Total Synthesis of the Gene for an Alanine Transfer Ribonucleic Acid from Yeast

Agarwal, Kan; Büchi, H.; Caruthers, Marvin H.; Gupta, Naba K.; Khorana, H. G.; Kleppe, Kjell; Kumar, Ashok; Ohtsuka, Eiko; RajBhandary, Uttam L.; Sande, J.H. van de; Sgaramella, Vittorio; Weber, Hans Hermann; Yamada, Toshiro

doi:10.1038/227027a0

Cited by 235 publications

(82 citation statements)

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“…In both cases the melting profiles are characteristic of specific hybrids, with tm values (in 0.1 X SSC) of 610 for unfractionated chloroplast tRNA and 63' for tRNAPhe. The tm of polynucleotide complexes is a function of (i) the G+C content (23,24), (ii) the number (up to approximately 50) of interacting nucleotides (24), (iii)' the sequence of the interacting nucleotides (25), and (iv) the number, type, and location of mismatched base pairs (26). The chloroplast tRNAPhe contains 54% G+C (9) and the calculated (23) tm in 0.1 X SSC for a DNA-DNA duplex containing 54% G+C is 760, whereas the observed value for the chloroplast tRNAPhE-DNA hybrid is 630.…”

Section: Resultsmentioning

confidence: 99%

“…The iodination mixture contained in 0. 25 ,ul of 0.1 M Na2SO3 and 1 ml of freshly prepared 1 M NH4 acetate-NH40H buffer (pH 8.0) were added and the mixture was incubated at 700 for 35 min. The iodinated RNA was dialyzed against 0.001 M potassium phosphate buffer (KPO4, pH 7.0) and then placed onto a 1.4 X 1.8 cm hydroxylapatite C (Clarkson Chemical Co.) column equilibrated with the same buffer.…”

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confidence: 99%

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Transcriptional origin of Euglena chloroplast tRNAs

Schwartzbach

Hecker

Barnett

1976

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

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tRNA*DNA hybridization studies indicate that Euglena chloroplast tRNAs are transcriptional products of the chloroplast genome, which contains approximately 28 tRNA cistrons. Hybridization with purified chioroplast tRNAPhe and tRNAAP shows that the chloroplast genome contains one cistron for each of these two species. No hybridization of chloroplast tRNA with nuclear DNA was observed. tRNAs from Euglena cytoplasm, Escherichia coli, and Agmenellum quadraduplicatum do not compete with chloroplast tRNA for hybridization with chloroplast DNA. Evidence is presented that photoinduction of chloroplast tRNAs is at the level of transcription rather than maturation of tRNA precursor molecules. The chloroplasts of Euglena gracilis contain tRNAs and aminoacyl-tRNA synthetases that are (i) induced by light and (ii) exclusively compartmentalized within these organelles (1-3). The synthetases are encoded by nuclear genes and are translated on cytoplasmic ribosomes (2). In the present report we have used tRNA-DNA hybridization to ascertain the intracellular localization of the structural genes for the chloroplast tRNAs.MATERIALS AND METHODS Euglena gracilis var. bacillaris and W3BUL, an ultravioletinduced mutant lacking detectable chloroplast structure and DNA, were used (4). The blue-green alga, Agmenellum quadraduplicatum (a gift from Dr. Lonnie 0. Ingram), was grown in ASP2 medium (5) and bubbled with 1% CO2 in air.Preparation of Nucleic Acids. Chloroplasts were isolated as described (2) except that the time of zonal centrifugation was 3 hr. They were stored at -80°. For the isolation of chloroplast DNA, the frozen chloroplasts were thawed in 0.15 M NaCl, 0.1 M Na2EDTA, and 0.1 M Tris-HCl (pH 8.0), and the DNA was extracted essentially as described (6) by Marmur using boiled pancreatic ribonuclease (100 Ag/ml, Sigma, Type IIIA) followed by treatment with predigested Pronase (500 gg/ml, Calbiochem). Nuclear DNA was isolated from W3BUL by the same procedure. After precipitation with isopropanol, the DNA was further purified by preparative CsCl gradient centrifugation (7). The gradients were fractionated so as to retain all DNA species. The amount of nuclear DNA in the chloroplast DNA fraction was determined from the density-gradient profiles.Chloroplast ribosomes and rRNA were extracted as described (8). rRNA was further purified by Sephadex G-100 chromatography in order to remove any residual tRNA. tRNA was isolated as described, using both DEAE-cellulose and Sephadex G-100 column chromatography (1, 3). Cytoplasmic tRNA was isolated from W3BUL, whereas chloroplast tRNA was extracted from purified chloroplasts (2). The chloroplast tRNA was free of contaminating cytoplasmic tRNA as determined by benzoylated DEAE-cellulose column chromaAbbreviations: SSC, 0.15 M NaCI-0.015 M sodium citrate (pH 7); x X SSC, concentration of the solution is x times that of SSC; tin, thermal dissociation ("melting") temperature.* A brief report of this work appeared in Plant Physiol. (1975) 56, S71. tography of the purified chloroplast...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Transcriptional origin of Euglena chloroplast tRNAs

Schwartzbach

Hecker

Barnett

1976

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

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“…Synthetic oligonucleotides of specitlc base sequence can be synthesized by organic chemical methods [21] and a combination of organic chemical and enzymic methods has recently led to the total synthesis of a gene [22]. One major aspect, under which the present study was undertaken, was to develop methods, which would allow also the enzymic synthesis of oligodeoxynucleotides of specific base sequences.…”

Section: Discussionmentioning

confidence: 99%

Synthetic Polynucleotides

Roychoudhury

Kössel

1971

European Journal of Biochemistry

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1. The use of chemically synthesized hexathymidylate as primer for terminal deoxynucleotidyl transferase in the presence of a ribonucleoside triphosphate and Mg2+ results in a limited addition of ribonucleotides a t the 3'-end of the primer. The incorporation is absolutely dependent upon the presence of Mg2+ and primer, and the pH optimum is similar to that observed for deoxynucleotide incorporation.2. The priming efficiency decreases when oligothymidylates of smaller chain length are used according to the order: hexa > penta > tetra > tri. The incorporation into the trinucleotide is very small. 3. Depending upon the primer: triphosphate ratio, the reaction mixture after incubation contains either a mono-or a diaddition product, or both and such products can be synthesized under controlled conditions. The upper limit of this terminal addition has been found to be up to two nucleotide units. A ribonucleotide to ribonucleotide linkage is formed in the case of the diaddition product.4. The ribonucleotide terminated hexathymidylates are capable of accepting dAMP residues from dATP in the presence of terminal deoxynucleotidyl transferase to give rise to a polymeric product whereby the newly synthesized polymer remains covalently linked to the original hexathymidylate by a ribonucleotide linkage.5. Due to the presence of this ribonucleotide linkage, the polymeric product can be cleaved from the primer by alkaline hydrolysis, resulting in a separation of the primer sequences from the product sequences. The use of ribonucleotide terminated oligodeoxynucleotides thus provides a valuable tool for enzymic synthesis of polydeoxynucleotides, whereby the material used as primer (necessary for an enzyme catalyzed reaction) can be removed from the product.Enzyme catalyzed reactions leading to the synthesis of polydeoxynucleotides are dependent on primers [l-31 which during the reaction are incorporated into the polymeric products. Since such primers remain covalently linked to the product material, the base sequences of the primer used thus will interfere with the nucleotide sequences of the desired products. Therefore, the ideal primer for the synthesis of specific polynucleotides would be the one which could be conveniently separated from the product, after the desired stretch of polynucleotides has been synthesized. With this objective in mind we have explored the possibility of addition of one ribonucleotide at the initiating end of a small oligodeoxynucleotide primer and subsequent addition of deoxynucleotides at the ribo-3I-end of the primer. The advantage of using small oligonucleotides as the starting material resides in the fact that upon limited synthesis, the newly synthesized products could be separated from the starting material and subsequently checked for correctness of additions. Insertion of one ribonucleotide between the primer and product sequences would enable their separation by alkaline hydrolysis.In this communication we wish to report the synthesis of ribonucleotide terminated oligodeoxynucleotides and t...

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“…It was an arduous task, taking Khorana and 17 co-workers years to assemble a very small gene (207 base-pairs). 6,7 Scientists had been "reading" the genetic code for years. Khorana and colleagues were the first to accomplish the next step: "writing" the code of the building blocks of life by making a small but functional gene.…”

Section: Early Milestonesmentioning

confidence: 99%