Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.

Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

rAAV2/5 gene-targeting to rods:dose-dependent efficiency and complications associated with different promoters.

A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken ß actin proximal promoter CBA were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor-specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, whereas the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 10(11)vg ml(-1) were used.

Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy.

PURPOSE: Diseased corneas are potential targets for viral-based gene therapy to normalize (stimulate or inhibit) the expression of specific proteins. The choice of viral vectors is important to achieve optimal effect. The purpose of this study was to compare the tropism to different corneal cells of recombinant adenovirus (rAV) and recombinant adeno-associated virus (rAAV) constructs using live rabbit and organ-cultured human corneas. METHODS: rAV constructs harbored the green fluorescent protein (GFP) gene under the control of major immediate early cytomegalovirus (CMV) promoter. rAAV constructs from virus serotypes 1, 2 5, 7, and 8 had GFP under the chicken beta-actin promoter and CMV enhancer. For organ culture, 16 healthy and diabetic postmortem human corneas were used. Five or fifteen microl rAV at 10(7) plaque forming units per 1 microl were added for 2 days to culture medium of uninjured corneas that were further cultured for 5-32 days. rAAV were added at 1.2-7.8x10(10) vector genomes per cornea for 3 days to each cornea; the culture then continued for another 14-23 days. Corneal cryostat sections were examined by immunohistochemistry. Live rabbit corneas were used following excimer laser ablation of the corneal epithelium with preservation of the basal cell layer. Equal numbers of rAAV particles (2x10(11) vector genomes) were applied to the cornea for 10 min. After seven days to allow for corneal healing and gene expression the animals were euthanized, the corneas were excised, and sections analyzed by immunohistochemistry. RESULTS: By direct fluorescence microscopy of live organ-cultured human corneas GFP signal after rAV transduction was strong in the epithelium with dose-dependent intensity. On corneal sections, GFP was seen in all epithelial layers and some endothelial cells but most keratocytes were negative. In rAAV-transduced organ-cultured human corneas GFP signal could only be detected with anti-GFP antibody immunohistochemistry. GFP was observed in the epithelium, keratocytes, and endothelium, with more pronounced basal epithelial cell staining with rAAV1 than with other serotypes. No difference in the GFP expression patterns or levels between normal and diabetic corneas was noted. The rabbit corneas showed very similar patterns of GFP distribution to human corneas. With all rAAV serotype vectors, GFP staining in the epithelium was significantly (p=0.007) higher than the background staining in non-transduced corneas, with a trend for rAAV1 and rAAV8 to produce higher staining intensities than for rAAV2, rAAV5 (p=0.03; rAAV5 versus rAAV1), and rAAV7. rAAV serotype vectors also transduced stromal and endothelial cells in rabbit corneas to a different extent. CONCLUSIONS: rAAV appears to reach many more corneal cells than rAV, especially keratocytes, although GFP expression levels were lower compared to rAV. rAV may be more useful than rAAV for gene therapy applications requiring high protein expression levels, but rAAV may be superior for keratocyte targeting.

Extramitochondrial citrate synthase activity in bakers' yeast.

We isolated the gene for citrate synthase (citrate oxaloacetate lyase; EC 4.1.3.7) from Saccharomyces cerevisiae and ablated it by inserting the yeast LEU2 gene within its reading frame. This revealed a second, nonmitochondrial citrate synthase. Like the mitochondrial enzyme, this enzyme was sensitive to glucose repression. It did not react with antibodies against mitochondrial citrate synthase. Haploid cells lacking a gene for mitochondrial citrate synthase grew somewhat slower than wild-type yeast cells, but exhibited no auxotrophic growth requirements.

Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria.

The terminal intron of the mitochondrial cob gene of Saccharomyces cerevisiae can undergo autocatalytic splicing in vitro. Efficient splicing of this intron required a high concentration of monovalent ion (1 M). We found that at a high salt concentration this intron was very active and performed many of the reactions described for other group I introns. The rate of the splicing reaction was dependent on the choice of the monovalent ion; the reaction intermediate, the intron-3' exon molecule, accumulated in NH4Cl but not in KCl. In addition, the intron was more reactive in KCl, accumulating in two different circular forms: one cyclized at the 5' intron boundary and the other at 236 nucleotides from the 5' end. These circular forms were able to undergo the opening and recyclization reactions previously described for the Tetrahymena rRNA intron. Cleavage of the 5' exon-intron boundary by the addition of GTP did not require the 3' terminus of the intron and the downstream exon. An anomalous guanosine addition at the 3' exon and at the middle of the intron was also detected. Hence, this intron, which requires a functional protein to splice in vivo, demonstrated a full spectrum of characteristic reactions in the absence of proteins.

Derepression of citrate synthase in Saccharomyces cerevisiae may occur at the level of transcription.

Pulse-chase labeling in whole cells and cell-free protein synthesis were used to establish that the mitochondrial enzyme citrate synthase is made as a larger precursor in Saccharomyces cerevisiae. A 54,000 Mr precursor form appeared to be a primary translation product since it could be labeled with N-[35S]formylmethionine in vitro. The induction of citrate synthase was monitored in S. cerevisiae cells grown on fermentable (glucose) and nonfermentable (ethanol and glycerol) carbon sources. The amount of citrate synthase activity and immune-reactive protein increased more than 15-fold as S. cerevisiae cells entered the stationary growth phase on glucose-containing medium. This increase was paralleled by an increase in translatable RNA for the enzyme. When cells were grown on a nonfermentable carbon source, no increase in either citrate synthase or its mRNA was detected. The results suggest that the release of citrate synthase from catabolite repression may occur at the level of transcription.

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.

Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.

Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein.

The nuclear CBP2 gene encodes a protein essential for the splicing of a mitochondrial group I intron in Saccharomyces cerevisiae. This intron (bI5) is spliced autocatalytically in the presence of high concentrations of magnesium and monovalent salt but requires the Cbp2 protein for splicing under physiological conditions. Addition of Cbp2 during RNA synthesis permitted cotranscriptional splicing. Splicing did not occur in the transcription buffer in the absence of synthesis. The Cbp2 protein appeared to modify the folding of the intron during RNA synthesis: pause sites for RNA polymerase were altered in the presence of the protein, and some mutant transcripts that did not splice after transcription did so during transcription in the presence of Cbp2. Cotranscriptional splicing also reduced hydrolysis at the 3' splice junction. These results suggest that Cbp2 modulates the sequential folding of the ribozyme during its synthesis. In addition, splicing during transcription led to an increase in RNA synthesis with both T7 RNA polymerase and mitochondrial RNA polymerase, implying a functional coupling between transcription and splicing.

Suppression of mouse rhodopsin expression in vivo by AAV mediated siRNA delivery.

PURPOSE: The purpose of this study is to demonstrate that the expression of rhodopsin can be down regulated in vivo by AAV-delivered siRNA. This is the first step in an RNA replacement strategy for the allele-independent treatment of Autosomal Dominant Retinitis Pigmentosa (ADRP). METHODS: HEK 293 cells were co-transfected with a plasmid carrying mouse RHO cDNA driven by the CMV promoter and a chemically synthesized siRNA duplex of 21 nucleotides. Reduction of RHO mRNA was confirmed by RT-PCR. One active siRNA and a control siRNA were embedded in a small hairpin RNA (shRNA) and cloned in Adeno-associated virus (AAV) vector under regulation of the H1 promoter and containing a GFP reporter. AAV5 expressing either active siRNA or an irrelevant siRNA were subretinaly injected into the right eyes of wild-type or RHO+/- heterozygote mice at post-natal day 16. At 1 and 2 months post-injection, animals were analyzed by electroretinography (ERG). Animals were then sacrificed, and retinas were examined by Western blot, RT-PCR, histology and immunohistochemistry. RESULTS: All of the siRNAs tested in HEK 293 cells caused degradation of RHO mRNA, although the efficiency varied from 25% to 80%. In vivo siRNA delivery to the retina led to more than 40% reduction of scotopic a- and b-wave amplitudes in RHO+/- heterozygotes. Although the reduction of RHO mRNA was estimated at 30% compared to control animals, Western blots revealed 60% decrease in rhodopsin content. Histological analysis showed significant reduction in the thickness of the ONL, ranging between 53% and 86%. CONCLUSIONS: AAV-siRNA delivery into the subretinal space resulted in the reduction of retinal function caused by diminished RHO mRNA and protein content. This level of reduction may permit the replacement of endogenous mRNA with siRNA-resistant mRNA encoding wild-type RHO.

Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea.

Usher syndrome type 3 is caused by mutations in the USH3A gene, which encodes the protein clarin-1. Clarin-1 is a member of the tetraspanin superfamily (TM4SF) of transmembrane proteins, expressed in the organ of Corti and spiral ganglion cells of the mouse ear. We have examined whether the AAV-mediated anti-clarin ribozyme delivery causes apoptotic cell death in vivo in the organ of Corti. We used an AAV-2 vector delivered hammerhead ribozyme, AAV-CBA-Rz, which specifically recognizes and cleaves wild type mouse clarin-1 mRNA. Cochleae of CD-1 mice were injected either with 1mul of the AAV-CBA-Rz, or control AAV vectors containing the green fluorescent protein (GFP) marker gene (AAV-CBA-GFP). Additional controls were performed with saline only. At one-week and one-month post-injection, the animals were sacrificed and the cochleae were studied by histology and fluorescence imaging. Mice injected with AAV-CBA-GFP displayed GFP reporter expression of varying fluorescence intensity throughout the length of the cochlea in the outer and inner hair cells and stria vascularis, and to a lesser extent, in vestibular epithelial cells. GFP expression was not detectable in the spiral ganglion. The pro-apoptotic effect of AAV-CBA-delivered anti-clarin-1 ribozymes was evaluated by TUNEL-staining. We observed in the AAV-CBA-Rz, AAV-CBA-GFP and saline control groups apoptotic nuclei in the outer and inner hair cells and in the stria vascularis one week after the microinjection. The vestibular epithelium was also observed to contain apoptotic cells. No TUNEL-positive spiral ganglion neurons were detected. After one-month post-injection, the AAV-CBA-Rz-injected group had significantly more apoptotic outer and inner hair cells and cells of the stria vascularis than the AAV-CBA-GFP group. In this study, we demonstrate that AAV-CBA mediated clarin-1 ribozyme may induce apoptosis of the cochlear hair cells and cells of the stria vascularis. Surprisingly, we did not observe apoptosis in spiral ganglion cells, which should also be susceptible to clarin-1 mRNA cleavage. This result may be due to the injection technique, the promoter used, or tropism of the AAV serotype 2 viral vector. These results suggest the role of apoptosis in the progression of USH3A hearing loss warrants further evaluation.

Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA.

Adenosine modulates a variety of cellular functions by interacting with specific cell surface G protein-coupled receptors (A1, A2A, A2B, and A3) and is a potential mediator of angiogenesis through the A2B receptor. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design a hammerhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its effect on retinal angiogenesis. Ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. Human embryonic kidney (HEK) 293 cells were transfected with these plasmids and A2B receptor mRNA levels were determined by quantitative real-time RT-PCR. Human retinal endothelial cells (HRECs) were also transfected and cell migration was examined. The effects of these ribozymes on the levels of preretinal neovascularization were determined using a neonatal mouse model of oxygen-induced retinopathy (OIR). We produced a ribozyme with a Vmax of 515+/-125 pmol/min and a Kcat of 36.1+/-8.3 min(-1) (P< or =1x10(-5)). Transfection of HEK293 cells with the plasmid expressing the ribozyme reduced A2B receptor mRNA levels by 45+/-4.8% (P=5.1x10(-5)). Transfection of HRECs reduced NECA-stimulated migration of cells by 47.3+/-1.2% (P=7x10(-4)). Intraocular injection of the constructs into the mouse model reduced preretinal neovascularization by 53.5+/-8.2% (P=4.5x10(-5)). Our results suggest that the A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor and provide further support for the role of A2B receptor in retinal angiogenesis.

Ribozyme gene therapy: applications for molecular medicine.

RNA enzymes--ribozymes--are being developed as treatments for a variety of diseases ranging from inborn metabolic disorders to viral infections and acquired diseases such as cancer. Ribozymes can be used both to downregulate and to repair pathogenic genes. In some instances, short-term exogenous delivery of stabilized RNA is desirable, but many treatments will require viral-mediated delivery to provide long-term expression of the therapeutic catalyst. Current gene therapy applications employ variations on naturally occurring ribozymes, but in vitro selection has provided new RNA and DNA catalysts, and research on trans-splicing and RNase P has suggested ways to harness the endogenous ribozymes of the cell for therapeutic purposes.

Ribozyme uses in retinal gene therapy.

In this chapter we discuss the design, delivery and preclinical testing of mutation-specific ribozymes for the treatment of dominantly inherited retinal disease. We focus particular attention on the initial screening of ribozymes in vitro, because the activity of RNA enzymes in cell-free systems can be used to predict their suitability for animal experiments. Current techniques for delivering genes of interest to cells of the retina using viral vectors are then briefly surveyed emphasizing vector properties that best match to the needs of a ribozyme-based therapy. Using these considerations, analysis of ribozyme gene therapy for an autosomal dominant RP-like disease in a rodent model is outlined emphasizing the desirability of combining biochemical, morphological and electrophysiological measures of therapy. Finally, we describe alternative, perhaps more general, ribozyme approaches that have yet to be tested in the context of retinal disease.

Selective autophagy of peroxisomes in methylotrophic yeasts.

The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha respond to a methanol substrate by synthesizing peroxisomal enzymes resulting in the formation of large peroxisomes. When the carbon source was changed from methanol to glucose, we observed a rapid loss of peroxisomes. In this comparative study, we utilized biochemical and morphological techniques to characterize the loss of peroxisomes in these yeasts. We used metabolic labeling and chase procedures to evaluate whether this loss was due to suppressed synthesis or enhanced degradation. The synthesis of alcohol oxidase was depressed 10-fold when cultures grown in methanol attained stationary growth. However, no further reduction of synthesis was observed upon transfer of these cultures to glucose medium. In stationary phase cultures maintained in methanol, two peroxisomal proteins, alcohol oxidase and dihydroxyacetone synthase, were degraded with a half-life of over 3 h. However, within 3 h of glucose repression, as much as 80% of the radiolabeled peroxisomal proteins were lost from both yeasts. This glucose-mediated degradative event appeared to be specific for peroxisomal proteins, since mitochondrial proteins were stable. Ultrastructural examination of both yeasts revealed that glucose induced the sequestration of peroxisomes into the yeast vacuole, the presumed site of degradation. These results suggest that peroxisome loss during glucose repression is due to a selective, enhanced degradation of whole peroxisomes by autophagic mechanisms.

Ribozyme-targeted destruction of RNA associated with autosomal-dominant retinitis pigmentosa.

PURPOSE: To design ribozymes--catalytic RNA molecules--to cleave the P23H and S334Ter mutant mRNA selectively and to test them in vitro to determine their potential as therapeutic agents in the prevention of autosomal dominant retinitis pigmentosa. METHODS: Synthetic RNA targets were used in cleavage assays to determine the catalytic efficiencies of the ribozymes in vitro. Cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis. Total retinal RNA was also used as a substrate, and opsin mRNA cleavage was assayed by reverse transcription-polymerase chain reaction. RESULTS: All three ribozymes cleaved the mutant target specifically. Substrate cleavage was seen in less than 5 mM magnesium and was detectable after 15 minutes of incubation. The most active ribozyme against the P23H target was the hammerhead (kcat:K(m) [Michaelis-Menton constant] ratio = 5 x 10(7) M/min), then the P23H hairpin ribozyme (kcat:K(m) ratio = 9 x 10(5) M/min) and the S334Ter hammerhead (kcat:K(m) ratio = 8 x 10(5) M/min). No cleavage activity was observed, when wild-type target sequences or inactive control ribozymes were used. The ribozymes bound and specifically digested the intact mutant opsin mRNA in the presence of all normal retinal RNA. CONCLUSIONS: Ribozymes can discriminate between the mutant and wild-type sequences of mRNA associated with autosomal dominant retinitis pigmentosa. The kinetics and specificity of ribozyme cleavage indicate that they should reduce the amount of aberrant rhodopsin in the rod cells and may have potential as therapeutic agents against genetic disease.

Noninvasive imaging by optical coherence tomography to monitor retinal degeneration in the mouse.

PURPOSE: Optical coherence tomography (OCT) is a high-resolution imaging technique that measures the intensity of backscattered light from biological microstructures in living tissue. The objective was to evaluate OCT as a routine, noninvasive technique for quantitative measurements of retinal thickness and detachment in small animal models of retinal degenerative diseases. METHODS: An OCT scanning unit was designed and built to visualize retinal tissue from rodents at high resolution in vivo. Several normal and retinal degeneration (rd) mouse strains with different pigmentation, as well as a transgenic mouse strain that carries a wild-type beta-PDE gene in an rd/rd background, were analyzed at different ages. Retinal detachment was induced by subretinal injection of saline. Retinal function was evaluated by full-field ERG, and then each retina was cross-sectionally scanned by OCT. OCT image analysis and measurements of retinal thickness were performed. Animals were then killed and retinal histology was documented. RESULTS: OCT images of the mouse retina revealed structural landmarks allowing assignment of retinal structures. There was no difference in the OCT pattern between pigmented and nonpigmented mice. Changes in the retinal thickness measured by OCT correlated very well with the loss in function measured by ERG and histology in rd/rd and rd/rd/tg(+) transgenic mice at a variety of ages. In addition, retinal detachment caused by surgery was easily visualized and observed by OCT imaging. CONCLUSIONS: OCT imaging is applicable to the mouse retina. There is excellent agreement between the retinal thickness measured by OCT, ERG amplitude, and retinal histology, thus validating OCT imaging as a sensitive and noninvasive tool for monitoring the structural progression of retinal diseases in rodent models. OCT also appears useful for visualizing retinal detachments in the mouse.

An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa.

PURPOSE: To develop a hammerhead ribozyme-based gene therapy for a porcine model of autosomal dominant retinitis pigmentosa (ADRP). METHODS: Hammerhead ribozymes were developed and assayed in vitro against RNA targets homologous to the opsin P347S mutants found in a transgenic porcine model and in humans. Both cloned and synthetic RNA oligonucleotide versions of ribozymes and targets were tested under multiple-turnover conditions using oligonucleotide RNA targets. Digestion of full-length P347S mRNA from porcine retina was performed. RESULTS: The porcine P347S hammerhead ribozyme was specific for the opsin P347S sequence. Multiple-turnover analysis yielded the following kinetic parameters: Vmax=7.3+/-0.5 nM/min, Km=2.1+/-0.6 mM, and kcat=1.5+/-0.4 min-1. The human P347S hammerhead ribozyme was substantially less active (~10,000 fold). CONCLUSIONS: We have developed a hammerhead ribozyme to use as a model for gene therapy of autosomal dominant retinitis pigmentosa in a transgenic porcine model. Based on kinetic characterization of this ribozyme compared to others used for gene therapy, this should be an effective reagent RNA. The allele specific ribozyme we tested for the human sequence, however, is not likely to be useful for gene therapy indicating that an alternative approach is necessary.

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase.

Spontaneous and ethyl methanesulfate induced mutants of Saccharomyces cerevisiae, with partial and complete deficiency of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), were isolated by selection for resistance to 8-azaadenine. Matings between totally deficient mutants and tester strain resulted in diploid heterozygotes that were sensitive to azaadenine. Upon sporulation and tetrad analysis, azaadenine resistance (and APRT deficiency) segregated as expected for a single Mendelian gene. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity in the mutants was similar to that in the wild-type cells. There was no detectable activity of adenine aminohydrolase (EC 3.5.4.2) in the wild-type or mutant cells.

Inhibition of gene expression by ribozymes.

RNA enzymes, or ribozymes, can be defined as RNA molecules that promote a variety of reactions involving RNA and DNA molecules. These include site-specific cleavage, ligation, polymerization, and phosphoryl exchange (1). The use of ribozymes for medical therapy was recognized soon after RNA catalysis was discovered in the early 1980s (2). Three broad classes, naturally occurring ribozymes have been recognized: (1) RNase P, required for tRNA processing; (2) self-splicing introns, including group I and II introns of bacteria, mitochondria, and chloroplasts; and (3) selfcleaving viral agents, including hepatitis delta virus and components of plant viroids that cleave the RNA genome during replication. Because of their small size and great specificity, the self-cleaving ribozymes have the greatest potential for medical applications. The ability of these ribozymes to cleave other RNA molecules at specific sites makes them useful as inhibitors of viral replication or of cell proliferation (3-8).