In vitro Synthesis of DNA Components of Human Genes for Globins

Kacian, D. L.; Spiegelman, S.; Bank, Arthur; Terada, Masaaki; Metafora, Salvatore; Dow, Lois W.; Marks, Paul A.

doi:10.1038/newbio235167a0

Cited by 152 publications

(55 citation statements)

References 19 publications

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“…Total cellular RNA greater than 7 S was recovered from the cells by phenol extraction and sucrose gradient centrifugation (7,19). Human and mouse globin mRNAs were prepared from reticulocytes (19) and their complementary DNAs (cDNAs) were synthesized (20)(21)(22)(23). RNA was annealed to cDNA and mRNA-cDNA hybrids were detected after exposure to micrococcal nuclease as described (7,(23)(24)(25) 2B).…”

Section: Methodsmentioning

confidence: 99%

Hemoglobin synthesis in somatic cell hybrids: coexpression of mouse with human or chinese hamster globin genes in interspecific somatic cell hybrids of mouse erythroleukemia cells.

Deisseroth¹,

Barker²,

Anderson³

et al. 1975

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

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Somatic cell hybrids were derived by fusion of mouse erythroleukemia cells with fractionated human marrow enriched in erythroblasts, or with chinese hamster fetal liver erythroid cells. Such interspecific hybrid cells, when isolated in suspension culture, had retained nearly all the mouse chromosomes and had lost most of the human or chinese hamster chromosomes. However, two such hybrids (one human, the other hamster) studied 4-6 weeks after fusion, were found to contain several non-mouse chromosomes. RNA extracted from the human marrow X erythroleukemia hybrid annealed equally to both human and mouse globin complementary DNA, indicating that coexpression of the globin genes of each species had occurred in the hybrid cells. Mouse Hybrids between somatic cells provide an experimental system in which the coexpression of specific structural genes of two parent cell types can be examined (1-3). To study the regulation of the globin genes, we have used cell hybrids formed with a murine erythroleukemia line. Induction of mouse hemoglobin synthesis by dimethylsulfoxide (Me2SO) has been observed in a number of these erythroleukemia cell lines (4-6), but fusion of erythroleukemia cells with fibroblasts of mouse or human origin produces hybrid cells in which extinction of globin mRNA synthesis has occurred at the level of transcription or mRNA processing (7-9). We have previously described a hybrid cell line, formed by fusion of human bone marrow to erythroleukemia cells (7), which had a low level of constitutive hemoglobin synthesis that was markedly amplified on exposure of the hybrid cells to Me2SO. This line, when studied 10 weeks after fusion, retained an average of 1 to 3 human chromosomes per metaAbbreviations: cDNA, complementary DNA; CM-cellulose, carboxymethyl cellulose; Me2SO, dimethylsulfoxide; HAT medium, F-14 medium supplemented with aminopterin, hypoxanthine, and thymidine as formulated by Littlefield (10); G-6-P dehydrogenase, glucose-6-phosphate dehydrogenase; 6-P-GA dehydrogenase, 6-phosphogluconate dehydrogenase; 1S = one diploid set of chromosomes; 2S = two diploid sets of chromosomes; CH, chinese hamster. *This is paper II of a series. Paper I is ref. 7. phase, and only mouse globin was produced. In this paper, we report further studies of interspecific somatic cell hybrids derived from fusion of the Friend mouse erythroleukemia cell with hematopoietic cells of human and chinese hamster origin. Two hybrid lines containing several nonmouse chromosomes exhibited co-expression of the mouse and hamster, or mouse and human globin genes on exposure of hybrid cells to Me2SO. METHODSCell Fusions. A 6-thioguanine-resistant clone of the Friend mouse erythroleukemia cell line (clone 745) was used. This line had a spontaneous reversion frequency of less than 10-8 (7), even under fusion conditions. Cells from human marrow aspirates were enriched in erythroid cells by centrifugation through a discontinuous albumin gradient (15-30%) to reduce the number of nonnucleated cells and then fractionated by gra...

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Section: Methodsmentioning

confidence: 99%

Hemoglobin synthesis in somatic cell hybrids: coexpression of mouse with human or chinese hamster globin genes in interspecific somatic cell hybrids of mouse erythroleukemia cells.

Deisseroth¹,

Barker²,

Anderson³

et al. 1975

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

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“…Transcriptase I was a gift of Dr M. A. Chirigos (Bethesda, Maryland) through Dr J. W. Beard (Gulfport, Florida) and transcriptase I1 was a separate preparation isolated by the method of Kacian and Spiegelman [5].…”

Section: Determination Of the Length Of The 3'-poly(a) Regionmentioning

confidence: 99%

“…Chicken globin cDNA approaching a nucleotide length equivalent to that of globin mRNA was synthesized using an avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase referred to as transcriptase1) which was the kind gift of Dr M. A. Chirigos and Dr J. W. Beard. Intermediate length (300 -400 nucleotides) cDNA was synthesized using an avian myeloblastosis virus RN A-dependent DNA polymerase (transcriptase 11) isolated by the procedure of Kacian and Spiegelman [5]. Using saturating amounts of each enzyme, globin cDNA was synthesized in a 50yl or 100-pl reaction volume containing 120 pM of the labelled deoxynucleotide triphosphate, 0.66 mM unlabelled deoxynucleotide triphosphates, 100 mM KC1, 8 mM MgC12, 8 mM dithiothreitol, 50 mM Tris-C1 pH 8.3, 100 pg/ml actinomycin D, 20 pg/ml (dT)12 -19 (P. L. Biochemicals) as primer, 0.5 pg globin mRNA, and 2 -20 units of enzyme.…”

Section: ____mentioning

confidence: 99%

Organization of Sequences in Avian Globin mRNA

Crawford

Scott

Wells

1977

European Journal of Biochemistry

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Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 650 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions.1. 45 -50 of these untranslated nucleotides are present as poly(A) at the 3' terminus. 2. The 3' untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lengths of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se. In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S 1 nuclease digestion. 3. It is probable that there is greater evolutionary divergence in the 3' untranslated region ofchicken and rabbit globin mRNA when compared with the coding regions of these molecules.The combined data is used to formulate a regional map of chicken globin mRNA.Molecular weight determinations of all specific mRNAs so far isolated indicate that they contain many more nucleotides than required for coding [I 1.

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“…We have extended these methods to the study of human globin mRNA. Because there is sufficient cross-hybridization between human globin mRNA and rabbit globin cDNA (24,25), we have been able to use the chain-specific rabbit globin cDNAs to quantitate the ratio of a to fi globin mRNA in the reticulocyte RNA of humans with different thalassemia syndromes.…”

mentioning

confidence: 99%

“…It is possible to synthesize a radioactive DNA copy of globin mRNA (cDNA) by using the RNA dependent-DNA polymerase of avian myeloblastosis virus (23)(24)(25). This DNA can be used in DNA-RNA hybridization assays to identify and quantitate globin mRNA in an RNA sample.…”

mentioning

confidence: 99%

Quantitative Deficiency of Chain-Specific Globin Messenger Ribonucleic Acids in the Thalassemia Syndromes

Housman

Forget²,

Skoultchi³

et al. 1973

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

114

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A hybridization assay procedure was devised that makes possible quantitation of the ratio of mRNA of alpha to mRNA of beta globin chains in an RNA sample. The assay uses the radioactive synthetic DNA copies obtained by incubation of RNA-dependent DNA polymerase of avian myeloblastosis virus with rabbit globin mRNA that is 80-90%4 enriched in mRNA specific for synthesis of alpha or beta globin chains. The rabbit alpha-chain mRNA is obtained from the postribosomal supernatant of rabbit reticulocyte lysates; the rabbit beta-chain mRNA is obtained from the largest polysomes of rabbit reticulocytes treated with L-O-methylthreonine. Sufficient homology exists between rabbit and human globin chains and globin mRNAs that the synthetic DNA copies of chainspecific rabbit globin mRNA hybridize with human globin mRNA. Applied to the study of globin mRNA isolated from reticulocytes of humans with alpha and beta thalassemia, the technique revealed marked quantitative deficiency of alpha-chain mRNA relative to beta-chain mRNA in alpha thalassemia and similar deficiency of beta-chain mRNA relative to alpha-chain mRNA in beta thalassemia. The thalassemia' syndromes are therefore characterized by true quantitative deficiency of the mRNA specific for the affected globin chain.

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In vitro Synthesis of DNA Components of Human Genes for Globins

Cited by 152 publications

References 19 publications

Hemoglobin synthesis in somatic cell hybrids: coexpression of mouse with human or chinese hamster globin genes in interspecific somatic cell hybrids of mouse erythroleukemia cells.

Hemoglobin synthesis in somatic cell hybrids: coexpression of mouse with human or chinese hamster globin genes in interspecific somatic cell hybrids of mouse erythroleukemia cells.

Organization of Sequences in Avian Globin mRNA

Quantitative Deficiency of Chain-Specific Globin Messenger Ribonucleic Acids in the Thalassemia Syndromes

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