Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.     

Total synthesis of a tyrosine suppressor transfer RNA gene. XIV. Chemical synthesis of oligonucleotide segments corresponding to the terminal regions.

Chemical syntheses of the two dodecanucleotides d(T-C-A-A-C-G-T-A-A-C-A-C) and d(A-C-G-T-T-G-A-G-A-A-A-G), the two undecanucleotides d(T-T-T-A-C-A-G-C-G-G-C) and d(T-G-T-A-A-A-G-T-G-T-T), the decanucleotide d(A-G-T-C-C-G-A-A-A-G), and the nonanucleotide d(A-A-T-T-C-T-T-T-C) are described. These deoxyribo-oligonucleotide segments, excluding the decanucleotide, represent the DNA duplex corresponding to the previously determined nucleotide sequence -30 to -51 of the promoter region of the gene for the tyrosine suppressor tRNA (Sekiya, T., Gait, M.J., Norris, K., Ramamoorthy, B., and Khorana, H.G. (1976) J. Biol. Chem. 251, 4481-4489) and include the EcoRI restriction endonuclease sequence at the appropriate 5'-end. The nona- and decanucleotide along with the previously synthesized deoxyribo-oligonucleotide segments 25 to 27 (Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G. (1976) J. Biol. Chem. 251, 676-694) together represent the DNA duplex corresponding to the natural nucleotide sequence 121 to 142 of the region adjoining the C-C-A end of the tyrosine tRNA gene and, in addition, a run of nine nucleotides which include the EcoRI restriction enzyme sequence at the 5'-end. The syntheses used protected mono- and oligonucleotides and stepwise condensation methods. A noteworthy feature of the present syntheses was the use of reverse phase high pressure liquid chromatography for the rapid and efficient separation of synthetic reaction mixtures.     

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 10. Enzymatic joining of chemically synthesized segments to form the DNA duplex corresponding to the nucleotide sequence 86-126.

The polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxypolynucleotides (segments 19 to 26), comprising the nucleotide sequence 86-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been investigated. Joining was studied using various combinations of 3, 4, or larger number of segments at a time. The extent of joining was in general low (0 to 40%) for the three-component as well as for the four-component systems. Joining of the five- and six- component systems was more satisfactory with yields from 25 to about 60%. The three duplexes [IVa] to [IVc]were prepared in single step reactions in yields of about 50% and were characterized. Duplex [IVd] could not be prepared in a single step reaction because of the failure of 5'-phosphorylated segment 26 to join to the rest of the duplex. Using a carefully annealed mixture of segments 24, 25, and phosphorylated segment 26, the joining of the latter to segment 24 could be realized in about 25% yield, much activated intermediate being concurrently present.     

Total synthesis of a tyrosine suppressor transfer RNA gene. XIII. Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence -1 to -29 in the promoter region.

Chemical syntheses of two tridecanucleotides, d(G-C-A-T-C-A-T-A-T-C-A-A-A) and d(G-C-G-T-C-A-T-T-T-G-A-T-A), and three undecanucleotides, d(G-G-A-A-G-C-G-G-G-G-C), d(T-G-A-T-G-C-G-C-C-C-C), and d(T-G-A-C-G-C-G-C-C-G-C), are described. These deoxyribo-oligonucleotide segments together represent the DNA duplex corresponding to the previously determined nucleotide sequence -1 to -29 of the promoter region of the tyrosine tRNA gene (Sekiya, T., van Ormondt, H., and Khorana, H.G. (1975) J. Biol. Chem. 250, 1087-1098). Chemical syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. The desired condensation products were isolated by solvent extraction methods in the case of di- and trincleotides and by anion exchange chromatography in the case of longer chains. All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling at the 5'-ends with a [32P]phosphate group.     

Total synthesis of a tyrosine suppressor tRNA gene. XV. Synthesis of the promoter region.

By use of polynucleotide kinase and polynucleotide ligase, the 10 deoxyoligonucleotide segments, whose syntheses have been described in accompanying papers, have been joined to form the 62-nucleotide-long DNA corresponding to the promoter region of an Escherichia coli suppressor tRNA gene. The following sequence in the joining reactions was used to obtain error-free and optimal yields of the products: 1) joining of Segment P-1 to P-3 in the presence of Segment P-2; 2) joining of Segments P-4 to P-7 to form Duplex [P4-7]; 3) joining of Segments P-8 to P-10 to Duplex [P4-7] to form Duplex [P4-10]; and finally, 4) joining of P-(1 + 3) and P-2 to Duplex [P4-10] to form the total promoter Duplex [P].     

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 3. Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence 27-51.

Chemical syntheses of the four deoxyribodecanucleotides, d(T-C-G-A-A-G-T-C-G-A), d(C-G-T-C-A-T-C-G-A-C), d(T-G-A-C-G-G-C-A-G-A), and d(C-T-A-A-A-T-C-T-G-C) are described. These polynucleotides form, respectively, segments 7 to 10 in the plan adopted for the total synthesis of the DNA corresponding to the precursor for the Escherichia coli tyrosine tRNA. The syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. Detailed schemes used in the present syntheses are shown in Diagrams 1 to 4 in the text. The final products were subjected to extensive chromatography and were characterized as pure by chemical and enzymatic procedures.     

Mechanism of suppression in Drosophila: II. Enzymatic discrimination of wild-type and suppressor tyrosine transfer RNA

Previous studies had shown that two principle forms of tyrosine transfer RNA of Drosophila melanogaster were present in wild-type adult flies but that the second form was virtually absent in a suppressor mutant, su(s)². Current results are at variance with the previous ones, in that the suppressor mutant has significant amounts of the second form of tRNA^Tyr. A second chromatography system for separating these forms of tRNA^Tyr is described, RPC-5, and is compared to the system used previously, RPC-2. Both systems indicate that wild-type flies contain the two forms of tRNA^Tyr in a ratio of $\text{40}\text{60}$, the suppressor mutant in a ratio of $\text{60}\text{40}$. The difference between current and previous results can be attributed to the procedures used in the preparation of the enzyme that is used as a source of tyrosyl-tRNA ligase. The enzyme activity can be separated into two fractions on DEAE-cellulose chromatography. With suppressor tRNA as substrate, one enzyme fraction charges both forms of tRNA^Tyr but the second enzyme fraction charges the first form preferentially or nearly exclusively in some cases, as was seen in the previous experiments. With wild-type tRNA as substrate both enzyme fractions charge both forms of tRNA^Tyr. Storage results in the loss of the enzyme's ability to discriminate against the second form of tRNA^Tyr from the suppressor mutant, while the enzymatic activity is retained. We postulate that the su(s)⁺ locus produces an enzyme that modifies the second isoacceptor of tRNA^Tyr and that, when such modification fails to occur (as in the su(s)² mutant), the tRNA is unable to accept tyrosine from one form of tyrosyl-tRNA ligase. How the discrimination against the second isoacceptor by the ligase may be important metabolically is not apparent.     

CXII. Total synthesis of the structural gene for an alanine transfer RNA from yeast. Enzymic joining of the chemically synthesized polydeoxynucleotides to form the DNA duplex representing nucleotide sequence 1 to 20

The DNA duplex (designated [A]) corresponding to the nucleotides 1 to 20 of the major yeast alanine transfer RNA (Fig. 1) has been synthesized. The first step involved the T4 ligase-catalyzed joining of d-(5′-³²P)-C-C-G-G-A-A-T-C (segment 4, Fig. 1) to the dodecanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T (segment 1, Fig. 1), in the presence of the complementary decanucleotide d-(5′-OH)-C-C-G-G-A-C-T-C-G-T (segment 3, Fig. 1). The resulting icosanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T-C-C-G-G-A-A-T-C, was isolated free from the decanucleotide (segment 3). The synthesis of [A] was then completed by the ligase-catalyzed joining of 5′-³²P or ³³P-labeled hexanucleotide d-(5′-P)-C-C-A-C-C-A (segment 2) to the 5′-³²P or ³³P-labeled decanucleotide, d-(5′-P)-C-C-G-G-A-C-T-C-G-T (segment 3), in the presence of the above icosanucleotide.     

The background of the total synthesis of yeast alanine transfer RNA

<正>The research findings concerning the total synthesis of yeast alanine transfer RNA (yeast alanine tRNA) were successively published in Chinese Science Bulletin (1982) and Science in     

RNA synthesis in synchronously growing populations of HeLa S3 cells. I. Rate of total RNA synthesis and its relationship to DNA synthesis

The rate of RNA synthesis in synchronously growing HeLa S3 cells was determined as a function of position in the cell generation cycle. Measurements throughout the cycle of both the rate of incorporation of radioactively-labeled uridine and of the total amount of RNA indicate that (1) the rate of RNA synthesis is constant (or increases only slightly) during G₁, approximately doubles during the first half of S, and then remains constant during the remainder of S and G₂, and (2) cells attain the average G₁ rate of RNA synthesis very early in G¹, and maintain the average G₂ rate until mitosis. If the initiation of DNA synthesis is blocked, the acceleration of RNA synthesis is markedly reduced or eliminated. Further experiments in which DNA synthesis was inhibited at different times in S, or to varying degrees from the beginning of S, suggest that the extent to which RNA synthesis is accelerated depends on the amount of DNA duplicated. These data also indicate that duplication of the first half, and in particular the first few per cent, of the DNA complement results in a disproportionate acceleration of RNA synthesis. The possibility that fluctuations in the sizes of precursor pools may lead to misinterpretation of labeled-uridine incorporation data was examined. Experiments indicate that in this system pool fluctuations do not cause invalid measures of RNA synthesis. It is concluded that RNA synthesis occurs throughout interphase, but undergoes a two-fold increase in rate which is dependent on the duplication of DNA.     

Purification of a hybrid molecule composed of tyrosine transfer RNA and its complementary DNA sequence

The transducing bacteriophage φ80psu_III⁺ carries one structural Escherichia coli gene specifying tyrosine tRNA.The r strand of bacteriophage φ80psu_III⁺ was hybridized with E. coli transfer RNA and the hybrid digested with Neurospora crassa endonuclease. The analysis of the products of enzymic digestion demonstrated the release of a cistron-hybrid composed of tyrosine tRNA and its complementary DNA sequence. The cistron-hybrid was purified from unhybridized DNA by cesium sulphate density-gradient centrifugation and gel filtration.The ratio between tyrosine tRNA and its complementary DNA sequence in the final product was 1:1 as demonstrated by radioisotopic analysis. This purification represents a 30,000-fold enrichment of the E. coli genome for a specific DNA sequence.