel 2.5 kb deletion which removes the 5' end of the A gamma-globin gene. Unusually, this deletion in the beta-globin gene cluster is not associated with increased fetal haemoglobin production. Sequence analysis of the deletion endpoints revealed no significant homology at the breakpoint and failed to support a role for a proposed recombination hotspot in IVS-2 in the generation of this illegitimate recombination event. The existence of small "silent" deletions in the beta-globin gene cluster emphasizes the importance of deletion size in altering expression of the fetal globin genes.     

Nonuniform recombination within the human beta-globin gene cluster.

Population genetic analysis of 15 restriction site polymorphisms demonstrates nonuniform recombination within the human beta-globin gene cluster. These DNA polymorphisms show two clusters of high nonrandom associations, one 5' and another 3' to the beta-globin structural gene, with no significant linkage disequilibrium between the two clusters. The 5'- and 3'-association clusters are 34.6 kilobases (kb) and 19.4 kb long, respectively, and are separated by 9.1 kb of DNA immediately 5' to the beta-globin gene. For each of these three DNA regions, we have observed a relationship between nonrandom associations and physical distance between the polymorphisms. However, this relationship differed for each of these regions. On the assumption that the effective population size (Ne) is 5,000-50,000, we estimate the total recombination rate to be 0.0017%-0.0002% in the 5' cluster, 0.0931%-0.0093% in the 3' cluster, and 0.2912%-0.0219% in the 9.1-kb region between them. The beta cluster thus shows nonuniformity in recombination. Moreover, the recombination rate in the 9.1-kb DNA segment is 3-30 times greater than expected and is thus a hot spot for meiotic recombination.     

Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus.

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5'-rho-beta H-beta A-epsilon-3'). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of rho to just downstream of epsilon. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the rho-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of rho, beta H, and epsilon. (v) A site 3' of beta A and 5' of epsilon was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo II sites. These data are consistent with the hypothesis that, in vivo, topo II preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.     

Characterization of a topoisomerase-like activity at specific hypersensitive sites in the Drosophila histone gene cluster

It is well known that treatment of DNA-topoisomerase complexes with SDS induces cleavage of the DNA by trapping a reactive intermediate in which the topoisomerase is covalently linked to the terminal phosphates of the cut DNA. I have used this technique to examine potential topoisomerase binding sites in the histone gene chromatin of Drosophila Kc cells. Treatment of Kc nuclei with SDS induces Mg++-dependent DNA cleavage near the borders of two nuclease-hypersensitive sites located 5' and 3' of histone H4. It is likely that the SDS-induced cleavage at these hypersensitive sites is due to a topoisomerase because protein becomes tightly bound to the ends of the cleaved DNA fragments. Preliminary experiments suggest that a type II topoisomerase may be responsible for the cleavage.     

Restriction site polymorphisms in the pig beta-globin gene cluster

A restriction fragment length polymorphism was detected in pig DNA digested with Hind III restriction endonuclease and probed with rabbit beta 1-globin gene. Eight different phenotypes were observed and for six of them family data demonstrated that they are determined by three alleles. As this polymorphism is not found with four other restriction endonucleases (Bam HI, Eco RI, Kpn I, and Pst I), single point mutations are proposed to explain the observed differences.     

Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement

The major distal regulatory sequence for the beta-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.     

Detection of a novel DNA polymorphism in the beta-globin gene cluster

Analysis of DNA from the beta-globin gene cluster in an Albanian family identified a novel RsaI site approximately 550 base pairs 5' to the beta-globin gene. In this family, two chromosomes carrying otherwise identical beta-globin haplotypes were found to differ at the RsaI site. Population screening demonstrated the presence and absence of the site in DNA from individuals of northern European, Mediterranean, Middle Eastern, Southeast Asian, African, and Asian Indian descent, indicating that this site is a DNA polymorphism common in many ethnic groups. The polymorphism is also present in DNA from individuals carrying different beta-globin alleles. Additional nucleotide sequence changes identified in an RsaI (+) genomic clone in the region immediately 3' to the RsaI site suggest a mechanism for the randomization of the site with respect to haplotype.     

Isolation and characterization of recombinant clones containing the chicken adult beta-globin gene.

We have isolated and characterized two independent clones containing the chicken adult beta-globin gene. Each clone contains a 6.2-kilobase-pair Eco RI restriction fragment of chicken erythrocyte DNA inserted into the vector, lambda gtWES . lambda B. The orientation of the inserted fragment is opposite in the two clones. Characterization of the clones by electron microscopic R-loop studies, by restriction enzyme mapping, and by filter hybridization shows that the adult beta-globin gene is interrupted by at least one small and one large intervening sequence. In addition to the complete adult beta-globin gene, at least part of a second beta-globin-like gene was identified about 2.7 kilobase pairs from the 3'-end of the adult gene. The two independent clones, while very similar, do differ at two Msp I restriction endonuclease sites in regions flanking the adult beta-globin gene.     

Hemoglobin K?ln: analysis of linkage relationships between the mutant gene and polymorphic restriction sites in the beta-globin gene cluster

Nuclear DNA has been analyzed by means of restriction endonuclease mapping procedure to identify chromosomes that carry mutant Hb K?ln beta-globin genes in a family with individuals heterozygous for this disease. Inherited DNA polymorphisms within the beta-globin gene cluster yielded a direct linkage of the Hb K?ln mutation to haplotype constellations that are diagnostic for further offspring.     

Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts.