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The structural features of nucleic acid probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implication for the hybridization process. We discuss how arrays of oligonucleotides provide powerful tools to study the molecular basis of these interactions on a scale which is impossible using conventional analysis.
Viral RNA, molecularly cloned proviral DNA, and virus-specific protein of avian retrovirus MH2 were analyzed. The complexity and sequence conservation of the transformation-specific v-myc sequences of MH2 RNA were compared with those of the other members of the MC29 subgroup of acute leukemia viruses, MC29, CMII, and OK10, and with chicken cellular c-myc sequences. All T1 oligonucleotides mapping within the 1.3-kilobase coding region of MC29 v-myc have homologous counterparts in the RNAs of all MC29 subgroup viruses and in c-myc. These counterparts are either identical in composition or altered by single point mutations. Hence, the 47,000-dalton carboxy-terminal sequences of the transforming proteins of these viruses and of the cellular gene product are probably highly conserved but may contain single amino acid substitutions. T1 oligonucleotide mapping of MH2 RNA indicated that the MH2 v-myc sequences map close to the 3' end of viral RNA. A genomic library of an MH2-transformed quail cell line was prepared by using the Charon 4A vector system. By screening with an myc-specific probe, a clone containing the entire MH2 provirus (lambda MH2-1) was isolated. Digestion of cloned DNA with KpnI yielded a 5.1-kilobase fragment hybridizing to both gag- and myc-specific probes. Further restriction mapping of lambda MH2-1 DNA showed that about 1.6 kilobases of the gag gene are present near the 5' end of proviral DNA, and the conserved part of v-myc, i.e., 1.3 kilobases, is present near the 3' end of proviral DNA. These two domains are separated by a segment of at least 1 kilobase of different genetic origin, including additional unique sequences unrelated to virion genes. Tryptic peptide analysis of the gag-related protein of MH2, p100, revealed gag-specific peptides and several unique methionine-containing peptides. One of the latter is possibly shared with the polymerase precursor protein Pr180gag-pol, but no myc-specific peptides, defined for the MC29 protein p110gag-myc, appear to be present in MH2 p100. The data on viral RNA, proviral DNA, and protein of MH2 reveal a unique genetic structure for this virus of the MC29 subgroup and suggest that its v-myc gene is not expressed as a gag-related protein.
Recently, hybrids of RNA and D-arabinonucleic acids (ANA) as well as the 2'-deoxy-2'-fluoro-D-arabinonucleic acid analog (2'F-ANA) were shown to be substrates of RNase H. This enzyme is believed to be involved in the primary mechanism by which antisense oligonucleotides cause a reduction in target RNA levels in vivo. To gain a better understanding of the properties of arabinose based oligonucleotides, we have prepared a series of 2'F-ANA sequences of homopolymeric (A and T) and mixed base composition (A, T, G and C). UV thermal melting and circular dichroic (CD) studies were used to ascertain the thermodynamic stability and helical conformation of 2'F-ANA/RNA and 2'F-ANA/DNA hybrids. It is shown that 2'F-ANA has enhanced RNA affinity relative to that of DNA and phosphorothioate DNA. The 2'-fluoroarabino modification showed favorable pairing to single-stranded DNA also. This is in sharp contrast to ANA, which forms weak ANA/DNA hybrids at best. According to the measured thermodynamic parameters for duplex formation, the increased stability of hybrids formed by 2'F-ANA (e.g., 2'F-ANA/RNA) appears to originate from conformational pre-organization of the fluorinated sugars and a favorable enthalpy of hybridization. In addition, NMR spectroscopy revealed a five-bond coupling between the 2'F and the base protons (H6/H8) of 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. This observation is suggestive of a through-space interaction between 2'F and H6/H8 atoms. CD experiments indicate that 2'F-ANA/RNA hybrids adopt an 'A-like' structure and show more resemblance to DNA/RNA hybrids than to the pure RNA/RNA duplex. This feature is believed to be an important factor in the mechanism that allows RNase H to discriminate between 2'F-ANA/RNA (or DNA/RNA) and RNA/RNA duplexes.
The immune protein, scolexin, a bacteria-induced, larva-specific protein from Manduca sexta, was shown to exist in the hemolymph in two isoelectric forms designated herein as scolexin-1 and scolexin-2 (native M(r) approximately 72 kd). These two charge isomers appeared to share the same amino acid composition. Scolexin is composed of two subunits (peptide M(r) approximately 36 kd) that possess the same N-terminus. Scolexin-2 was subjected to glycosyl composition analysis, revealing the presence of galactose, glucose, mannose, xylose, and sialic acid residues. Hybridization of epidermal RNA with oligonucleotides deduced from the scolexin N-terminal sequence showed a continuous decline in mRNA following day 0 of the 5th larval instar. By employing in vitro protein labelling, it was found that organ cultures of the epidermis from immune larvae showed a greater ability over that of naive epidermal cultures to synthesize scolexin; these data reflected the inducible response seen in the hemolymph, and confirm other data indicating that the epidermis is an important site of scolexin biosynthesis.
Two biofilm reactors operated with hydraulic retention times of 0.8 and 5.0 h were used to study the links between population dynamics and reactor operation performance during a shift in process operation from pure nitrification to combined nitrification and organic carbon removal. The ammonium and the organic carbon loads were identical for both reactors. The composition and dynamics of the microbial consortia were quantified by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes combined with confocal laser scanning microscopy, and digital image analysis. In contrast to past research, after addition of acetate as organic carbon nitrification performance decreased more drastically in the reactor with longer hydraulic retention time. FISH analysis showed that this effect was caused by the unexpected formation of a heterotrophic microorganism layer on top of the nitrifying biofilm that limited nitrifiers oxygen supply. Our results demonstrate that extension of the hydraulic retention time might be insufficient to improve combined nitrification and organic carbon removal in biofilm reactors.
Methanol dehydrogenase has been purified from the type I marine methanotroph Methylomonas sp. strain A4 and found to be similar to other methanol dehydrogenase enzymes in subunit composition, molecular mass, and N-terminal sequence of the two subunits. A heterologous gene probe and a homologous oligonucleotide have been used to identify a DNA fragment from Methylomonas sp. strain A4 which contains moxF, the gene encoding the large subunit of methanol dehydrogenase. Protein expression experiments with Escherichia coli, immunoblotting of expression extracts, and partial DNA sequence determination have confirmed the presence of moxF on this DNA fragment. In addition, expression and immunoblot experiments have shown the presence of the genes for the small subunit of methanol dehydrogenase (moxI) and for the methanol dehydrogenase-specific cytochrome c (moxG). The moxG gene product has been shown to be cytochrome c552. The expression experiments have also shown that two other genes are present on this DNA fragment, and our evidence suggests that these are the homologs of moxJ and moxR, whose functions are unknown. Our data suggest that the order of these genes in Methylomonas sp. strain A4 is moxFJGIR, the same as in the facultative methylotrophs. The transcriptional start site for moxF was mapped. The sequence 5' to the transcriptional start does not resemble other promoter sequences, including the putative moxF promoter sequence of facultative methylotrophs. These results suggest that although the order of these genes and the N-terminal amino acid sequence of MoxF and MoxI are conserved between distantly related methylotrophs, the promoters for this gene cluster differ substantially.
Denitrification is applied in the tertiary treatment of wastewater to reduce N-pollutants. Fluorescence in situ hybridisation (FISH), CARD (catalyzed reporter deposition)-FISH, cloning, and scanning electron microscopy (SEM) were applied to follow the evolution of the microbial composition and structure of granular sludge in autotrophic denitrifying bioreactors fed with nitrate and thiosulfate. With this goal, FISH oligonucleotide probes for the autotrophic denitrifiers, Thiobacillus denitrificans and Thiomicrospira denitrificans, were designed and their utility tested. CARD-FISH and cloning data showed that bacterial diversity changed with bioreactor operation time. After 110 days of operation, the abundance of Thiobacillus denitrificans cells increased considerably: from 1 to 35% of total DAPI-stained cells and from no isolated clones to 30% of the total positives clones. This fact strongly suggests that this microorganism played a dominant role in the autotrophic denitrification. The Archaeal diversity remained almost unchanged and it was mainly represented by Methanosaeta soehngenii. Scanning electron microscopy results indicated a considerable loss in the integrity of the sludge granules during the operation, with risk of sludge buoyancy.
Hybridization of synthetic oligodeoxynucleotides with single-stranded phage M13mp2 DNA has been studied in terms of temperature, ionic strength, oligonucleotide molar excess and chain length, and DNA secondary structure. Combination of two decadeoxynucleotides corresponding to a nicked eicosamer (composite primer) was found to be efficient in the template-directed DNA polymerase-catalyzed chain elongation, where both decamers separately failed. Circular SS DNA was specifically linearized by BamHI cleavage of SS DNA-tetradecadeoxynucleotide duplex.
Total messenger RNA (mRNA) was isolated from adult human liver and copied to give complementary DNA (cDNA) with reverse transcriptase. Double stranded cDNA was cloned by GC tailing into the PstI site of the plasmid pAT153; approximately 2000 recombinant clones were isolated. The sensitivity of the bacterial cells to ampicillin was used as a marker to study the stability of the library during consecutive overnight growth in mixed liquid culture. A significant change in the composition of the library was observed after three overnight cultures. A heterologous rat albumin cDNA recombinant and two specific oligonucleotides were used to screen the library, and clones containing sequences of the human mitochondrial rRNA- and the alpha-1 antitrypsin-genes were isolated.
Bulky DNA adducts, previously termed type II I-compounds, are detected by 32P-postlabeling following treatment of DNA with several Fenton-type oxygen radical-generating reagents, i.e., mixtures of Fe(II) or Ni(II) and H2O2. In an attempt to characterize the chemical nature and mechanism(s) of formation of these novel adducts, 16 single-stranded deoxyribooligonucleotides (20- and 21-mers) of known sequence were oxidized with Fe(II) or Ni(II) and H2O2, and the products were analyzed by 32P-postlabeling. Eight adducts were obtained reproducibly by oxidation of DNA and test oligonucleotides in a sequence-dependent manner. One major adduct (2) was formed only if the test oligonucleotide contained two adjacent adenine residues. Similarly, adducts 3 and 8 specifically originated in AC and CA sequences, respectively. Adduct 6 required a 5'-C-purine-3' sequence. On the other hand, GN sequences (where N is any normal nucleotide) gave rise to adduct 1, another major product, and adduct 7. Similarly, adducts 4 and 5 were produced by the oxidation of AN sequences. These observations are most readily explained if the oxidation reactions caused intrastrand cross-links between adjacent nucleotides, leading to dimer formation. The observation that adducts 1, 4, 5, and 7 did not require a specific 3'-nucleotide was consistent with the notion that these nucleotides lacked a 3'-base, suggesting the presence of a 5'-->3' purine-sugar cross-linked in the oxidized products. The majority of the lesions came from AA and 5'-purine-N-3' sequences. The effects of Fe(II) and Ni(II) were qualitatively similar; however, higher yields of products were observed with Fe(II) as the catalyst. The definition of the chemical origins of these bulky DNA modifications, which represent a new type of DNA damage, is expected to contribute to a better understanding of the mechanism of metal carcinogenesis and to shed light upon the origins of certain endogenous DNA lesions. Recently, some of the major oxidative DNA adducts characterized here were detected by 32P-postlabeling in the renal DNA of male rats treated with ferric nitrilotriacetate, a known potent prooxidative kidney carcinogen in these animals.
We have studied the binding of the octanucleotide (5'-3')d(AAGGAGGT) which is fully complementary to the 3' end of 16S ribosomal RNA, to ribosomes and to the isolated target sequence (5'-3') (ACCUCCUUA). The binding constant for 30S or 70S ribosomes is (5 +/- 2) X 10(7) mol-1, whereas the duplex containing the octa- and the nonanucleotide has an association constant of (6 +/- 3) X 10(7) mol-1. The two values are the same within the experimental error. This result suggests that basepairing at the 3' end of 16S rRNA is not stabilized by ribosomal proteins.
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To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50 degrees C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.
Synthetic oligonucleotides were used to introduce mutations into the lacPUV5 promoter. Four mutations were obtained at positions -13, -14, and -15, with respect to the transcriptional start site. The effects of these mutations were measured in vivo and the results are discussed with respect to the consensus sequence and other promoter mutations located in this region.
Human DNA polymerase alpha holoenzyme follows an ordered sequential terreactant mechanism of substrate recognition and binding (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S.-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We confirmed this mechanism for the DNA polymerase alpha holoenzyme purified from Drosophila melanogaster embryos and studied the interaction of Drosophila pol alpha with synthetic oligonucleotide template-primers containing modified tetrahydrofuran moieties as model abasic lesions chemically engineered at a number of defined sites. Abasic lesions in the template had relatively little effect on the polymerase incorporation reaction at sites proximal to the lesion. However, incorporation opposite an abasic site was undetectable relative to that which occurred opposite a normal template nucleotide. Moreover, abasic residues in the primer region of the template-primer construct as far as 4 base pairs removed from the 3'-primer terminus prevented detectable nucleotide incorporation relative to that seen on an unmodified template-primer. Primer-region lesions had qualitatively similar effects whether they were located on the primer strand itself or on the complementary template strand. Data from polymerase incorporation experiments were corroborated by competitive binding assays performed under steady state reaction conditions. Results of these experiments suggested that polymerase binding to synthetic oligonucleotide template-primers was essentially unaffected by lesions located at sites that did not block incorporation. Lesions that did block incorporation apparently did so by abrogating template-primer binding. These observations have implications for understanding the mechanisms whereby DNA polymerase alpha recognizes noninformational template sites in vivo and prevents DNA synthesis from proceeding past these points.
The temperature dependence of the formation of a complex between an alpha-d(CCTTCC) hexanucleotide and its complementary beta-d(GGAAGG) sequence was studied and compared to the formation of the beta-d(CCTTCC):beta-d(GGAAGG) complex. Such alpha-beta complex is more stable than the regular beta:beta complex. The Tm value for the alpha:beta complex is 28 degrees C (delta G degrees = -7.3 kcal/mole) while Tm = 20, 1 degree C (delta G degrees = -6.3 kcal/mole) for the beta:beta complex. The stoechiometry of the alpha:beta complex corresponds to the formation of a 1:1 duplex. However, when the alpha- strand is made of alpha-purines: alpha-d(GGAAGG), the stability of the alpha:beta complex, alpha-d(GGAAGG):beta-d(CCTTCC) is found to be lower (Tm = 13.8 degrees C) than the stability of the regular beta-beta complex, leading to the conclusion that the nature of the alpha-sequence is important in terms of stability when considering the synthesis of such a sequence for using it as antisense oligonucleotide.
In order to clarify the role of the purine 2-amino group in the recognition of DNA by small molecules we have examined the binding of actinomycin D and echinomycin to artificial DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine (DAP) in one of the complementary strands. These DNAs, prepared by a method based upon PCR, present various potential sites for antibiotic binding, including several containing only a single purine 2-amino group in different configurations. The results show unambiguously that the presence of two 2-amino groups is mandatory for binding of actinomycin D to double-stranded DNA. In the case of echinomycin only one purine 2-amino group is required for remarkably strong binding to the asymmetric TpDAP.TpA dinucleotide step, but the CpDAP.TpI step (which also contains only a single purine-2 amino group) does not afford a binding site. Evidently, removing a 2-amino group (G-->I substitution) is dominant over adding one (A-->DAP substitution). No sequences containing just a single guanine residue are acceptable. The possibility is raised that replacing guanosine with inosine may do more than remove a group endowed with hydrogen bonding capability and interfere with ligand binding in other ways. The new methodology developed to construct asymmetrically substituted DNA substrates for this work provides a novel strategy that should be generally applicable for studying ligand-DNA interactions, beyond the specific interest in drug binding to DNA, and may help to elucidate how proteins and oligonucleotides recognize their target sites.
By using appropriately modified oligonucleotides, a gap-structure is formed in substrate DNA, and 2-6 ethylenediamine-N,N,N'-triacetate residues are placed near this gap. When these composites are treated with homogenous Ce(IV)/EDTA complex, the phosphodiester linkages in the gap-site are selectively hydrolyzed at much greater rates than achieved previously by using unmodified oligonucelotides.