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T R Cech

Publications and source records attributed to T R Cech.

230 records · Page 13Linked to original sources
PubMed1979

Localization of transcribed regions on extrachromosomal ribosomal RNA genes of Tetrahymena thermophila by R-loop mapping.

R-loop hybridization and electron microscopy were used to map the RNA transcription products of the extrachromosomal rRNA genes of Tetrahymena thermophila. The mature 17S and 26S rRNAs and the nuclear 35S pre-rRNA and pre-26S rRNA were located with a precision of approximately 100 base pairs. A 370-base pair intervening sequence was found in the 26S coding region. It has the same size and relative location as that found in Tetrahymena pigmentosa [Wild, M.A. & Gall, J.G. (1979) Cell 16, 565-573]. One class of R-loop structures formed by nuclear pre-rRNA provided preliminary evidence for a primary transcript that contains the intervening sequence. The results suggested a processing scheme in which splicing of the intervening sequences is followed by a series of strand cleavages to give the mature 17S and 26S rRNAs. Analysis of the data also showed that RNA . DNA and DNA . DNA duplexes, when mounted for electron microscopy by the R-loop procedure, have the same length per base pair within 4%.

Animals↗
PubMed1976

Partial denaturation of mouse DNA in preparative CsCl density gradients at alkaline pH.

A new technique--partial denaturation of DNA in equilibrium CsCl density gradients at pH 11.4--is used to determine the distribution of intermediate states in the melting of mouse DNA. When the technique is applied in the preparative ultracentrifuge, the DNA is fractionated according to stability. Neutralization of the partially denatured fractions results in the recovery of most of the DNA in its native form. The individual fractions are more homogeneous than the total DNA: they have decreased density heterogeneity (smaller band widths), neutral CsCl buoyant densities that differ from the average, and more homogeneous melting profiles with melting temperatures that differ from the average.

Animals↗
PubMed1976

Electron microscopy of DNA crosslinked with trimethylpsoralen: test of the secondary structure of eukaryotic inverted repeat sequences.

It has been suggested that inverted repeat (palindrome) sequences, which are widespread in eukaryotic genomes, exist in two alternate configurations, a linear form and a cruciform. To investigate the relative frequency of these forms, the DNA of intact mouse tissue culture cells was covalently crosslinked with 4,5',8-trimethylpsoralen (me3-psoralen) in order to prevent rearrangement of the DNA secondary structure during DNA isolation. The distribution of me3-psoralen crosslinks was determined by electron microscopy after denaturation of the DNA in the presence of glyoxal. Because of the high frequency and the relatively uniform distribution of the me3-psoralen crosslinks, it could be concluded that almost all of the inverted repeat sequences had been crosslinked. In spite of this, no significant number of cruciforms was detected by electron microscopy. To determine whether the me3-psoralen might itself be disrupting cruciform structures, cruciforms were first produced in isolated Tetrahymena rDNA by heat treatment and then crosslinked in vitro. The crosslinking was found to stabilize rather than disrupt these cruciforms. We conclude that the inverted repeat sequences of the mouse tissue culture cells we tested are predominantly in linear forms rather than in cruciform structures inside the cell.

Base Sequence↗
PubMed1975

An electron microscopic study of mouse foldback DNA.

Foldback DNA is defined by its rapid, concentration-independent renaturation, consistent with intramolecular base pairing of inverted repeat sequences. Foldback DNA, isolated from renatured mouse main band DNA by hydroxyapatite chromatography, is spread for electron microscopy by the formamide isodenaturing technique. A large fraction of the molecules can be recognized as intramolecular "hairpins"--structures in which complementary sequences on a single DNA strand form base-paired "stem" regions analogous to tRNA stems. The stem regions of the hairpins have a wide distribution of lengths, averaging about 1000 base pairs. About 60% of the stem regions terminate in single-stranded loops, ranging from 400 to many thousands of nucleotides in length, while 40% of the hairpins do not have discernible loops. There are about 40,000 hairpin-forming sequences in the main band portion of the mouse haploid genome. They appear to be either clustered in groups or confined to about one third of the DNA, rather than uniformly or randomly distributed. Another large fraction of the molecules seen in foldback DNA consists of linear structures, some of which are probably also hairpins. The electron microscopic results, along with simple theoretical considerations, make possible a better interpretation of our previous studies of the yield and S1 nuclease resistance of mouse foldback DNA.

Animals↗
PubMedDate not supplied

Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction.

The intervening sequence (IVS) of the Tetrahymena ribosomal RNA precursor is excised as a linear RNA molecule which subsequently cyclizes itself in a protein-independent reaction. Cyclization involves cleavage of the linear IVS RNA 15 nucleotides from its 5' end and formation of a phosphodiester bond between the new 5' phosphate and the original 3'-hydroxyl terminus of the IVS. This recombination mechanism is analogous to that by which splicing of the precursor RNA is achieved. The circular molecules appear to have no direct function in RNA splicing, and we propose the cyclization serves to prevent unwanted RNA from driving the splicing reactions backwards.

Animals↗
PubMedDate not supplied

Specific interaction between the self-splicing RNA of Tetrahymena and its guanosine substrate: implications for biological catalysis by RNA.

Splicing of the ribosomal RNA precursor of Tetrahymena has previously been shown to 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.

Animals↗
PubMedDate not supplied

Mechanism of recognition of the 5' splice site in self-splicing group I introns.

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.

Animals↗
PubMedDate not supplied

The Tetrahymena ribozyme acts like an RNA restriction endonuclease.

A shortened form of the Tetrahymena self-splicing ribosomal RNA intervening sequence acts as an endoribonuclease, catalysing the cleavage of large RNA molecules by a mechanism involving guanosine transfer. The sequence specificity approaches that of the DNA restriction endonucleases. Site-specific mutagenesis of the enzyme active site alters the substrate sequence specificity in a predictable manner, so that endoribonucleases can be synthesized to cut at a variety of tetranucleotide sequences.

Animals↗