Enzymatic synthesis of a segment of bacteriophage Qbeta coat protein gene.

The oligoribonucleotide, A-A-A-C-U-U-U-Gp, constituting a segment of RNA bacteriophage Qbeta coat protein gene was efficiently synthesized at a milligram scale by a combination of enzymatic methods using bacteriophage T4 RNA ligase and the thermophilic polynucleotide phosphorylase. A-A-A-Cp was synthesized from A-A-A and pCp by the newly developed mononucleotide addition method using T4 RNA ligase in a yield of 83%, followed by dephosphorylation with bacterial alkaline phosphatase to obtain A-A-A-C. pU-U-U-Gp was synthesized from pU-U-U and GDP by the simultaneous action of polynucleotide phosphorylase and RNase T1 in a yield of 32%. finally, the two oligonucleotides (A-A-A-C and pU-U-U-Gp) were ligated with T4 RNA ligase and the octanucleotide, A-A-A-C-U-U-U-Gp, was obtained in a yield of 85%.     

Continuous production of homopolynucleotides by immobilized polynucleotide phosphorylase

A polynucleotide phosphorylase was immobilized with glutaraldehyde, via an aminopropyl spacer, on porous glass. The specific activity of the immobilized enzyme was effectively increased by the addition of an appropriate ribonucleoside diphosphate on immobilization.A homopolynucleotide could be synthesized continuously by passing a nucleoside diphosphate solution through the immobilized enzyme column. The chain length of the product depended upon the temperature and the flow rate. Polyinosinic acid, poly(I), was continuously synthesized with the immobilized enzyme for about one month without appreciable loss of activity.Polyinosinic acid-polycytidylic acid, poly(I)·poly(C), prepared from poly(I) and poly(C) synthesized with the immobilized polynucleotide phosphorylase, induced interferon-β (IFN-β) in human cultured cells as effectively as that prepared from homopolynucleotides synthesized with the free enzyme.     

Total synthesis of a structural gene for the human peptide hormone angiotensin II.

Seven oligonucleotide chains containing between 6 and 11 nucleotide units were synthesized. The segments were phosphorylated by T4 polynucleotide 5'-hydroxyl-kinase and joined by T4 polynucleotide synthetase (ATP) to give the double-stranded DNA consisting of 33 base pairs. The DNA sequence was deduced from the known peptide sequence according to the genetic code.     

Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase.

In crude extracts of T2L phage-infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate. Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well-known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165 000 with three subunits of 55 000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane-bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 X 10(4) min-1 at 37 degrees C. Even at 0 degree C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate-limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)-synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.     

Enzymatic synthesis of polyuridylic acid containing modified bases.

5'Mercaptouridine-5'-diphosphate (hs5UDP) has been synthesized and investigated as a substrate of the polynucleotide phosphorylase of Micrococcus luteus. While hs5UDP is not utilized alone, it can be copolymerized with UDP; however, unusually for this enzyme, the ratio of 5'mercaptouridylate vs. uridylate residues in the polynucleotide product (MPU) is always lower than the ratio of hs5UDP v. UDP in the substrate mixture. Furthermore, hs5UDP decreases the rate of the enzymic polymerization reaction. The MPU product forms two-stranded and three-stranded complexes with poly(A). The circular dichroic spectra of these complexes are similar to those formed between poly(U) and poly(A), but their melting profiles indicate somewhat lower stability. The physicochemical and biochemical properties of the enzymic product are qualitatively similar to those of MPU prepared by chemical modification; both are potent inhibitors of a DNA-dependent RNA polymerase.     

Template-free ribosomal synthesis of polypeptides from aminoacyl-tRNA. Polyphenylalanine synthesis from phenylalanyl-tRNALys

Misacylated phenylalanyl-tRNALys, just as lysyl-tRNALys, but not phenylalanyl-tRNAPhe, have been shown to serve as substrates for ribosomal synthesis of polypeptides (polyphenylalanine and polylysine, respectively) in the absence of a template polynucleotide (poly(A)). The conclusion was made that it is the structure of tRNA that determines the ability of the aminoacyl-tRNALys to participate in peptide elongation on ribosomes without codon-anticodon interactions.     

Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3' sequences of RNA.

A simple procedure for purifying polynucleotide phosphorylase from Escherichia coli cells by means of affinity chromatography on an RNA-Sepharose column is described. The purified enzyme preparation has a specific activity 3500-fold that of the crude extract and is essentially homogeneous, as determined by ultracentrifugation, polyacrylamide gel electrophoresis under denaturing conditions, isoelectric focusing and serological assays. It is virtually free of nuclease contamination, a property which permits its use in the synchronous phosphorolysis of RNA chains. The enzyme molecule is composed of three identical subunits of Mr = 84,000. Each subunit contains three cysteine residues, one of which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) whereas the two other groups are only exposed on denaturation of the protein. All three enzyme subunits participate in the processive phosphorolysis of the poly(A) tail of each globin mRNA chain. An advantageous method was developed for synchronous phosphorolysis of RNA molecules using a molar excess of polynucleotide phosphorylase immobilized onto Sepharose.     

Enzymatic synthesis of deoxyribo-oligonucleotides of defined sequence. Deoxyribo-oligonucleotide synthesis*

An enzyme, which is probably identical with polynucleotide phosphorylase, was prepared from Escherichiacoli B. In the presence of Mn(2+) it catalyzes the addition of one (and to a slight extent more) residue of deoxyribonucleotide residue from the diphosphate to an oligodeoxyribonucleotide primer. The shortest effective primers contained three phosphate residues. Ribodinucleotides were effective as primers and accepted two or three deoxyribonucleotide residues under these conditions. The application of the procedures to the convenient synthesis of certain defined oligodeoxyribonucleotides up to nine residues long is discussed.     

The synthesis and properties of N6-substituted 2-amino-purine derivatives.

1. The synthesis, ultraviolet absorption spectra, and behaviour in alkali of N6-methoxy-, N6-methyl, hydroxy-, and N6-hydroxy-2-aminopurines have been described 2. N6-Methoxy-2-aminopurine riboside 5'-pyrophosphate has been prepared and used for polymerization with polynucleotide phosphorylase. 3. The copolymer containing N6-methoxy-2-aminopurine riboside and adenosine residues has been obtained; attempts to synthesize the homopolymer have not been successful. 4. All the purine analogues synthesized have been tested and shown to act mutagenically on Salmonella typhimurium TA1530.     

Immobilization of DNA via oligonucleotides containing an aldehyde or carboxylic acid group at the 5'' terminus.

A general method for the immobilization of DNA through its 5'-end has been developed. A synthetic oligonucleotide, modified at its 5'-end with an aldehyde or carboxylic acid, was attached to latex microspheres containing hydrazide residues. Using T4 polynucleotide ligase and an oligonucleotide splint, a single stranded 98mer was efficiently joined to the immobilized synthetic fragment. After impregnation of the latex microspheres with the fluorescent dye, Nile Red and attachment of an aldehyde 16mer, 5 X 10(5) bead-DNA conjugates could be detected with a conventional fluorimeter.     

Enzymatic synthesis of polymers containing nicotinamide mononucleotide.

Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.     

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].     

Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence. Polynucleotide phosphorylase catalysed synthesis using pyrimidine analog-containing deoxyribonucleoside 5''-diphosphates.

The E. coli polynucleotide phosphorylase-catalysed reaction of the deoxynucleoside 5'-diphosphates of 5-methyldeoxycytidine, N4-hydroxydeoxycytidine, deoxyuridine and 5-mercurideoxyuridine with the primers d(pT-T-A-G) and d(pT-T-T-T-T-T) have been studied under conditions where the primer is extended, predominantly, by one or two nucleotide residues. In experiments with 5-mercurideoxyuridine 5'-diphosphate, no 5-mercurideoxy-uridine-containing oligonucleotides were produced. The other three nucleotide analogs were found to be good substrates for E. coli PNPase and the conditions established for synthesis with these analogs will allow the construction of a number of biologically useful types of oligodeoxyribonucleotide.     

Specific oligoribonucleotide synthesis using primer-independent polynucleotide phosphorylase.

Oligoribonucleotides of a predetermined base sequence beginning with adenylyl-3', 5'-adenosine at the 5' end have been synthesized in yields varying between 13% and 42%. The synthesis was carried out using primer-independent polynucleotide phosphorylase from E. coli in the presence of high concentrations of primer and of sodium chloride.     

Quaternary structure and proteolysis of the polynucleotide phosphorylase from C. perfringens]

This report describes structural studies on purified polynucleotide phosphorylase from C. perfringens. A method is described for the purification of the enzyme which yields a product equivalent in activity to the native polynucleotide phosphorylase from E. coli. These studies revealed a molecular heterogeneity arising from successive stages of proteolysis, to which this enzyme is especially sensitive; unusally, the enzyme is obtained as a mixture of variable proportions of the native and proteolysed forms. We found in all cases a trimeric basic structure composed of the native (alpha) or proteolysed (lapha) or proteolysed (alpha', alpha") catalytic sub-units, However, the enzyme is rather easily dissociated into its sub-units, a phenomenon which seems to accompany proteolysis (Table). Under the action of either endogenous proteases or trypsin, two enzymatic forms are obtained: their quaternary structures seem analogous, but they differ in their catalytic properties from each other and from the initial enzyme. With some care at each step of purification, the polynucleotide phosphorylase of E. coli can be obtained exclusively in its native form. The greater susceptibility to proteolysis of the enzyme from C. perfrigens and the relationship between such degradation and quaternary structure seem to be at the origin of the peculiar behavior of this polynucleotide phosphorylase.     

Effects of low concentrations of actinomycin D on the initiation of DNA synthesis in rapidly proliferating and stimulated cell cultures.

Analysis of harlequin chromosomes obtained by the fluorescent plus Gimesa (FPG) technique indicates that the ratio of single to twin sister chromatid exchanges (SCE's) approximates the 2:1 ratio that would be expected if the chromosomal subunits had opposite polarity. The greater resolution obtained with the FPG technique than with autoradiography enables us to conclude that exceptional segregation of label, such as would lead to isolabelling of chromosomes after two rounds of replication or heterolabelling after one round, does not occur. The data indicate that Chinese hamster ovary (CHO) chromosomes most likely consist of only 1 DNA double helix, i.e., are mononemic. No evidence for single polynucleotide strand exchange was found.     

Inhibition of protein synthesis by polypeptide antibiotics.. II. In vitro protein synthesis

Ennis, Herbert L. (St. Jude Children's Research Hospital, Memphis, Tenn.). Inhibition of protein synthesis by polypeptide antibiotics. II. In vitro protein synthesis. J. Bacteriol. 90:1109-1119. 1965.-This investigation has shown that the polypeptide antibiotics of the PA 114, vernamycin, and streptogramin complexes are potent inhibitors of the synthetic polynucleotide-stimulated incorporation of amino acids into hot trichloroacetic acid-insoluble peptide. The antibiotics inhibited the transfer of amino acid from aminoacyl-soluble ribonucleic acid (s-RNA) to peptide. The A component of the antibiotic complex was active alone in inhibiting in vitro protein synthesis, whereas the B fraction was totally inactive. However, the A component, when in combination with the B component, gave a greater degree of inhibition than that observed with the A fraction alone. On the other hand, the endogenous incorporation of amino acid was much less susceptible to inhibition than the incorporation of the corresponding amino acid in a system stimulated by synthetic polynucleotide. In addition, synthesis of polyphenylalanine stimulated by polyuridylic acid was inhibited to a greater extent when the antibiotics were added before the addition of polyuridylic acid to the reaction mixture than when the antibiotics were added after the polynucleotide had a chance to attach to the ribosomes. However, the antibiotics apparently did not inhibit the binding of C(14)-polyuridylic acid or C(14)-phenylalanyl-s-RNA to ribosomes. The antibiotics did not affect the normal release of nascent protein from ribosomes and did not disturb protein synthesis by causing misreading of the genetic code. The antibiotics bind irreversibly to the ribosome, or destroy the functional identity of the ribosome. The antibiotic action is apparently a result of the competition between antibiotic and messenger RNA for a functional site(s) on the ribosome.     

DNA synthesis in Pseudomonas acidovorans infected with mutants of bacteriophage phi W-14 defective in the synthesis of alpha-putrescinylthymine.

Normal levels of the hypermodified pyrimidine, alpha-putrescinylthymine, which is formed from hydhydroxymethyluracil at the polynucleotide level (Maltman et al., J. Virol. 34:354-359, 1984), are not required in bacteriophage luminal diameterW-14 DNA for the DNA to serve as a replicative template in luminal diameterW-14-infected cells.     

On the free-energy changes in the synthesis and degradation of nucleic acids.

Standard free-energy changes for reactions involving single- and double-stranded nucleic acids have been related to that for polynucleotide synthesis from ribonucleoside diphosphates for which deltaG degrees' approximately O. For polynucleotide formation from triphosphates this quantity is about -1 kcal. In the replication reaction the base pairing interactions are quantitatively of comparable importance. Production of a hydrolytic break in a double strand is substantially less favorable than in a single strand. The resealing of breaks utilizing ATP and NAD+ have similar free-energy changes and are entropy driven processes. The highly exergonic hydrolysis of pyrophosphate is maintained to be of significance for both in vivo and in vitro polymerizations.