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Biomedical subjects

A Bank

Publications and source records attributed to A Bank.

At least 109 records · Page 6Linked to original sources
PubMed1985

Genetic defects in the thalassemias.

In summary, the beta-thalassemias are models for the study of human genetic disease. Defining the genetic defects in and surrounding the beta-globin gene in the beta(+)- and the beta(0)-thalassemias has resulted in new insights into the relationships between changes in gene structure and abnormalities in gene function. The region 5' to the beta-gene, the coding regions within the gene, and the IVS have all been found to contain single nucleotide defects which diminish or abolish beta-globin mRNA production and the production of beta-globin. The ability to isolate beta-globin genes by cloning, and to express these beta-globin genes in cells, has given remarkable insights into the relationship between globin gene structure and globin gene function. In addition, new technology is available for the antenatal diagnosis of the beta-thalassemias based on the knowledge of the specific defects in these genes, with the use of oligomers to detect single nucleotide changes. Finally, recent advances have suggested new approaches to gene therapy in these disorders using gene transfer.

Cloning, Molecular↗
PubMed1985

Structure and expression of two beta genes in a beta thalassemia homozygote.

Two beta globin gene alleles have been cloned and characterized from a patient with beta + thalassemia. Both beta genes have single base mutations in the small intervening sequence (IVS 1); one 6 nucleotides and the other 110 nucleotides from the 5' end of IVS 1. Both genes lead to abnormal splicing of beta globin mRNA precursors when expressed in HeLa cells. Despite the fact that both alleles produce some normal beta globin mRNA transcripts, the patient has clinically severe beta + thalassemia (Cooley's anemia).

Base Sequence↗
PubMed1984

Gene analysis in delta beta and delta (0) thalassemia.

Restriction mapping of the globin genes from a homozygous delta beta thalassemia patient from Israel indicates that at least a 10-kilobase deletion is present extending 3' from within the large intervening sequence (IVS 2) of the delta globin gene and including the entire beta globin gene. Unique bands are seen when cellular DNA from this patient is digested with a variety of restriction endonucleases and hybridized with a probe specific for the delta IVS 2. Extensive analysis of the Israeli delta beta thalassemia DNA as well as material from an Italian delta beta thalassemia homozygote with enzymes which cleave more frequently in delta IVS 2 has localized the 5' end of the deletion to a 107-base pair region within delta IVS 2. This region contains a unique repetitive sequence (TG)4 which has been reported to be a specific recognition signal for recombination and may be involved in the formation of these mutant genes. Two homozygous delta(0) thalassemia DNA samples from Japan were also analyzed for gene rearrangements or other changes by restriction enzyme mapping. No changes from normal were seen using 14 different enzymes indicating the absence of large deletions in the region around the delta globin gene. More specifically, both the 5' and 3' splice junctions of the IVS 2 appear to be normal from hybridization of restriction fragments generated by HphI and AluI, respectively, with a delta IVS 2 specific probe. We have also shown that point mutations which could lead to termination codons are not present at codons 35, 37, 43, 61, and 121, since restriction enzymes which recognize these sites produce normal patterns. The delta(0) thalassemia phenotype in these two subjects is most likely due to a point mutation either at one of the other 24 potential termination codons not accessible to restriction analysis or to other single nucleotide changes which could either decrease delta globin gene transcription or lead to abnormal processing or transport of delta globin mRNA.

Base Sequence↗
PubMed1984

Hemoglobin Miyada: DNA analysis of the anti-Lepore beta delta fusion gene.

Hemoglobin Miyada, an anti-Lepore hemoglobin, represents the protein product of a nonhomologous crossover between beta and delta genes. The mutant globin is beta-like from the N terminus to amino acid 12, and delta-like from amino acid 22 through the C terminus, thus predicting a crossover site in the first coding region of the gene. DNA analysis, using multiple restriction endonucleases and hybridization to delta and beta globin gene-specific probes, confirms that the beta delta hybrid gene 1) is located on a single chromosomal fragment between normal delta and beta genes, and 2) has 5' beta promoter sequences, delta IVS 1 and 2, and 3' coding and flanking sequences.

Base Sequence↗
PubMed1984

Stable transfer and expression of exogenous human globin genes in human erythroleukemia (K562) cells.

To study the expression of globin genes in human cells, human epsilon-globin genes were transferred into a K562 cell line, Bos, which synthesizes very low amounts of epsilon-globin mRNA. A plasmid (pSV2neo-epsilon) containing a complete epsilon-globin gene and 2 kilobases (kb) of 5' flanking DNA as well as a neomycin-resistance gene and a simian virus 40 origin of replication was transfected into Bos cells; the compound G418, a neomycin analogue, was used to select transformed cells. The presence of unique bands by DNA restriction analysis shows that 11 of 14 of the G418-resistant clones have at least one copy of an integrated epsilon-globin gene. RNA expression measured by RNA blotting shows significantly more epsilon-globin mRNA sequences than in untransfected Bos cells in 10 of 11 lines; in most lines, epsilon-globin mRNA was additionally increased in the presence of hemin. In two lines, epsilon-globin mRNA expression with hemin was comparable to that of a high epsilon-globin producing cell line, K562 clone 2. The one G418-resistant line without epsilon-globin genes had no epsilon-mRNA expression. The high epsilon-mRNA expression in several of the lines suggests that exogenous epsilon-globin genes with only 2-kb 5' flanking DNA may be sufficient to be appropriately expressed in these homologous erythroid cells. These results have implications for the potential success of transfer of normal human genes to human bone marrow cells as an approach to the treatment of inherited anemias.

Base Sequence↗
PubMed1984

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.

Cloning, Molecular↗
PubMed1984

Specific globin mRNAs in human erythroleukemia (K562) cells.

Specific globin mRNA accumulation was quantitated in several lines of K562 cells in the absence and the presence of hemin. Using specific cloned DNA probes, the amounts of zeta, alpha, epsilon and gamma mRNAs were shown to be increased 2-3-fold in the presence of 20 microM hemin. No delta- or beta-globin mRNAs were detectable in any of the lines. In one line, Bos, there was a marked decrease in epsilon-globin mRNA, which increased with hemin, although still to much lower levels than in the other lines. The decreased epsilon-globin mRNA accumulation in Bos is shown to be due to decreased epsilon-globin gene transcription.

Cell Line↗
PubMed1983

The genetics of thalassemia.

Two newly developed techniques have greatly enlarged our knowledge of the basic genetic defects of the beta-thalassemias: 1) Restriction enzyme digestion of cellular DNA followed by analysis of the cleaved fragments. 2) Cloning of beta-globin genes from patients with beta-thalassemia and subsequent determination of the nucleotide sequence of these genes. Some of the results are presented. Only rarely a deletion of the beta-globin gene was found. In most cases, a mutation within the beta-globin gene was discovered as basis for the beta-thalassemia syndrome. These and other findings are a starting point for planning a therapy of beta-thalassemia by means of gene technology: correction of the defect within the abnormal beta-globin gene, replacement of the beta-globin gene, stimulation of gamma-globin synthesis.

DNA Restriction Enzymes↗
PubMed1983

Abnormal splice in a mutant human beta-globin gene not at the site of a mutation.

We have studied the expression of a cloned mutant human beta-globin gene in tissue culture cells. The gene, which was previously isolated from the chromosomal DNA of an individual with a low level of normal beta-globin expression (beta+-thalassemia), contains five mutations inside the large intervening sequence (IVS2), as well as a silent change in codon 2. This beta-thalassemia gene (thal) was inserted into a plasmid that is replicated and transcribed in a line of monkey kidney cells in culture. S1 nuclease mapping of the beta-globin RNA transcribed from this gene indicates that some of the beta-globin RNA is spliced abnormally by using a cryptic 3' splice sequence normally present in IVS2 but not used in processing the normal beta-globin transcript. The cryptic 3' splice site is not the site of a mutation in the thal gene. Because neither the 5' or 3' splice junction nor the cryptic site is mutated in this gene, it is most likely that the mutation at position 705 of IVS2, the only nonpolymorphic change in the gene, interferes indirectly with normal processing. These results suggest that certain sequences within IVS must be conserved to prevent abnormal splicing and loss of gene function.

Base Sequence↗
PubMed1982

Effect of methanol extraction residue of Bacillus Calmette-Guerin in advanced Hodgkin's disease.

One-hundred-ninety-six patients with Stage III and IV Hodgkin's disease were prospectively randomized to receive either treatment with the methanol extraction residue of Bacillus Calmette-Guerin (MER/BCG) or no immunotherapy. Prior to the MER/BCG randomization, patients received six courses of induction and two years of maintenance chemotherapy so that a group with a presumptively low tumor burden could be established. Only patients achieving a complete remission were evaluated. During the first two years of immunotherapy, the MER/BCG group had a relapse frequency twice that of controls. The overall crude relapse frequency and disease-free survival were similar between the two treatment groups. The MER/BCG dose schedule used in this study was associated with a high frequency of unacceptable toxicity. Ulcerations of greater than 1 cm occurred in one-third of the patients with associated pain, fever, and occasional lymphadenopathy. A high degree of patient noncompliance (36%) was observed. Age (P = 0.002), prior radiotherapy (P = 0.032), and chemotherapy (P = 0.044) were prognostic factors found to significantly influence remission duration. These factors were balanced between patients treated with immunotherapy and those who were not. MER/BCG therapy did not significantly delay or prevent relapse.

Adult↗
PubMed1982

Five nucleotide changes in the large intervening sequence of a beta globin gene in a beta+ thalassemia patient.

A beta globin gene from a patient with homozygous beta+ thalassemia has been cloned and completely sequenced. No changes from normal are found in the 200 nucleotides 5' to the cap site, in the 3' untranslated region up to the poly A addition site, in the small intervening sequence (IVS 1), or in the coding sequence except for a third base change in codon 2. The only other differences are in the large intervening sequence (IVS 2). One of these, at a position 16 nucleotides from the 5' end of IVS 2, has been reported previously in normal individuals, and is probably a polymorphism. Four other changes, at positions 74, 81, 666, and 705 are also seen in IVS 2. Abnormal beta globin mRNA precursors detected in the bone marrow cells of this patient, and abnormal beta globin RNA splicing observed when this gene is transcribed in a tissue culture system taken together with these IVS 2 changes, suggest that the beta+ thalassemia phenotype is produced by a decrease in normal beta globin mRNA processing.

Base Sequence↗
PubMed1981

Two cloned beta thalassemia genes are associated with amber mutations at codon 39.

Two beta globin genes from patients with the beta(+) thalassemia phenotype have been cloned and sequenced. A single nucleotide change from CAG to TAG (an amber mutation) at codon 39 is the only difference from normal in both genes analyzed. The results are consistent with the assumption that both patients are doubly heterozygous for beta(+) and beta degrees thalassemia, and that we have isolated and analyzed the beta degrees thalassemia gene.

Base Sequence↗
PubMed1981

Restriction endonuclease sensitivity of DNA containing globin genes in different murine erythroleukemia cell lines.

The arrangement of the globin structural genes has been examined in murine erythroleukemia cells, strain DS19, and several related inducer-resistant variant cell lines. One fragment larger than 20 kilobases and six globin gene-containing fragments between 10 and 1.9 kilobases in size are detected in EcoRI-cleaved purified DNA prepared from strain DS19. By comparison, when isolated nuclei are digested with EcoRI, only two globin gene-containing fragments are detected, one greater than 20 kilobases and the other 1.9 kilobases. Of seven cell lines derived from DS19 and resistant to inducers, six had similar patterns to DS19 of globin gene-containing EcoRI-generated DNA fragments from nuclei and from purified DNA. One cell line, DR10, a DMSo-resistant cell line, lacks the 1.9 kilobase fragment after digestion of either nuclei or purified DNA. The 1.9 kilobase fragment hybridizes with alpha-globin cDNA but not with the beta-globin cDNA, suggesting either rearrangement or deletion of an alpha-globin gene-like fragment in DR10 DNA.

Animals↗
PubMed1981

A nucleotide change at a splice junction in the human beta-globin gene is associated with beta 0-thalassemia.

beta 0-Thalassemia is a heterogeneous group of disorders associated with absence of beta-globin. In a survey of DNAs from patients with beta 0-thalassemia of diverse ethnic origins, a change at the splice junction at the 5' end of the large intervening sequence (IVS 2) of the human beta-globin gene has been found in one patient of Italian and another two of Iranian ethnic origins. The enzyme Hph I recognizes a change at this site and generates a large-than-normal fragment of DNA, which hybridizes specifically to a beta-globin IVS 2 probe. No other changes in beta-globin gene DNA structure or organization are detectable by extensive restriction endonuclease analysis. The enzyme HinfI which recognizes a sequence beginning three nucleotides from the 5' end of the IVS 2 splice junction, produces normal fragments and localizes the defect to a G-G-T sequence at the 5'-end IVS 2 splice junction. This sequence is known to be remarkably conserved in all globin genes from many species and in most other genes examined to date. Thus, in at least some beta 0-thalassemia patients, the beta 0-thalassemia defect is associated with a nucleotide change at a splice junction. These patients provide unique examples of naturally occurring defects in splice junctions of eukaryotic genes associated with absence of specific gene function.

Base Sequence↗
PubMed1981

Localization of the site of recombination in formation of the Lepore Boston globin gene.

The site of crossover between the delta- and beta-globin gene sequences resulting in Lepore Boston globin gene formation has been localized at the DNA Level. Probes specific for detecting the intervening sequences (IVS) of the delta- and beta-globin genes were used to map the origin of cellular DNA fragments of a patient homozygous for hemoglobin Lepore Boston. Restriction endonuclease analysis and hybridization of this DNA to IVS specific probes show that most, if not all, of the large intervening sequences (IVS 2) in Lepore DNA are derived from the beta-globin gene IVS 2. In addition, the crossover occurs in a region of DNA in which the delta- and beta-genes have almost complete nucleotide homology, and might be expected to pair most tightly during meiosis.

Base Sequence↗
PubMed1981

A new polymorphism in the human beta-globin gene useful in antenatal diagnosis.

A new polymorphism in the beta-globin is described, using the restriction enzyme Asu I. A radioactive probe specifically representing the large intervening sequence (IVS 2) of the beta-globin gene has been used to detect this polymorphism. Normally, a 0.8-kilobase fragment containing beta-IVS 2 is generated by Asu I; however, a 1.0-kilobase fragment is seen in association with 18% of beta A-genes, and 38% of beta-thalassemia genes in an Israeli population studied. By contrast, the Asu I polymorphism has rarely been seen in blacks examined to date. An additional Asu I change is seen the the delta-globin gene with a delta-IVS probe. The beta-Asu I polymorphism is shown to be useful in the antenatal diagnosis of beta-thalassemia.

Base Sequence↗