Synthesis and characterization of a d(ApG) platinated nonanucleotide duplex.

The nonamer 5'd(CTCAGCCTC) 3' 1 has been reacted with cis-diamminediaquaplatinum(II) in water at pH 4.2. The major reaction product was shown by enzymatic digestion and 1H NMR to be the d(ApG)cis-Pt(NH3)2 chelate [cis-Pt(NH3)2[d(CTCAGCCTC)-N7(4),N7(5)]] 1-Pt. When mixed with its complementary strand 2, 1-Pt forms a B DNA type duplex 3-Pt with a Tm of 35 degrees C (versus 58 degrees C for the unplatinated duplex). The NMR study of the exchangeable protons of 3-Pt revealed that the helix distortion is localized on the CA*G*-CTG moiety (the asterisks indicating the platinum chelation sites) with a strong perturbation of the A*(4)T(15) base pair related to a large tilt of A*(4).     

Interaction of trans and cis regulatory elements in the INO1 promoter of Saccharomyces cerevisiae.

Electrophoretic mobility shift assays (EMSA) were used to define the regions of the INO1 promoter that interact with factors present in extracts prepared from the yeast, Saccharomyces cerevisae. These experiments identified three different types of protein:DNA complexes that assemble with the INO1 promoter. Formation of one type of complex depended on functional alleles of previously described regulatory genes, INO2 and INO4, that encode positive regulatory factors. Formation of the INO2/INO4-dependent complexes was increased when extracts prepared from cells grown under derepressing conditions (i.e. absence of inositol and choline). A second type of complex was dependent on the OPI1 gene, that encodes a negative regulator. Computer-aided sequence analysis of the promoters of several genes encoding phospholipid biosynthetic enzymes revealed a highly conserved nine basepair element (5'-ATGTGAAAT-3'), designated 'nonamer' motif, that is similar to the octamer motif identified in the promoters of mammalian immunoglobulin genes. The nonamer motif was shown to form a specific complex with a factor, designated nonamer binding factor (NBF). Extracts prepared from Schizosaccharomyces pombe formed a nonamer-specific complex.     

Statistical considerations on the formation of circular photosynthetic light-harvesting complexes from Rhodopseudomonas palustris

Depending on growth conditions, some species of purple photosynthetic bacteria contain peripheral light-harvesting (LH2) complexes that are heterogeneous owing to the presence of different protomers (containing different αβ-apoproteins). Recent spectroscopic studies of Rhodopseudomonas palustris grown under low-light conditions suggest the presence of a C ₃-symmetric LH2 nonamer comprised of two distinct protomers. The software program Cyclaplex, which enables generation and data-mining of virtual libraries of molecular rings formed upon combinatorial reactions, has been used to delineate the possible number and type of distinct nonamers as a function of numbers of distinct protomers. The yield of the C ₃-symmetric nonamer from two protomers (A and B in varying ratios) has been studied under the following conditions: (1) statistical, (2) enriched (preclusion of the B-B sequence), and (3) seeded (pre-formation of an A-B-A block). The yield of C ₃-symmetric nonamer is at most 0.98 % under statistical conditions versus 5.6 % under enriched conditions, and can be dominant under conditions of pre-seeding with an A-B-A block. In summary, the formation of any one specific nonamer even from only two protomers is unlikely on statistical grounds but must stem from enhanced free energy of formation or a directed assembly process by as-yet unknown factors.     

Dissecting the Dynamics of HIV-1 Protein Sequence Diversity

The rapid mutation of human immunodeficiency virus-type 1 (HIV-1) and the limited characterization of the composition and incidence of the variant population are major obstacles to the development of an effective HIV-1 vaccine. This issue was addressed by a comprehensive analysis of over 58,000 clade B HIV-1 protein sequences reported over at least 26 years. The sequences were aligned and the 2,874 overlapping nonamer amino acid positions of the viral proteome, each a possible core binding domain for human leukocyte antigen molecules and T-cell receptors, were quantitatively analyzed for four patterns of sequence motifs: (1) “index”, the most prevalent sequence; (2) “major” variant, the most common variant sequence; (3) “minor” variants, multiple different sequences, each with an incidence less than that of the major variant; and (4) “unique” variants, each observed only once in the alignment. The collective incidence of the major, minor, and unique variants at each nonamer position represented the total variant population for the position. Positions with more than 50% total variants contained correspondingly reduced incidences of index and major variant sequences and increased minor and unique variants. Highly diverse positions, with 80 to 98% variant nonamer sequences, were present in each protein, including 5% of Gag, and 27% of Env and Nef, each. The multitude of different variant nonamer sequences (i.e. nonatypes; up to 68%) at the highly diverse positions, represented by the major, multiple minor, and multiple unique variants likely supported variants function both in immune escape and as altered peptide ligands with deleterious T-cell responses. The patterns of mutational change were consistent with the sequences of individual HXB2 and C1P viruses and can be considered applicable to all HIV-1 viruses. This characterization of HIV-1 protein mutation provides a foundation for the design of peptide-based vaccines and therapeutics.     

Sequence analysis of chitooligosaccharides by matrix-assisted laser desorption ionization postsource decay mass spectrometry

Chitin/chitosan oligosaccharides composed of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and/or 2-amino-2-deoxy-D-glucopyranose (GlcN) were prepared by chemical degradation of chitin or chitosan and separated by gel permeation chromatography. Oligosaccharides obtained after enzymatic hydrolysis of chitosan [F(A) 0.19] with a fungal chitinase were derivatized by reductive amination with 2-aminoacridone and sequenced by matrix-assisted laser desorption ionization time-of-flight postsource decay (PSD) mass spectrometry (MS). The sequence of a trimer, D1A2, was established as D-A-A. The composition of a hexamer D3A3 was ca. 65% D-A-D-D-A-A and 35% D-D-A-D-A-A. The PSD MS of a nonamer D5A4-amac revealed four isobaric species D-X-Y-D-X-Y-D-A-A, where A is GlcNAc, D is GlcN, and X and Y (X not equal Y) are mutually either D or A. This structure motif was also observed in a dodecamer D7A5 which was composed of eight isobaric sequences of the general formula (D-X-Y)(3)-D-A-A.     

The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process.     

N1,N5,N10-Tris(4-hydroxycinnamoyl)spermidines from Microdesmis keayana roots

Three new N1,N5,N10-tris(4-hydroxycinnamoyl)spermidines were isolated from a methanolic root extract of Microdesmis keayana. They were identified as N5,N10-di(p-coumaroyl)-N1-feruloylspermidine,N5-(p-coumaroyl)-N1,N10-diferuloylspermidine, and N1,N5,N10-triferuloylspermidine, and were named keayanidines A, B, and C (1-3), respectively. Their structures were established by spectral techniques(electrospray mass spectrometry, one- and two-dimensional NMR). A 4',4',4'-trimethylated derivative was prepared by methylation of keayanidine C, and the same compound was synthesized fromspermidine and 3,4-dimethoxycinnamic acid to confirm the spectral attributions of the NMR data of the natural compounds. Radical-scavenging properties of all compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical spectrophotometric assay.     

Chemical synthesis and sequence studies of deoxyribooligonucleotides which constitute the duplex sequence of the lactose operator of Escherichia coli.

We have synthesized the deoxyribooligonucleotide fragments, constituting the sequence of the lac operator of Escherichia coli. Two of these fragments, d(pApApTpTpGpTpTpApT) (nonamer) and d(pApApTpTpGpTpGpApG) (nonamer), corresponding to the 5' termini of lac operator have been synthesized by the phosphodiester method. The remaining four fragments, d(ApCpApApTpT) (hexamer), d(ApTpApApCpApApTpT) (nonamer), d(ApApTpTpGpTpGpApGpCpGpG) (dodecamer), and d(ApApTpTpGpTpTpApTpCpCpGpCpTpC) (pentadecamer), have been synthesized by an improved phosphotriester method. All of the compounds were first characterized by venom and spleen phosphodiesterase digestion to obtain their base composition. The sequence of these oligonucleotides was fully confirmed by the characteristic mobility shifts of their partial venom phosphodiesterase digestion products on two-dimensional homochromatography. A comparative study of the two methods for the synthesis of oligonucleotides has revealed that the phosphotriester method is more convenient than the phosphodiester method because of higher yields and ease of handling large scale preparations.     

Solution conformation of the type I collagen alpha-2 chain telopeptides studied by 1H and 13C NMR spectroscopy

The high-field 1H and 13C NMR studies of the N- and C-terminal telopeptides of the alpha-2 chain of collagen were carried out in CD3OH/H2O solutions. All proton assignments are based on two-dimensional phase-sensitive COSY and ROESY experiments. The conformation of the N-telopeptide (nonamer) is predominantly extended with a small proportion of the molecules existing in a type I beta turn. The four residues involved in this turn are D3-A4-K5-G6 which is stabilized by a C = O(D3)-NH(G6) hydrogen bond. The C-terminal telopeptide is extended throughout. A model is proposed involving charge-charge and hydrophobic interactions between the extended alpha-2 chain N-telopeptide and the adjacent segments of triple-helix. A similar model is proposed for the C-telopeptide.     

DNA binding features of human POT1: a nonamer 5'-TAGGGTTAG-3' minimal binding site, sequence specificity, and internal binding to multimeric sites

The human telomeric protein POT1 is known to bind single-stranded telomeric DNA in vitro and to participate in the regulation of telomere maintenance by telomerase in vivo. We examined the in vitro DNA binding features of POT1. We report that deleting the oligosaccharide/oligonucleotide-binding fold of POT1 abrogates its DNA binding activity. The minimal binding site (MBS) for POT1 was found to be the telomeric nonamer 5'-TAGGGTTAG-3', and the optimal substrate is [TTAGGG](n (n > or = 2)). POT1 displays exceptional sequence specificity when binding to MBS, tolerating changes only at position 7 (T7A). Whereas POT1 binding to MBS or [TTAGGG](2) was enhanced by the proximity of a 3' end, POT1 was able to bind to a [TTAGGG](5) array when positioned internally. These data indicate that POT1 has a strong sequence preference for the human telomeric repeat tract and predict that POT1 can bind both the 3' telomeric overhang and the displaced TTAGGG repeats at the base of the t-loop.     

Synthesis of aminoglycoside conjugates of 2'-O-methyl oligoribonucleotides

Aminoglycoside conjugates of 2'- O-methyl oligoribonucleotides have been synthesized entirely on a solid phase using conventional phosphoramidate chemistry. For this purpose, appropriately protected neamine-derived phosphoramidites, viz., 2-cyanoethyl [6,3',4'-tri- O-levulinoyl- N (1), N (3), N (2) (') , N (6) (') -tetra(trifluoroacetyl)neamine-5- O-ethyl] N,N-diisopropylphosphoramidite, 1, and 2-cyanoethyl [6,3',4',2',3'-penta- O-levulinoyl- N (1), N (3), N (2) (') , N (6) (') -tetra(trifluoroacetyl) ribostamycin-5'-yl] N, N-diisopropylphosphoramidite, 2, have been prepared and attached via phosphodiester linkage to an appropriate 2'- O-methyl oligoribonucleotide. Levulinoyl esters are used to cap the hydroxyl groups of the aminoglycoside moieties, since they may be selectively removed prior to ammonolysis. In this manner, the potential O-->N acyl migration is excluded. Applicability of the strategy has been demonstrated by the synthesis of eight different aminoglycoside conjugates, in which 1 and 2 are attached directly to the 5'-end ( 6 and 10) or, alternatively, to an inserted non-nucleosidic hydroxyalkyl armed branching unit ( 3, 4, or 5), which results in intrachain conjugates ( 7- 9, 11- 13). The potential of these conjugates to act as a sequence-selective artificial nuclease has been studied.     

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.     

Modeling of a propagation mechanism of infectious prion protein; a hexamer as the minimum infectious unit

To construct a new model of the propagation mechanism of infectious scrapie-type prion protein (PrP(Sc)), here we conducted a disruption simulation of a PrP(Sc) nonamer using structure-based molecular dynamics simulation method based on a hypothetical PrP(Sc) model structure. The simulation results showed that the nonamer disrupted in cooperative manners into monomers via two significant intermediate states: (1) a nonamer with a partially unfolded surface trimer and (2) a hexamer and three monomers. Dimers and trimers were rarely observed. Then, we propose a new PrP(Sc) propagation mechanism where a hexamer plays an essential role as a minimum infectious unit.     

Rearrangement reactions of 1,N2-isopropenoguanine cyclonucleosides

Under acid-catalyzed transglycosylation conditions 5',8-cyclo-8-oxo-1,N2-isopropenoguanine nucleosides (1, 10) undergo a ring-opening reaction to 8-oxo-1,N2-isopropenoguanosine derivatives (4, 11) followed by recyclization to fluorescent 5',3-cyclonucleosides (2, 12).     

A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2''-hydroxyl groups.

Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide.     

Some observations relating to the oximate ion promoted unblocking of oligonucleotide aryl esters.

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.     

The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.     

DNA adduct bypass polymerization by Sulfolobus solfataricus DNA polymerase Dpo4: analysis and crystal structures of multiple base pair substitution and frameshift products with the adduct 1,N2-ethenoguanine

1,N(2)-Etheno(epsilon)guanine is a mutagenic DNA lesion derived from lipid oxidation products and also from some chemical carcinogens. Gel electrophoretic analysis of the products of primer extension by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) indicated preferential incorporation of A opposite 3'-(1,N(2)-epsilon-G)TACT-5', among the four dNTPs tested individually. With the template 3'-(1,N(2)-epsilon-G)CACT-5', both G and A were incorporated. When primer extension was done in the presence of a mixture of all four dNTPs, high pressure liquid chromatography-mass spectrometry analysis of the products indicated that (opposite 3'-(1,N(2)-epsilon-G)CACT-5') the major product was 5'-GTGA-3' and the minor product was 5'-AGTGA-3'. With the template 3'-(1,N(2)-epsilon-G)TACT-5', the following four products were identified by high pressure liquid chromatography-mass spectrometry: 5'-AATGA-3', 5'-ATTGA-3', 5'-ATGA-3', and 5'-TGA-3'. An x-ray crystal structure of Dpo4 was solved (2.1 A) with a primer-template and A placed in the primer to be opposite the 1,N(2)-epsilon-G in the template 3'-(1,N(2)-epsilon-G)TACT 5'. The added A in the primer was paired across the template T with classic Watson-Crick geometry. Similar structures were observed in a ternary Dpo4-DNA-dATP complex and a ternary Dpo4-DNA-ddATP complex, with d(d)ATP opposite the template T. A similar structure was observed with a ddGTP adjacent to the primer and opposite the C next to 1,N(2)-epsilon-G in 3'-(1,N(2)-epsilon-G)CACT-5'. We concluded that Dpo4 uses several mechanisms, including A incorporation opposite 1,N(2)-epsilon-G and also a variation of dNTP-stabilized misalignment, to generate both base pair and frameshift mutations.     

Specificity of the N1 and N2 sialidase subtypes of human influenza A virus for natural and synthetic gangliosides

Sialyl-linkage specificity of sialidases of the human influenza A virus strains, A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1) were studied using natural and synthetic gangliosides. The sialidase of the A/Aichi/2/68 strain hydrolyzed the terminal Neu5Acalpha2-3Gal sequence but not the Neu5Acalpha2-3 linkage on the inner Gal of GM1a, which is a ganglioside that has the gangliotetraose chain (Galbeta1-3GalNAcbeta1-4- (Neu5Acalpha2-3)Galbeta1++ +-4Glcbeta1-Cer). The sialidase hydrolyzed the Neu5Ac on the inner Gal of GM2, which had a shorter gangliotriose chain. GM4, which had the shortest chain (Neu5Acalpha2-3Galbeta1-Cer) of the gangliosides, had a lower substrate specificity. The N1 and N2 sialidase subtypes of the human influenza A virus had no significant variation in their substrate specificity for the gangliosides. Analysis of 11 synthetic gangliosides, which contained various ceramide or sialic acid moieties, demonstrated that A/Aichi/2/68 (H3N2) sialidase recognized the ceramide and sialic acid moiety and the length and structure of the sialyl sugar chain.