Insertion of synthetic copies of human globin genes into bacterial plasmids

Wilson, John T.; Wilson, Lois B.; Deriel, J. K.; Villa‐Komaroff, Lydia; Efstratiadis, Argiris; Forget, Bernard G.; Weissman, Sherman M.

doi:10.1093/nar/5.2.563

Cited by 332 publications

(88 citation statements)

References 25 publications

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“…As shown in Fig. 5, the complement of the deduced sequence is strikingly homologous to rabbit and human ,B-globin mRNA sequences between amino acids 61 and 100 (30 (1) (2) (5) (6) (7) (1) (2) 5). These homologies in fragment size and nucleotide sequence may well lead investigators to incorrectly argue for the presence of a d3 gene in thalassemia when, in fact, the gene is deleted.…”

Section: G-a-a-g-t-c-a-n-c-a-c-c-mentioning

confidence: 69%

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Molecular cloning of human epsilon-globin gene.

Proudfoot¹,

Baralle²

1979

Proc. Natl. Acad. Sci. U.S.A.

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Human a-like globin genes were investigated by use of rabbit P-globin cDNA plasmid as a cross-species hybridization probe. Normal and 2°/SB thalassemic DNA were compared by filter hybridization procedures. It roved possible to demonstrate that the rabbit probe detected ad A7, 6, a, a0, and 6PB human globin genes as well as an additional unidentified a-like globin gene. By use of an agarose gel elution procedure, fractions of HindIII-digested DNA enriched for a-like globin genes were purified. One of these fractions, 8.0 kilobases in size, was cloned into X788, and EK2 X HindIII vector. A positive clone was obtained and characterized by restriction mapping and sequence analysis. The sequence data obtained predicted an amino acid sequence that exactly matches a part of human &globin. The human non--globin locus is now nearly complete. 6, A, and y human globin genes have already been cloned and analyzed. We describe here the cloning of the remaining non-x-globin gene, e.The globin gene system of humans provides an excellent model system for the study of gene expression in eukaryotes, both during and after development. In the yolk sac of early embryos two genes, E and A, produce embryonic hemoglobin ({2e2). Later in the fetal liver there is a switch to the expression of y and a genes, producing fetal hemoglobin (a2Y'2). Finally, at birth there is a second switch to the bone marrow synthesis of adult hemoglobins a2#2 and a262 (ratio 40:1) by the (3, 6, and a genes (1). The genetic defects that cause thalassemia are associated with this gene system and could prove very useful in establishing how these genes are regulated during development and in adult life (2).Great advances have been made in the structural analysis of this gene system. The 13, 3, and a part of the y genes (3, 4) have been cloned from genomic DNA. Their linkage and restriction site maps have been established by analysis of these clones and also by direct analysis of genomic DNA (5, 6) by use of human globin cDNA plasmids as probes (7,8). Similarly, the two linked a-globin genes have been mapped by genomic DNA analysis (9). However, no information has been obtained on the genes coding for the embryonic hemoglobin.We describe in this paper the molecular cloning of an 8-kilobase (kb) human DNA fragment containing the -globin gene.This clone was isolated by use of a rabbit /3-globin cDNA plasmid (10) as a crosshybridization probe. MATERIALS AND METHODSAnalysis of Genomic and Cloned DNAs by Restriction, Filter Hybridization, and Sequence Determination. Genomic (both normal and 10/3130 thalassemic) and cloned DNAs were digested with various restriction enzymes-EcoRI, HindIII, BamHI, and Pst I (Boehringer, Mannheim, W. Germany); Xba I and Bgl II (BioLabs)-under standard conditions. Digests were fractionated on 0.8-1.5% agarose gels, with a HindIII digest of wild-type X DNA (11) and a Hae III digest of PBR322 (12) as size markers. Gels for filter hybridization were denatured in alkali (0.2 M NaOH/0.6 M NaCl, 1 hr, 200C), neutralized (1.0 M Tris-HCI, ...

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Section: G-a-a-g-t-c-a-n-c-a-c-c-mentioning

confidence: 69%

“…The 13, 3, and a part of the y genes (3,4) have been cloned from genomic DNA. Their linkage and restriction site maps have been established by analysis of these clones and also by direct analysis of genomic DNA (5,6) by use of human globin cDNA plasmids as probes (7,8). Similarly, the two linked a-globin genes have been mapped by genomic DNA analysis (9).…”

mentioning

confidence: 99%

Molecular cloning of human epsilon-globin gene.

Proudfoot¹,

Baralle²

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…The extracted RNA was aliquoted, precipitated in 2 volumes of absolute ethanol and stored at -80˚C, washed once with cold 75% ethanol, dried and dissolved in DEPC-treated water before use. Total RNA (10 μg) was loaded onto 1% agarose gel, electrophoresed, transferred to a nylon membrane (Gene Screen Plus) and hybridized using a 32 P-labelled JW151 plasmid probe containing human Á-globin coding gene (26). The ß-actin probe was produced by RT-PCR (reverse transcriptase polymerase chain reaction).…”

Section: Rna Isolation and Northern Blottingmentioning

confidence: 99%

Bis-epoxyethyl derivatives of distamycin A modified on the amidino moiety: Induction of production of fetal hemoglobin in human erythroid precursor cells

Bianchi

Borgatti²,

Fibach³

et al. 1998

Int J Mol Med

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Abstract.Derivatives of distamycin A modified at the Cterminal amidine moiety and tethered to bis-epoxyethyl moieties at the N-terminal position were tested for their ability to induce erythroid differentiation in the human erythroleukemic cell line K562. None of the compounds without bis-epoxyethyl moiety were active. A comparison of the biological activity of diepoxy compounds containing different non-basic amidine-modified moieties, showed low activity of amidoxime, carbamoyl and N-methyl carbamoyl derivatives as differentiation agents. In contrast, a cyanamidine derivative, compound 3, was able to induce erythroid differentiation of K562 cells. In addition, the cyanamidine derivative 3 was able to induce HbF accumulation following treatment of cultures of erythroid precursor cells isolated from the peripheral blood of normal subjects. IntroductionThe identification and characterization of potential therapeutic agents for the treatment of ß-thalassemia and sickle cell anemia is based on the pharmacologically mediated regulation of the expression of human Á-globin genes (1-8). It is well established that an increased production of fetal hemoglobin (HbF, · 2 Á 2 ) leads to a significant improvement of the clinical status of the patients (4,5,7). Therefore, many published experiments were designed to find hormones, cytotoxic agents, hemopoietic cytokines and short fatty acids as agents able to increase HbF levels in humans (1-3,9-11).In this respect, DNA-binding drugs are very interesting, since we reported that mithramycin, cis-platinum and tallimustine analogs are potent inducers of erythroid differentiation (12-14). The same compounds have been demonstrated to increase HbF production in human erythroid precursor cells obtained from peripheral blood of normal subjects and thalassemic patients (9). In particular, we found that the distamycin analog tallimustine and some related compounds are powerful inducers of differentiation and Á-globin mRNA accumulation in normal human erythroid progenitors (10).In this study, we report on the biological characterization of potential HbF inducers of the hybrid molecules 3-6 that include the amidine-modified tripeptide tris-N-methyl-pyrrolecarboxamide distamycin A, which contains two electrophile epoxydic functions on the amino end. In the bis-epoxyethyldistamycin A hybrids, the amidino moiety has been replaced by various non-basic groups of a different electronic nature, lipophilicity and bulk, such as cyanamidine (compound 3), amidoxime (compound 4), carbamoyl (compound 5) and Nmethyl carbamoyl (compound 6).As was previously reported, the synthesis of amidinomodified analogs provides a number of advantages (15-18). First, they are not hygroscopic and are easy to handle. Moreover, these derivatives are uncharged. Thus, products and intermediates can be readily purified by column chromatography and recrystallized.These compounds were investigated for potential erythrodifferentiation ability, in order to verify whether some of them were able to increase Á-globin gene express...

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“…Following hybridization, blots were washed at 65°C with four changes of 2 x SSC, 0.5% SDS for approximately 2 h. Autoradiography times ranged from a few hours to several days. Reprobing of blots was performed after blots were stripped at 70°C in the hybridization buffer for 1 h. Probes used in this work were murine c-myc [40], rat actin plasmid 749 [41], human VH2 plasmid HA2 [42], the constant region of mouse Igp plasmid 3741 [43], human a-globin [44], rat transferrin plasmid PFIO [45], rat albumin plasmid PRSA13 [46] rat aldolase B plasmid A.4C9 [47] and chicken glyceraldehyde-Zphosphate dehydrogenase pGAD28 [48]. These plasmids were kind gifts of A. Kimchi, …”

Section: Proh Ing With Nick-translated Probesmentioning

confidence: 99%

Differential mRNA stability to reticulocyte ribonucleases correlates with 3′ non‐coding (U)_nA sequences

Wreschner

Rechavi

1988

European Journal of Biochemistry

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The stabilities of different mRNA species were analyzed in a reticulocyte lysate system under protein‐synthesizing conditions. In all cases examined the relative mRNA degradation by reticulocyte ribonucleases as well as by the interferon‐modulated (2′‐5′) (A)n‐dependent endonuclease correlated with the extent of (U)nA sequences within the 3′ non‐coding region. The experimental data presented indicate that according to their stabilities at least three major mRNA groups may be identified: (a) (U)nA‐poor mRNAs (e.g. globin) are essentially stable and are only slightly degraded by the (2′‐5′)(A)n‐dependent endonuclease; (b) mRNA species with intermediate (U)nA levels (e.g. Igα and Igμ heavy‐chain mRNAs) are partially degraded by general ribonuclease activity and further degraded by the (2′‐5′)(A)n‐dependent endonuclease and (c) (U)nA‐rich mRNA species (such as c‐myc and non‐skeletal actin mRNAs) are inherently unstable and are extremely sensitive to degradation by general ribonuclease activity. A survey of mRNA nucleotide sequences demonstrated that without exception (U)nA‐rich stretches appeared more frequently within the 3′ non‐coding region than in the coding or 5′ non‐coding regions. A comparison of 3′ non‐coding region sequences from 92 different mRNAs revealed that transiently expressed mRNAs, such as the interleukins, nerve growth factor, epidermal growth factor receptor, c‐myc, c‐fos, c‐myb and several other oncogenes as well as interferons α, β and γ were exceptially (U)nA‐rich. It is postulated that differential mRNA stability may be partly determined by the primary nucleotide sequence and in particular by (U)nA sequences within the 3′ non‐coding region. This may represent a novel post‐transcriptional strategy employed by the cell to selectively retain or destroy discrete mRNA species.

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Insertion of synthetic copies of human globin genes into bacterial plasmids

Cited by 332 publications

References 25 publications

Molecular cloning of human epsilon-globin gene.

Molecular cloning of human epsilon-globin gene.

Bis-epoxyethyl derivatives of distamycin A modified on the amidino moiety: Induction of production of fetal hemoglobin in human erythroid precursor cells

Differential mRNA stability to reticulocyte ribonucleases correlates with 3′ non‐coding (U)_nA sequences

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Insertion of synthetic copies of human globin genes into bacterial plasmids

Cited by 332 publications

References 25 publications

Molecular cloning of human epsilon-globin gene.

Molecular cloning of human epsilon-globin gene.

Bis-epoxyethyl derivatives of distamycin A modified on the amidino moiety: Induction of production of fetal hemoglobin in human erythroid precursor cells

Differential mRNA stability to reticulocyte ribonucleases correlates with 3′ non‐coding (U)nA sequences

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Differential mRNA stability to reticulocyte ribonucleases correlates with 3′ non‐coding (U)_nA sequences