The Synthesis of Hemoglobin in a Cell-Free System

Schweet, Richard S.; Lamfrom, Hildegarde; Allen, Esther H.

doi:10.1073/pnas.44.10.1029

Cited by 129 publications

(24 citation statements)

References 19 publications

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“…Several points, however, argue in favor of the purity of our globin mRNA and the authenticity of its putative DNA complement. The globin mRNA is derived from purified reticulocyte polysomes involved almost exclusively in the synthesis of rabbit globin (29). The usual purification scheme yields an mRNA that has a unique "fingerprint" after degradation with specific nucleases (30).…”

Section: Discussionmentioning

confidence: 99%

In Vitro Synthesis of DNA Complementary to Purified Rabbit Globin mRNA

Ross

Aviv

Scolnick

et al. 1972

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

187

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Several properties of the viral RNA-dependent DNA polymerases and of rabbit globin mRNA make it possible to consider synthesis of the globin gene in vitro. These enzymes copy an RNA template using a short sequence of complementary nucleotides as a primer. Furthermore, globin mRNA has a 3'-terminal sequence of adenylic acid residues that make it particularly suitable as a template, since oligo(dT) can be annealed to a specific site on the mRNA. This small primer could phase the DNA polymerase, possibly ensuring that replication is initiated from that end of the globin message. We have used this approach and find that purified mRNA is an efficient template for the polymerase enzyme. The reaction requires the RNA template and the four deoxyribonucleoside triphosphates, and it is markedly stimulated by the addition of oligo(dT). Consistent with the expectation that the oligo(dT) uniquely phases the polymerase at an adenine-rich region in the globin message, oligo(dG), oligo(dC), and oligo(dA) fail to serve as primers. The product has a density intermediate between that of DNA and RNA, and shifts to a lighter DNA density after treatment with base. Further, it is specifically complementary to globin mRNA and sediments slightly faster in an alkaline sucrose gradient than a DNA standard that has a molecular weight of 129,000. The data suggest that a major portion of the DNA product is a sequence of at least 500 bases, about 50 more than would be necessary to encode rabbit globin. The potential usefulness of this interesting product is discussed.

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Section: Discussionmentioning

confidence: 99%

In Vitro Synthesis of DNA Complementary to Purified Rabbit Globin mRNA

Ross

Aviv

Scolnick

et al. 1972

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

187

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“…Circulating reticulocytes, nonnucleated cells capable of prolonged protein synthesis, are derived from cells in the bone marrow that proceed from an apparently multipotential nucleated stem cell through various stages of increasingly mature or differentiated cell types (2)(3)(4)(5). Protein synthesis in reticulocytes proceeds on ribosomes (6,7), in particular those ribosomes characterized by sedimentation coefficients in excess of 110 S, referred to as polyribosomes (8)(9)(10). In addition to ribosomal RNA, transfer RNA and, presumably, "messenger" RNA are required for protein synthesis in reticulocytes.…”

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confidence: 99%

Synthesis of Ribonucleic Acid in Rabbit Blood Cells In Vivo*

DeBellis¹,

Gluck²,

Marks³

1964

J. Clin. Invest.

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Erythroid cells constitute a model particularly suitable for study of the relationship between cell maturation and nucleic acid and protein synthesis. Circulating reticulocytes, nonnucleated cells capable of prolonged protein synthesis, are derived from cells in the bone marrow that proceed from an apparently multipotential nucleated stem cell through various stages of increasingly mature or differentiated cell types (2-5). Protein synthesis in reticulocytes proceeds on ribosomes (6, 7), in particular those ribosomes characterized by sedimentation coefficients in excess of 110 S, referred to as polyribosomes (8-10). In addition to ribosomal RNA, transfer RNA and, presumably, "messenger" RNA are required for protein synthesis in reticulocytes. It vitro studies have shown that reticulocytes cannot synthesize RNA (8). The present investigations were designed to determine when, in the course of in vivo maturation of erythroid cells, the RNA of reticulocytes is synthesized.In these experiments, the synthesis of RNA in rabbit erythroid cells in vivo was evaluated by following the time course of incorporation of administered PS2 into cellular RNA. The findings indicate that RNA of reticulocytes is synthesized in a relatively early precursor cell stage of erythroid cell maturation. RNA, once synthesized, can remain functional through the reticulocyte stage. Our study also provides evidence that nucleated leukocytes, in contrast to reticulocytes, can synthesize RNA.*Submitted for publication January 3, 1964; accepted February 17, 1964. This work was supported in part by grants CY 2332 and GM 07368 from the U. S. Public Health Service and by grant NSF GB 310 from the National Science Foundation.A portion of these data was presented at the Annual Meeting of the American Federation for Clinical Investigation, Atlantic City, N. J., May 1963, and appeared in abstract form (1). MethodsInduction of reticulocytosis in rabbits. Two-to 3-kg rabbits were made anemic by subcutaneous injection of 25 mg phenylhydrazine per day for 4 days (11). This regularly resulted in a reticulocytosis of 60 to 90%.In vivo labeling of RNA with P". Twenty-four hours after the last injection of phenylhydrazine, 3 mc of carrier-free P's (approximately 3 mc per ml) was injected intravenously. Samples of blood were then obtained at intervals of from 30 minutes to 17 days. A total of 14 rabbits was studied. In 10 animals, a single sample of blood was obtained at the times indicated below. In 3 experiments, blood was obtained from the same animal at two different time intervals, and in 1 experiment, at three different time intervals. Reticulocyte ribosomal RNA was purified and analyzed for each of these blood samples. The RNA of cells remaining unlysed after "shock" lysis was analyzed for 7 of the blood samples. These samples were drawn at 30 minutes and 1 (twice), 3,20,22, and 42 hours.Preparation of blood samples. Blood was obtained by cardiac puncture and collected into heparinized syringes.A sample was removed for enumeration of the erythrocytes, le...

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“…How could a cell with no genes synthesize a specific protein? Somewhat later, Richard Schweet, who had moved from Borsook's laboratory to the City of Hope, demonstrated the cell-free synthesis of hemoglobin (47). The isolation of messenger RNA was around the corner, and its association with ribosomes was soon demonstrated for both bacteriophage-specific RNA and host-specific RNA (4,13).…”

Section: Knudsonmentioning

confidence: 99%