Simple and rapid procedure for synthesis of deoxynucleoside 3'-2-cyanoethyl-N,N-diisopropyl-amino phosphites.

2-Cyanoethyl-bis/N,N-diisopropyl/phosphoamidite can be prepared from PCl3 by a very fast and efficient procedure. Without purification it was used for the phosphitilation of suitably protected deoxynucleosides which after simple purification are obtained with a high yield, in a stable and easy to manage form.

Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs.

By the use of glucoamylase-specific synthetic oligodeoxyribonucleotides and molecular cloning of cDNA synthesized from Aspergillus niger total poly(A) + RNA, the primary structure of the glucoamylase G1 mRNA was determined. Glucoamylase G1 is synthesized as a precursor of 640 amino acid residues containing a putative signal peptide of 18 residues, a short propeptide of six residues and the 616 residues long mature enzyme. In vitro translations of mRNA and immunoprecipitations with glucoamylase-specific antisera showed that two glucoamylase polypeptides are synthesized. The larger form with an apparent mol. wt. of 71 000 corresponds to the precursor of glucoamylase G1, and the shorter form with an apparent mol. wt. of 61 000 corresponds to the precursor of glucoamylase G2. From the nucleotide sequencing data of several glucoamylase-specific cDNA recombinants it is shown that the G1 mRNA contains a 169 bp long intervening sequence that can be spliced out to generate a G2 mRNA. Only the 3' part of the G1 mRNA is modified by this splicing event. This kind of differential mRNA processing to give different protein products from one primary transcript has previously only been demonstrated in higher eukaryotes.

A cDNA encoding a small common precursor for human pancreatic polypeptide and pancreatic icosapeptide.

A cDNA for the hormone, human pancreatic polypeptide (PP), was isolated by oligodeoxynucleotide screening from a cDNA library constructed from normal human pancreatic mRNA. The primary structure of the precursor protein as deduced from the cDNA sequence is 95 amino acids long and is composed of a typical, but rather long signal peptide of 29 residues, followed by the sequence of the 36 amino acid human pancreatic polypeptide, which again is separated from the human pancreatic icosapeptide sequence by a classic cleavage and amidation site, Gly-Lys-Arg. The precursor terminates in a heptapeptide which is cleaved from the icosapeptide at a monobasic processing site. Both the size and the structure of the PP precursor was supported by the results of peptide analysis of biosynthetically labeled pro-PP isolated from canine PP cells in which processing was prevented by the arginine analogue canavanine. It is concluded that the precursor for mammalian PP gives rise to two peptide products, the well preserved, carboxyamidated PP and an icosapeptide which is preserved only in its COOH-terminal end, plus a small highly variable COOH-terminal oligopeptide.

Solid phase synthesis of oligonucleotides on a crosslinked polyacrylmorpholide support.

A suitable support for the synthesis of oligonucleotides by the phosphodi- and triester approaches was prepared by treatment of commercially available crosslinked polyacrylmorpholide beads with piperazine. The resulting polymer was used as a support for the synthesis of the self complementary decanucleotide d(T-C-G-G-A-T-C-C-G-A). An analog of dDMTrt in which one of the methoxy groups had been replaced by a carboxymethyloxy group was anchored to the polymer by an amide addition of an excess of protected nucleotide using phosphodiester methodology. Polymer supported synthesis by the phosphotriester approach of the octanucleotide d(T-T-T-T-T-T-T-T) and the hexanucleotide d(G-C-C-C-A-T) is also described. In this case 5'-O-dimethoxytritylthymidine 3'-O-monophthalate was synthesized and linked to the piperazinylated polymer. For the chain extension(3' to 5') a very simple deprotection and coupling cycle was devised.

Asymmetric linker molecules for recombinant DNA constructions.

Asymmetric EcoRI DNA linkers consisting of an AATTC(A)7 dodecamer and a complementary G(T)7 octamer were synthesized. Ligation of such linkers to DNA fragments obviates the need for EcoRI digestion prior to cloning in EcoRI-cleaved vectors.

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 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 transfer RNA gene. XVI. Enzymatic joinings to form the total 207-base pair-long DNA.

The total synthesis of a 207-base pair-long DNA, which is biologically functional as a tyrosine suppressor transfer RNA gene, has been completed. The synthesis involved the enzymatic joining of the previously synthesized duplexes. Thus, the duplex corresponding to the promoter region [P] (Sekiya, T., Brown, E.L., Ramamoorthy, B., Fritz, H.-J., Gait, M.J., Lees, R.G., Ryan, M.J., Khorana, H.G., and Norris, K.E. (1979) J. Biol. Chem. 254, 5781-5786) was jointed to Duplex [I] (Caruthers, M.H., Kleppe, R., Kleppe, K., and Khorana, H.G. (1976) J. Biol Chem. 251, 658-666) to form [P + I]. Separatively, Duplex [III + IV + Vb] was prepared from the previously described Duplexes [III], [IV], and [Vb]. (Loewen, P.C., Miller, R.C., Panet, A., Sekiya, T., and Khorana, H.G. (1976) J. Biol. Chem. 251, 642-650; Sekiya, T., Besmer, P., Takeya, T., and Khorana, H.G. 1976) J. Biol. Chem. 251, 634-641; Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G., (1976) J. Biol Chem. 251, 676-694). The product [P + I], was joined to Duplex [II] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H.G. (1976) J. Biol. Chem. 251, 651-657) and then to [III + IV + Vb] without isolation of the intermediates. In all the above joinings, the duplexes carried 32P-labeled phosphate groups at the appropriate 5'-ends. The total DNA and the intermediate duplexes were all characterized by their relative mobilities in electrophoresis on polyacrylamide gel slabs, by nearest neighbor analysis, and by degradation to 5'-nucleotides of radioactively labeled joined products. Two succeeding papers describe the transcription in vitro and the suppressor activity in vivo, of the synthetic gene now described.

Catalytic oxidation of two ribonucleosides in the 5'-position.

Guanosine and 7-beta-D-ribofuranosyltheophylline was oxidized to the corresponding nucleoside 5'-carboxylic acids by molecular oxygen and a platinum catalyst.