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K B Chapman

Publications and source records attributed to K B Chapman.

13 recordsLinked to original sources
PubMed1998

Telomerase reverse transcriptase genes identified in Tetrahymena thermophila and Oxytricha trifallax.

Telomerase reverse transcriptase (TERT) has been identified as the catalytic subunit of the chromosome end-replicating enzyme in Euplotes, yeasts, and mammals. However, it was not reported among the protein components of purified Tetrahymena telomerase, the first telomerase identified and the most thoroughly studied. It therefore seemed possible that Tetrahymena used an alternative telomerase that lacked a TERT protein. We now report the cloning and sequencing of a Tetrahymena thermophila gene whose encoded protein has the properties expected for a TERT, including large size (133 kDa), basicity (calculated pI = 10.0), and reverse transcriptase sequence motifs with telomerase-specific features. The expression of mRNA from the Tetrahymena TERT gene increases dramatically at 2-5 h after conjugation, preceding de novo addition of telomeres to macronuclear DNA molecules. We also report the cloning and sequencing of the ortholog from Oxytricha trifallax. The Oxytricha macronuclear TERT gene has no introns, whereas that of Tetrahymena has 18 introns. Sequence comparisons reveal a new amino acid sequence motif (CP), conserved among the ciliated protozoan TERTs, and allow refinement of previously identified motifs. A phylogenetic tree of the known TERTs follows the phylogeny of the organisms in which they are found, consistent with an ancient origin rather than recent transposition. The conservation of TERTs among eukaryotes supports the model that telomerase has a conserved core (TERT plus the RNA subunit), with other subunits of the holoenzyme being more variable among species.

Amino Acid Sequence↗
PubMed1997

Telomerase catalytic subunit homologs from fission yeast and human.

Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

Amino Acid Sequence↗
PubMed1995

Isolation of a ribozyme with 5'-5' ligase activity.

BACKGROUND: Many new ribozymes, including sequence-specific nucleases, ligases and kinases, have been isolated by in vitro selection from large pools of random-sequence RNAs. We are attempting to use in vitro selection to isolate new ribozymes that have, or can be evolved to have, RNA polymerase-like activities. As phosphorimidazolide-activated nucleosides are extensively used to study non-enzymatic RNA replication, we wished to select for a ribozyme that would accelerate the template-directed ligation of 5'-phosphorimidazolide-activated oligonucleotides. RESULTS: Ribozymes selected to perform the desired template-directed ligation reaction instead ligated themselves to the activated substrate oligonucleotide via their 5'-triphosphate, generating a 5'-5' P1,P4-tetraphosphate linkage. Deletion analysis of one of the selected sequences revealed that a 54-nucleotide RNA retained activity; this small ribozyme folds into a pseudoknot secondary structure with an internal binding site for the substrate oligonucleotide. The ribozyme can also synthesize 5'-5' triphosphate and 5'-5' pyrophosphate linkages. CONCLUSIONS: The emergence of ribozymes that accelerate an unexpected 5'-5' ligation reaction from a selection designed to yield template-dependent 3'-5' ligases suggests that it may be much easier for RNA to catalyze the synthesis of 5'-5' linkages than 3'-5' linkages. 5'-5' linkages are found in a variety of contexts in present-day biology. The ribozyme-catalyzed synthesis of such linkages raises the possibility that these 5'-5' linkages originated in the biochemistry of the RNA world.

Base Sequence↗
PubMed1995

Multiple molecular determinants for retrotransposition in a primer tRNA.

Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.

Base Sequence↗
PubMed1994

Yeast lariat debranching enzyme. Substrate and sequence specificity.

Yeast RNA lariat debranching enzyme has been purified to near homogeneity using a bacterial overproducer of the enzyme. The enzyme is capable of digesting a variety of branched nucleic acid substrates, including group II intron lariats, multicopy single-stranded DNAs (msDNAs), and a variety of synthetic branched RNAs. A trinucleotide release assay using radiolabeled msDNA substrates was developed and used to determine the basic biochemical parameters for the enzyme. The debranching enzyme shows a strong preference for purines at the 2'-position in both msDNA and synthetic branched RNA substrates, in accord with the structure of its native substrate, which always has a 2'-G residue. The use of small synthetic branched RNA substrates will allow systematic mechanistic and structural studies of this unique enzyme.

Base Sequence↗
PubMed1994

In vitro selection of catalytic RNAs.

In vitro selection techniques are poised to allow a rapid expansion of the study of catalysis by RNA enzymes (ribozymes). This truly molecular version of genetics has already been applied to the study of the structures of known ribozymes and to the tailoring of their catalytic activity to meet specific requirements of substrate specificity or reaction conditions. During the past year, in vitro selection has been successfully used to isolate novel RNA catalysts from random sequence pools.

Catalysis↗
PubMed1994

Mapping of DBR1 and YPK1 suggests a major revision of the genetic map of the left arm of Saccharomyces cerevisiae Chromosome XI.

The Saccharomyces cerevisiae dbr1 mutation has been mapped on the left arm of chromosome XI. XIL is a chromosome arm that was until now rather sparsely populated with accurately mapped markers. On the basis of physical data, the overall order of markers is inverted relative to the existing genetic map of XI. We present tetrad analyses using a variety of markers on XI that indicate that the existing genetic map of XIL should be inverted, at least for the strains in which our mapping was carried out, and probably for other S. cerevisiae strains.

Chromosome Mapping↗
PubMed1992

SDH1, the gene encoding the succinate dehydrogenase flavoprotein subunit from Saccharomyces cerevisiae.

We describe the isolation, sequence and construction of a disruption of the yeast SDH1 gene, encoding the flavoprotein subunit of succinate dehydrogenase. This is the first eukaryotic flavoprotein subunit-encoding gene to be fully sequenced. The deduced amino acid (aa) sequence is 50% identical to the Escherichia coli enzyme sequence. The yeast gene encodes an N-terminal extension of 45 aa relative to the E. coli sequence which may act as a mitochondrial targeting signal. Disruption of the gene results in the inability to respire, assayed as the inability to utilize the nonfermentable carbon source, glycerol. This is the expected phenotype for disruption of an essential component of the yeast citric acid cycle.

Amino Acid Sequence↗
PubMed1992

Initiator methionine tRNA is essential for Ty1 transposition in yeast.

The yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism similar to that of retroviral reverse transcription and integration. Ty1 RNA contains a putative minus strand primer binding site (-PBS) that is complementary to the 3' acceptor stem of the initiator methionine tRNA (tRNA(iMet)). Here we demonstrate that the tRNA(iMet) is used as a primer for Ty1 reverse transcription. Mutations in the Ty1 element that alter 5 of 10 nucleotides that are complementary to the tRNA(iMet) abolish Ty1 transposition, even though they are silent with regard to Ty1 protein coding. We have constructed a yeast strain lacking wild-type tRNA(iMet) that is dependent on a mutant derivative of tRNA(iMet) that has an altered acceptor stem sequence, engineered to restore homology with the Ty1 -PBS mutant. The compensatory mutations made in the tRNA(iMet) alleviate the transposition defect of the Ty1 -PBS mutant. The mutant and wild-type tRNA(iMet) are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1 -PBS. Thus, complementarity between the Ty1 -PBS and tRNA(iMet) is essential for transposition but is not necessary for packaging of the tRNA inside virus-like particles.

Base Sequence↗
PubMed1991

Isolation and characterization of the gene encoding yeast debranching enzyme.

Using a genetic screen aimed at identifying cellular factors involved in Ty1 transposition, we have identified a mutation in a host gene that reduces Ty1 transposition frequency. The mutant, dbr1, is also defective in the process of intron turnover. In dbr1 cells, excised introns derived from a variety of pre-mRNAs are remarkably stable and accumulate to levels exceeding that of the corresponding mRNA. The stable excised introns accumulate in the form of a lariat that is missing the linear sequences 3' of the branchpoint. The DBR1 gene has been isolated by complementation of the transposition phenotype. DBR1 is shown to encode debranching enzyme, an RNA processing activity that hydrolyzes the 2'-5' phosphodiester linkage at the branchpoint of excised intron lariats. In Saccharomyces cerevisiae, debranching enzyme plays a requisite role in the rapid turnover of excised introns, yet its function is not essential for viability.

Amino Acid Sequence↗
PubMed1991

Retrotransposition mechanisms.

Recent developments in the area of the transposition mechanisms used by retrotransposons and related retroviral pathways are discussed. In particular, advances in the areas of retrotransposon gene expression, virus-like particle assembly, reverse transcription, and integration are reviewed.

Animals↗
PubMed1988

Determination of male-specific gene expression in Drosophila accessory glands.

The developmental regulation of three male-specific somatic transcripts is investigated. These RNAs are synthesized exclusively in the adult male accessory gland, an internal tissue derived from the genital disk of Drosophila melanogaster. The expression of these male-specific transcripts (msts) is under the control of the sex determination regulatory hierarchy, as demonstrated by the expression of all three msts in chromosomal females carrying mutant alleles at the doublesex (dsx), intersex (ix), or transformer-2 (tra2) loci. Although transcription of all three male RNAs is initiated late in pupation, temperature shifts of X/X; tra2ts2 homozygotes during development indicate that this expression is irreversibly determined earlier, during the third larval instar. A shift of X/X; tra2ts2 homozygotes to the male-determining temperature only for the duration of the late larval period is sufficient to elicit the expression of the msts during the adult stage. This critical period for the determination of these transcripts appears to correlate with the time of morphological determination of the accessory gland in these animals. Thus, the expression of these genes could be specified by the morphological determination of the male-specific tissue in which they are active.

Alleles↗