Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.

Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.     

DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth.

The nucleic acid fraction from cells of 6 species of bacterium and 2 kinds of vertebrate, calf and salmon, was extracted and purified by the same procedures as described previously. When the spleen cells from BALB/c mice were incubated with the nucleic acid fraction from either of the bacteria, natural killer (NK) activity of the cells was remarkably elevated and the cells produced factors to activate macrophages and to inhibit viral growth. It was shown that the factor to activate macrophages was interferon (IFN)-gamma and that to inhibit viral growth was IFN-alpha/beta. On the other hand, the nucleic acid fraction from either of the vertebrate cells did not show such activities. Pretreatment of the bacterial nucleic acid fraction with DNase, but not with RNase, abrogated completely the biological activities. The activities of the bacterial nucleic acid were not influenced by the presence of polymyxin B, an inhibitor of lipopolysaccharide (LPS), and the spleen cells from not only BALB/c mice but also LPS-insensitive C3H/HeJ mice were activated, indicating that the activities of the fraction were not ascribed to LPS contaminated possibly into the fraction, but to DNA itself. Intralesional injection with the bacterial DNA fraction caused regression of mouse IMC tumors, but the injection with the vertebrate DNA fraction did not. These findings prompted us to examine the biological activities of DNA samples from a variety of animals and plants, which were provided from other laboratories or purchased from manufacturers. All of the DNA samples from cells of 5 kinds of bacterium, 2 of virus and 4 of invertebrate augmented NK activity and induced IFN, more or less, in mouse spleen calls, while the DNA from 10 kinds of vertebrate, including 3 of fish and 5 of mammal, showed no such activities. The DNA from 2 species of plants, were also inactive. Possible mechanisms to explain the different biological activities of DNA from different cell sources were discussed based on our previous finding that the particular palindromic sequences with a G-C motif(s) are required for induction of IFNs and activation of NK cells with synthetic 30-mer oligonucleotides.     

Lipofection of Synthetic Oligodeoxyribonucleotide Having a Palindromic Sequence of AACGTT to Murine Splenocytes Enhances Interferon Production and Natural Killer Activity

A synthetic 22-mer oligodeoxyribonucleotide having an AACGTT palindrome, AAC-22, induced interferon (IFN) production and augmented the natural killer (NK) activity in murine splenocytes, whereas its analogue, ACC-22, having an ACCGGT palindrome, did not. The binding of AAC-22 to splenocytes was not different from that of ACC-22. Lipofection of AAC-22 to splenocytes remarkably enhanced IFN production and NK cell activity, whereas that of ACC-22 caused little enhancement. These results strongly suggest that the prerequisite for IFN production is not the binding of AAC-22 to the cell surface receptors, but its penetration into the spleen cells.     

Nuclease resistance and RNase H sensitivity of oligonucleotides bridged by oligomethylenediol and oligoethylene glycol linkers

The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated. These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present. It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli. The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers. Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers. Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H. As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy.     

The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage.

The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a variety of lesions with diverse chemical structures and the presence of helicase activity in the UvrAB complex which can displace short oligonucleotides annealed to single-stranded DNA led to a model in which this activity moves UvrAB along undamaged DNA to damaged sites where the lesion blocks further translocation and the protein-DNA pre-incision complex is formed. To evaluate this mechanism for damage recognition, we constructed substrates with oligonucleotides of different lengths annealed to single-stranded DNA circles and placed a single 2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the circle. For the substrates with no lesion, the UvrAB complex effectively displaced a 22-mer but not a 27-mer or longer fragments. The presence of AAF on the oligonucleotide significantly increased the release of the 27-mer but oligomers of 30 or longer were not separated. Placing the lesion on the circular strand did not block the release of the fragments. Instead, the releasing activity of UvrAB was stimulated and also depended on the length of the annealed oligonucleotide. These observations do not agree with the predictions of a damage recognition mechanism that depends on helicase-driven translocation. Most likely, the strand-separating activity of UvrAB is a consequence of local changes occurring during the formation of a DNA-protein pre-incision complex at the damaged site and is not due to translocation of the protein along undamaged DNA to locate a lesion.     

Non-radioactive automated sequencing of oligonucleotides by chemical degradation

A non-radioactive sequencing of fluorescently labelled oligonucleotides by solid-phase chemical degradation is described. Although non-radioactive methods have been reported for the dideoxy chain termination technique, such a method has not yet been developed for the chemical degradation sequencing of DNA fragments. A 21-mer fluorescein labelled M13 sequencing primer was sequenced in an on-line automated system in about 30 minutes. The fluorescent dye and its bond to the oligonucleotide were stable during the chemical reactions used for the base specific degradations. As the sequence is determined on-line during electrophoresis, reloading and running 10 fragments simultaneously allows us to use one gel for sequencing of about 50 different oligonucleotides.     

Reducing the number of microlocations in oligonucleotide microchip matrices by the application of degenerate oligonucleotides

The application of degenerate oligonucleotides to DNA Sequencing by Hybridisation with Oligonucleotide Matrix (SHOM) is proposed. The use of degenerate oligonucleotides is regarded as an example of pooling methods that are suitable for various laboratory procedures requiring numerous samples to be assayed. As each DNA sequence coded by four letters (A, G, C, T) may be defined by two sequences: a sequence coded by W and S (W-weak-A or T, S-strong-G or C) and a sequence coded by R and Y (R-purine-A or G, Y-pirymidine-T or C), n4n -nucleotide sequences may be defined with the help of 2xn2sequences. In the place of the originally described microchip matrix composed of all possible unambiguous octanucleotides (4(8)=65 536) attached to the equal number of 65 536 microlocations a matrix composed of 512 microlocations containing 256 2(8)-degenerate octanucleotides is proposed. The matrix contains all 256 possible octanucleotides coded by W and S variations and all 256 possible octanucleotides coded by R and Y variations. The 512 256-degenerate octanucleotides allows to retrieve the same information as 65 536 unambiguous octanucleotides. A variant of the DNA sequence reconstruction method applicable to this system is presented. The use of degenerate oligonucleotides also gives the possibility to apply matrices composed of longer oligonucleotides without increasing the number of microlocations in matrices, which would enable increasing the length of unambiguously reconstructed sequence, e.g. a matrix comprising 131 072 16-mer oligonucleotides i.e. 65 536 65 536-fold degenerate oligonucleotide coded by W and S variations and 65 536 65 536-fold degenerate oligonucleotide coded by R and Y variations could replace one matrix comprising all possible unambiguous 16-mer oligonucleotides (ca. 4.3x10(9)).     

The binding in vitro of the intermediate filament protein vimentin to synthetic oligonucleotides containing telomere sequences

The ability of the intermediate filament subunit protein vimentin to bind synthetic oligonucleotide telomere models containing repeat sequences from Oxytricha (T4G4), Saccharomyces (TGTGTG3), or Tetrahymena (T2G4) was investigated in vitro with a filter binding assay and a gel overlay assay. At low ionic strength, vimentin bound these oligonucleotides with high affinity. At higher ionic strength, the vimentin-oligonucleotide complex was less stable, such that approximately 30% of the initial binding remained at 150 mM KCl. One mole of vimentin tetramer bound approximately 1 mol of telomere oligonucleotide. Vimentin bound well oligonucleotides containing either a random duplex or random 3'-overhang, but showed a reduced affinity for a blunt-ended oligonucleotide. A control random sequence oligonucleotide was not bound by vimentin. The oligonucleotide-binding site of vimentin was shown to be localized in the non-alpha-helical N-terminal domain by assays employing purified proteolytic fragments of vimentin. Preliminary results in the gel overlay assay show that other members of the intermediate filament family, nuclear lamins A-C, all bind the synthetic oligonucleotide containing the telomere repeat sequence of Oxytricha.     

Solid-phase oligonucleotide synthesis and flow cytometric analysis with microspheres encoded with covalently attached fluorophores

A novel combinatorial approach to synthesize oligonucleotides on fluorescently encoded microspheres based on flow sorting and segmental solid-phase synthesis is described. BODIPY dyes were covalently attached to polystyrene (8.8 microm, 55% DVB) microsphere particles to generate four fluorescently encoded sets. 20-mer oligonucleotide sequences can be synthesized on these microspheres with yields comparable to conventional CPG supports (80% overall yield, average stepwise yield = 99%). The concept of segmental solid-phase synthesis by flow sorting was demonstrated by synthesizing unique 20-mer oligonucleotide sequences on each of four fluorescently encoded microsphere sets by including a flow sorting step (after first eight base additions) and flow cytometric detection of sequences synthesized on each microsphere set by hybridization with fluorescently labeled complementary sequence.     

Hybridization methods for DNA sequencing.

I have conducted a general analysis of the practicability of using oligonucleotide hybridization to sequence DNA. Any DNA sequence may be sequenced by hybridization with a complete panel of oligonucleotides. However, sequencing DNA segments over 2 kb long requires an unrealistic number of hybridization reactions. The optimal protocol is to hybridize 7-mer or 8-mer mixed oligonucleotide probes to immobilized DNA fragments 80 bp long: should this prove impractical, hybridization of labeled 270-bp fragments to immobilized mixed 10-mers is a potential alternative. Both protocols require no more experiments to sequence large regions of DNA than conventional m13-based sequencing and are much easier to automate, thus reducing the requirements for skilled personnel. In the ideal case, hybridization sequencing reduces the number of experiments required to sequence megabase DNA by 90%.     

Fragments of bovine insulin-like growth factors I and II stimulate proliferation of rat L6 myoblast cells

The active sites of bovine insulin-like growth factor (IGF) I and II fragments were studied. Overlapping fragments of IGF I (residues 1-25, 11-35, 21-45, 31-55, and 41-70) and of IGF II (residues 1-24, 10-34, 20-44, 30-54, and 40-67) were chemically synthesized. The activity of the fragments was measured by stimulating the proliferation of rat L6 myoblast cells. Two fragments of IGF I (residues 21-45 and 31-55) and two fragments of IGF II (residues 20-44 and 30-54) were active while the other fragments were inactive in stimulating cell proliferation. Although the activity of these fragments was observed only at a high concentration of 0.1 mM, the results imply that the active site is located around residues 31-45 for IGF I fragments and residues 30-44 for IGF II fragments. Consequently, an IGF I fragment (residues 26-50) having a five-residue extension to both the N- and C-terminal sites of residues 31-45 also stimulated the proliferation of L6 myoblast cells. Furthermore, the substitution of Ile-35 in two IGF II fragments (residues 21-45 and 31-55) by Ser inactivated these fragments. This suggests that Ile-35 is an essential residue for IGF II fragment activity. Ser-35, which was reported in the original sequencing of bovine IGF II, is incorrect in the sequence and furthermore has been consistently found to be an Ile-35 in our hands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oligodeoxynucleotides containing palindrome sequences with internal 5'-CpG-3' act directly on human NK and activated T cells to induce IFN-gamma production in vitro.

Previous studies have shown that the action of bacterial or synthetic oligodeoxynucleotide (oligo-DNA) on mouse NK cells to produce IFN-gamma is mediated mostly by monocytes/macrophages activated by olig-DNA. However, its action on human IFN-gamma-producing cells has not been well investigated. In the present study, we examined the effect of oligo-DNAs on highly purified human NK and T cells. Bacillus Calmette-Guérin-derived or synthetic oligo-DNAs induced NK cells to produce IFN-gamma with an increased CD69 expression, and the autocrine IFN-gamma enhanced their cytotoxicity. The response of NK cells to oligo-DNAs was enhanced when the cells were activated with IL-2, IL-12, or anti-CD16 Ab. T cells did not produce IFN-gamma in response to oligo-DNAs but did respond independently of IL-2 when they were stimulated with anti-CD3 Ab. In the action of oligo-DNAs, the palindrome sequence containing unmethylated 5'-CpG-3' motif(s) appeared to play an important role in the IFN-gamma-producing ability of NK cells. The changes of base composition inside or outside the palindrome sequence altered its activity: The homooligo-G-flanked GACGATCGTC was the most potent IFN-gamma inducer for NK cells. The CG palindrome was also important for activated NK and T cells in their IFN-gamma production, although certain nonpalindromes acted on them. Among the sequences tested, cell activation- or cell lineage-specific sequences were likely; i.e., palindrome ACCGGT and nonpalindrome AACGAT were favored by activated NK cells but not by unactivated NK cells or activated T cells. These results indicate that oligo-DNAs containing CG palindrome act directly on human NK cells and activated T cells to induce IFN-gamma production.     

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.     

Multiple domains exist within the upstream activator sequence of the octopine synthase gene.

It is known that a 16-base pair palindrome (ACGTAAGCGCTTACGT) located upstream of the ocs gene can activate a maize adh1 promoter in a transient expression system [Ellis et al. (1987). EMBO J. 6, 11-16; Ellis et al. (1987). EMBO J. 6, 3203-3208]. We have determined that this palindrome is also essential for ocs promoter activity in tobacco calli. In addition, sequences immediately adjacent to this palindrome, both 5' and 3', modulate its activity. The palindrome is sensitive to the differentiated state of the plant cells in which it resides; it is active in calli and the leaves of small shoots but is inactive in the leaves of rooted plants. We have tested upstream sequences from two other T-DNA genes that have homology to this palindrome for their ability to activate the octopine synthase promoter in tobacco calli. The upstream region from the mannopine synthase gene can activate the octopine synthase promoter, but an upstream region from the gene implicated in octopine and nopaline secretion cannot activate the promoter.     

Evaluation of 50-mer oligonucleotide arrays for detecting microbial populations in environmental samples

Microarrays fabricated with oligonucleotides longer than 40 bp have been introduced for monitoring whole genome expression but have not been evaluated with environmental samples. To determine the potential of this type of microarray for environmental studies, a 50-mer oligonucleotide microarray was constructed using 763 genes involved in nitrogen cycling: nitrite reductase (nirS and nirK), ammonia monooxygenase (amoA), nitrogenase (nifH), methane monooxygenase (pmoA), and sulfite reductase (dsrAB) from public databases and our own sequence collections. The comparison of the sequences from pure cultures indicated that the developed microarrays could provide species-level resolution for analyzing microorganisms involved in nitrification, denitrification, nitrogen fixation, methane oxidation, and sulfite reduction. Sensitivity tests suggested that the 50-mer oligonucleotide arrays could detect dominant populations in the environments, although sensitivity still needs to be improved. A significant quantitative relationship was also obtained with a mixture of DNAs from eight different bacteria. These results suggest that the 50-mer oligonucleotide array can be used as a specific and quantitative parallel tool for the detection of microbial populations in environmental samples.     

Molecular cloning of a cDNA coding for human leukotriene A4 hydrolase. Complete primary structure of an enzyme involved in eicosanoid synthesis

We have isolated a near full-length cDNA encoding human leukotriene A4 hydrolase, which synthesizes a potent chemotactic and spasmogenic compound, leukotriene B4. A human spleen cDNA library was screened with a 48-mer oligonucleotide probe, synthesized according to the partial amino acid sequence of the human leukocyte enzyme. The nucleotide sequence of the cDNA had an open reading frame of 1,833 base pairs, which contained regions coding for the N-terminal amino acid sequence, the amino acid sequence for the probe design, and several other peptide sequences of the enzyme. The complete primary structure of the enzyme composed of 610 amino acid residues (molecular weight, 69,153) was deduced from the cDNA.