Cloned Beta-globin Gene Research Articles

Correction of murine beta-thalassemia by gene transfer into the germ line.

A murine beta-thalassemia was corrected by the transfer of cloned beta-globin genes into the mouse germ line. The cloned mouse beta maj-globin gene or the cloned human beta-globin gene was introduced into mice deficient in beta-globin synthesis because of a deletion of the beta maj-globin gene. Both introduced genes produced functional beta-globin chains, leading to a reduction in one case, and elimination in another case, of the anemia and associated abnormalities of the red blood cells.

Erythroid-specific expression of human beta-globin genes in transgenic mice.

Transgenic mice carrying human beta-globin genes were produced by microinjecting linear DNA molecules containing cloned beta-globin genes with up to 4300 bp of 5'-flanking sequence and 1700 bp of 3'-flanking sequence. Most (15 of 20) of these transgenic mice expressed the human beta-globin genes in blood cells and the level of expression in some mice was comparable with that obtained from endogenous beta-globin genes. Human beta-globin gene expression appeared to be restricted to cells of the erythroid lineage and was first detected between 11 and 14 days of development, in parallel with mouse beta-globin. Constructs with as little as 48 bp of 5'-flanking sequence also appeared to be expressed appropriately. The mRNA transcripts had correct 5' ends and directed human beta-globin synthesis in reticulocyte lysates. Human beta-globin protein was detectable in mature erythrocytes from progeny of one of these mice. The frequency and extent of expression was severely depressed when the procaryotic vector DNA was not removed prior to microinjection.

Functional analysis of a beta-globin gene containing a TATA box mutation from a Kurdish Jew with beta thalassemia.

We recently reported a TATA box mutation (ATAAAA to ATACAA) in a cloned beta-globin gene from a Kurdish Jew with homozygous beta thalassemia (Poncz, M., Ballantine, M., Solowiejczyk, D., Barak, I., Schwartz, E., and Surrey, S. (1982) J. Biol. Chem. 257, 5994-5996). We have now introduced this gene into HeLa cells after CaPO4 precipitation of the DNA and studied expression by analyzing globin-gene transcripts with a novel S1 nuclease mapping assay. Quantitative and qualitative comparison with the normal beta-globin gene revealed a promoter-down phenotype in the TATA box mutant, with normal RNA processing, and a normal start site for initiation of the primary transcript. Decreased transcriptional efficiency was confirmed directly by analysis of run-off transcripts using assays in vitro. The patient's phenotype of beta thalassemia major is probably the result of two different mutations since haplotype analysis of the beta-like globin gene clusters in genomic DNA from this patient shows heterozygosity for the Mediterranean-type haplotypes I and VII, with the TATA box mutation on a haplotype I chromosomal background.

Erythroleukemia (K562) cells contain a functional beta-globin gene.

K562 cells are human erythroid cells that synthesize embryonic and fetal globins but not adult beta-globin. A cloned beta-globin gene was isolated from K562 cells and transfected into HeLa cells. The RNA transcripts produced were comparable in both amount and size to those obtained with a normal beta-globin gene.

Inactivation of an acceptor RNA splice site by a short deletion in beta-thalassemia.

The cloned beta-globin gene of an Indian patient with beta-thalassemia revealed a 25-nucleotide deletion at the 3'-end of the first intervening sequence, including the acceptor RNA splicing site. RNA transcripts of this mutant gene produced following transfection into HeLa cells remained unspliced at both the first intervening sequence donor and acceptor sites. This beta-thalassemic gene is the first in which critical sequences of an acceptor splice junction are mutated and associated with abnormal RNA processing.

ATA box transcription mutation in beta-thalassemia.

DNA sequence analysis of a cloned beta-globin gene from a Chinese patient with beta-thalassemia revealed a single nucleotide substitution (A leads to G) within the ATA box homology and 28 base pairs upstream from the cap site. The patient was homozygous for this particular allele based on restriction mapping at nine different polymorphic sites in the beta-globin gene cluster. Upon transient expression in HeLa cells this gene directed the production of 3-5-fold less beta-globin mRNA than the normal beta-gene. In RNA isolated from the patient's erythroid cells beta-RNA was 10-fold less abundant relative to alpha-RNA than normal, indicating close approximation of the heterologous cell expression results and the in vivo state. These findings support the validity of such transient expression assays for analysis of phenotypes associated with naturally occurring mutant genes and establish the functional significance of nucleotide substitutions at position -28 for human beta-globin gene transcription.

Nonsense and frameshift mutations in beta 0-thalassemia detected in cloned beta-globin genes.

The molecular basis for deficiency of beta-globin synthesis in beta-thalassemia was investigated by gene cloning and DNA sequencing. beta-Globin genes of two patients with beta 0-thalassemia were cloned in a phage lambda vector. Both beta-genes transcribed normally in vitro. The gene of an Italian individual had a single nucleotide substitution (C leads to T) in the codon for amino acid 39 that resulted in formation of a nonsense codon. In a Turkish individual, the cloned beta-globin gene had a dinucleotide deletion in the codon for amino acid 8. This frameshift mutation produced a termination codon at the position of the new 21st codon. Mutations that lead to premature termination of beta-globin synthesis appear to be among the common causes of beta 0-thalassemia in man.

Induction of premature termination of transcription of the mouse beta-globin gene by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

Hybridization of pulse-labeled RNA from DRB-treated Friend cells to mouse beta-globin cDNA revealed that the appearance of beta-globin mRNA in the cytoplasm was inhibited by greater than 87%. To examine the effect of DRB (125 microM) on HnRNA synthesis, nuclear RNA was electrophoresed in methyl mercuric hydroxide gels, transferred to nitrocellulose, and hybridized with beta-globin specific probes. Full-length nuclear transcripts, while present in untreated cells, were not detected in DRB-treated cells. Using restriction enzymes, the cloned beta-globin gene was divided into fragments proceeding from the 5' gene region to the 3' gene region. RNA labeled in vitro by transcription in nuclei isolated from DRB-treated cells hybridized only to the promoter proximal DNA fragment. Transcripts hybridizing to fragments from both the 5' and 3' regions of the gene were produced in nuclei from untreated cells. Together these results indicate that DRB causes premature termination of transcription within the beta-globin gene.

The organization of the gamma-delta-beta gene complex in normal and thalassemia cells.

Restriction enzyme digestion analysis and direct human globin gene cloning have permitted analysis of the physical arrangement of nucleotide sequences within and surrounding the human globin genes. With these methods it has been shown that the linear arrangement 5' to 3' of the globin genes is G gamma-A gamma-delta-beta. The G gamma and A gamma genes are separated by about 3.5 kilobases (kb), while the A gamma and delta genes are 15 kb apart, and the delta and beta 6.5 kb apart. Each of these genes contains a large intervening sequence (IVS) of approximately 1 kb in precisely the same position between condons 104 and 105. In addition, each of these genes has a small IVS between codons 30 and 31. In homozygous delta beta thalassemia DNA, there is deletion of all of the normal delta and beta gene fragments. However, a new fragment 4.2 kb in size containing the 5' end of the delta globin gene is retained. Retention of this fragment in delta beta thalassemia, but not in HPFH is consistent with a role for sequences in this region for limiting gamma globin gene expression. Studies to date suggest that the beta + and beta 0 thalassemias will be due to a heterogeneous group of DNA defects affecting either beta globin gene transcription or beta mRNA processing. In most cases of beta + and beta 0 thalassemia DNA analyzed, there is no detectable deletion of beta or delta genes. In three India beta 0 patients, deletion of the 3' end of the beta gene has been found. Analysis of cloned beta globin genes from a patient with beta + thalasseia shows differences from normal in the fragments generated by restriction enzymes which cut frequently. Whether these differences are responsible for the defect in thalassemia or are polymorphisms unrelated to thalassemia remains to be determined.

Comparison of cloned rabbit and mouse β-globin genes showing strong evolutionary divergence of two homologous pairs of introns

Cloned beta-globin genes of both mouse and rabbit each contain a large and a small intervening sequence (intron) of about equal length at precisely the same positions relative to the coding sequence. The homologous introns show some sequence similarity, particularly at the junctions with the coding sequence. They most probably arose from a common ancestral sequence and diverged substantially during evolution.