High-resolution chromosomal localization of the beta-gene of the human beta-globin gene complex by in situ hybridization

A 4.4-kb PstI fragment containing the entire beta-gene of the human beta-globin gene cluster plus both 5'- and 3'-flanking sequences was used as a probe to study the chromosomal localization of the beta-gene by in situ hybridization. Using random oligonucleotides as primers, the beta-gene DNA was 3H-labeled with the large fragment of DNA polymerase I (Klenow fragment) to a specific activity of 1.2 X 10(8) cpm/micrograms. Almost 80% of hybridization grains observed were located on the distal short arm of chromosome 11. High-resolution chromosome analysis suggests a more precise location of the beta-gene to region 11p15.4----p15.5.     

High-mobility group protein 2 may be involved in the locus control region regulation of the beta-globin gene cluster.

Expression regulation of the beta-globin gene cluster is a result of synergistic interactions between cis-elements and trans-acting factors. Previous studies usually concentrated on the core sequence of each hypersensitive site in the locus control region of the beta-globin gene cluster. But more and more evidence illustrates that the flanking regions are indispensable also. Using electrophoretic mobility shift assay and solid-phase DNase I footprinting methods, we identified a small nuclear protein from K562 cells that binds specifically to the first AT-rich region flanking the hypersensitive site 2 core sequence of the human beta-globin gene locus control region. N-terminal sequencing of the enriched protein proved that it is a member of the high-mobility group protein 2 family. This indicates that the AT-rich region in human hypersensitive site 2 may take part in the regulation of the beta-globin gene cluster by facilitating DNA bending, which is a prerequisite for the looping mechanism in this region.     

The human myoglobin gene: a third dispersed globin locus in the human genome.

Human myoglobin is specified by a single gene. Unique sequence DNA probes were isolated from the cloned gene and used to test for the presence of the human myoglobin gene in a series of human rodent somatic cell hybrids containing various complements of human chromosomes. The myoglobin gene cosegregated with human chromosome 22. Somatic cell hybrids containing translocation chromosomes carrying part of chromosome 22 were used to locate the myoglobin gene to the region 22q11----22q13. The myoglobin gene is therefore not linked to the alpha-globin gene cluster on chromosome 16 or the beta-globin cluster on chromosome 11, and represents a third dispersed globin locus in the human genome.     

Beta-globin gene haplotypes in Polynesians are predominantly southern Chinese in type

beta-Globin gene haplotypes obtained in Polynesian Samoans were similar to those described in Southern Chinese. An atypical HindIII restriction fragment length polymorphism detected with pRK29, a 3' beta-globin gene probe, was present at a gene frequency of 7% in Samoans. Haplotype patterns suggest that this polymorphism may have arisen by 1 or 2 mutational events. DNA haplotypes derived from the beta-globin gene cluster confirm nuclear and mitochondrial DNA data that Polynesian precursor populations were East Asian in origin.     

An intron nucleotide sequence variant in a cloned beta +-thalassaemia globin gene.

A 7.5 kb Hsu I restriction fragment of genomic DNA containing a beta-globin gene has been isolated from a patient doubly heterozygous for beta + thalassaemia and a delta beta (Lepore globin fusion gene. This fragment must be derived from the chromosome carrying the beta +-thalassaemia determinant. The gross structure of the cloned gene plus flanking sequences is indistinguishable from that of a normal beta-globin gene. Within in 1606 base-pair transcribed region of the gene there is only one nucleotide difference from the normal beta-globin gene sequence. This is a G leads to A replacement 21 nucleotides upstream from the 3' terminus of the small intron. This nucleotide lies within a 10 base-pair sequence repeated in an inverted configuration near the 5' terminus of the small intron. The nucleotide replacement may result in a precursor mRNA less amenable to RNA splicing than its normal counterpart.     

Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning the oxazolomycin biosynthetic gene cluster from Streptomyces albus JA3453

Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. Using degenerate primers to clone genes encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from Streptomyces albus JA3453 was cloned and found to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned genes in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment containing six genes for methoxymalonyl-ACP biosynthesis from the specific region of the chromosome, as well as deletion of the ozmC gene within this region, to generate OZM-nonproducing mutants.     

Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.     

Variability in nuclear DNA among nonhuman primates: Application of molecular genetic techniques to intra- and inter-species genetic analyses

Nuclear DNA clones and sequence information derived from human genetic analyses were used to detect and characterize intra- and inter-species DNA variation at several nuclear loci in hominoids and cercopithecoids. Restriction fragment length polymorphisms were found at five loci among captive rhesus monkeys. Cross-species polymerase chain reaction (PCR) amplification detected an insertion within the beta-globin gene cluster in hylobatids. The combined use of cross-species PCR and denaturing gradient gel electrophoresis detected both species differences and intra-species polymorphism in the homeobox cluster 2 of hominoids. These results a) demonstrate that DNA clones and nucleotide sequence information from human molecular genetics can be used to facilitate studies of the molecular genetics of nonhuman primates, and b) document specific examples of intra- and inter-species molecular variability at several loci. © 1992 Wiley-Liss, Inc.     

The detection of a cluster of beta-globin genes in the long arm of chromosome 2 in the domestic hen by using DNA-DNA in situ hybridization]

Using isotopic and nonisotopic methods of in situ hybridization, the localization the beta-like globin gene cluster was established. Molecular hybridization of DNA probes containing embryonic epsilon-globin and adult beta-globin genes revealed the localization of the cluster of the long arm on chromosome 2 (2q).     

Signals in chicken beta-globin DNA influence chromatin assembly in vitro.

We have confirmed the result that chicken beta-globin gene chromatin, which possesses the characteristics of active chromatin in erythroid cells, has shortened internucleosome spacings compared with bulk chromatin or that of the ovalbumin gene, which is inactive. To understand how the short (approximately 180-bp) nucleosome repeat arises specifically on beta-globin DNA, we have studied chromatin assembly of cloned chicken beta-globin DNA in a defined in vitro system. With chicken erythrocyte core histones and linker histone H5 as the only cellular components, a cloned 6.2-kb chicken beta-globin DNA fragment assembled into chromatin possessing a regular 180 +/- 5-bp repeat, very similar to what is observed in erythroid cells. A 2-kb DNA subfragment containing the beta A gene and promoter region, but lacking the downstream intergenic region between the beta A and epsilon genes, failed to generate a regular nucleosome array in vitro, suggesting that the intergenic region facilitates linker histone-induced nucleosome alignment. When the beta A gene was placed on a plasmid that contained a known chromatin-organizing signal, nucleosome alignment with a 180-bp periodicity was restored, whereas nucleosomes on flanking plasmid sequences possessed a 210-bp spacing periodicity. Our results suggest that the shortened 180-bp nucleosome spacing periodicity observed in erythroid cells is encoded in the beta-globin DNA sequence and that nucleosome alignment by linker histones is facilitated by sequences in the beta A-epsilon intergenic region.     

Selective enrichment of a large size genomic DNA fragment by affinity capture: an approach for genome mapping.

A method to enrich large size DNA fragments obtained by digestion with rare cutting restriction endonucleases was developed and applied for the isolation of a 150 kb SfiI fragment containing the beta-globin gene cluster. The digested DNA is rendered single stranded at the ends by diffusing a strand specific exonuclease into an agarose plug containing DNA. The plug is melted and solution hybridization is then performed with a bridge RNA containing specific sequences from the end of a desired fragment linked to a common probe sequence. The common probe sequence is annealed to a biotinylated RNA and the resulting tripartite hybrid is retained onto a solid matrix containing avidin and specifically released by ribonuclease action. Enrichments of greater than 350 fold have been achieved consistently. Such directed purification of large DNA fragments without cloning can considerably expedite mapping and gene localization in a complex genome and facilitate the construction of sublibraries from defined regions of the genome.     

In vitro methylation of the 5'-flanking regions of the mouse beta-globin gene

The enzymatic methylation of the 5'-flanking region of the mouse beta-globin (major) gene containing putative regulatory regions has been investigated. In vitro methylation of this 368-base pair regulatory DNA by a DNA methyltransferase obtained from mouse erythroleukemia cells yields an asymmetric methylation pattern. Of the 10 available CG pairs, only 5-6 are modified, leading to one hemimethylated site and two apparently fully methylated sites. Only CG pairs which are localized in a 29-base pair cluster are methylated. The data suggest that a CG cluster approximately 100 base pairs upstream from the CAP site may be the in vivo site of methylation in the 5'-regulator region of the mouse beta-globin gene.     

Site-specific transfer of an intact beta-globin gene cluster through a new targeting vector

The ideal gene-therapy vector for treating genetic disorders should deliver intact therapeutic genes and their essential regulatory elements into the specific "safe genomic site" and realize long-term, self-regulatory expression. For beta-thalassemia gene therapy, viral vectors have been broadly used, but the accompanying insertional mutation and immunogenicity remain problematic. Hence, we aimed to develop new non-viral vectors that are efficient and safe in treating diseases. As previous studies have demonstrated that physiological expression of beta-globin genes requires both a 5' locus control region and 3' specific elements, we constructed a new human chromosome-derived targeting vector to transfer the intact beta-globin gene cluster into K562 cells. The whole beta-globin gene cluster was precisely integrated into the target site and expressed in a self-regulatory pattern. The results proved that the human chromosome-derived vector was specifically targeted to the human genome and this could provide a novel platform for further gene therapy research.     

Genomic and phylogenic comparisons of the alpha-globin and beta-globin intergenic sequences between zebra fish (Danio rerio) and six closely related Cyprinindae species

The alpha-globin and beta-globin genes of the zebrafish are tightly linked on the same chromosome in a 3'-5' and 5'-3' configuration, respectively. Although the location of the controlling sequences has been mapped to the intergenic region, analysis determining the uniqueness of this unusual arrangement to zebrafish has not been undertaken. To explore this, we isolated, sequenced, and phylogenetically analyzed the intergenic region between globin gene families of seven Cyprinindae species including zebrafish. These species were grouped into an in group (immediate relatives, not so distant relatives), and an out group (distant relative). Cellulose acetate electrophoresis of hemoglobin (Hb) detected multiple variants in each species, but a band with electrophoretic mobility (EM) of 6.7 x 10(-5) cm(2).volt(-1).sec(-1) was shared between species. Polymerase chain reaction (PCR) amplification of the intergenic globin gene region also detected a 1.0-kb fragment that was repeated in the in group and a 1.2-kb fragment in the out group. Sequence comparison confirmed that the genetic orientation and controlling sequences location were conserved throughout this region in all seven species. This phylogenic footprinting indicated that the configuration was not exclusive to zebrafish. To confirm sequence alignment, maximum parsimony phylogenic analysis, was performed. Only one member of that group the giant danio, was not closely clustered, being located almost equidistance between the immediate relative and the species of the other clusters. This may represent an ancestral configuration prior to transposition of the alpha globin and beta-globin genes families to nonsynteny.     

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.     

Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II

We have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene. The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion. We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA. The sensitivity factors were determined by a least squares curve fitting method based on target analysis. In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II. In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences. There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity. This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain. Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain. These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.     

Molecular evolution of intergenic DNA in higher primates: pattern of DNA changes, molecular clock, and evolution of repetitive sequences

A 3.1-kb intergenic DNA fragment located between the psi beta-globin and delta-globin genes in the beta-globin gene cluster was cloned from gorilla, orangutan, rhesus monkey, and spider monkey, and the nucleotide sequence of each fragment was determined. The phylogeny of these four sequences, together with two previously published allelic sequences from humans and one from chimpanzee, was constructed, and the accumulation of mutations in the region was analyzed. The sites of base substitutions are not evenly distributed within the region: two Alu repeats have accumulated 0.21 + 0.02 substitutions/site with 0.15 + 0.008 substitutions/site in the remainder of the fragment. The occurrence of substitutions at neighboring sites is more frequent than would be expected if they were independent. The observed excesses disappear when ancestral -CG- dinucleotide sites are excluded. The phylogenetic relationships of the sequences indicate that the human sequence shares a most recent coancestor with the chimpanzee sequence. The data also show that great apes have accumulated fewer mutations in this part of the genome than has the rhesus monkey. The relative rates of accumulation of 12 kinds of nucleotide substitution in the region during primate evolution are asymmetric in the DNA strands. From these rates of accumulation, the origin of a simple stretch of sequence near the 3' end of the 3.1-kb fragment was deduced to be a sequence comprising 50% T and 50% C on one strand. The two oppositely oriented Alu sequences in the 3.1-kb region were inserted at their present positions before the divergence of the New-World monkeys from other lineages. Our analysis shows that the nucleotide sequences of the two Alu repeats in spider monkey are unexpectedly similar both to each other and to the deduced ancestral sequence of Alu repeats. The data suggest that there has been some type of recombinational event between the spider monkey Alu repeats but that it was not a simple gene conversion.