Changes in restricted human cellular DNA fragments containing globin gene sequences in thalassemias and related disorders

Human cellular DNA fragments from cells of normal subjects and patients with thalassemia obtained by restriction enzyme digestion were analyzed for their globin gene content. The fragments were separated on agarose gels, transferred to nitrocellulose filters, hybridized to globin [(32)P]cDNA, and radioautographed. One to ten picograms of globin gene sequences were detectable. With EcoRI digestion, eight to nine cellular DNA fragments were found to contain globin genes. Three of these contained beta-like gene sequences assayed with beta globin cDNA probe. One beta-like fragment was absent in DNA from a homozygous subject for hemoglobin Lepore. Two of the three beta gene-containing fragments present in normal DNA were absent in DNA from a patient with hereditary persistence of fetal hemoglobin. The same two fragments containing beta-like genes were absent from deltabeta thalassemic DNA and one new fragment containing beta-like genes was found. Together with results obtained by hybridization of these DNAs in solution, the data are consistent with deletion of specific restriction human DNA fragments in subjects with these disorders and a greater deletion of beta-like gene sequences in subjects with hereditary persistence of fetal hemoglobin than in those with deltabeta thalassemia.     

A general method for cloning eukaryotic structural gene sequences.

Complementary DNA, transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded, has been inserted into Escherichia coli plasmids pSC101 and pMB9 by the poly(dT)/poly(dA) "tailing" and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. An estimate of the amount of inserted globin sequences has been provided by fingerprint analysis of globin mRNA sequences hybridized to the purified plasmid chimeras. Inserted sequences so far subjected to detailed analysis have been ascribed to the rabbit beta globin chain. The susceptibility of inserted beta globin, sequences to the restriction endonuclease EcoRI confirms the existence of a site already found through previous nucleotide sequence analysis.     

Presence of tadpole and adult globin RNA sequences in oocytes of Xenopus laevis

Complementary DNA transcribed from adult Xenopus laevis globin mRNA was used to assay ovary RNA from Xenopus for the presence of globin sequences by RNA.cDNA hybridization. These sequences are present at approximately the same concentration as the majority of poly(A)-containing ovary sequences. The sequences are also found at approximately 200,000 copies per cell in poly(A)-containing RNA extracted from mature oocytes.To rule out contamination of the oocytes with somatic cells, two additional experiments were performed. First, RNA isolated from ovulated unfertilized eggs, which are devoid of somatic cells, was also shown to contain the globin sequences. Second, globin mRNA was isolated from Xenopus tadpoles. Adult globin mRNA is free of the tadpole sequence and no homology was detected between adult and tadpoles globin RNA. The ovary was shown to contain tadpole globin RNA at nearly the same concentration as the adult sequences. Thus, the results cannot be explained by contamination with erythroid cells which should contain only the adult sequence.The swimming tadpole, which possesses an active circulatory system, was also assayed for the tadpole and adult globin sequences. Whereas the adult sequences are present at approximately the same concentration as in the mature oocyte, the concentration of the tadpole sequences increases at least 300-fold in the first 3 days following fertilization.     

Nucleotide Sequences of Human Globin Messenger RNA

Globin messenger RNA, isolated from human peripheral blood reticulocytes, was transcribed into complementary DNA by use of the RNA-dependent DNA polymerase of avian myeloblastosis virus. The complementary DNA was then transcribed into (32)P-labeled complementary RNA by E. coli RNA polymerase in the presence of alpha-(32)P-labeled ribonucleoside triphosphates. The fingerprint pattern obtained from ribonuclease T1 digests of human globin complementary RNA was specific and reproducible. Different patterns were obtained from digests of duck, mouse, and rabbit globin complementary RNA. The fingerprint patterns obtained from digests of purified natural human 10S globin messenger RNA, labeled in vitro with (125)I or with [gamma-(32)P]ATP and polynucleotide kinase, were similar to that of the complementary RNA but contained some additional oligonucleotides. Sufficient nucleotide sequence information has been obtained from about 50% of the intermediate sized oligonucleotides (8-14 base residues long), to make possible examination of correspondence between these nucleotide sequences and globin amino-acid sequences. Approximately 70% of these oligonucleotide sequences can be matched to unique amino-acid sequences in the alpha- or beta-globin chains. The other 30% do not match known amino-acid sequences and presumably correspond to untranslated portions of the mRNA; some of these sequences, however, can be matched to amino-acid sequence in the abnormally long segment of the alpha chain of hemoglobin Constant Spring, which is thought to result from a chain-termination mutation.     

Restriction endonuclease mapping of globin genomic regions of HEL (human erythroleukemia) line

The restriction endonuclease map of the alpha and beta globin genomic region of the new human erythroleukemia line, HEL, was compared with that of normal human DNA. The HEL line, which produces mainly fetal (G gamma and A gamma) but no adult (delta and beta) globin chains, was shown to have the same pattern of DNA fragments as that of normal human DNA. This suggests that the selective expression of the gamma globin genes observed in HEL cells is not due to a major deletion or rearrangement in the epsilon-G gamma-A gamma-delta-beta gene complex. Thus, the HEL line provides a model for studying the control of globin developmental switching in cells with structurally intact globin gene regions.     

The detection and use of hemoglobin mutants in the direct analysis of human globin genes

Many human globin-chain mutants contain amino acid replacements that result from single base changes in the corresponding globin gene. Using recombinants, the coding sequences of each of the alpha-, beta-, Ggamma- , and Agamma-globin genes have now been determined. Those sequences of DNA that are cleaved by a number of specific restriction endonucleases have been identified and accurately positioned. Mutations at these sequences abolish the restriction site, and therefore, the pattern of DNA fragments containing hybridizing globin-gene sequences is altered compared to DNA from normal persons. This allows the identification of one of a pair of cross-hybridizing human globin-gene sequences, as is shown here for the two alpha-globin, the two gamma-globin, and the delta- and beta-globin genes.     

Replication of alpha and beta globin DNA sequences occurs during early S phase in murine erythroleukemia cells.

Murine erythroleukemia cells (MELC) can be induced to express the characteristics of erythroid differentiation by a variety of agents. Previous studies indicate that an action of inducer, occurring during early S phase, may be critical to the expression of differentiated characteristics such as initiation of accumulation of newly synthesized alpha and beta globin mRNAs. In this investigation, the time of replication of globin genes in MELC was studied. DNA was isolated from synchronous populations of cells obtained by centrifugal elutriation. Newly replicated DNA sequences were prepared from synchronized cells cultured for 1 1/2 hr with 5-bromodeoxyuridine; bromodeoxyuridine-containing DNA was isolated by CsCl gradient centrifugation. By employing cloned probes for hybridization to newly synthesized DNA, it was found that alpha and beta globin gene sequences are replicated early in S phase, while ribosomal RNA gene sequences are replicated to about the same extent in early, middle, and late S phases.     

Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells.

Nucleated erythroid cells were incubated for 10 min in the presence of [5-3H]uridine, and the total RNA was isolated by three different extraction procedures. RNA containing globin messenger RNA sequences was purified from other cellular RNAs by selective hybridization to globin complementary DNA cellulose. Depending upon the extraction procedure employed, 0.4-0.6% of the radioactively-labeled total cellular RNA applied to the column annealed to globin complementary DNA cellulose. The annealed RNA was treated with formaldehyde and analyzed by formaldehyde/polyacrylamide gel electrophoresis. Mature globin mRNA and an RNA migrating at approximately 15 S were observed. No globin mRNA containing sequences larger than 20 S were present. The 15S RNA was partially resolved from mature globin mRNA by neutral sucrose density gradient centrifugation. The RNA isolated from the heavy region of this gradient migrated as 15 S in the formaldehyde/polyacrylamide gels and retained its ability to quantitatively anneal to globin complementary DNA cellulose. On the basis of these observations, we conclude that nucleated erythroid cells obtained from the spleens of anemic mice have a 15S RNA which contains globin mRNA sequences. The 15S RNA is not an aggregate and is a good candidate for a globin mRNA precursor.     

Analysis of globin gene structure in patients with beta thalassemia by restriction endonuclease mapping

Twenty-six DNA samples from individuals either heterozygous or homozygous for beta thalassemia were analyzed by restriction endonuclease digestion, agarose gel electrophoresis, and Southern blot analysis to define DNA fragments containing portions or all of the beta globin gene. A total of twenty-seven genes affected by a beta thalassemia mutation and twenty-seven genes affected by a beta thalassemia mutation and twenty-two normal beta globin genes were examined in Italian, Greek, or Asian individuals. With all four restriction endonucleases used, the fragments generated from DNA of thalassemic individuals were identical to those found in DNA from normal. Thus, gross rearrangement or deletion within the genomic region containing the beta globin gene is not characteristic of mutations which cause a thalassemia. A third patient homozygous for pancellular hereditary persistence of fetal hemoglobin was shown to have complete deletion of the delta and beta globin genes.     

Rous sarcoma virus activates embryonic globin genes in chicken fibroblasts.

Complementary DNA (cDNA) specific for chick globin mRNA sequences fails to hybridize to total RNA extracted from chicken fibroblasts. After infection by Rous sarcoma virus, RNA complementary to globin cDNA is detectable in 100-500 copies per cell. Infection of fibroblasts with the transformation defective (td) deletion mutant of Rous sarcoma virus leads to normal virus production, but not to host cell transformation or accumulation of RNA sequences complementary to globin cDNA. Our evidence shows that the globin genes activated by Rous sarcoma virus are those specified by embryonic chick red cells; adult-specific globin sequences were not detected.     

alpha-Globulin sequences are located in a region of early-replicating DNA in murine erythroleukemia cells.

The time of replication in the S phase of regions of the mouse genome including the alpha-globin genes was determined in the murine erythroleukemia cell line transformed by Friend virus. Cells grown for short times in the presence of BrdUrd were fractionated into synchronous populations by centrifugal elutriation. The DNA was cleaved by restriction endonucleases, and fragments containing bromouracil (BrU-DNA) were isolated in density gradients of Cs2SO4. BrU-DNA fractions replicated during selected S-phase intervals were subjected to electrophoresis in agarose gels, transferred to diazobenzyloxymethyl-paper, and hybridized to an alpha-globin probe. Reconstruction experiments using a cloned mouse EcoRI fragment including one alpha-globin gene demonstrated that the extent of hybridization provides an accurate measurement that the extent of hybridization provides an accurate measurement of the concentrations of specific fragments in a DNA sample. The alpha-globin fragments were detected primarily in the BrU-DNA replicated during early S phase (approximately the first quarter of S). This result was confirmed in other synchrony experiments and by Cot analysis. (Cot is the initial concentration of DNA in mol of nucleotide per liter multiplied by the time in sec.) The temporal replication of mouse satellite sequences, already known from previous studies, was used as an internal control for cell synchrony. To show that the globin sequences were not lost from the cells in late S phase during isolation of the DNA, we quantitated th alpha-globin fragments in BrU-DNA prepared from a mixture of cells in early and late S phase. The results demonstrate that the alpha-globin gene regions in these cells are replicated during early S phase.     

Cloning and characterization of DNA sequences surrounding the human gamma-, delta-, and beta-globin genes.

The human gamma-, delta-, and beta-globin genes are located within a 30-kilobase (kb) region of DNA, of which only 20% represents the globin genes. We have attempted to define the nature of flanking and intergenic sequences by isolating recombinants containing the human epsilon, both gamma-, or the 3' end of the beta-globin gene from a bacteriophage library of cloned human DNA. Comparison of these recombinants and a recombinant containing the delta- and beta-globin genes (H beta G1) has provided the following results. The epsilon-globin gene is located 14 kb 5' to the G gamma gene. DNA sequence homology between the region containing the two G gamma genes and the delta nd beta gene region is limited to only a few hundred nucleotides which include the globin coding sequences. Repetitive DNA sequences have been found in the region 3' to the beta-globin gene. Sequences located adjacent to the beta-globin gene are repeated in the globin gene region. A repetitive DNA sequence more than 3.2 kb long is repeated frequently in the human genome but is not repeated in the globin gene region in the clones examined.     

Enzymatic Synthesis of Solid Phase-Bound DNA Sequences Corresponding to Specific Mammalian Genes

With oligo(dT)-cellulose as primer, RNA-dependent DNA polymerase catalyzes the synthesis of cellulose-bound DNA that is complementary to mouse globin mRNA. The resulting cellulose-bound or solid phase complementary DNA hybridizes specifically with globin mRNA and permits the recovery of intact globin mRNA. This simple technique for the synthesis of solid phase-bound complementary DNA provides an additional and convenient method for the purification of specific genetic sequences.     

Demonstration of Globin Messenger Sequences in Giant Nuclear Precursors of Messenger RNA of Avian Erythroblasts

Highly purified globin mRNA from ducks was copied with RNA-directed DNA polymerase from avian myeloblastosis virus into anti-messenger DNA. With excess RNA, more than 90% of this DNA annealed back to its template with a C(o)t/2 value of 7.5 x 10(-4) mol.sec. liter(-1); the melting temperature of the hybrid was 86 degrees . Giant nuclear RNA fractions with sedimentation coefficients of more than 50 S formed hybrids of almost equal stability at C(o)t/2 values of 0.05-0.42 mol.sec. liter(-1), indicating amRNA content of 0.3-1.5%. 12S RNA from the same polyribosomes and nuclear giant RNA from HeLa cells did not cross-hybridize. Although a large part of the giant RNA broke down in 99% dimethylsulfoxide gradients, RNA fractions sedimenting faster than 28S rRNA still were found to consist of up to 0.03% globin mRNA sequences. Thus, the mRNA sequences are contained in the covalent structure of giant nuclear precursors, which are termed precursor-mRNA.     

Silent delta-globin gene in Old World monkeys.

The delta polypeptide chain is present in the adult hemoglobin of all higher primates except Old World monkeys. Because Old World monkeys have evolved from higher primate ancestors, it can be concluded that the ability to synthesize this polypeptide has been lost relatively recently. It is shown here that the gene for delta globin exists in two of these species, the rhesus monkey (Macaca mulatta) and the baboon (Papio papio). Restriction endonuclease fragments of monkey genomic DNA bearing the delta- and beta-globin genes were detected after hybridization of human globin cDNA probes to filter-bound primate DNAs according to the Southern method. A restriction map prepared for rhesus DNA was identical in overall organization to the map of the human region. This indicates that large deletions or additions of DNA are not responsible for the Old World monkeys' lack of delta globin.