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PubMed2002

Functionality versus strength -- has functional selection taken place in the case of the ecdysteroid receptor response element?.

Nuclear receptors are ligand-dependent transcription factors responsible for controlling differentiation, growth and development of higher eukaryotes. Three amino acids within the recognition alpha-helix of the DNA-binding domain of the nuclear receptors constitute the so-called "P-box" which determines response element specificity. In the ultraspiracle (Usp) protein, which together with EcR forms the heterodimeric ecdysone receptor, the P-box residues are E19, G20 and G23. Substitution of E19, the most characteristic amino acid for estrogen receptor-like P-boxes, with alanine showed that the mutation did not appreciably alter the affinity of the wild-type Usp DNA-binding domain (UspDBD(WT)) for a probe containing natural ecdysone response element (hsp27(wt)). Since in many cases E19 contacts a G/C base pair in position -4, which is absent in hsp27(wt), we analysed the interaction of UspDBD(WT), E19A and other P-box region mutants with the hsp27(wt) derivative which contains a G/C instead of an T/A base pair in position -4. UspDBD(WT) exhibited higher affinity for this element than for hsp27(wt). Moreover, a different interaction pattern of P-box region mutants was also observed. Thus we conclude that the E19 residue of UspDBD is not involved in any hsp27(wt) sequence-discerning contacts. However, substitution of the hsp27(wt) T/A base pair in position -4 with G/C generates target sequence with distinct functional characteristics and possibly with a new specificity. These results could serve as a basis for understanding the role of the presence of a T/A or G/C base-pair in the position -4 in the two types of ecdysone response elements found in nature.

Amino Acid Sequence↗
PubMed1993

Binding to DNA, cellular uptake and biological activity of a distamycin-ellipticine hybrid molecule.

A hybrid molecule which conjugates the minor groove binding agent distamycin and an ellipticine derivative was synthesized and evaluated for cytostatic and cytotoxic activities against L1210 leukaemia cells in vitro. The binding of the hybrid molecule, named 'Distel', to a range of natural DNAs and synthetic polynucleotides with different base pair arrangements was studied by electric linear dichroism. The interaction with DNA simultaneously implicates binding of the distamycin part in the minor groove and intercalation of the ellipticine chromophore. The drug binds to DNA without any apparent preference for AT or GC polynucleotides, and can accommodate both homopolymeric and co-polymeric sequences as a binding site. However, the geometry of the drug-DNA complex varies depending on the targeted sequence. The lower activity of the hybrid as compared to the ellipticine derivative cannot be explained in terms of DNA binding. Taking advantage of the fluorescence of the pyridocarbazole chromophore, fluorescence microscopy was used to map cellular uptake of the hybrid molecule compared to the ellipticine derivative. Both the conjugate and the ellipticine derivative preferentially accumulate in the nuclei of HeLa cells rather than in the cytoplasm. Nuclei of ellipticine derivative-treated cells appear markedly more fluorescent than those of cells treated with the hybrid, which seems to be preferentially located in the nucleoli. Therefore, we consider the possibility that the difference in cytotoxicity between the two ellipticine-containing drugs is due to different intranuclear concentrations of these two compounds.

Animals↗
PubMed2001

Comparison of performance in successive CASP experiments.

As the number of completed CASP (Critical Assessment of Protein Structure Prediction) experiments grows, so does the need for stable, standard methods for comparing performance in successive experiments. It is critical to develop methods for determining the areas in which there is progress and in which areas are static. We have added an analysis of the CASP4 results to that previously published for CASPs 1, 2, and 3. We again use a unified difficulty scale to permit comparison of performance as a function of target difficulty in the different CASPs. The scale is used to compare performance in aligning target sequences to a structural template. There was a clear improvement in alignment quality between CASP1 (1994) and CASP2 (1996). No change is apparent between CASP2 and CASP3 (1998). There is a small barely detectable improvement between CASP3 and the latest experiment (CASP4, 2000). Alignment remains the major source of error in all models based on less than about 30% sequence identity. Comparison of performance in the new fold modeling regime is complicated by issues in devising an objective target difficulty scale. We have found limited numerical support for significant progress between CASP3 and CASP4 in this area. More subjectively, most observers are convinced that there has been substantial progress. Progress is dominated by a single group.

Models, Molecular↗
PubMed2006

Multiple mapping method: a novel approach to the sequence-to-structure alignment problem in comparative protein structure modeling.

A major bottleneck in comparative protein structure modeling is the quality of input alignment between the target sequence and the template structure. A number of alignment methods are available, but none of these techniques produce consistently good solutions for all cases. Alignments produced by alternative methods may be superior in certain segments but inferior in others when compared to each other; therefore, an accurate solution often requires an optimal combination of them. To address this problem, we have developed a new approach, Multiple Mapping Method (MMM). The algorithm first identifies the alternatively aligned regions from a set of input alignments. These alternatively aligned segments are scored using a composite scoring function, which determines their fitness within the structural environment of the template. The best scoring regions from a set of alternative segments are combined with the core part of the alignments to produce the final MMM alignment. The algorithm was tested on a dataset of 1400 protein pairs using 11 combinations of two to four alignment methods. In all cases MMM showed statistically significant improvement by reducing alignment errors in the range of 3 to 17%. MMM also compared favorably over two alignment meta-servers. The algorithm is computationally efficient; therefore, it is a suitable tool for genome scale modeling studies.

Algorithms↗
PubMed1997

Effect of nucleotide sequence and metal ions at the active site on a short ribozyme activation.

Leadzyme, CUGGGAGUCC, is a ribozyme that requires Pb2+. We have investigated effects of target sequences and metal ions on a catalytic reaction of the leadyzme. The leadzyme plays as a catalyst for one site cleavage of a mutant substrate, GGAC decrease GAGCCAG, and a wild-type substrate, GGACC decrease GAGCCAG. The addition of 25 microM Nd3+ in the presence of 25 microM Pb2+ led to significant increase in the cleavage yield for the mutant substrate. But this increase of the yield is less than that for the wild-type substrate. The result suggests that the effect of Nd3+ on the leadzyme reaction depends on substrate sequence.

Base Sequence↗
PubMed2002

Purification and characterization of yeast Sco1p, a mitochondrial copper protein.

The present studies were undertaken to further characterize the properties of Sco1p, a constituent of the mitochondrial inner membrane implicated in copper transfer to cytochrome oxidase. We report a procedure capable of yielding Sco1p of >95% purity. Sco1p has been purified from strains of Saccharomyces cerevisiae that overexpress the protein. The amino-terminal sequence of purified Sco1p indicates that the first 40 amino acids of the primary translation product constitute a mitochondrial targeting sequence that is proteolytically cleaved during import. We estimate that Sco1p constitutes 0.08% total mitochondrial proteins in wild type yeast and 5% in the transformant used for the purification. Sco1p contains approximately 1 mol of copper/mol protein. The copper is not removed by the treatment of Sco1p with EDTA, indicating that it is bound with high affinity. Purified Sco1p sediments identical to Sco1p in crude extracts of mitochondria from wild type yeast or from a strain transformed with SCO1 on a high copy plasmid. Native Sco1p has an estimated mass of 88 kDa, suggesting that it is a homotrimer. Sco1p expressed as a soluble protein lacking the internal 17 amino acids of the membrane-anchoring domain has been localized in the matrix. The protein has also been targeted to the intermembrane space. Neither soluble matrix nor intermembrane-localized Sco1p is able to complement a sco1 mutant, suggesting that only the membrane form with the carboxyl-terminal domain facing the intermembrane space is able to exert its normal function.

Biological Transport↗
PubMed1993

Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.

Amino Acid Isomerases↗
PubMed2006

A multivariate prediction model for microarray cross-hybridization.

BACKGROUND: Expression microarray analysis is one of the most popular molecular diagnostic techniques in the post-genomic era. However, this technique faces the fundamental problem of potential cross-hybridization. This is a pervasive problem for both oligonucleotide and cDNA microarrays; it is considered particularly problematic for the latter. No comprehensive multivariate predictive modeling has been performed to understand how multiple variables contribute to (cross-) hybridization. RESULTS: We propose a systematic search strategy using multiple multivariate models [multiple linear regressions, regression trees, and artificial neural network analyses (ANNs)] to select an effective set of predictors for hybridization. We validate this approach on a set of DNA microarrays with cytochrome p450 family genes. The performance of our multiple multivariate models is compared with that of a recently proposed third-order polynomial regression method that uses percent identity as the sole predictor. All multivariate models agree that the 'most contiguous base pairs between probe and target sequences,' rather than percent identity, is the best univariate predictor. The predictive power is improved by inclusion of additional nonlinear effects, in particular target GC content, when regression trees or ANNs are used. CONCLUSION: A systematic multivariate approach is provided to assess the importance of multiple sequence features for hybridization and of relationships among these features. This approach can easily be applied to larger datasets. This will allow future developments of generalized hybridization models that will be able to correct for false-positive cross-hybridization signals in expression experiments.

Algorithms↗
PubMedDate not supplied

Multi-colour brightfield in situ hybridisation on tissue sections.

We describe the brightfield microscopical detection of multiple DNA target sequences in cell and tissue preparations. For this purpose, chromosome-specific DNA probes labelled with biotin, digoxigenin or fluorescein were simultaneously hybridised and detected by enzyme cytochemistry using two horseradish peroxidase (PO) reactions and one alkaline phosphatase (APase) reaction. For triple-colour detection on single cell preparations, the combination of the enzyme precipitates PO/diaminobenzidine (DAB, brown colour), APase/fast red (FR, red colour) and PO/tetramethylbenzidine (TMB, green colour) resulted in an accurate detection of DNA targets. Embedding of the preparations in a thin cross-linked protein layer further stabilised the enzyme reaction products. For in situ hybridisation on tissue sections, however, this detection procedure showed some limitations with respect to both the stability of the APase/FR and PO/TMB precipitates, and the sequence of immunochemical layers in multiple-target procedures. For this reason, the APase/FR reaction was replaced by the APase/new fuchsin (NF, red colour) reaction and the washing steps after the PO/TMB reaction were restricted to the use of phosphate buffer pH 6.0. Furthermore, to improve the efficiency of the ISH reaction, APase/NF was applied in an avidin-biotin complex detection system and, to avoid target shielding in the triple-target ISH, the third primary antibody was applied prior to the second enzyme cytochemical reaction. These adaptations resulted in stable, well contrasting brown, red and green coloured precipitates. After quick haematoxylin counterstaining, the tissue preparations were directly mounted in phosphate buffer and, optionally, embedded in the cross-linked protein layer.

Color↗
PubMed1988

Drosophila homoeotic genes encode transcriptional activators similar to mammalian OTF-2.

Homoeotic genes in Drosophila melanogaster are active in spatially restricted metameric domains and control the morphogenesis of segment-specific features such as legs or wings within these domains. They exert their function, according to the 'selector gene' hypothesis, by regulating the expression of subordinate genes. Homoeotic genes also control their own expression and the expression of each other. The proteins encoded by these genes contain a domain, called a homoeodomain, that is strongly conserved, and that shows homologies to proteins that bind DNA and regulate transcription. Homoeoproteins have been shown to bind specific DNA sequences. We show here that the Drosophila homoeotic genes Ultrabithorax (Ubx) and Abdominal-B (Abd-B) code for proteins that are capable of activating transcription of reporter genes linked to specific cis-regulatory target sequences in transfected mammalian cells. Their activity, as well as their target specificity, is similar to that of a mammalian lymphoid-specific octamer transcription factor, OTF-2, which was recently found to contain a homoeodomain.

Animals↗
PubMed2004

Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements.

Penelope-like elements are a class of retroelement that have now been identified in >50 species belonging to at least 10 animal phyla. The Penelope element isolated from Drosophila virilis is the only transpositionally active representative of this class isolated so far. The single ORF of Penelope and its relatives contains regions homologous to a reverse transcriptase of atypical structure and to the GIY-YIG, or Uri, an endonuclease (EN) domain not previously found in retroelements. We have expressed the single ORF of Penelope in a baculovirus expression system and have shown that it encodes a polyprotein with reverse transcriptase activity that requires divalent cations (Mn2+ and Mg2+). We have also expressed and purified the EN domain in Escherichia coli and have demonstrated that it has EN activity in vitro. Mutations in the conserved residues of the EN catalytic module abolish its nicking activity, whereas the DNA-binding properties of the mutant proteins remain unaffected. Only one strand of the target sequence is cleaved, and there is a certain degree of cleavage specificity. We propose that the Penelope EN cleaves the target DNA during transposition, generating a primer for reverse transcription. Our results show that an active Uri EN has been adopted by a retrotransposon.

Animals↗
PubMed2005

The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation.

Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID-RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.

Amino Acid Sequence↗
PubMed1992

Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase.

The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs. Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins. To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA. Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals. A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal. Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity. Both could have resulted from third-base-codon substitutions. A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme. The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme. The complete sequence of chicken retinal GS is also identical to that of the liver enzyme. GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s).

Amino Acid Sequence↗
PubMed1991

Sp1-dependent activation of a synthetic promoter by human immunodeficiency virus type 1 Tat protein.

The Tat protein coded by human immunodeficiency virus (HIV) is a strong activator of viral gene expression from the long terminal repeat (LTR). It appears that Tat-mediated trans-activation of the HIV LTR is predominantly a transcriptional event. It has been reported that Tat acts at the level of both transcriptional initiation and elongation through interaction with a nascent RNA target sequence termed TAR (for trans-activation response element). However, the precise mechanism(s) by which Tat mediates TAR-dependent transcriptional activity is not known. To determine whether Tat functions similarly to other eukaryotic transcriptional activators through any of the conventional promoter elements, we tested Tat activity on synthetic promoters containing consensus sequences required for binding transcription factor Sp1 and a TATA box. Here, we report that a chimeric Tat protein targeted to the promoter region by the DNA-binding domain of yeast transcription factor GAL4 activates the synthetic promoter. Because this trans-activation depends on Sp1-binding sites, Tat can apparently mediate transcriptional activation through its interaction with Sp1. Mutational analysis of the gal4-tat chimeric gene reveals that the N-terminal 48-amino acid region of Tat constitutes the activation region for Sp1-dependent trans-activation. This region of Tat exhibits substantially more activity than the N-terminal 58 amino acids of Tat, which includes the arginine-rich basic region. Effects of specific mutations in the 48-amino acid Tat region of GAL4-Tat on trans-activation of the synthetic promoter mimic the effects of these specific mutations on Tat-mediated trans-activation of the HIV-1 LTR, suggesting that trans-activation of both the synthetic promoter and the intact LTR occurs by a common mechanism.

Base Sequence↗
PubMed1996

Characterization of the hydroxymethylglutaryl-CoA lyase precursor, a protein targeted to peroxisomes and mitochondria.

We previously showed that human liver hydroxymethylglutaryl-CoA (HMG-CoA) lyase (HL; EC 4.1.3.4) is found in both mitochondria and peroxisomes. HL contains a 27-residue N-terminal mitochondrial targeting sequence which in cleaved on mitochondrial entry, as well as a C-terminal Cys-Lys-Leu peroxisomal targeting motif. Because peroxisomal HL has a greater molecular mass and more basic pI value than mitochondrial HL, we predicted that peroxisomal HL retains the mitochondrial leader. To test this hypothesis, we expressed both the precursor (pHL) and mature (mHL) peptides in Escherichia coli and studied their properties. pHL purified by ion-exchange and hydrophobic chromatography had a pI of 7.6 on FPLC chromatofocusing and a molecular mass of 34.5 kDa on SDS/PAGE, similar to our findings for peroxisomal HL. For purified mHL, pI (6.2) and molecular mass (32 kDa) values resemble those of mitochondrial HL. Purified pHL is similar to mHL in K(m) for HMG-CoA (44.8 microM), k(cat) (6.3 min(-1)) and pH optimum (9.0-9.5). However, the quaternary structures of pHL and mHL differ. On Superose 12 FPLC gel filtration and also on ultrafiltration, both in the presence and in the absence of HMG-CoA), pHL behaves as a monomer whereas mHL migrates as a dimer. We conclude that the HL percursor is probably identical to peroxisomal HL, that its catalytic properties resemble those of mature mitochondrial HL, and that the mitochondrial leader peptide prevents dimerization on pHL.

Amino Acid Sequence↗
PubMed1988

Identification of sequence-specific DNA-binding factors by label transfer: application to the adenovirus-2 major late promoter.

A method of affinity labelling proteins specifically associated with DNA target sequences is proposed. The method utilizes covalent UV-crosslinking of proteins to highly labelled DNA (e.g. in crude cell or nuclear extracts) followed by degradation of the DNA to short oligonucleotides. Proteins selectively labelled by attached residual oligonucleotides are readily amenable to molecular mass determination. Using this approach, we have characterized a HeLa polypeptide specifically bound to a short segment of the adenovirus-2 major late promoter (Ad2 MLP). A molecular mass value (approximately 51 kD) and precise location of the crosslinking site(s) of the protein within the MLP (-55 with respect to the cap site) were determined.

Adenoviruses, Human↗
PubMed2025

SMART-RNA-Metavirome: a practical RNA metavirome platform compatible with high-throughput sequencing of both short and long reads.

BACKGROUND: The RNA virosphere's extensive diversity and its role in emerging infectious diseases underscore the importance of non-targeted sequencing for identifying unknown or rare pathogens, including co-infections. However, enriching low-abundance viral sequences in RNA metaviromics, particularly in the preparation of cDNA libraries and their compatibility with next-generation sequencing (NGS) and third-generation sequencing (TGS), remains challenging. Therefore, our objective is to develop and systematically assess a practical RNA metavirome methodology specifically tailored for the enrichment of low-abundance viral sequences within samples. METHODS: We developed the SMART-RNA-Metavirome platform, integrating SMART-9n library preparation with NGS and TGS technologies. Total RNA was extracted from two field-collected wild Aedes albopictus pools, along with one laboratory-infected Ae. albopictus pool harboring dengue virus (DENV). This RNA was subjected to reverse transcription using both this optimized protocol and random primer-based methods, followed by high-throughput sequencing on Illumina, Oxford Nanopore, and QitanTech Nanopore technologies. Welch's t-test was employed for comparative analysis of the subsequent RNA metavirome data, specifically to evaluate differences in viral species composition and abundance of viral reads between experimental groups. Furthermore, the effectiveness of this platform was systematically validated via RT-qPCR and SMART-RNA-Metavirome-based Oxford Nanopore sequencing across multiple sample types, including mosquito specimens from DENV-infected Ae. albopictus, serum samples from dengue patients and viral isolates of Japanese encephalitis virus (JEV) and Zika virus (ZIKV). RESULTS: The SMART-RNA-Metavirome platform has been systematically validated to excel in enriching the composition and diversity of the RNA virome (P = 0.04), providing sufficient coverage for the complete reconstruction of viral genomes. When employed in the detection of DENV-infected Ae. albopictus, clinical serum samples, and viral isolates of JEV and ZIKV, this technique exhibits a robust correlation with RT-qPCR (r2 > 0.95). Notably, it demonstrates exceptional sensitivity, ensuring sufficient coverage even in samples of DENV-infected Ae. albopictus with a Ct-value of 35.3, attaining an impressive 99.88% genome coverage. Furthermore, this platform possesses the capability to identify virus species and determine their serotypes. CONCLUSIONS: In our study, the SMART-RNA-Metavirome platform outperforms traditional methods, enriching RNA virome composition and diversity, enabling practical compatibility with both NGS and TGS technologies. It demonstrates significant proficiency in detecting both known and unknown arboviruses, even in low-titer samples such as those from wild mosquitoes and clinical sera. This platform facilitates comprehensive monitoring, risk assessment, and early warning of RNA virus transmissions, enhancing our understanding of RNA virome diversity and ecological patterns.

High-Throughput Nucleotide Sequencing↗
PubMed1999

Ribozyme-mediated decrease in mumps virus nucleocapsid mRNA level and progeny in infected vero cells.

The effects of endogenously expressed ribozymes directed to the mumps virus nucleocapsid (NP) mRNA were studied during viral infection. To this end, eukaryotic expression vectors encoding ribozymes or controls of passive hybridization effects were constructed and used to transfect mumps permissive Vero cells. Transcripts spanning trans-acting ribozymes of the hammerhead and hairpin types were designed to hydrolyze the first 5'GUC-3' sequence downstream from the initiation site and to hybridize to a 16 base sequence containing the putative cleavage site. Control vectors encoded mutated and catalytically inactive forms of the ribozymes or a 16 base antisense version of the target sequence. When stably expressed in cells, both ribozymes and passive control RNAs reduced viral yields. A ribozyme-mediated effect on viral growth was, however, observed, as both ribozyme types reduced viral titers by approximately 80%, well above the highest inhibition level of approximately 35% found when noncatalytic RNAs were expressed. In addition, levels of NP mRNA were generally lower in cells expressing catalytic RNAs, supporting the observed inhibition of viral growth. Although cleavage in vitro of a synthetic analog of the NP mRNA was demonstrated using RNAs isolated from ribozyme-expressing cells, in vivo cleavage products were not detectable despite the use of sensitive methods, possibly because of degradation phenomena. We also suggest here that additional controls should be conducted when semicompetitive RT-PCR methods are used to evaluate intracellular cleavage by ribozymes, as the results may depend on the initial target RNA concentration.

Animals↗