Gail Dinter-Gottlieb | Acadia University (original) (raw)
Papers by Gail Dinter-Gottlieb
Antisense research and development, 1992
RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage,... more RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage, and thus have the potential for serving as ribozymes in a trans-cleaving reaction. Because the catalytic core of such an enzymatic RNA was not evident from phylogenetic data, we took a step-wise approach to identifying the core, reducing the RNA in size, and characterizing various properties for each size class. Thus, a 186-nucleotide antigenomic RNA (termed Ag180) was found to be capable of cleaving well in 20 M formamide (Smith and Dinter-Gottlieb, 1991), and this unusual stability in formamide was lost by reducing the 3' end of the molecule, leaving a 140-nucleotide RNA (Ag 140). Both RNAs showed only intramolecular cleavage at a wide range of concentrations, and a number of conformers could be seen in the Ag140 RNA, some of which were resistant to cleavage at 37 degrees C. Since Ag140 could not cleave in 20 M formamide, the 5' and 3' termini of Ag180 were truncated and produced Ag5-84, which cleaved to 100% at 37 degrees C in less than 0.25 min. Internal deletions of the Stem IV region resulted in Ag5-73, still capable of efficient cleavage, although with a lessened stability in formamide. A trans-cleaving enzyme-substrate pair was finally derived from this RNA, and it consisted of a 67-nucleotide enzyme that cleaved a 13-nucleotide RNA substrate.
Tosynchronize SV40replicons, simiancellsinfected witha tsAmutantwere restricted at400,tocomplete ... more Tosynchronize SV40replicons, simiancellsinfected witha tsAmutantwere restricted at400,tocomplete ongoing replication andreturnedto320,to activatenewreplicons inthepresence oftheDNAchainelongation inhibitor aphidicolin.Uponfurther incubation at400withoutthedrug,3H-dTwasincorporated intoSV40FIDNA,almosttotheextentseenwithcellsrecovered intheabsence ofthedrug.Todetermine whetherDNAsynthesis wouldbeginfromtheorigin, following thetemperature-shifts-aphidicolin regimen, chainssubsequently pulselabeled with(a-3P)dGTP inisolated nucleiwereanalyzed forsizedistribution andgenomiclocation.Thesechainsreachedupto 300-400nucleotides insize, unlikethecontrolwhichfeatured comparable amountsoflabelinlongchainsand Okazakipieces.ThenascentDNAofthedrug-treated systemcouldbechasedinto longerchains,indicating thatitwasa replicative intermediate; andithybridizedpreferentially to anoriginproximal fragment ofAtuj- restricted SV40DNA, demonstrating partialreplicon synchronization. Thedataprove thatT-antigen ...
Nucleic Acids Research, 1996
The Hepatitis Delta Virus (HDV) ribozyme self-cleaving activity in 20 M formamide solutions is un... more The Hepatitis Delta Virus (HDV) ribozyme self-cleaving activity in 20 M formamide solutions is unique. Does this catalytic activity result from the conservation of its tertiary structure in 20 M formamide? We followed the ribozyme structure in formamide solutions by monitoring the amount of bound Ethidium Bromide (EB). We were able to measure the quantity of dye bound using time-resolved fluorescence spectroscopy, as an estimate of the ribozyme double helical content. This method, calibrated by using oligonucleotides with defined tertiary structure and denaturing solvents, parallels NMR and UV measurements as a function of temperature. Measurements with the HDV ribozyme lead to three conclusions: (a) both the precursor and product RNAs are structured to 24 M (95% w/w) formamide or 4 M H 2 O solutions which is equivalent to 4 M H 2 O; (b) the HDV ribozyme is the only RNA sequence investigated in this study that retains so much structure in formamide; and (c) DNA analogs of formamide resistant HDV ribozyme sequences lose their structure at less than 15 M formamide. Thus, the structural integrity of the HDV ribozyme is an intrinsic property of the RNA molecule and its sequence. MATERIALS AND METHODS Nucleic acid synthesis
The optimistic predictions of a number of microbiologists notwithstanding, the past decade has no... more The optimistic predictions of a number of microbiologists notwithstanding, the past decade has not signaled the end of infectious disease, but rather an introduction to a host of new and complex microorganisms and their resulting depredations on humanity. The identification of new pathogens, such as the causative agent of Lyme disease and the Human Immuno-deficiency Virus (HIV), as well as the Hepatitis Delta Virus (HDV) has not only revealed new forms of clinical pathology, but new and unexpected variations on the life cycle and the molecular biology of the pathogens. In this volume a number of the leaders in the field of Hepatitis Delta virus research, ranging from clinicians and virologists to molecular biologists and biochemists describe what in their experience typifies some of these unique features.
Biochemistry, 1996
The arrangement and stacking of noncovalently contiguous double-helical sections are increasingly... more The arrangement and stacking of noncovalently contiguous double-helical sections are increasingly invoked in single-stranded DNA and RNA tertiary structure. These tertiary structures of nucleic acids are defined by their double stranded regions, and their orientation in the molecular frame constitutes an important component of the nucleic acid structure. A direct view of these tertiary structures can be obtained by fluorescence polarization anisotropy of bound ethidium bromide (EB). The orientation of the dye in the molecular frame of the nucleic acid yields the orientation of the helix. The complete anisotropy function for EB intercalated in genome-derived DNA duplexes was derived by Allison and Schurr (1979) and accounts for base-pair twisting and DNA bending. Single-stranded ribozymes, ribosomal and transfer RNAs, and model DNA junctions contain double-stranded regions shorter than 35 bp in length, for which bending is not significant. We developed and experimentally verified an expression of the anisotropy function for short DNA duplexes which is theoretically compatible with the existing theory, originally developed for long nucleic acids (Schurr et al., 1992). Simulations showed that for DNA duplexes shorter than 35 bp, our expression of the anisotropy function is equivalent to Schurr's and is consistent with experiments carried out on eight DNA duplexes. Modeling the eight duplexes as cylinders, we calculate a duplex diameter of 1.91 (0.15 nm when EB makes a 90°angle with the DNA helix axis and undergoes anisotropic wobbling and 1.97 (0.15 nm when EB makes a 70.5°angle and undergoes isotropic wobbling, respectively. We used this treatment to establish the conformation of five DNA oligonucleotides made of single and tethered hairpins, some designed to exhibit coaxial stacking. Analysis of the fluorescence anisotropy decays shows that the tethered hairpins take an extended rather than parallel conformation. It also shows that the DNA oligonucleotides made of two tethered hairpins exhibit freedom compatible with two independent hairpins. When the linker between hairpins is shortened, the two hairpins are not independent anymore as probed by fluorescence anisotropy, suggesting coaxial stacking of the two helices.
Proceedings of the National Academy of Sciences, 1986
Group I introns are found in nuclear rRNA genes, mitochondrial mRNA and rRNA genes, and chloropla... more Group I introns are found in nuclear rRNA genes, mitochondrial mRNA and rRNA genes, and chloroplast tRNA genes. The hallmarks of this intron class are a 16nucleotide consensus sequence and three sets ofcomplementary sequences. The viroids (circular pathogenic plant RNAs) and the virusoids (plant satellite RNAs) also contain the consensus sequence and the three sets of complementary bases. Pairing of the complementary bases would generate a viroid structure resembling a group I intron, which might be stabilized in vivo through interactions with proteins. The Tetrahymena selfsplicing rRNA intron further has sequenceshomologous with regions of potato spindle tuber viroid associated with the
Molecular Plant-Microbe Interactions, 1991
Journal of Biological Chemistry
The replicative DNA polymerase alpha is an intracellular target of aphidicolin. In vitro this dru... more The replicative DNA polymerase alpha is an intracellular target of aphidicolin. In vitro this drug inhibits DNA polymerase alpha reversibly. Yet, its in vivo effect on SV40 DNA replication, which depends on DNA polymerase alpha, was found to be irreversible. Thus, exposure of infected cells to aphidicolin led to a progressive loss in their ability to incorporate [3H]dT into viral DNA in a subsequent pulse without drug. This loss was time-dependent (t1/2 at 37 degrees C at 2 microgram/ml of drug was approximately 20 min) and increased with drug concentration. Likewise, replicating SV40 DNA, pulse-labeled prior to exposure, lost the ability to mature into form I DNA upon removal of the drug. No degradation of replicating SV40 DNA molecules was detected by neutral sucrose gradient analysis during or up to 1 h after aphidicolin exposure. However, longer incubations resulted in breakdown of the arrested replicative intermediate, concomitant with the resumption of viral DNA synthesis. Ori...
Progress in clinical and biological research
Journal of Virology
Recently we reported that in vitro RNA transcripts complementary to the genome of hepatitis delta... more Recently we reported that in vitro RNA transcripts complementary to the genome of hepatitis delta virus (HDV) contain a unique site at which self-cleavage can occur. Subsequent studies showed that a similar self-cleavage site was present on in vitro RNA transcripts of genomic HDV RNA. The same self-cleavage reactions were also found to occur on HDV RNAs from the livers of infected chimpanzees. Using the in vitro RNA it was also possible to determine that the minimum length of contiguous sequence needed for self-cleavage of genomic RNA was 30 bases 5' and 74 bases 3' of the cleavage site. This sequence was not compatible with the "hammerhead" structure hypothesized to be important in the self-cleavage reactions of other RNAs.
Progress in clinical and biological research
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1993
A chemical modification-interference assay was used to evaluate the sequence requirements for sel... more A chemical modification-interference assay was used to evaluate the sequence requirements for self-cleavage of a 73-nucleotide self-cleaving RNA from the genomic hepatitis delta virus (HDV). Twenty-two nucleotides were categorized as individually essential for self-cleavage, shown by loss of activity when modified. All of these required nucleotides fell within 38 nucleotides downstream of the cleavage site, suggesting an essential structural or functional role for this region. Lesser effects were seen for nucleotides further 3' of the cleavage site, and a small number of nucleotides had a negligible effect on the extent of self-cleavage when modified. Several modifications increased the extent of self-cleavage, suggesting these nucleotides may act to inhibit the reaction when unmodified. The functional requirements for certain nucleotides are discussed in the light of structural probing data and conventional mutational analysis available for other HDV RNAs.
Journal of virology, 1988
The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus... more The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus (HDV) are unique relative to those of known animal viruses, and yet there are real similarities between HDV and certain infectious RNAs of plants. Therefore, since some of the latter RNAs have been shown to undergo in vitro site-specific cleavage and even ligation, we tested the hypothesis that similar events might also occur for HDV RNA. In partial confirmation of this hypothesis, we found that in vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location. This cleavage was found to produce junction fragments consistent with a 5'-hydroxyl and a cyclic 2',3'-monophosphate. Since the observed cleavage was both site-specific and occurred only once per genome length, we propose that the site may be relevant to the normal intracellular replication of the H...
Antisense research and development, 1992
RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage,... more RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage, and thus have the potential for serving as ribozymes in a trans-cleaving reaction. Because the catalytic core of such an enzymatic RNA was not evident from phylogenetic data, we took a step-wise approach to identifying the core, reducing the RNA in size, and characterizing various properties for each size class. Thus, a 186-nucleotide antigenomic RNA (termed Ag180) was found to be capable of cleaving well in 20 M formamide (Smith and Dinter-Gottlieb, 1991), and this unusual stability in formamide was lost by reducing the 3' end of the molecule, leaving a 140-nucleotide RNA (Ag 140). Both RNAs showed only intramolecular cleavage at a wide range of concentrations, and a number of conformers could be seen in the Ag140 RNA, some of which were resistant to cleavage at 37 degrees C. Since Ag140 could not cleave in 20 M formamide, the 5' and 3' termini of Ag180 were truncated and p...
Nucleic Acids Research, 1997
Phosphorothioate (NTPαS) analogues were incorporated into the HDV genomic ribozyme by transcripti... more Phosphorothioate (NTPαS) analogues were incorporated into the HDV genomic ribozyme by transcription with T7 polymerase. The introduction of a sulfur in place of the pro-Rp oxygen at the phosphate 5′ to positions A 64 , A 63 , A 43 , U 27 , G 62 , C 61 , C 44 , C 41 , C 22 and C 21 appeared to inhibit self-cleavage activity of the G73 genomic ribozyme. Except for position C 22 , elevated levels of Mg 2+ rescued the reaction to various extents. When the sites were identified in the RNA sequence, they were clustered in three distinct regions that, in the secondary structure models, are predicted to be primarily single-stranded. Two of these regions have been proposed to form extensive interactions that are thought to involve a homopurine base pair. The third region is thought to be directly associated with assembly of the cleavage site.
Nucleic Acids Research, 1991
The ribozymes derived from Hepatitis delta virus (HDV) RNA appear unique in their sequence requir... more The ribozymes derived from Hepatitis delta virus (HDV) RNA appear unique in their sequence requirements for self-cleavage. While truncating the 1679 nucleotlde antigenomic HDV RNA, we have characterized the cleavage requirements of a number of ribozymes of intermediate length. Two of these, containing 186 and 106 HDV nucleotides respectively, cleaved to completion in the presence of 18 M formamide. The 186 nucleotlde ribozyme also cleaved to completion in 10 M urea. Removal of an additional 10 nts from the 3' terminus of the 106 nt ribozyme resulted in a loss of the ability to cleave In high concentrations of the denaturants. The interaction of nucleotides near the cleavage site with a sequence within this 10 base region may confer unusual stability on these ribozymes.
Nucleic Acids Research, 1991
Analysis of the self-cleavage of ribozymes derived from the genomic RNA of Hepatitis delta virus ... more Analysis of the self-cleavage of ribozymes derived from the genomic RNA of Hepatitis delta virus (HDV) has revealed that certain co-transcribed vector sequences significantly affect the activity of the ribozyme.
Nucleic Acids Research, 1982
To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 400, to... more To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 400, to complete ongoing replication and returned to 320, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 400 without the drug, 3H-dT was incorporated into SV40 FI DNA, almost to the extent seen with cells recovered in the absence of the drug. To determine whether DNA synthesis would begin from the origin, following the temperature-shifts-aphidicolin regimen, chains subsequently pulselabeled with (a-3 P)dGTP in isolated nuclei were analyzed for size distribution and genomic location. These chains reached up to 300-400 nucleotides in size, unlike the control which featured comparable amounts of label in long chains and Okazaki pieces. The nascent DNA of the drug-treated system could be chased into longer chains, indicating that it was a replicative intermediate; and it hybridized preferentially to an origin proximal fragment of Atuj-restricted SV40 DNA, demonstrating partial replicon synchronization.
Antisense research and development, 1992
RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage,... more RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage, and thus have the potential for serving as ribozymes in a trans-cleaving reaction. Because the catalytic core of such an enzymatic RNA was not evident from phylogenetic data, we took a step-wise approach to identifying the core, reducing the RNA in size, and characterizing various properties for each size class. Thus, a 186-nucleotide antigenomic RNA (termed Ag180) was found to be capable of cleaving well in 20 M formamide (Smith and Dinter-Gottlieb, 1991), and this unusual stability in formamide was lost by reducing the 3' end of the molecule, leaving a 140-nucleotide RNA (Ag 140). Both RNAs showed only intramolecular cleavage at a wide range of concentrations, and a number of conformers could be seen in the Ag140 RNA, some of which were resistant to cleavage at 37 degrees C. Since Ag140 could not cleave in 20 M formamide, the 5' and 3' termini of Ag180 were truncated and produced Ag5-84, which cleaved to 100% at 37 degrees C in less than 0.25 min. Internal deletions of the Stem IV region resulted in Ag5-73, still capable of efficient cleavage, although with a lessened stability in formamide. A trans-cleaving enzyme-substrate pair was finally derived from this RNA, and it consisted of a 67-nucleotide enzyme that cleaved a 13-nucleotide RNA substrate.
Tosynchronize SV40replicons, simiancellsinfected witha tsAmutantwere restricted at400,tocomplete ... more Tosynchronize SV40replicons, simiancellsinfected witha tsAmutantwere restricted at400,tocomplete ongoing replication andreturnedto320,to activatenewreplicons inthepresence oftheDNAchainelongation inhibitor aphidicolin.Uponfurther incubation at400withoutthedrug,3H-dTwasincorporated intoSV40FIDNA,almosttotheextentseenwithcellsrecovered intheabsence ofthedrug.Todetermine whetherDNAsynthesis wouldbeginfromtheorigin, following thetemperature-shifts-aphidicolin regimen, chainssubsequently pulselabeled with(a-3P)dGTP inisolated nucleiwereanalyzed forsizedistribution andgenomiclocation.Thesechainsreachedupto 300-400nucleotides insize, unlikethecontrolwhichfeatured comparable amountsoflabelinlongchainsand Okazakipieces.ThenascentDNAofthedrug-treated systemcouldbechasedinto longerchains,indicating thatitwasa replicative intermediate; andithybridizedpreferentially to anoriginproximal fragment ofAtuj- restricted SV40DNA, demonstrating partialreplicon synchronization. Thedataprove thatT-antigen ...
Nucleic Acids Research, 1996
The Hepatitis Delta Virus (HDV) ribozyme self-cleaving activity in 20 M formamide solutions is un... more The Hepatitis Delta Virus (HDV) ribozyme self-cleaving activity in 20 M formamide solutions is unique. Does this catalytic activity result from the conservation of its tertiary structure in 20 M formamide? We followed the ribozyme structure in formamide solutions by monitoring the amount of bound Ethidium Bromide (EB). We were able to measure the quantity of dye bound using time-resolved fluorescence spectroscopy, as an estimate of the ribozyme double helical content. This method, calibrated by using oligonucleotides with defined tertiary structure and denaturing solvents, parallels NMR and UV measurements as a function of temperature. Measurements with the HDV ribozyme lead to three conclusions: (a) both the precursor and product RNAs are structured to 24 M (95% w/w) formamide or 4 M H 2 O solutions which is equivalent to 4 M H 2 O; (b) the HDV ribozyme is the only RNA sequence investigated in this study that retains so much structure in formamide; and (c) DNA analogs of formamide resistant HDV ribozyme sequences lose their structure at less than 15 M formamide. Thus, the structural integrity of the HDV ribozyme is an intrinsic property of the RNA molecule and its sequence. MATERIALS AND METHODS Nucleic acid synthesis
The optimistic predictions of a number of microbiologists notwithstanding, the past decade has no... more The optimistic predictions of a number of microbiologists notwithstanding, the past decade has not signaled the end of infectious disease, but rather an introduction to a host of new and complex microorganisms and their resulting depredations on humanity. The identification of new pathogens, such as the causative agent of Lyme disease and the Human Immuno-deficiency Virus (HIV), as well as the Hepatitis Delta Virus (HDV) has not only revealed new forms of clinical pathology, but new and unexpected variations on the life cycle and the molecular biology of the pathogens. In this volume a number of the leaders in the field of Hepatitis Delta virus research, ranging from clinicians and virologists to molecular biologists and biochemists describe what in their experience typifies some of these unique features.
Biochemistry, 1996
The arrangement and stacking of noncovalently contiguous double-helical sections are increasingly... more The arrangement and stacking of noncovalently contiguous double-helical sections are increasingly invoked in single-stranded DNA and RNA tertiary structure. These tertiary structures of nucleic acids are defined by their double stranded regions, and their orientation in the molecular frame constitutes an important component of the nucleic acid structure. A direct view of these tertiary structures can be obtained by fluorescence polarization anisotropy of bound ethidium bromide (EB). The orientation of the dye in the molecular frame of the nucleic acid yields the orientation of the helix. The complete anisotropy function for EB intercalated in genome-derived DNA duplexes was derived by Allison and Schurr (1979) and accounts for base-pair twisting and DNA bending. Single-stranded ribozymes, ribosomal and transfer RNAs, and model DNA junctions contain double-stranded regions shorter than 35 bp in length, for which bending is not significant. We developed and experimentally verified an expression of the anisotropy function for short DNA duplexes which is theoretically compatible with the existing theory, originally developed for long nucleic acids (Schurr et al., 1992). Simulations showed that for DNA duplexes shorter than 35 bp, our expression of the anisotropy function is equivalent to Schurr's and is consistent with experiments carried out on eight DNA duplexes. Modeling the eight duplexes as cylinders, we calculate a duplex diameter of 1.91 (0.15 nm when EB makes a 90°angle with the DNA helix axis and undergoes anisotropic wobbling and 1.97 (0.15 nm when EB makes a 70.5°angle and undergoes isotropic wobbling, respectively. We used this treatment to establish the conformation of five DNA oligonucleotides made of single and tethered hairpins, some designed to exhibit coaxial stacking. Analysis of the fluorescence anisotropy decays shows that the tethered hairpins take an extended rather than parallel conformation. It also shows that the DNA oligonucleotides made of two tethered hairpins exhibit freedom compatible with two independent hairpins. When the linker between hairpins is shortened, the two hairpins are not independent anymore as probed by fluorescence anisotropy, suggesting coaxial stacking of the two helices.
Proceedings of the National Academy of Sciences, 1986
Group I introns are found in nuclear rRNA genes, mitochondrial mRNA and rRNA genes, and chloropla... more Group I introns are found in nuclear rRNA genes, mitochondrial mRNA and rRNA genes, and chloroplast tRNA genes. The hallmarks of this intron class are a 16nucleotide consensus sequence and three sets ofcomplementary sequences. The viroids (circular pathogenic plant RNAs) and the virusoids (plant satellite RNAs) also contain the consensus sequence and the three sets of complementary bases. Pairing of the complementary bases would generate a viroid structure resembling a group I intron, which might be stabilized in vivo through interactions with proteins. The Tetrahymena selfsplicing rRNA intron further has sequenceshomologous with regions of potato spindle tuber viroid associated with the
Molecular Plant-Microbe Interactions, 1991
Journal of Biological Chemistry
The replicative DNA polymerase alpha is an intracellular target of aphidicolin. In vitro this dru... more The replicative DNA polymerase alpha is an intracellular target of aphidicolin. In vitro this drug inhibits DNA polymerase alpha reversibly. Yet, its in vivo effect on SV40 DNA replication, which depends on DNA polymerase alpha, was found to be irreversible. Thus, exposure of infected cells to aphidicolin led to a progressive loss in their ability to incorporate [3H]dT into viral DNA in a subsequent pulse without drug. This loss was time-dependent (t1/2 at 37 degrees C at 2 microgram/ml of drug was approximately 20 min) and increased with drug concentration. Likewise, replicating SV40 DNA, pulse-labeled prior to exposure, lost the ability to mature into form I DNA upon removal of the drug. No degradation of replicating SV40 DNA molecules was detected by neutral sucrose gradient analysis during or up to 1 h after aphidicolin exposure. However, longer incubations resulted in breakdown of the arrested replicative intermediate, concomitant with the resumption of viral DNA synthesis. Ori...
Progress in clinical and biological research
Journal of Virology
Recently we reported that in vitro RNA transcripts complementary to the genome of hepatitis delta... more Recently we reported that in vitro RNA transcripts complementary to the genome of hepatitis delta virus (HDV) contain a unique site at which self-cleavage can occur. Subsequent studies showed that a similar self-cleavage site was present on in vitro RNA transcripts of genomic HDV RNA. The same self-cleavage reactions were also found to occur on HDV RNAs from the livers of infected chimpanzees. Using the in vitro RNA it was also possible to determine that the minimum length of contiguous sequence needed for self-cleavage of genomic RNA was 30 bases 5' and 74 bases 3' of the cleavage site. This sequence was not compatible with the "hammerhead" structure hypothesized to be important in the self-cleavage reactions of other RNAs.
Progress in clinical and biological research
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1993
A chemical modification-interference assay was used to evaluate the sequence requirements for sel... more A chemical modification-interference assay was used to evaluate the sequence requirements for self-cleavage of a 73-nucleotide self-cleaving RNA from the genomic hepatitis delta virus (HDV). Twenty-two nucleotides were categorized as individually essential for self-cleavage, shown by loss of activity when modified. All of these required nucleotides fell within 38 nucleotides downstream of the cleavage site, suggesting an essential structural or functional role for this region. Lesser effects were seen for nucleotides further 3' of the cleavage site, and a small number of nucleotides had a negligible effect on the extent of self-cleavage when modified. Several modifications increased the extent of self-cleavage, suggesting these nucleotides may act to inhibit the reaction when unmodified. The functional requirements for certain nucleotides are discussed in the light of structural probing data and conventional mutational analysis available for other HDV RNAs.
Journal of virology, 1988
The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus... more The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus (HDV) are unique relative to those of known animal viruses, and yet there are real similarities between HDV and certain infectious RNAs of plants. Therefore, since some of the latter RNAs have been shown to undergo in vitro site-specific cleavage and even ligation, we tested the hypothesis that similar events might also occur for HDV RNA. In partial confirmation of this hypothesis, we found that in vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location. This cleavage was found to produce junction fragments consistent with a 5'-hydroxyl and a cyclic 2',3'-monophosphate. Since the observed cleavage was both site-specific and occurred only once per genome length, we propose that the site may be relevant to the normal intracellular replication of the H...
Antisense research and development, 1992
RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage,... more RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage, and thus have the potential for serving as ribozymes in a trans-cleaving reaction. Because the catalytic core of such an enzymatic RNA was not evident from phylogenetic data, we took a step-wise approach to identifying the core, reducing the RNA in size, and characterizing various properties for each size class. Thus, a 186-nucleotide antigenomic RNA (termed Ag180) was found to be capable of cleaving well in 20 M formamide (Smith and Dinter-Gottlieb, 1991), and this unusual stability in formamide was lost by reducing the 3' end of the molecule, leaving a 140-nucleotide RNA (Ag 140). Both RNAs showed only intramolecular cleavage at a wide range of concentrations, and a number of conformers could be seen in the Ag140 RNA, some of which were resistant to cleavage at 37 degrees C. Since Ag140 could not cleave in 20 M formamide, the 5' and 3' termini of Ag180 were truncated and p...
Nucleic Acids Research, 1997
Phosphorothioate (NTPαS) analogues were incorporated into the HDV genomic ribozyme by transcripti... more Phosphorothioate (NTPαS) analogues were incorporated into the HDV genomic ribozyme by transcription with T7 polymerase. The introduction of a sulfur in place of the pro-Rp oxygen at the phosphate 5′ to positions A 64 , A 63 , A 43 , U 27 , G 62 , C 61 , C 44 , C 41 , C 22 and C 21 appeared to inhibit self-cleavage activity of the G73 genomic ribozyme. Except for position C 22 , elevated levels of Mg 2+ rescued the reaction to various extents. When the sites were identified in the RNA sequence, they were clustered in three distinct regions that, in the secondary structure models, are predicted to be primarily single-stranded. Two of these regions have been proposed to form extensive interactions that are thought to involve a homopurine base pair. The third region is thought to be directly associated with assembly of the cleavage site.
Nucleic Acids Research, 1991
The ribozymes derived from Hepatitis delta virus (HDV) RNA appear unique in their sequence requir... more The ribozymes derived from Hepatitis delta virus (HDV) RNA appear unique in their sequence requirements for self-cleavage. While truncating the 1679 nucleotlde antigenomic HDV RNA, we have characterized the cleavage requirements of a number of ribozymes of intermediate length. Two of these, containing 186 and 106 HDV nucleotides respectively, cleaved to completion in the presence of 18 M formamide. The 186 nucleotlde ribozyme also cleaved to completion in 10 M urea. Removal of an additional 10 nts from the 3' terminus of the 106 nt ribozyme resulted in a loss of the ability to cleave In high concentrations of the denaturants. The interaction of nucleotides near the cleavage site with a sequence within this 10 base region may confer unusual stability on these ribozymes.
Nucleic Acids Research, 1991
Analysis of the self-cleavage of ribozymes derived from the genomic RNA of Hepatitis delta virus ... more Analysis of the self-cleavage of ribozymes derived from the genomic RNA of Hepatitis delta virus (HDV) has revealed that certain co-transcribed vector sequences significantly affect the activity of the ribozyme.
Nucleic Acids Research, 1982
To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 400, to... more To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 400, to complete ongoing replication and returned to 320, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 400 without the drug, 3H-dT was incorporated into SV40 FI DNA, almost to the extent seen with cells recovered in the absence of the drug. To determine whether DNA synthesis would begin from the origin, following the temperature-shifts-aphidicolin regimen, chains subsequently pulselabeled with (a-3 P)dGTP in isolated nuclei were analyzed for size distribution and genomic location. These chains reached up to 300-400 nucleotides in size, unlike the control which featured comparable amounts of label in long chains and Okazaki pieces. The nascent DNA of the drug-treated system could be chased into longer chains, indicating that it was a replicative intermediate; and it hybridized preferentially to an origin proximal fragment of Atuj-restricted SV40 DNA, demonstrating partial replicon synchronization.