Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The K562 human leukemia cell is an erythroid-like cell that may serve as a model for the study of globin gene expression in transcriptionally active human erythroid cells. K562 cells express all globin genes with the exception of that for beta-globin; failure to produce beta-globin could result from an acquired mutation in each of the beta-globin genes or from an alteration in the regulatory factor environment of the beta-globin gene. To uncover a possible acquired mutation, restriction endonuclease analysis of genomic K562 DNA and expression studies of a cloned K562 beta-globin gene were carried out. Restriction endonuclease analysis revealed no structural alteration of the K562 beta-globin genes. Analysis of the polymorphic Ava II site in intervening sequence 2 of the beta-globin gene showed that K562 cells contain two different beta-globin alleles, both of which are inactive. A K562 beta-globin gene was cloned, ligated into the expression vector pLTN3B, and introduced into COS cells. Transcripts were analyzed by RNA blot, dot blot, S1 nuclease mapping, and primer extension assay. The cloned K562 beta-globin gene was transcribed in COS cells as efficiently as a normal beta-globin gene introduced into COS cells; the mRNA was 10 S and polyadenylylated; the 5' and 3' termini and the processing of transcripts were identical to that of mRNA transcribed from a normal gene. Based on these data we suggest that the absence of beta-globin gene expression results not from an alteration in the beta-globin gene, but from a quantitative or qualitative alteration in a trans-acting factor important in beta-globin gene expression.
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PMID:A beta-globin gene, inactive in the K562 leukemic cell, functions normally in a heterologous expression system. 620 98

Genomic DNA from a fetal sheep homozygous for the beta A gene was used to construct a library of one million cloned DNA fragments using the bacteriophage vector, Charon 4A. Screening of 150,000 plaques from this library using radioactive beta-globin gene sequences resulted in the isolation of two recombinant bacteriophage containing globin genes. One of these, S beta AG-21, contains the complete adult beta A-globin gene as demonstrated by hybridization and restriction endonuclease analysis. In common with adult globin genes from other species, the beta A gene contains small (105 base pairs) and large (900 base pairs) intervening sequences. The second recombinant bacteriophage, SG-4, contains a complete embryonic beta-like globin gene which is expressed in the sheep embryo as demonstrated by hybridization analysis with cDNA made from sheep embryonic globin mRNA. Although differing in its restriction endonuclease map from the adult beta-globin genes, SG-4 appears to contain a large intervening sequence of at least 750 base pairs in length. Finally, preliminary evidence is discussed which indicates that a Pvu II site just 5' to the Cap site may be a common feature of sheep globin genes.
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PMID:Hemoglobin switching in sheep. Cloning and characterization of the beta A and beta-like embryonic globin genes from genomic DNA. 624 12

Restriction endonuclease mapping permitted identification of a form of beta 0-thalassemia in which a partial deletion of the beta-globin structural gene occurred [Orkin, S. H., Old, J. M., Weatherall, D. J. & Nathan, D. G. (1979) Proc. Natil. Acad. Sci. USA 76, 2400-2404]. To analyze its structure more directly, this abnormal human gene has now been cloned in bacteriophage lambda gtWES. Restriction mapping of the cloned gene and of a normal beta-globin gene contained in the phage H beta G1 confirmed previous findings regarding the presence of a deletion toward the 3' end of the gene but could not establish its position more accurately. Electron microscopy of the hybrid of the beta-thalassemia gene with globin RNA (R-loop analysis) provided unequivocal evidence for a deletion with the beta-globin structural gene. Hybridization of restriction fragments of the mutant gene with homologous fragments of H beta G1 (heteroduplex analysis) revealed a continuous, internal deletion of about 0.6 kilobase of DNA in the mutant gene and permitted its localization within the beta-globin gene region. This deletion removed the terminal third of the large intervening sequence within the beta-globin gene, the entire 3' coding block (extending from codon 105 to the end of the gene), and approximately 150 base pairs of DNA past the end of the normal globin gene.
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PMID:Cloning and direct examination of a structurally abnormal human beta 0-thalassemia globin gene. 625 66

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.
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PMID:Silent delta-globin gene in Old World monkeys. 625 67

In order to assess the applicability of multiple restriction endonuclease analyses of amniocyte DNA to the prenatal diagnosis of beta-thalassemias in general, we studied 12 consecutive couples at risk. DNA of both members of the 12 couples and a previous offspring of each was analyzed for the presence of 4 polymorphic restriction endonuclease sites: the Hpa I site 3' to the beta-globin gene, the Hind III site in the G gamma gene, the Hind III site in the A gamma gene, and the Bam HI site 3' to the beta-gene. Linkage disequilibrium between these sites and beta A or beta thal genes was not found, presumably due to the heterogeneity of beta thal genes. However, the high frequency of polymorphism at these sites allowed differentiation of beta A-bearing chromosomes from beta thal or beta S-bearing chromosomes in both members of 6 couples. In these couples, complete prenatal diagnosis by linkage analysis of amniocyte DNA would be possible. In the remaining 6 couples, beta A and beta thal chromosomes could be discriminated in one member. In about 50% of the pregnancies of these couples, exclusion of beta-thalassemia is possible by this analysis. These data suggest that when linkage analysis of polymorphic restriction endonuclease sites is carried out, prenatal diagnosis of beta-thalassemia states can be accomplished by amniocentesis alone in 75% of pregnancies at risk.
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PMID:Prenatal diagnosis of beta-thalassemias by amniocentesis: linkage analysis using multiple polymorphic restriction endonuclease sites. 625 93

K562 human leukemia cells synthesize embryonic hemoglobins after culture in the presence of hemin. We have rigorously identified these hemoglobins by globin chain analysis and peptide mapping. No adult hemoglobin could be detected, and beta-globin synthesis was less than 2 ppm of total protein synthesis. Persistent embryonic globin gene expression is known to occur as a consequence of globin gene deletions. However, restriction endonuclease mapping showed that the globin gene complexes in K562 cells are indistinguishable from normal. Hemin increased the rate of embryonic globin synthesis. The pattern of hemoglobin synthesis proved to be stable when cells from different laboratories were compared. One line, however, synthesized large amounts of Hb X and very little Hb Portland in response to hemin. Hb X has been previously detected in human embryos; we show here that it has the composition epsilon 2 gamma 2 and is diagnostic of imbalanced chain synthesis or "zeta thalassemia." We have identified several agents that induce hemoglobin synthesis in K562 cells. Different inducers induced different patterns of embryonic hemoglobin synthesis but never any adult hemoglobin synthesis.
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PMID:Embryonic erythroid differentiation in the human leukemic cell line K562. 626 39

A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150 000 recombinant-DNA-containing colonies were screened for the presence of the human beta-globin related genes. Five recombinants were isolated containing the human beta-globin locus and encompassing approx. 70 kb of human DNA.
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PMID:Isolation of beta-globin-related genes from a human cosmid library. 626 15

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.
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PMID:A nucleotide change at a splice junction in the human beta-globin gene is associated with beta 0-thalassemia. 627 Jun 63

Several reports have been published on the use of polymorphisms found in the human hemoglobin genes as a means for prenatal diagnosis of sickle cell anemia. The disadvantages of this approach reside in its limited application and the need for family analysis. Here we report that, by use of restriction endonuclease Dde I and diazobenzyloxymethyl-paper transfer procedures, a direct analysis can be made. Individuals with normal hemoglobin (AA) show two bands (175 and 201 base pairs) complementary to a 5'-specific beta-globin gene probe. Sickle cell trait individuals (AS) exhibit an additional band (376 base pairs). Individuals with sickle cell anemia (SS) show the band at 376 base pairs with a concomitant loss of the 175-base pair band. We interpret these changes in banding pattern to be the result of the elimination of a restriction site for Dde I in the altered codon associated with the sickle cell allele. Because an analysis can be performed on as little as 20 micrograms of cellular DNA, the application to prenatal diagnosis of sickle cell anemia should be possible.
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PMID:Direct identification of sickle cell anemia by blot hybridization. 627 89

We have reported the direct analysis of the allele for beta 2-globin by using restriction endonuclease Dde I coupled with blot-hybridization analysis. In that report we predicted that a major use of our analysis could be for the prenatal diagnosis of sickle cell anemia. Here we present such an analysis. In addition, this report also describes the use of a new enzyme Mst II, which also distinguish the beta s allele from the normal beta-globin allele. Blot-hybridization analysis with restriction endonuclease Mst II shows the 5' end of the normal beta-globin gene to reside on a fragment of approximately 1.14 kilobases, whereas the 5' end of the beta s-globin gene resides on a fragment of approximately 1.34 kilobases. Because the fragment sizes generated by Mst II are significantly larger than those generated by Dde I, one can easily perform a prenatal diagnosis for sickle cell by standard blot hybridizations onto nitrocellulose filters.
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PMID:Use of restriction endonucleases for mapping the allele for beta s-globin. 628 54


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