Synthesis and modification of genes through artificial splicing by directed ligation (ASDL).

An approach to the directed genetic recombination in vitro mediated by synthetic oligodeoxynucleotides and polymerase chain reaction (PCR) is devised, which allows the joining, in a predetermined chemical-enzymatic way, of a series of DNA segments to give a precisely spliced polynucleotide sequence (Artificial Splicing by Directed Ligation, ASDL). The approach can thus lead to the totally processed eukaryotic genes using genomic DNA, with no mRNA needed. This approach has been used for the synthesis of artificial genes of interleukin-1 alpha and, in combination with PCR on the mRNA-cDNA duplex as template, of interleukin-1 receptor antagonist and their analogues, as well as for the modified genes.     

Synthesis and properties of poly 5-methylthiouridylic acid.

In an effort to search for good methods for the enzymatic synthesis of polynucleotide analogs with antitemplate activity, 5-methylthiouridine-5'-diphosphate (ms5UDP) has been synthesized and investigated as a substrate for polynucleotide phosphorylase. While ms5UDP was polymerized at a very low rate to give a 6% yield of polynucleotides by the polynucleotide phosphorylase of Micrococcus luteus, it was utilized more efficiently by the corresponding enzyme of Escherichia coli resulting in a 15% yield of poly (5-methylthiouridylic) acid. Results of the co-polymerization of ms5UDP and UDP revealed that the ratio of 5-methylthiouridylate to uridylate residues in the polynucleotide product was lower than the ratio of ms5UDP to UDP in the substrate mixture. The 5-methylthio group conferred only minute changes on the conformation of the modified polyuridylic acid, and the complexes formed between poly-(5-methylthiouridylic) acid and poly(adenylic) acid possessed slightly higher Tm values than did the unmodified counterparts. Poly(5-methylthiouridylic) acid was a potent inhibitor of calf thymus DNA polymerase alpha.     

Artificial DNA splicing using directed ligation]

An approach to the directed genetic recombination in vitro has been devised, which allows for joining, in a predetermined chemical-enzymatic way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA Splicing by Directed Ligation, SDL). The approach makes use of amplification, by several polymerase chain reactions (PCR), of the chosen DNA segments. The corresponding primers contain recognition sites of the class IIS restriction endonucleases, yielding protruding ends of unique primary structures. The protruding ends of the segments to be joined together are structurally predetermined to make them mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1 alpha.     

Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases

Repair replication of short synthetic DNA's corresponding to parts of the gene for the major yeast alanine transfer RNA has been studied. The enzymes used were the Escherichia coli, Microccua luteus and T4 DNA polymerases, similar results being obtained with all the three enzymes. The DNA's used (Fig. 1) were: four double-stranded DNA's with the termini containing the 5′-hydroxyl group protruding by one, three, four or ten nucleotide units and two single-stranded DNA's 29 units long which are capable of folding back on themselves. The aspects inve tigated were: (1) completion of repair, (2) the minimum size of the polynucleotide chains required as primers and those which can serve as templates and (3) the minimum size of the primers which can abolish hairpin formation so as to give the “normal” repair replication. Repair replication of DNA's (DNA-I to DNA-IV: Fig. 1) was characterized to be essentially complete. The minimum size of the primer for repair replication of an extended single-stranded deoxypolynucleotide was ascertained to be about five to seven units long, while the primers required to overcome the hairpin formation in DNA-V and DNA-VI were found to be about twelve units long. Thus, in the presence of a suitable primer, the nucleotide incorporation which occurs in DNA-V in the absence of added primer was completely replaced by incorporation expected for normal repair replication. The minimum size of a chain which can serve as a template was also found in the present work to be about 12 units long.     

Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase.

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.     

Isolation and characterization of a polynucleotide phosphorylase from Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellular enzymes, and the synthesis of these proteins appears to be dependent upon abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyl transferase) was identified, purified, and characterized from this strain. The purification scheme involved cell disruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gel filtration, and diethylaminoethyl-Sephadex chromatography. The purified enzyme demonstrated the reactions characteristic of polynucleotide phosphorylase: polymerization, phosphorolysis, and inorganic phosphate exchange with the beta-phosphate of a nucleotide diphosphate. The enzyme was apparently primer independent and required a divalent cation. The reactions for the synthesis of the homopolyribonucleotides, (A)n and (G)n, were optimized with respect to pH and divalent cation concentration. The enzyme is sensitive to inhibition by phosphate ion and heparin and is partially inhibited by rifamycin SV and synthetic polynucleotides.     

Sequence analysis of the putative regulatory region of rat alpha 2-macroglobulin gene

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase reactant, concentration of which in serum increases more than 100-fold in the course of inflammation. Glucocorticoid and some protein factors such as interleukin 1 (IL-1) have been known to be involved in the regulation of this plasma protein synthesis. To understand the regulatory mechanism of alpha 2M production at the molecular level, we isolated genomic DNA clones of rat alpha 2M gene and characterized the promoter region of the gene by comparing the nucleotide sequence with those of other acute-phase reactant genes. Several possible regulatory signals were identified. Particularly, a sequence (T/A)T(C/G)TGGGA(A/T) was found about at 170 bp upstream from a putative capping site, which was also found in the 5'flanking region of various acute-phase reactant genes.     

DNA polymerase alpha cofactors C1C2 function as primer recognition proteins

Most, if not all, of the DNA polymerase alpha activity in monkey and human cells was complexed with at least two proteins, C1 and C2, that together stimulated the activity of this enzyme from 180- to 1800-fold on low concentrations of denatured DNA, parvovirus DNA, M13, and phi X174 DNA or RNA-primed DNA templates, and poly(dT):oligo(dA) or oligo(rA). These primer-template combinations, which have from 200 to 5000 bases of template/primer, were then 7- to 50-fold more effective as substrates than DNase I-activated DNA. C1C2 specifically stimulated alpha polymerase, and only from the same cell type. Alpha X C1C2-polymerase reconstituted from purified alpha polymerase and the C1C2 cofactor complex behaved the same as native alpha X C1C2-polymerase and C1C2 had no effect on the sensitivity of alpha polymerase to aphidicolin, dideoxythymidine triphosphate, and N-ethylmaleimide. In the presence of substrates with a high ratio of single-stranded DNA template to either DNA or RNA primar, C1C2 increased the rate of DNA synthesis by decreasing the Km for the DNA substrate, decreasing the Km for the primer itself, increasing the use of shorter primers, and stimulating incorporation of the first deoxyribonucleotide. In contrast, C1C2 had no effect on the Km values for deoxyribonucleotide substrates (which were about 150-fold higher than for DNA replication in isolated nuclei), the ability of specific DNA sequences to arrest alpha polymerase, or the processivity of alpha polymerase. Accordingly, C1C2 function as primer recognition proteins. However, C1C2 did not reduce the comparatively high Km values or stimulate DNA synthesis by alpha polymerase on lambda DNA ends and DNase I-activated DNA, substrates with 12 and about 30-70 bases of template/primer, respectively. DNA restriction fragments with 1 to 4 bases of template/primer were substrates for neither alpha nor alpha X C1C2-polymerase. Therefore, we propose that C1C2 enhances the ability of alpha polymerase to initiate DNA synthesis by eliminating nonproductive binding of the enzyme to single-stranded DNA, allowing it to slide along the template until it recognizes a primer.     

An improved chemico-enzymatic method of synthesizing long DNA segments

A simple and economy method of the biochemical assembling of long double-stranded DNA segments is described. A single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates. This single-stranded polynucleotide was converted, in the presence of DNA polymerase 1 and a 12-meric primer, in to the full-length double-stranded DNA (the beta-interferon gene segment). It was cloned into an E. coli plasmid vector pBR322 and its sequence confirmed.     

The acute-phase induction of alpha 2-macroglobulin in rat hepatocyte primary cultures: action of a hepatocyte-stimulating factor, triiodothyronine and dexamethasone

During inflammation a number of liver-derived plasma proteins increases in concentration. In the rat these so-called acute-phase proteins are mainly proteinase inhibitors, such as alpha 1-proteinase inhibitor, alpha 1-acute-phase globulin and alpha 2-macroglobulin. At present, the mechanisms responsible for the enhanced synthesis of acute-phase proteins are poorly understood. Therefore, we have studied the induction of alpha 2-macroglobulin synthesis in rat hepatocyte primary cultures. Adrenaline, triiodothyronine, estradiol and progesterone were tested for their ability to stimulate alpha 2-macroglobulin synthesis. Only triiodothyronine induced alpha 2-macroglobulin synthesis markedly. However, the presence of dexamethasone was a prerequisite for alpha 2-macroglobulin induction indicating a permissive action of glucocorticoids. Besides glucocorticoids and triiodothyronine a non-dialyzable factor (HSF) derived from rat Kupffer cells or human peripheral blood monocytes was found to be able to stimulate alpha 2-macroglobulin synthesis in hepatocytes. Equal amounts of HSF activity were found in conditioned media from lipopolysaccharide-stimulated and unstimulated rat Kupffer cells as well as in human monocytes. Since the supernatants of unstimulated rat Kupffer cells or human monocytes did not exhibit interleukin 1 activity, HSF activity distinct from interleukin 1 must exist. No HSF activity was found in media conditioned by rat Kupffer cells which had been treated with dexamethasone. Hepatocyte primary cultures were incubated with [35S]methionine-labeled proteins secreted by rat Kupffer cells. A 30 kDa polypeptide was found to be bound to or internalized by rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of DNA with human DNA primase monitored with photoactivatable cross-linking agents: implications for the role of the p58 subunit.

Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the primase subunits with DNA were examined. After primase synthesizes a primer that DNA polymerase alpha (pol alpha) can readily elongate, further primase activity is negatively regulated. This occurs within both the context of the four-subunit pol alpha-primase complex and in the p49-p58 primase complex, indicating that the newly generated primer-template species need not interact with pol alpha to regulate further primase activity. Photo-cross-linking of single-stranded DNA-primase complexes revealed that whereas the isolated p49 and p58 subunits both reacted with DNA upon photolysis, only the p58 subunit reacted with the DNA when photolysis was performed using the p49-p58 primase complex. After primer synthesis by the complex, p58 was again the only subunit that reacted with the DNA. These results suggest a model for regulation of primer synthesis in which the newly synthesized primer-template species binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer-template species suggests that p58 mediates the transfer of primers from the primase active site to pol alpha.     

Proteolytic removal of the COOH terminus of the T4 gene 32 helix-destabilizing protein alters the T4 in vitro replication complex

The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. 1. Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32I protein for this synthesis. 2. Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. 3. Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. 4. The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3'-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities.     

Interleukin 1 and tumor necrosis factor synergistically stimulate prostaglandin synthesis and phospholipase A2 release from rat renal mesangial cells

Treatment of rat glomerular mesangial cells with recombinant human interleukin 1 alpha (rIL-1 alpha), recombinant human interleukin 1 beta (rIL-1 beta) or recombinant human tumor necrosis factor (rTNF) induces prostaglandin E2 (PGE2) synthesis and the release of a phospholipase A2 (PLA2) activity. rIL-1 beta is significantly more potent than rIL-1 alpha or rTNF in stimulating PGE2 as well as PLA2 release from mesangial cells. When given together, rTNF interacts in a synergistic fashion with rIL-1 alpha and rIL-1 beta to enhance both, PGE2 synthesis and PLA2 release. The released PLA2 has a neutral pH optimum and is calcium-dependent. Pretreatment of cells with actinomycin D or cycloheximide inhibits basal and cytokine-stimulated PGE2 and PLA2 release.     

Bypass of pyrimidine dimers in DNA of bacteriophage T4 via induction of primer RNA

Bacteriophage T4 has a third pathway for repair of damaged DNA besides excision repair and recombination repair. This pathway is a mechanism for the toleration of lesions rather than the repair of lesions. The substrate for this process is gapped DNA copied from a damaged template. Evidence indicates that these gaps are filled, giving rise to daughter strands that are sensitive to heat and to treatments with RNAase. These daughter strands subsequently serve as templates for DNA that is resistant to RNAase. This third pathway is dependent upon gene 41 (RNA-priming protein), gene uvsZ (function unknown) and gene 30 (polynucleotide ligase) and is presumed to consist of 4 steps: (1) induction of primer RNA opposite the lesion in the template; (2) elongation of primers by DNA polymerase; (3) ligation of daughter-strand fragments, without removal of primer RNA; (4) replication of DNA carrying RNA sequences, giving homogeneous DNA strands. We have called this process 'Re-initiation repair'.     

New Reaction of <Emphasis Type="Italic">O</Emphasis>-Benzoquinone at the Thioether Group of Methionine

THERE are two biochemical systems which probably evolved before the development of accurate polynucleotide-specified protein synthesis: these are the system for polynucleotide replication and the machinery of protein synthesis itself^{1, 2}. Before accurately specified proteins became available, these processes were perhaps catalysed by polynucleotide enzymes. Both tRNA and rRNA, which can be viewed as polynucleotide enzymes, have persisted as indispensable components of the contemporary apparatus. This has led me to wonder whether polynucleotide enzymes might still be operative in DNA replication. Moreover, in view of the complexity which would have been required for even a rudimentary form of protein synthesis, it seems unlikely that tRNA and rRNA arose by chance in a single evolutionary step¹. More probably they have evolved from the replicative machinery for polynucleotides and thus it seems likely that the machinery of DNA replication may have many features in common with the polynucleotide components of protein synthesis.     

Interleukin 1- and tumor necrosis factor-stimulation of prostaglandin E2 synthesis in MDCK cells, and potentiation of this effect by cycloheximide

The effects of interleukin (IL)-1 alpha, IL-1 beta and TNF alpha on prostaglandin-E2 synthesis in Madin-Darby canine kidney (MDCK) cells were investigated. IL-1 beta time- and dose-dependently stimulated prostaglandin-E2 synthesis. While TNF alpha produced a comparatively small but significant stimulation of PGE2 release, coincubation of IL-1 beta with TNF alpha produced a marked synergistic stimulation of PGE2 release. The effect of IL-1 beta and of IL-1 beta and TNF alpha was apparent as early as after 2 h of incubation. The enhanced PGE2 synthesis was inhibited by indomethacin as well as actinomycin D, while cycloheximide surprisingly potentiated PGE2 synthesis in response to both IL-1 beta and TNF alpha. IL-1 alpha alone was ineffective in stimulating a significant release of PGE2 at concentrations as high as 10 nM. However, it also showed a marked synergistic interaction with TNF alpha in stimulating PGE2 release.     

Synthesis, conformation and enzymatic properties of 1-(beta-D-allofuranosyl)uracil and some derivatives.

A new route for the synthesis of 1-(beta-D-allofuranosyl)uracil ("allo-uridine") and the corresponding 6'-deoxy-derivative ("6'-deoxy-allo-uridine") as well as for 1-(beta-D-altrofuranosyl) uracil ("altro-uridine") is described. NMR studies of allo-uridine revealed a preferred conformation with the base in anti-position, C-2'-endo-pucker of the sugar moiety, the 5'-OH-group above the furanose ring and the 5'-CH2OH-group in a gt position with the OH-group in the plane of the furanose ring. The same conformation is found for the 5'- and 6'-phosphate, indicated by the influence of the phosphate group on the H-6 signal. Allo-uridine is phosphorylated by the phosphotransferases from carrot and from malt sprouts only in the 6'-position. The phosphate ester is hydrolysed by unspecific phosphatases but not by 5'-nucleotidase. A (3' leads to 6')-dinucleoside phosphate is formed by pancreatic ribonuclease with 2',3'-cyclic cytidylic acid and allo-uridine. It is split by nuclease S1, but not by snake-venom phosphodiesterase. It has no primer activity for polynucleotide phosphorylase. All-uridine 6'-diphosphate could not be prepared enzymatically by nucleotide kinase or by chemical methods, where 5',6'-cyclic phosphates are formed, which are hydrolysed exclusively to 6'-monophosphates.     

DNA polymerase switching: II. Replication factor C abrogates primer synthesis by DNA polymerase alpha at a critical length

A crucial event in DNA replication is the polymerase switch from the synthesis of a short RNA/DNA primer by DNA polymerase alpha/primase to the pro?cessive elongation by DNA polymerase delta. In order to shed light on the role of replication factor C (RF-C) in this process, the effects of RF-C on DNA polymerase alpha were investigated. We show that RF-C stalls DNA polymerase alpha after synthesis of approximately 30 nucleotides, while not inhibiting the polymerase activity per se. This suggested that RF-C and the length of the primer may be two important factors contributing to the polymerase switch. Furthermore the DNA binding properties of RF-C were tested. Band shift experiments indicated that RF-C has a preference for 5' recessed ends and double-stranded DNA over 3' ends. Finally PCNA can be loaded onto a DNA template carrying a RNA primer, suggesting that a DNA moiety is not necessarily required for the loading of the clamp. Thus we propose a model where RF-C, upon binding to the RNA/DNA primer, influences primer synthesis and sets the conditions for a polymerase switch after recruiting PCNA to DNA.