http://cimb.colorado.edu These are the search results for the query, showing results 1 to 15. http://cimb.colorado.edu/directory/cech/tom-cechs-publications/SullivanCech1985 The Tetrahymena rRNA intervening sequence (IVS) excises itself from the pre-rRNAand then mediates its own cyclization. We now find that certain di- and trinucleotides with free 3' hydroxyl groups reopen the circular IVS at the cyclization junction, producing a linear molecule with the oligonucleotide covalently attached to its 5' end. This linear molecule recyclizes with release of the added oligonucleotide. Thus the IVS RNA, like an enzyme, lowers the activation energy for both forward and reverse cleavage-ligation reactions. Certain combinations of pyrimidines are required for circle reopening. The most reactive oligonucleotide is UCU. This sequence resembles those preceding the major and minor cyclization sites in the linear IVS RNA (UUU and CCU) and the 5'splice site in the pre-rRNA (UCU). We propose that an oligopyrimidine binding site within the IVS binds the sequences upstream of each of these target sites for cleavage-ligation. No publisher admin 2009-07-06T21:06:37Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/PriceCech1985 Splicing of the Tetrahymena ribosomal RNA precursor is mediated by the folded structure of the RNA molecule and therefore occurs in the absence of any proteinin vitro. The Tetrahymena intervening sequence (IVS) has been inserted into the gene for the alpha-donor fragment of beta-galactosidase in a recombinant plasmid. Production of functional beta-galactosidase is dependent on RNA splicing in vivoin Escherichia coli. Thus RNA self-splicing can occur at a rate sufficient to support gene expression in a prokaryote, despite the likely presence of ribosomes on the nascent RNA. The beta-galactosidase messenger RNA splicing system provides a useful method for screening for splicing-defective mutations, several of whichhave been characterized. No publisher admin 2009-07-06T21:06:37Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/PriceEtAl1985 The sequence requirements for splicing of the Tetrahymena pre-rRNA have been examined by altering the rRNA gene to produce versions that contain insertions and deletions within the intervening sequence (IVS). The altered genes were transcribed and the RNA tested for self-splicing in vitro. A number of insertions (8-54 nucleotides) at three locations had no effect on self-splicing activity. Two of these insertions, located at a site 5 nucleotides preceding the 3'-end ofthe IVS, did not alter the choice of the 3' splice site. Thus the 3' splice siteis not chosen by its distance from a fixed point within the IVS. Analysis of deletions constructed at two sites revealed two structures, a hairpin loop and astem-loop, that are entirely dispensable for IVS excision in vitro. Three other regions were found to be necessary. The regions that are important for self-splicing are not restricted to the conserved sequence elements that define this class of intervening sequences. The requirement for structures within the IVS for pre-rRNA splicing is in sharp contrast to the very limited role of IVS structure in nuclear pre-mRNA splicing. No publisher admin 2009-07-06T21:06:37Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/InoueCech1985 The structure of the intervening sequence (IVS) of the Tetrahymena rRNA precursor mediates cleavage-ligation reactions that result in pre-rRNA splicing and IVS cyclization. We have developed a method for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynucleotide primers was found tostop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. In general, the sites of modification were consistent with a previous model of the secondary structure ofthe linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations, and phylogenetic comparison. Thus, IVS RNA autocyclization does not involve major rearrangements of the secondary structure, although thereis evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules. No publisher admin 2009-07-06T21:06:37Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/Cech1985 No publisher admin 2009-07-06T21:06:37Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/GottschlingCech1984 Oxytricha macronuclear DNA exists as approximately 24 X 10(6) gene-sized molecules terminating with a C4A4 repeat. DNA-protein interactions at the ends of bulk macronuclear molecules were probed with micrococcal nuclease and methidiumpropyl-EDTA X Fe(II) (MPE X Fe[II]). The ends were indirectly labeled by hybridizing with (C4A4)2. Alternatively, a novel method using MPE X FE(II) as a probe and directly labeling the 3' ends with terminal transferase was implemented. A terminal complex involving approximately 100 bp with nucleosomes phased inward from the complex was found to be characteristic of most or all of the ends. Analysis of two specific genes confirmed the pattern and showed that the special structure was on both ends of each molecule. We conclude that a DNA-protein complex involving 100 bp and terminating with the C4A4 repeat can besufficient to provide the fundamental functions of telomeres, allowing linear DNA replication and conferring stability of linear DNA. No publisher admin 2009-07-06T21:06:37Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/ZaugEtAl1984 The excised intervening sequence of the Tetrahymena ribosomal RNA precursor mediates its own covalent cyclization in the absence of any protein. The circular molecule undergoes slow reopening at a single phosphodiester bond, the one that was formed during cyclization. The resulting linear molecule has 5'-phosphate and 3'-hydroxyl termini; these are unusual products for RNA hydrolysis but are typical of the other reactions mediated by this molecule. The reopened circle retains cleavage-ligation activity, as evidenced by its ability to undergo another round of cyclization and reopening. The finding that an RNA molecule canbe folded so that a specific phosphate can be strained or activated helps to explain how the activation energy is lowered for RNA self-splicing. The proposedmechanisms may be relevant to several other RNA cleavage reactions that are RNA-mediated. No publisher admin 2009-07-06T21:06:37Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/BassCech1984 Splicing of the ribosomal RNA precursor of Tetrahymena has previously been shownto require no protein in vitro; the cleavage-ligation activity is intrinsic to the RNA molecule. Analysis of the reaction kinetics with guanosine, which is a substrate in the reaction, and with several guanosine analogues suggests that guanosine binds to a specific site on the pre-rRNA. It appears that the RNA, like an enzyme, binds its substrate to promote the rate and specificity of a biological reaction. No publisher admin 2009-07-06T21:06:37Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/PalenCech1984 The chromatin structure of regulatory regions of the extrachromosomal rRNA genesof Tetrahymena thermophila was probed by nuclease treatment of isolated nuclei. The chromatin near the origins of replication contains hypersensitive sites for micrococcal nuclease, DNAase I, and DNAase II. These sites persist in starved cells, consistent with the origins' being maintained in an altered chromatin structure independent of DNA replication. The region between the two origins of replication is organized into a phased array of seven nucleosomes, the fourth ofwhich is centered at the axis of symmetry of the palindromic rDNA. The entire transcribed region and 150 bp upstream from the initiation site are generally accessible to nucleases; any histone proteins associated with these regions are clearly not in a highly organized nucleosomal array as seen in the central region. Comparison of the chromatin structures of the central spacer of T. thermophila and T. pyriformis rDNA reveals that deletion or insertion of DNA hasoccurred in increments of 200 bp. This is taken to imply that there are constraints on the evolution of spacer DNA sequences at the level of the nucleosome. No publisher admin 2009-07-06T21:06:38Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/PardueEtAl1984 The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila virilis was investigated by Me3 psoralen photoreaction of mitochondria isolated from embryos. After photoreaction the mtDNA was purified and the pattern of DNA cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The transcribed regions of the mtDNA molecule contained some uncross-linked regions, but such regions were infrequent and randomly distributed. In contrast, the A + T-rich region around the origin of replicationof the mtDNA was usually protected from psoralen cross-linking. The data were best fit by two protected sites, each approximately 400 base pairs, compared to the four 400 base pair sites observed in the equivalent region of D. melanogaster mtDNA [Potter et al. (1980) Proc. Nat. Acad. Sci. USA 77, 4118-4122]. Thus this region of the mtDNA appears to be involved in a DNA-protein structure that is highly conserved even though the DNA sequence has diverged rapidly relative to protein-coding sequences. No publisher admin 2009-07-06T21:06:38Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/Cech1983 No publisher admin 2009-07-06T21:06:38Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/CechEtAl1983 Splicing of the ribosomal RNA precursor of Tetrahymena is an autocatalytic reaction, requiring no enzyme or other protein in vitro. The structure of the intervening sequence (IVS) appears to direct the cleavage/ligation reactions involved in pre-rRNA splicing and IVS cyclization. We have probed this structureby treating the linear excised IVS RNA under nondenaturing conditions with various single- and double-strand-specific nucleases and then mapping the cleavage sites by using sequencing gel electrophoresis. A computer program was then used to predict the lowest-free-energy secondary structure consistent with the nuclease cleavage data. The resulting structure is appealing in that the ends of the IVS are in proximity; thus, the IVS can help align the adjacent coding regions (exons) for ligation, and IVS cyclization can occur. The Tetrahymena IVShas several sequences in common with those of fungal mitochondrial mRNA and rRNAIVSs, sequences that by genetic analysis are known to be important cis-acting elements for splicing of the mitochondrial RNAs. In the predicted structure of the Tetrahymena IVS, these sequences interact in a pairwise manner similar to that postulated for the mitochondrial IVSs. These findings suggest a common origin of some nuclear and mitochondrial introns and common elements in the mechanism of their splicing. No publisher admin 2009-07-06T21:06:38Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/BrehmCech1983 In previous studies of RNA splicing in vitro, we have shown that the interveningsequence (IVS) of the Tetrahymena rRNA precursor is excised as a unique linear RNA molecule and subsequently cyclized. In the present work, we have investigated the occurrence and stability of these RNA species in vivo. RNA was separated by gel electrophoresis, transferred to diazotized paper, and hybridized with 32P-labeled DNA probes. RNA molecules containing the IVS were found to reside within the nucleus and not in the cytoplasm. The species found in nucleus include both the linear and circular forms of the excised IVS RNA, as well as the unspliced precursor. On the basis of quantitation of the hybridization, the half-lives of the IVS-containing pre-rRNA and the excised IVS RNA in rapidly growing cells were estimated as 2 and 6 s, respectively. We conclude that splicing is not a rate-limiting step in rRNA maturation and that the IVS RNA is quickly degraded after its excision. When the deproteinized nuclear RNA was incubated at 37 degrees C in a Mg2+-containing solution, a substantial portion of the linear IVS RNA was converted to the circular form. Autocyclization, previously characterized with IVS RNA produced by splicing in vitro, is therefore also a property of IVS RNA produced in vivo. No publisher admin 2009-07-06T21:06:38Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/GottschlingEtAl1983 The chromatin structure of the palindromic macronuclear ribosomal RNA genes of Tetrahymena thermophila was probed with micrococcal nuclease. Independent of thestate of transcriptional activity, the transcribed region had a shorter nucleosome repeat (184 +/- 3 base pairs) than the non-transcribed central spaceror bulk chromatin (both 200 base pairs). The transcribed region displayed an increased sensitivity to micrococcal nuclease in rapidly growing cells, which suggested an altered chromatin structure during transcription. At early stages of nuclease digestion, the central spacer appeared to be in a highly structured nucleosomal array. Based on the differences in nucleosome repeat distance and sensitivity to nuclease, we conclude that quite different chromatin structures are maintained in two adjacent regions of the Tetrahymena ribosomal RNA gene. The DNA of the non-transcribed terminal spacer was found to contain sequences which are highly susceptible to micrococcal nuclease, precluding any conclusions aboutnucleosome structure in this region. No publisher admin 2009-07-06T21:06:38Z Article Reference http://cimb.colorado.edu/directory/cech/tom-cechs-publications/PalenCech1983 DNA renaturation kinetics was used to examine the relative accessibility of various regions of the Tetrahymena ribosomal RNA gene (rDNA) chromatin to micrococcal nuclease. In nuclei from cells active in rRNA transcription, the transcribed region of the rDNA chromatin was as much as 5-fold more accessible than the average of the total chromatin. As few as 20% inactive genes in the population could have accounted for all of the hybridization, so the transcribedregion of the active units may be totally unprotected from nuclease degradation.The terminal non-transcribed spacer downstream from the transcription unit was also preferentially digested, but to a smaller degree. The central non-transcribed spacer was degraded to the same extent as total chromatin after a high degree of nuclease digestion. In nuclei from starved cells, which have 96% reduced rRNA transcription, the transcribed and terminal spacer regions of the rDNA were again more accessible than the total chromatin from the same nuclei, but the difference did not exceed 2-fold. We conclude that transcriptional activation is accompanied by major changes in the structure of the ribosomal gene chromatin, and that the extent and/or type of structural alteration differs in each functionally defined region of the rDNA. No publisher admin 2009-07-06T21:06:38Z Article Reference