N-terminal amino acid sequences of intact and cleaved forms of mung bean nuclease.

We report, for the first time, the N-terminal amino acid sequences of both intact and cleaved forms (fragments A and B) of Mung bean nuclease, purified from sprouts of Vigna radiata or purchased from Amersham Biosciences. The N-terminal sequence of Mung bean nuclease shows high similarity with the putative bifunctional nuclease from Arabidopsis thaliana (AC: AAM63596).

A novel transcriptome subtraction method for the detection of differentially expressed genes in highly complex eukaryotes.

We have designed a novel transcriptome subtraction method for the genome-scale analysis of differential gene expression in highly complex eukaryotes, in which suppression subtractive hybridization (SSH) is performed first to enrich the target and, after exchange of adapters, negative subtraction chain (NSC) is then used to eliminate the remaining background. NSC evolved from differential subtraction chain (DSC). We designed novel adapters which make the subtraction system more robust. SSH and NSC were then combined to successfully detect differentially expressed genes in Solanum. The combined technique improves qualitatively upon SSH, the only commercially available transcriptome subtraction system, by detecting target genes in the middle abundance class, to which most differentially expressed genes in highly complex eukaryotes are expected to belong. The main advantage of the combined technique with SSH/NSC is its ability to isolate differentially expressed genes quickly and cost-efficiently from non-standard models, for those microarrays are unavailable.

Tissue-specific attenuation of endogenous DNA I-compounds in rats by carcinogen azoxymethane: possible role of dietary fish oil in colon cancer prevention.

I-compounds are bulky covalent DNA modifications that are derived from metabolic intermediates of nutrients. Some I-compounds may play protective roles against cancer, aging, and degenerative diseases. Many carcinogens and tumor promoters significantly reduce I-compound levels gradually during carcinogenesis. Colon cancer is the second leading cause of cancer death in the United States, whereas cancer of the small intestine is relatively rare. Here we have studied levels of I-compounds in DNA of colon and duodenum of male Sprague-Dawley rats treated with azoxymethane. The effects of dietary lipids (fish oil or corn oil) on colon and duodenal DNA I-compounds were also investigated. Rats fed a diet containing fish oil or corn oil were treated with 15 mg/kg azoxymethane. Animals were terminated 0, 6, 9, 12, or 24 hours after injection. I-compound levels were analyzed by the nuclease P1-enhanced (32)P-postlabeling assay. Rats treated with azoxymethane displayed lower levels of I-compounds in colon DNA compared with control groups (0 hour). However, I-compound levels in duodenal DNA were not diminished after azoxymethane treatment. Animals fed a fish oil diet showed higher levels of I-compounds in colonic DNA compared with corn oil groups (mean adduct levels for fish and corn oil groups were 13.35 and 10.69 in 10(9) nucleotides, respectively, P = 0.034). Taken together, these results support claims that fish oil, which contains a high level of omega-3 polyunsaturated fatty acids, may have potent chemopreventive effects on carcinogen-induced colon cancer. The fact that duodenal I-compounds were not diminished by azoxymethane treatment may have been due to the existence of tissue-specific factors protecting against carcinogenesis. In conclusion, our observations show that endogenous DNA adducts may serve not only as sensitive biomarkers in carcinogenesis and cancer prevention studies, but are also helpful to further our understanding of the chemopreventive properties of omega-3 fatty acids and mechanisms of carcinogenesis.

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA.

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.

Mismatch cleavage by single-strand specific nucleases.

We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S1) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using higher pH, temperature and divalent cation conditions than are routinely used for digestion of single-stranded DNA. Surprisingly, crude plant extracts performed as well as the highly purified preparations for this application. These observations suggest that diverse members of the S1 family of sss nucleases act similarly in cleaving non-specifically at bulges in heteroduplexes, and single-base mismatches are the least accessible because they present the smallest single-stranded region for enzyme binding. We conclude that a variety of sss nucleases and extracts can be effectively used for high-throughput mutation and polymorphism discovery.

Combination of S1 nuclease and PNA for site-selective hydrolysis of double-stranded DNA. Comparison with the site-selective hydrolysis using Ce(IV)/EDTA.

The potential of the combination of SI nuclease and pseudo-complementary PNA (pcPNA) for site-selective scission of double-stranded DNA has been investigated. Through strand invasion of two pcPNAs, single-stranded portions were formed in both strands of substrate DNA. In the initial stage of the enzymatic digestion, two scission fragments were obtained due to the hydrolysis at these two gap-like sites. On prolonged reactions, however, these products (as well as the substrate DNA) were further digested to smaller fragments. Under the conditions employed here, only Ce(IV)/EDTA is available for the preparation of desired fragments from double-stranded DNA.

Transcriptional control of the arginine/lysine transporter, cat-1, by physiological stress.

Cells respond to physiological stress by phosphorylating the alpha subunit of the translation initiation factor eIF2. This adaptive response inhibits protein synthesis and up-regulates genes essential for cell survival. Cat-1, the transporter for the essential amino acids, arginine and lysine, is one of the up-regulated genes. We previously showed that stress increases cat-1 expression by coordinated stabilization of the mRNA and increased mRNA translation. This induction is triggered by amino acid depletion and the unfolded protein response (UPR), which is caused by unfolded proteins in the endoplasmic reticulum. We show here that cat-1 gene transcription is also increased by cellular stress. Our studies demonstrate that the cat-1 gene promoter/regulatory region is TATA-less and is located in a region that includes 94 bases of the first exon. Transcription from this promoter is stimulated 8-fold by cellular stress. An amino acid response element within the first exon is shown to be required for the response to amino acid depletion but not to the UPR. The stimulation of transcription by amino acid depletion requires activation of GCN2 kinase, which phosphorylates eIF2alpha. This phosphorylation also induces translation of the cat-1 mRNA, demonstrating that stress-induced transcriptional and translational control of cat-1 are downstream targets of a signaling pathway initiating with eIF2alpha phosphorylation. Our studies show that the increase in cat-1 gene expression by cellular stress involves at least three types of coordinate regulation: regulation of transcription, regulation of mRNA stability, and regulation of mRNA translation.

High-level expression of ampC beta-lactamase due to insertion of nucleotides between -10 and -35 promoter sequences in Escherichia coli clinical isolates: cases not responsive to extended-spectrum-cephalosporin treatment.

Two Escherichia coli isolates were recovered from the blood of two cancer patients and were demonstrated to produce high levels of the AmpC beta-lactamase with isoelectric points of >9.0. The hypertranscription of ampC RNA was observed by Northern blot hybridization in both isolates. One isolate (isolate EC44) had a point mutation (G-->A at position -28) and insertion of thymidine between positions -20 and -19 of the ampC promoter gene (GenBank accession no. AE000487). The single nucleotide insertion of T between positions -19 and -20 created an optimal distance (17 bp) in the Pribnow box for ampC hyperproduction. The other isolate (isolate EC38) had two point mutations (G-->A at position -28 and C-->T at position +58) and a 2-base (GT) insertion between positions -14 and -15. Although the insertion of GT between positions -14 and -15 may create a new promoter next to the original promoter, cloning of the ampC region with truncated nucleotides of the original -35 region of EC38 failed to verify the hypothesis that a new promoter would be created by such a nucleotide insertion. Instead, multiple start sites for ampC transcription at -1, +1, +2, and +3 were observed in an S1 nuclease protection assay. These results suggest that the RNA polymerase is flexible in the selection of a start site in ampC hypertranscription. In conclusion, nucleotide insertions between the -35 and -10 ampC promoter sequences was the mechanism for the hyperproduction of AmpC beta-lactamase and resistance to oxyimino-cephalosporins. The failure of the two patients to respond to treatment with oxyimino-cephalosporins highlights the important role of such a resistance mechanism in the clinical setting.

The function of SECIS RNA in translational control of gene expression in Escherichia coli.

The incorporation of selenocysteine into proteins is directed by specific UGA codons and mRNA secondary structures, designated SECIS elements. In bacteria, these elements are positioned within the reading frame of selenoprotein mRNAs immediately downstream of the triplet coding for selenocysteine, and they tether a complex of the selenocysteine-specific elongation factor SelB, GTP and selenocysteyl-tRNA(Sec) to the site of UGA decoding. A SECIS-like structure was identified in the 5' non-translated region of the selAB transcript, encoding selenocysteine synthase and SelB. It specifically binds to SelB and the formation of a SelB.GTP.selenocysteyl-tRNA(Sec) complex on the SECIS-like element represses expression of the downstream gene. This effect is abolished by mutations preventing formation of the complex. The regulatory pattern observed correlated with the levels of sel gene products. As quaternary complex formation on the SECIS-like element did not influence the transcription rate and only slightly reduced the level of selAB mRNA, it was concluded that the structure is involved in regulating translation initiation efficiency, thereby coupling selenocysteine biosynthesis to the availability of the trace element selenium.

Fragmentation of photomodified oligodeoxynucleotides adducted with metal ions in an electrospray-ionization ion-trap mass spectrometer.

We report the effect of metal-ion adduction on the fragmentation of oligodeoxynucleotides (ODNs) bearing DNA photoproducts. When protons on backbone phosphates of ODNs are completely replaced with metal ions, cleavages occur readily within the photoproduct moiety, whereas those cleavages do not occur in photomodified ODNs in which the phosphates are associated with protons. For example, thymine/adenine (TA*) photoproducts revert to their undamaged precursors upon collisional activation, the pyrimidine(6-4)pyrimidone product and its Dewar valence isomer show a characteristic neutral loss of C4H3NO3, and dimeric adenine photoproducts show a distinctive loss of NH2CN from the adenine six-membered ring. The product-ion mass spectra of photodamaged ODNs that are adducted to metal ions are complementary in terms of structure information to those spectra of ODNs in which the phosphates are associated with protons. The results also demonstrate that the energy required for strand cleavages is higher for ODNs adducted with metal ions than that for ODNs bound with protons. Furthermore, the loss of a pyrimidine is more favorable than the loss of a purine in the fragmentation of ODNs associated with metal ions.

Enzymatic characterization of organic phosphorus in animal manure.

Information on the forms of P present in animal manure may improve our ability to manage manure P. In most investigations of manure P composition, only inorganic and total P are determined, and the difference between them is assigned as organic P. In this study, we explored the possibility of identifying and quantifying more specific organic P forms in animal manure with orthophosphate-releasing enzymes. Pig (Sus scrofa) manure and cattle (Bos taurus) manure were first sequentially fractionated into water-soluble P, NaHCO3-soluble P, NaOH-soluble P, HCl-soluble P, and residual P. The fractions were separately incubated with wheat phytase, alkaline phosphatase, nuclease P1, nucleotide pyrophosphatase, or their combinations. The released orthophosphate was determined by a molybdate blue method. Part of the organic P in those fractions could be identified by the enzymatic treatments as phytate (i.e., 39% for pig manure and 17% for cattle manure in water-soluble organic P), simple phosphomonoesters (i.e., 43% for pig manure and 15% for cattle manure in NaOH-soluble organic P), nucleotide-like phosphodiesters (2-12%), and nucleotide pyrophosphate (0-4%). Our data indicate that the enzymatic treatment is an effective approach to identify and quantify the organic P forms present in animal manures.

Distribution analysis of the two chicken estrogen receptor-alpha isoforms and their transcripts in the hypothalamus and anterior pituitary gland.

Estrogen plays a key role in the control of reproductive behavior and in the regulation of the neuroendocrine system. To elucidate the mechanisms by which it controls these functions it is important to understand how estrogenic effects are mediated. We have investigated the distribution of the two isoforms of the chicken estrogen receptor alpha (cER-alpha) protein; the previously characterized cER-alpha 66 and a new N-terminal truncated isoform, cER-alpha 61. Immunolocalization demonstrated the presence of cER-alpha 66 protein in hypothalamic areas, principally the nucleus septalis lateralis, bed nucleus striae terminalis medialis, nucleus preopticus medialis, and nucleus infundibuli hypothalami, and in the anterior pituitary gland. When the distribution of ER-alpha immunoreactive cells was compared using the antibodies H 222 (directed against the hormone-binding domain) and ER 221 (directed against the 21-amino acid N-terminus), no apparent differences could be detected. Because this immunocytochemical approach was not able to distinguish whether full-length cER-alpha 66 is the only isoform observed in the ER-positive regions or whether both cER-alpha receptor isoforms are present, SI nuclease assays were performed to compare the relative abundance in these regions of the two distinct classes of cER-alpha mRNA variants (A1-D and A2), which encode the cER-alpha 66 and cER-alpha 61 protein isoforms, respectively. In cockerels and hens, both variants of cER-alpha mRNA are expressed in the anterior pituitary gland and basal hypothalamus with a dominance of the mRNA that encodes cER-alpha 66, whereas the mRNA that encodes cER-alpha 61 was not detectable in the anterior hypothalamus. Therefore, because both receptor isoforms differ in their ability to modulate estrogen target gene expression in a promoter and cell type-specific manner, these differences may mediate the pleiotropic actions of estrogen in reproductive behavior and neuroendocrine functions.

Leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum.

We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S(1) nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from -50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the -1 position and a BRE element with more frequent A bases at position -29 to -32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.

Activation of the alfalfa mosaic virus genome by viral coat protein in non-transgenic plants and protoplasts. The protection model biochemically tested.

In non-transgenic host plants and protoplasts alfalfa mosaic virus displays a strong need for coat protein when starting an infection cycle. The "protection model" states that the three viral RNAs must have a few coat protein subunits at their 3' termini in order to protect them in the host cell against degradation by 3'- to- 5' exoribonucleases [Neeleman L, Van der Vossen EAG, Bol JF (1993) Virology 196: 883-887]. We demonstrated that the naked genome RNAs are slightly infectious, if the inoculation is done at very high concentrations, or if it is preceded by an additional inoculation with the RNAs 1 and 2 (encoding subunits for the viral RNA polymerase). This could mean that the necessity for protection by coat protein is lost if the RNAs in large quantities can overcome the activity of the degrading enzymes, or are protected by association with the RNA polymerase, respectively. However, after having tested in protoplasts the survival of separately preinoculated naked RNA 1 during several hours before RNA 2 was inoculated, on the one hand, or of simultaneously inoculated RNAs 1 and 2, with cycloheximide in the medium during the first hours after inoculation, on the other hand, we had to conclude that the viral genome RNAs are quite stable in the cell in the absence of coat protein or RNA polymerase, respectively. This invalidates the protection model. Accommodation of the above findings by our published "messenger release model" for genome activation [Houwing CJ, Jaspars EMJ (1993) Biochimie 75: 617-621] is discussed.

Syncollin is differentially expressed in rat proximal small intestine and regulated by feeding behavior.

Gradients of gene expression are maintained along the proximal-distal axis of the mammalian small intestine despite a continuously regenerating epithelium. To study the molecular mechanisms responsible for this phenomenon, we utilized a subtractive hybridization strategy to isolate genes differentially expressed in the duodenum but not ileum. We isolated and sequenced 15 clones. The clones were fragments of genes encoding lipases, proteases, and an esterase. A novel clone was characterized and subsequently shown to encode syncollin, a secretory granule protein that binds to syntaxin in a calcium-sensitive manner. RT-PCR and S1 nuclease protection assay were used to clarify the 5'-end of syncollin. Syncollin was expressed in the rat pancreas, spleen, duodenum, and colon. In situ hybridization localized syncollin expression in the pancreas to acinar cells and in the duodenum to villus epithelial cells.

Increase production of lactonizing lipase (LipL) from Pseudomonas sp. strain 109 by lipids and detergents.

LipL of Pseudomonas sp. strain 109 is a unique lipase capable of catalyzing macrocyclic lactone synthesis using omega-hydroxyfatty acid esters as substrates. Several fatty acid esters were tested as inducers of LipL production. The addition of either soybean oil or a non-ionic detergent (Noigen HC) resulted in a 44 to 45-fold increase in extracellular LipL, and the presence of both resulted in a further 56-fold increase. Among the triglycerides tested, triolein was the most effective, with a 50-fold increase in LipL production. A Northern blot hybridization analysis found that the lipL transcript increased in the presence of soybean oil or Noigen HC, indicating that the production of LipL is regulated at the transcriptional level.

Glucocorticoid regulation of hypothalamic proopiomelanocortin.

Although glucocorticoids clearly inhibit proopiomelanocortin (POMC) gene transcription and peptide synthesis in the anterior pituitary, the effects of glucocorticoids on POMC in the hypothalamus are still unclear, even though most POMC neurons in the arcuate nucleus are known to have glucocorticoid receptors. In this study, we have therefore examined the effect of adrenalectomy (ADX) and glucocorticoid replacement on POMC mRNA and peptide (beta-EP and alpha-MSH) levels in the medial basal hypothalamus (MBH) of the rat. POMC mRNA was measured by a sensitive solution hybridization S1 nuclease protection assay, and beta-EP and alpha-MSH were measured by radioimmunoassay. In a first experiment, animals were studied 7 days after ADX or sham surgery. The mean POMC mRNA concentration was 1.01+/-0.14 pg/microg RNA (means+/-SE) in the intact animals and decreased to 0.55+/-0.07 pg/microg RNA in the MBH of the ADX animals (p < 0.005). Beta-EP levels decreased in parallel from 4.30+/-0.18 to 3.36+/-0.11 ng/mg protein (p < 0.001); alpha-MSH levels decreased from 3.25+/-0.21 to 2.41+/-0.16 ng/mg protein (p < 0.005). In a second experiment, animals were studied 2 weeks after ADX. POMC mRNA levels again fell significantly from 1.15+/-0.19 pg/microg RNA in the intact animals to 0.51+/-0.06 pg/microg in the ADX animals (p < 0.01). Beta-EP levels fell also, but this was not significant. In a third experiment, all animals underwent ADX, and half of them received daily subcutaneous injections of dexamethasone (20 microg). Nine days after ADX, the mean POMC mRNA level was 0.66+/-0.04 pg/microg RNA in the saline-treated animals and increased to 0.98+/-0.08 pg/microg RNA in the dexamethasone-treated animals (p < 0.005). A parallel increase in beta-EP levels from 5.03+/-0.41 to 6.01+/-0.53 ng/mg protein was also noted, but this was not statistically significant. We conclude that POMC gene expression is significantly inhibited in the MBH at 1 and 2 weeks after ADX. This effect was reversed by glucocorticoid replacement with doses close to the physiological range. The parallel changes in POMC mRNA and peptide levels strongly suggest that, in contrast to the anterior pituitary, low doses of glucocorticoids stimulate the biosynthesis of POMC in the MBH of ADX rats.

Synthesis and evaluation of some properties of chimeric oligomers containing PNA and phosphono-PNA residues.

In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs), particularly water solubility and cellular uptake, the synthesis of chimeric oligomers consisted of PNA and phosphono-PNA analogues (pPNAs) bearing the four natural nucleobases has been accomplished. To produce these chimeras, pPNA monomers of two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers representing derivatives of N-(2-aminoethyl)glycine, or N-(2-hydroxyethyl)glycine. The oligomers obtained were composed of either PNA and pPNA stretches or alternating PNA and pPNA monomers. The examination of hybridization properties of PNA-pPNA chimeras to DNA and RNA complementary strands in comparison with pure PNAs, and pPNAs as well as DNA-pPNA hybrids and DNA fragments confirmed that these chimeras form stable complexes with complementary DNA and RNA fragments. They were found to be resistant to degradation by nucleases. All these properties together with good solubility in water make PNA-pPNA hybrids promising for further evaluation as potential therapeutic agents.

Correlation of mutagenic potencies of various petroleum oils and oil coal tar mixtures with DNA adduct levels in vitro.

An in vitro system was utilized to measure DNA adduct-forming ability of petroleum oils and oil coal tar mixtures to define correlations between DNA adduct levels and their mutagenic potencies. The system consisted of reaction of dimethyl sulfoxide extracts of oils with calf thymus DNA in the presence of Aroclor-induced hamster liver microsomes for 30 min. Following DNA extraction, DNA adducts were measured by the nuclease P1-enhanced postlabeling assay coupled with two-dimensional polyethyleneimine (PEI)-cellulose TLC. Thin layer plates showed putative aromatic DNA adducts, with levels ranging from 60 to 1400 adducts per 10(9) DNA nucleotides. TLC mobilities suggested adducts to be aromatic compounds containing 4 or more rings. A good correlation (coefficient of correlation = 0.91) was observed between DNA adduct levels and Salmonella mutagenicity for 19 oils. All 19 samples tested produced DNA adducts. To expedite the TLC procedure, adducts were resolved by one-dimensional TLC and the radioactivity measured using a mechanical scanner. Results were comparable to those obtained by two-dimensional TLC and quantification after scraping. Our data show that the in vitro incubation system coupled with the postlabeling adduct assay is a useful screening method to identify mutagenic and potentially carcinogenic oils.

Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nuclease-resistance properties.

We report here the site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides (ODNs). ODNs containing both 5-( N-aminohexyl)-carbamoyl-2'-deoxyuridine (H), which serves as a tether for the further conjugation of functional groups, and 5-(N,N-dimethylaminohexyl)carbamoyl-2'-deoxyuridine (D), which contributes to the thermal stability of the duplex and to the resistance to nucleolytic hydrolysis by nucleases, were synthesized. Functional groups such as folic acid and palmitic acid were site-specifically introduced into the terminus of the aminohexyl-linker of H. The thermal stability and resistance toward nuclease digestion of the modified ODNs were studied. We found that ODNs containing D and H formed stable duplexes with both the complementary DNA and RNA strands even when a bulky functional group such as folic acid, palmitic acid or cholesterol was attached to the terminus of the amino-linker. We also found that ODN analogues which contained D were more resistant to nucleolytic degradation by exo- and endonuclease than the unmodified ODN. Furthermore, duplexes formed by ODNs containing D and the complementary RNA could elicit RNase H activity.