Synthesis of oligodeoxyribonucleotides containing oleylamine moieties]

A method for directional introduction of oleylamine residues to any position of oligodeoxyribonucleotides during their automated synthesis was developed. The presence of oleylamine residues in 3'- or 5'-terminal nucleotides was shown to have no effect on the thermodynamic stability of DNA duplexes formed by such oligonucleotides and the complementary sequences. The rate of the snake venom phosphodiesterase hydrolysis of oligonucleotides containing oleylamine residues in the 3'-terminal units was shown to be markedly lower than that of natural oligonucleotides.     

Defective interfering particles of poliovirus: mapping of the deletion and evidence that the deletions in the genomes of DI(1), (2) and (3) are located in the same region.

The deletions in RNAs of three defective interfering (DI) particles of poliovirus type 1 have been located and their approximate extent determined by three methods. (1) Digestion with RNase III of DI RNAs yields the same 3′-terminal fragments as digestion with RNase III of standard virus RNA. The longest 3′-terminal fragment has a molecular weight of 1.55 × 10⁶. This suggests that the deletions are located in the 5′-terminal half of the polio genome. (2) Fingerprints of RNase T₁-resistant oligonucleotides of all three DI RNAs are identical and lack four large oligonucleotides as compared to the fingerprints of standard virus, an observation suggesting that the deletions in all three DI RNAs are located in the same region of the viral genome. The deletion-specific oligonucleotides have also been shown to be within the 5′-terminal half of the viral genome by alkali fragmentation of the RNA and fingerprinting poly (A)-linked (3′-terminal) fragments of decreasing size. (3) Virion RNA of DI(2) particles was annealed with denatured double-stranded RNA (RF) of standard virus and the hybrid heteroduplex molecules examined in the electron microscope. A single loop, approximately 900 nucleotides long and 20% from one end of the molecules, was observed. Both the size and extent of individual deletions is somewhat variable in different heteroduplex molecules, an observation suggesting heterogeneity in the size of the deletion in RNA of the DI(2) population. Our data show that the DI RNAs of poliovirus contain an internal deletion in that region of the viral genome known to specify the capsid polypeptides. This result provides an explanation as to why poliovirus DI particles are unable to synthesize viral coat proteins.     

Detection of DNA hybridization on a liposome surface using ultrasound velocimetry and turbidimetry methods

19-mer oligonucleotides with oleylamine tethered at 3' and 5' terminal, respectively, were incorporated into unilamellar liposomes of dioleoylphosphatidylcholine (DOPC). Addition of complementary nucleotide resulted in hybridization with oligonucleotides located on different liposomes and caused liposome aggregation. Significant changes of sound velocimetry and turbidity were readily observed at 10 nM concentration of the complementary chain.     

The complete primary structure of a proline-rich phosphoprotein from human saliva.

The complete amino acid sequence of a calcium-binding "proline-rich phosphoprotein," named Protein A, from human saliva was determined by automated and manual Edman degradation of peptides obtained by enzymatic and chemical cleavage of the intact protein. The NH2-terminal pyrrolidone carboxylic acid was identified by means of NMR. The protein consists of 106 amino acids, including 24 residues of proline. The NH2-terminal 32 residues contain 13 of the 15 negatively charged residues including 2 phosphoserines, but only 1 proline. In spite of a high concentration of proline in the COOH-terminal part of the molecule, the longest oligoproline sequence is tetraproline. The protein contains a number of repeated sequences and there are also several sequences of 3 or 4 residues identical with known sequences of collagen, but the characteristic occurrence of glycine in every third position in collagen is not found in salivary Protein A.     

A ubiquitin-protein ligase specific for type III protein substrates

A previously studied species of ubiquitin-protein ligase contains specific sites for the binding of basic (Type I) and bulky hydrophobic (Type II) NH2-terminal amino acid residues of protein substrates. We now describe another enzyme that ligates ubiquitin specifically to proteins that have NH2-terminal residues other than the above two categories (Type III substrates). The new species of ligase, that we call E3 beta, is separable from the formerly described ligase (termed E3 alpha) by affinity chromatography on protein substrate columns. E3 beta was partially purified from extracts of rabbit reticulocytes and was shown to be required for the breakdown of Type III proteins. Apart from its different substrate specificity, it resembles E3 alpha in some physical properties, in a requirement for ubiquitin carrier protein (E2) for conjugate formation, and in its action to ligate multiple ubiquitin units to the substrate protein. The denatured derivative of bovine pancreatic ribonuclease is a specific substrate for E3 alpha, while that of ribonuclease S-protein is a good substrate for E3 beta. Since S-protein is formed by the removal from ribonuclease of NH2-terminal S-peptide, it is suggested that E3 beta interacts with an NH2-terminal determinant exposed in ribonuclease S-protein.     

Post-translational addition of an arginine moiety to acidic NH2 termini of proteins is required for their recognition by ubiquitin-protein ligase

Recent studies have shown that selection of proteins for degradation by the ubiquitin system occurs most probably by binding to specific sites of the ubiquitin-protein ligase, E3. A free alpha-NH2 residue of the substrate is one important determinant recognized by the ligase. Selective binding sites have been described for basic and bulky-hydrophobic NH2 termini (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698) and for alanine, serine, and threonine at the NH2-terminal position (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). Proteins with acidic NH2-terminal residues are degraded by the ubiquitin system only following conversion of the acidic residue to a basic residue by the addition of an arginine moiety (Ferber, S., and Ciechanover, A. (1987) Nature 326, 808-811). Although the enzymes involved in this post-translational modification have been characterized, the underlying mechanism has been obscure. By using a chemical cross-linking technique, we demonstrate that proteins with acidic NH2 termini do not bind to E3 without prior modification of this residue by the addition of arginine. In contrast, proteins with a basic NH2-terminal residue bind to the ligase without any modification. The recognition of acidic NH2-terminal substrates by E3 is dependent upon the addition of all the components of the modifying machinery, arginyl-tRNA-protein transferase, arginyl-tRNA synthetase, tRNA, and arginine. The ligase-bound modified proteins are converted to ubiquitin conjugates in a "pulse-chase" experiment, indicating that the binding is functional and that the enzyme-substrate complex is an obligatory intermediate in the conjugation process. Chemical modification of the carboxyl groups, which results in their neutralization, generates substrates that bind to E3 without modification. This finding suggests that the amino-terminal binding site of E3 is negatively charged, and only positively charged amino-terminal residues may bind to it. Negatively charged (acidic) NH2-terminal residues will bind only following neutralization or reversal of the charge.     

Interchain disulfide bonding in the regulatory subunit of cAMP-dependent protein kinase I

The two protomers of the purified regulatory subunit from porcine cAMP-dependent protein kinase I have been shown to be covalently cross-linked by interchain disulfide bonding. Limited proteolysis which cleaves the polypeptide chain into two fragments demonstrated that the disulfide bonding was associated exclusively with the fragment that corresponded to the NH2-terminal region of the polypeptide chain. This NH2-terminal fragment accounted for approximately 15 to 20% of the molecule. The disulfide bonding was further characterized by alkylating the cysteines in the native regulatory subunit. Following oxidation with performic acid, each regulatory subunit contained 7 cysteic acid residues; however, under denaturing conditions, but without prior reduction, only 5 cysteine residues could be alkylated with iodoacetic acid. Following limited proteolysis, all five of these cysteines were associated with the larger COOH-terminal, cAMP binding domain. In contrast, if the denatured subunit was first reduced prior to alkylation, all 7 cysteine residues were alkylated. The 2 cysteines that were only accessible to alkylation after prior reduction were both associated with the NH2-terminal end of the polypeptide chain ultimately with a 5,400 peptide. Alkylation of the isolated, denatured NH2-terminal domain with iodoacetic acid resulted in no covalent modification unless the fragment was first reduced with dithiothreitol. The NH2-terminal and COOH-terminal domains were shown to be linked by a region of the polypeptide chain that is rich in both proline and arginine. It is the arginine-rich site that is readily prone to proteolytic cleavage.     

Synthesis and Properties of Modified Oligodeoxyribonucleotides Containing 2"-O-(2,3-Dihydroxypropyl)uridine and 2"-O-(2-Oxoethyl)uridine

A new uridine derivative, 2"-O-(2,3-dihydroxypropyl)uridine, and its 3"-phosphoramidite were obtained for direct introduction into oligonucleotides during automated chemical synthesis. Oligonucleotides 10 to 15 nt long harboring 2"-O-(2,3-dihydroxypropyl)uridine residues were synthesized; periodate oxidation of these oligomers gave oligonucleotides containing 2"-O-(2-oxoethyl)uridine residues. The presence of a reactive aldehyde group in 2" position of the carbohydrate moiety was confirmed by the interaction withp-nitrophenylhydrazine and methionine methyl ester. The thermostability of DNA duplexes containing modified units does not practically differ from that of the natural analogues.     

Purification and properties of UDP-glucuronyltransferase from kidney microsomes of beta-naphthoflavone-treated rat

Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5-fold by treatment of the animal with beta-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of beta-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography:FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed "GT-2," was highly purified as judged from the single protein band (Mr 54,000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50,000 by endo-beta-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50,000 Da polypeptide carrying "high mannose" type oligosaccharide chain(s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu- Ile-Val . It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

Isolation and characterization of a cDNA clone encoding rat nucleoside diphosphate kinase

A cDNA clone for cytosolic nucleoside diphosphate (NDP) kinase was isolated from a cDNA library of rat skeletal muscle using synthetic oligonucleotides as probes. The clone constitutes a 621-base pair cDNA sequence including the 456-base pair coding region and 137-base pair 3'-untranslated one with polyadenylation site. The complete primary structure of NDP kinase was deduced from the coding sequence. An NH2-terminal amino acid sequence analysis suggested that the translated enzyme protein suffered proteolytic cleavage followed by modification at the alpha-NH2 group of the newly produced NH2-terminal amino acid residue. Taking this into account, it was tentatively concluded that the mature NDP kinase consists of 147 amino acid residues with a molecular weight of 16,724. Northern blot hybridization analysis showed that NDP kinase mRNA could be detected in total RNA fractions of brain, spleen, heart, lung, liver, kidney, testis as well as skeletal muscle, and that there was no difference in the size of mRNAs from these tissues. Tissue distribution of the mRNA nearly paralleled those of protein moiety and activity of the enzyme.     

Substrate specificity of a multifunctional calmodulin-dependent protein kinase

The substrate specificity of the multifunctional calmodulin-dependent protein kinase from skeletal muscle has been studied using a series of synthetic peptide analogs. The enzyme phosphorylated a synthetic peptide corresponding to the NH2-terminal 10 residues of glycogen synthase, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH2, stoichiometrically at Ser-7, the same residue phosphorylated in the parent protein. The synthetic peptide was phosphorylated with a Vmax of 12.5 mumol X min-1 X mg-1 and an apparent Km of 7.5 microM compared to values of 1.2 mumol X min-1 X mg-1 and 3.1 microM, respectively, for glycogen synthase. Similarly, a synthetic peptide corresponding to the NH2-terminal 23 residues of smooth muscle myosin light chain was readily phosphorylated on Ser-19 with a Km of 4 microM and a Vmax of 5.4 mumol X min-1 X mg-1. The importance of the arginine 3 residues NH2-terminal to the phosphorylated serine in each of these peptides was evident from experiments in which this arginine was substituted by either leucine or alanine, as well as from experiments in which its position in the myosin light chain sequence was varied. Positioning arginine 16 at residues 14 or 17 abolished phosphorylation, while location at residue 15 not only decreased Vmax 14-fold but switched the major site of phosphorylation from Ser-19 to Thr-18. It is concluded that the sequence Arg-X-Y-Ser(Thr) represents the minimum specificity determinant for the multifunctional calmodulin-dependent protein kinases. Studies with various synthetic peptide substrates and their analogs revealed that the specificity determinants of the multifunctional calmodulin-dependent protein kinase were distinct from several other "arginine-requiring" protein kinases.     

Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the second step in the biosynthesis of tetrahydrobiopterin, was purified approximately 18,000-fold to apparent homogeneity from rat liver. The molecular mass of the native enzyme was estimated to be 83 kDa by gel filtration. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 17 kDa. Up to 24 residues of the NH2-terminal sequence were determined by Edman degradation, which released a single amino acid at each step. These results indicate that the enzyme consists of identical subunits. The purified enzyme was digested with lysyl endopeptidase or V8 protease, and 11 peptide fragments were isolated. On the basis of the sequences of these peptides, oligonucleotides were synthesized and used to screen a rat liver cDNA library, and one cDNA clone was isolated. The complete nucleotide sequence of the 1176-base pair cDNA was then determined. The deduced amino acid sequence contained 144 amino acid residues, but a NH2-terminal four-amino acid sequence was not found in the purified protein. Therefore, the mature protein consists of 140 amino acids. A single mRNA band of 1.3 kilobases was obtained by RNA blot analysis of rat liver. The predicted amino acid sequence of 6-pyruvoyl-tetrahydropterin synthase was compared with the Protein Sequence Database of the National Biomedical Research Foundation, revealing significant local similarity to large T antigens from the polyomavirus family.     

Disulfide-linked oligonucleotide phosphorothioates: Novel analogues of nucleic acids

Summary The synthesis of phosphorothioate analogues of oligonucleotides by the oxidation of deoxyadenosine 3,5-bisphosphorothioate (3) was attempted. Cyclization of3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5-terminal phosphorothioate group undergoes efficient chain-extension when oxidized in the presence of3.     

A serine protease inhibitor from the anemone <Emphasis Type="Italic">Radianthus macrodactylus</Emphasis>: Isolation and physicochemical characteristics

A serine protease inhibitor with a molecular mass of 6106±2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed α/β-or α + β polypeptides. It was established that InhVJ is highly specific toward trypsin (K _i 2.49 × 10^?9 M) and α-chymotrypsin (K _i 2.17 × 10^?8 M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties.     

Structural relationship between "glutamic acid" and "lysine" forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography.

Urokinase digestion of maleinated plasminogen results in cleavage of the single peptide bond Arg-68-Met-69, which is one of the bonds normally cleaved during the first step of the activation procedure. The inactive intermediate compound formed in this way was subjected to NH2-terminal amino acid sequence analysis, which clearly demonstrates the structural relationship between the forms of plasminogen with different NH2-terminal amino acids. It is thus shown that lysine-78 and valine-79 in the "glutamic acid" plasminogen actually are the NH2-terminal amino acids in "lysine" and "valine" plasminogen respectively. The forms with glutamic acid in NH2-terminal position are called plasminogen A, while all other forms lacking the NH2-terminal part of the molecule and which can be activated in a single step are called plasminogen B. By affinity chromatographic studies of the NH2-terminal activation peptide on insolubilized plasminogen B, it was demonstrated that this peptide has specific affinity for plasminogen B. It was also shown that this noncovalent interaction is broken by 6-aminohexanoic acid in two concentration. The tryptic heptapeptide (Ala-Phe-Gln-Tyr-His-Ser-Lys) which occupies the positions number 45 to 51 in the NH2-terminal activation peptide (as well as in the intact plasminogen molecule) is importance for the conformational state of the plasminogen molecule.     

Pyrimidine-rich oligonucleotides from rat-liver ribosome surface.

The distribution of oligonucleotides which are released from rat liver ribosomes by treatment with pancreatic ribonuclease has been studied. Rat liver monoribosomes lost from 15 to 17% of their nucleotides by treatment with pancreatic ribonuclease. This quantity was highly reproducible and did not depend significantly on the temperature (0-20 degrees C) and time (10-120 min) of incubation or on the concentration of enzyme (1:5000-1:50). Whereas the amounts of oligonucleotides liberated was 16%, it was shown by column chromatography that they consisted of 71% mononucleotides, 16% dinucleotides, 6% trinucleotides, 4% tetranucleotides and 2% pentanucleotides and that these oligonucleotides were enriched in uridine, containing approximately half of the uridine residues present in the high-molecular-weitht ribosomal RNA. The high molecular weight of the RNA from ribonuclease-treated ribosomes was preserved until it was heated; after heating, RNA fragments having sedimentation coefficients of 5 S and less were present. It is inferred that the olignucleotides are derived from pyrimidine-rich clusters located in single-stranded "hairpin" loops on the outside surface of the ribosome.     

Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene.

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.