Abstract: New and useful intermediate nucleotides bound to an inorganic polymer support, including the preparation thereof, and processes for the conversion to oligonucleotides which are especially useful for the synthesis of polynucleotides, particularly ribonucleic (RNA) and deoxyribonucleic acids (DNA).

Abstract: The substrate specificity of micrococcal nuclease (EC 3.1.4.7.) has been studied. The enzyme recognises features of nucleotide composition, nucleotide sequence and tertiary structure of DNA. Kinetic analysis indicates that the rate of cleavage is 30 times greater at the 5' side of A or T than at G or C. Digestion of end-labelled linear DNA molecules of known sequence revealed that only a limited number of sites are cut, generating a highly specific pattern of fragments. The frequency of cleavage at each site has been determined and it may reflect the poor base overlap in the 5' T-A 3' stack as well as the length of contiguous A and T residues. The same sequence preferences are found when DNA is assembled into nucleosomes. Deoxyribonuclease 1 (EC 3.1.4.5.) recognises many of the same sequence features. Micrococcal nuclease also mimics nuclease S1 selectively cleaving an inverted repeat in supercoiled pBR322. The value of micrococcal nuclease as a "non-specific" enzymatic probe for studying nucleosome phasing is questioned.

Abstract: The oligonucleotides pppA2'p5'A2'p5'A and related oligomers (2-5A) are synthesized by an enzyme that is widely distributed in a variety of cells, the activity of which varies with interferon treatment, growth and hormone status. Because significant amounts of 2-5A have recently been detected in interferon-treated cells, it has been suggested that the oligonucleotides may be involved in interferon action and in the control of cell metabolism. In both intact cells and cell-free systems 2-5A has been shown to activate a ribonuclease. We report here investigations of the sequence specificities of the 2-5A-dependent ribonucleases in extracts of rabbit reticulocytes, mouse ascites tumour cells and human lymphoblastoid cells in conditions of partial digestion using terminally labelled RNA substrates. The enzymes cleaved on the 3'-side of UN sequences to yield UpNp terminated products. Cleavage was observed predominantly at UA and UU sequences.

Abstract: A series of nonionic oligonucleotide analogues, the deoxyribooligonucleoside methylphosphonates, were synthesized. The base sequences of these compounds, d(ApGpGp), d(ApGpGp)(2), and d[(ApGpGp)(2)T], are complementary to the Shine-Dalgarno sequence (-A-C-C-U-C-C-U-) found at the 3' end of bacterial 16S rRNA. These nonionic oligonucleotide analogues were tested for their ability to inhibit the in vitro translation of mRNAs in cell-free systems of Escherichia coli and rabbit reticulocyte. In the E. coli system, both d(ApGpGp)(2) and d[(ApGpGp)(2)T] effectively inhibited MS-2 RNA-directed protein synthesis but they had much less effect on either poly(U)- or poly(A)-directed polypeptide synthesis. In the reticulocyte system, these compounds had no significant effect on the translation of globin mRNA. The observation that d[(ApGpGp)(2)[(3)H]T)] binds to 70S ribosomes (association constant, 2.0 x 10(4) M(-1), 37 degrees C) together with the specificity of the inhibitory action of these compounds on protein synthesis strongly suggests that inhibition of translation is a consequence of analogue binding to Shine-Dalgarno sequence of 16S rRNA. The oligonucleoside methylphosphonates inhibited both protein synthesis (without concurrent inhibition of RNA synthesis) and colony formation by E. coli ML 308-225 (a permeable mutant) whose cell wall contains negligible quantities of lipopolysaccharide but had no effect on wild-type E. coli B. Our preliminary results on the uptake of oligodeoxyribonucleoside methylphosphonates by E. coli B show that these cells are not permeable to oligomers longer than 4 nucleotidyl units. Although oligodeoxyribonucleoside methylphosphonates are taken up by mammalian cells in culture, this series of analogues had negligible inhibitory effects on colony formation by transformed human cells. This study indicates that this class of nonionic oligonucleotide analogues can be used to probe and regulate the function and structure of nucleic acids of defined sequence within living cells.

Abstract: The gene 4 protein of bacteriophage T7 recognizes specific sequences on single-stranded DNA and then catalyzes the synthesis of tetraribonucleotide primers complementary to the template. With phi X174 DNA as a template, the gene 4 protein enables T7 DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) to initiate DNA synthesis at 13 major sites. DNA sequence analysis of the 5' termini of the newly synthesized DNA shows the predominant recognition sequences for the gene 4 protein to be 3'-C-T-G-G-G-5' or 3'-C-T-G-G-T-5'; the products of synthesis at these sites are RNA primers having the sequences pppA-C-C-C or pppA-C-C-A. The gene 4 protein can also synthesize primers at the sequences 3'-C-T-G-G-AC-5' and 3'-C-T-G-T-N-5', although these sites are used less than 10% as frequently as the predominant sites. Comparison of the utilization of primer sites suggests that the gene 4 protein binds randomly to single-stranded DNA and then translocates along the DNA in a unidirectional 5'-to-3' direction with regard to the DNA strand in search of recognition sequences. Models are presented for the role of the gene 4 protein in the initiation of lagging-strand synthesis and in the initiation of DNA replication at the origin.

Abstract: An in vitro adenovirus DNA replication system catalyzed the formation of a covalent complex between an 80,000-dalton protein and 5'-dCMP in the presence of [alpha-32P-dCTP, MgCl2, ATP, and adenovirus (Ad) DNA with a protein covalently bound to the 5' end of each strand (Ad DNA-prot). The requirement for Ad DNA-prot in this reaction was similar to that for in vitro DNA replication. When dATP, dTTP, and the 2',3'-dideoxynucleoside triphosphate (ddNTP) ddGTP were included in the reaction mixture, an elongated complex was detected, which consisted of an 80,000-dalton protein bound to a 26-base oligonucleotide. Formation of the elongated product, but not of the protein-dCMP complex, was inhibited by ddATP, ddCTP, or ddTTP. The requirements for formation of the protein-dCMP complex, the nature of the linkage between protein and dCMP, the size of the protein, and the existence of elongated forms indicated that the protein associated with the complex was identical to the 80,000-dalton Ad terminal protein found on replicating DNA molecules as described by Challberg et al. [Challberg, M. D., Desiderio, S. V. & Kelly, T. J., Jr. (1980) Proc. Natl. Acad. Sci. USA 77, 5105-5109].

Abstract: A rapid solid phase method of oligonucleotide synthesis based on monomeric protected nucleosides has been developed.

Abstract: The action of the N1, N1, N3, N3-tetramethylguanidinium salts of a number of oximes on 5'-O-methoxytetrahydropyranylthymidylyl-(3' leads to 5')-3'-O-methoxytetrahydropyranylthmidine aryl esters (7a-c) in dioxan-water (1:1 v/v) has been investigated. The O-chlorophenyl ester (7a) was unblocked by 4-nitrobenzaldoximate ion ca. 2.5 times as rapidly as the p-chlorophenyl ester (7b) and ca. 25 times as rapidly as the phenyl ester (7c). syn-2-Nitrobenzaldoxime (15a) is the unblocking agent of choice; its conjugate base reacts with 7a and 7b ca. 4 and 2.5 times, respectively, as rapidly as does the conjugate base of its 4-isomer (1). Internucleotide cleavage cannot be detected in the reaction between 2-nitrobenzaldoximate ion and 7a; its extent has been estimated to be no greater than 0.1%. Experiments with the corresponding fully-protected tetranucleoside triphosphate (14a) confirm the greater reactivity of 2- and 4-nitrobenzaldoximate ion and suggest that, if the molecular concentrations both of protected oligonucleotide and oximate ions are maintained, rates of unblocking may not decrease significantly with increasing oligonucleotide chain lengths.

Abstract: The complex formation between the single-strand DNA binding protein (ssB protein) from Escherichia coli and oligonucleotides and single-stranded DNA has been studied by using fluorescence titrations, ultracentrifugation measurements, and fast kinetic techniques. Determination of the stoichiometries of oligo(dT)--ssB complexes shows that each of the four subunits of the ssB protein represents a binding site for an oligonucleotide about eight residues long. Occupation of all four binding sites with oligo(dT) or poly(dT) leads to 80% quenching of the intrinsic protein fluorescence. The binding sites are nearly equivalent and independent. For d(pT)16, the intrinsic binding constant is 6 X 10(5) M-1, and for d(pT)30-40, which is long enough to extend continuously over the ssB tetramer, the binding constant is higher than 5 X 10(8) M-1. Oligoadenylates bind about 2 orders of magnitude weaker than the corresponding oligo(dT) species. The binding of oligo(dT) is very weakly dependent on ionic strength, in contrast to the oligo(dA)--ssB complex formation. For d(pT)8, d(pT)16, and d(pT)30-40, the complex formation can be described by a simple one-step reaction. The strength of the interaction is mainly expressed in the rate constant of dissociation. In the cooperative complexes with poly(dT) or poly(dA), all four binding sites on the ssB tetramer are also occupied. It is concluded that single-stranded DNA is coiled around the ssB molecule. Fluorescence melting experiments of the complexes show that the conformation of the single-stranded DNA has a strong influence on the stability of the complexes.

Abstract: Hemimethylated duplex DNA of the bacteriophage phi X 174 was synthesized using primed repair synthesis is in vitro with E. coli DNA polymerase I followed by ligation to produce the covalently closed circular duplex (RFI). Single-stranded phi X DNA was used as a template, a synthetic oligonucleotide as primer and 5-methyldeoxycytidine-5'-triphosphate (5mdCTP) was used in place of dCTP. The hemimethylated product was used as substrate for cleavage by various restriction enzymes. Out of the 17 enzymes tested, only 5 (BstN I, Taq I, Hinc II, Hinf I and Hpa I) cleaved the hemimethylated DNA. Two enzymes (Msp I and Hae III) were able to produce nicks on the unmethylated strand of the cleavage site. Msp I, which is known to cleave at CCGG when the internal cytosine residue is methylated, does not cleave when both cytosines are methylated. Another enzyme, Apy I, cleaves at the sequence CCTAGG when the internal cytosine is methylated, but is inactive on hemimethylated DNA in which both cytosines are methylated. Hemimethylated molecules should be useful for studying DNA methylation both in vivo and in vitro.

Abstract: The complete sequence of the mouse immunoglobulin kappa light chain MOPC 21 messenger RNA has been determined using a chain termination method and chemically synthesised deoxyoligonucleotides to initiate the synthesis of a DNA molecule complementary to the mRNA template. Five such oligonucleotide primers have been used for the sequence analysis of this messenger RNA. The approach is excellent for comparative studies of mouse k-chain mRNAs because they can be made on impure mRNA preparations. The MOPC 21 light chain mRNA is 943 nucleotides in length excluding the poly(A) region. An unexpected finding was that there are only three bases in the 5' non-coding region and its significance in terms of ribosome binding is discussed; 87 code for the precursor or leader sequence of the protein, 642 for the mature protein and 211 for the 3' non-coding region. The codons for the precursor region allows the previously undetermined amino acid sequence to be predicted. In common with other precursor regions a high proportion of the predicted amino acids are hydrophobic.

Abstract: When E. coli ribosomal subunits are reacted with 2-iminothiolane and then subjected to a mild ultraviolet irradiation, an RNA-protein cross-linking reaction occurs. About 5% of the total protein in each subunit becomes cross-linked to the RNA, and a specific sub-set of proteins is involved in the reaction. In the case of the 50S subunit, the sites of cross-linking to the 23S RNA have been determined for six of these proteins: protein L4 is cross-linked within an oligonucleotide comprising positions 613-617 in the 23S sequence, L6 within positions 2473-2481, L21 within positions 540-548, L23 within positions 137-141, L27 within positions 2332-2337 and L29 within positions 99-107.

Abstract: A 514-base pair fragment of double-stranded DNA coding for human interferon-α1 (166 amino acid residues), and containing initiation and termination signals plus appropriate restriction enzyme sites for plasmid insertion, has been totally synthesized. The synthesis involved preparation of 66 oligodeoxyribonucleotides, ranging in size from 14 to 21 residues, plus 1 deoxydecanucleotide, by rapid, solid phase procedures, and enzymatic ligation of the oligonucleotides. After ligation of the synthetic gene to a plasmid vector and transformation of Escherichia coli, clones containing the anticipated gene sequence were obtained.

Abstract: When we work out the structure of DNA molecules, we examine the fundamental level that underlies all process in living cells. DNA is the information store that ultimately dictates the structure of every gene product, delineates every part of the organism. The order of the bases along DNA contains the complete set of instructions that make up the genetic inheritance. We do not know how to interprete those instructions; like a child, we can spell out the alphabet without understanding more than a few words on a page. I came to the chemical DNA sequencing by accident. Since the middle sixties my work had focussed on the control of genes in bacteria, studying a specific gene product, a protein repressor made by the control gene for the lac operon (the cluster of genes that metabolize the sugar lactose. Benno Müller-Hill and I had isolated and characterized this molecule during the late sixties and demonstrated that this protein bound to bacterial DNA immediately at the beginning of the first gene of the three-gene cluster that this repressor controlled (1, 2). In the years since then, my laboratory had shown that this protein acted by preventing the RNA polymerase from copying the lac operon genes into RNA. I had used the fact that the lac repressor bound to DNA at a specific region, the operator, to isolate the DNA of this region by digesting all of the rest of the DNA with DNase to leave only a small fragment bound to the repressor, protected from the action of the enzyme. This isolated a twenty-five base-pair fragment of DNA out of the 3 million base pairs in the bacterial chromosome. In the early seventies, Allan Maxam and I worked out the sequence of this small fragment (3) by copying this DNA into short fragments of RNA and using on these RNA copies the sequencing methods that had been developed by Sanger and his colleagues in the late sixties. This was a laborious process that took several years. When a student, Nancy Maizels, then determined the sequence of the first 63 bases of the messenger RNA for the lac operon genes, we discovered that the lac repressor bound to DNA immediately after the start of the messenger RNA (4), in a region that lies under the RNA polymerase when it binds to DNA to initiate RNA synthesis. We continued to characterize the lac operator …

Abstract: Publisher Summary Exonuclease V (exoV) enzymes are ubiquitous in bacteria. These enzymes belong to a larger group of ATP-dependent DNases and DNA-dependent ATPases, which also includes enzymes such as type-I restriction endonucleases, helicases, and gyrases. All these enzymes exhibit ATP-dependent double-stranded DNA exonuclease and DNA-dependent ATPase activities. In addition, ATP-dependent single-stranded DNA exonuclease, ATP-stimulated single-stranded DNA endonuclease, and ATP-dependent DNA unwinding may also be associated with the enzyme. Mechanism of action of the exoV enzyme indicates that DNA unwinding is fundamental to several of the enzyme's activities and can be its most important function in the cell. The complete degradation of DNA to acid-soluble oligonucleotides may only serve a function in restriction. The observation of decreased or undetectable exoV activity in recombination and repair mutants in certain bacteria has sparked a great deal of interest in these enzymes. Recombination and repair lesions in bacteria that lack exoV are not absolute, so the deficiency must be carefully quantitated with well-defined strains.

Abstract: Although the antibiotic rifampicin inhibits the transcription of poly[d(A-T)] by E.coli RNA polymerase, a series of short oligonucleotides is produced. It is claimed that the overall inhibition of RNA synthesis by rifampicin is caused by a destabilising effect on the binding of the intermediate oligonucleotides to the active enzyme-DNA complex. Rifampicin itself can only interact specifically with RNA polymerase if the enzyme is free or in a binary complex with DNA. However, the enzyme is not susceptible in a ternary complex, even if the "RNA" is as short as a trinucleotide.

Abstract: — Hybrid cell lines (hybridomas) have been isolated from fusions between P3-NS1-1-Ag4-1 mouse myeloma cells and spleen cells from BALB/c mice hyperimmunised with UV-irradiated single-stranded DNA (UVssDNA) and UV-irradiated polydeoxythymidylic acid (UVpolydT) Monoclonal antibodies from two different hybridomas are characterised in the present report by competitive inhibition with different synthetic polynucleotides and oligonucleotides The first antibody, designated αUVssDNA-1, recognises thymidine dimers in a polynucleotide or an oligonucleotide sequence at least four nucleotides long but not isolated thymidine dimers, suggesting that it recognises the conformational change associated with thymidine dimers The second antibody, designated αssDNA-2, recognises unirradiated or UV-irradiated tracts of thymidine, but will not crossreact with tracts of other nucleotides (A, G, C, AT, GC, CU, U) Inhibition of binding of αUVssDNA-1 to [3H]-UVssDNA by calf thymus UVssDNA is dependent on UV exposure and wavelength as expected from the antigenic determinant

Abstract: A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described. Pulse-labeled RNA isolated by this procedure for Bacillus brevis, Bacillus subtilis, and Escherichia coli B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo(dT)-cellulose. This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA. The presence of polyadenylated RNA in E. coli was confirmed by an independent method which involved hybridization with [3H]polyuridylic acid. Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-treated cells of B. brevis, B. subtilis, and E. coli.