Biomedical subjects

J L Etheredge

Publications and source records attributed to J L Etheredge.

7 recordsLinked to original sources

PubMed1995

Transactivation by Rtg1p, a basic helix-loop-helix protein that functions in communication between mitochondria and the nucleus in yeast.

Rtg1p is a basic helix-loop-helix transcription factor in the yeast Saccharomyces cerevisiae that is required for basal and regulated expression of CIT2, the gene encoding a peroxisomal isoform of citrate synthase. In respiratory incompetent rho degree petite cells, CIT2 transcription is elevated as much as 30-fold compared with respiratory competent rho + cells. Here we provide evidence that Rtg1p interacts directly with a CIT2 upstream activation site (UASr) and that the rho degree/rho + regulation is not due to a change in the levels of Rtg1p. A fusion protein consisting of the DNA binding domain of Gal4p fused to the NH2 terminus of the full-length wild-type Rtg1p was able to transactivate an integrated LacZ reporter under control of the Gal4p-responsive GAL1 UASG in a rho degree/rho(+)-dependent manner. Other Gal4p fusions to deletions or mutations of Rtg1p indicate that the helix-loop-helix domain is essential for transactivation. Regulated expression of CIT2 also requires the RTG2 gene product. The Gal4-Rtg1p fusion was unable to transactivate the LacZ reporter gene in a strain deleted for RTG2, suggesting that the RTG2 product does not act independently of Rtg1p in the rho degree/rho + transcriptional response.

Alleles↗

PubMed1992

Characterization of a complementary DNA for rat liver aryl sulfotransferase IV and use in evaluating the hepatic gene transcript levels of rats at various stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

A complementary DNA (cDNA) for rat hepatic aryl sulfotransferase IV (AST IV) was isolated, characterized, and used as a hybridization probe to evaluate the molecular basis for the differential expression of AST IV during 2-acetylaminofluorine (2AAF)-induced hepatocarcinogensis. The AST IV cDNA clone was obtained by immunochemical screening of a male Sprague-Dawley rat liver cDNA library. The AST IV cDNA was found to be 1.3 kilobases long and to encode a fusion protein which was reactive with an antibody to AST IV and enzymatically able to generate the sulfuric acid ester of N-hydroxy-2AAF. Sequence analysis of the AST IV cDNA showed it to be 1127 residues in length and to have essentially complete homology with PST-I cDNA, a previously reported (S. Ozawa, et al., Nucleic Acids Res., 18: 4001, 1990), 1028-base cDNA for an uncharacterized rat liver aryl sulfotransferase. Comparison of the PST-I/AST IV cDNA-deduced amino acid sequence with data from a partial (51%) amino acid sequence analysis of purified AST IV showed complete amino acid homology, confirming the identity of the cDNA and establishing that AST IV was an N-blocked, 291-amino acid protein with a molecular mass of 33,909 daltons. The AST IV cDNA sequence differed from the PST-I cDNA in two principal ways: the 5' end lacked 18 coding bases, and the 3' end contained a 190-base extention in the untranslated region, including a consensus sequence for signalling polyadenylation. Studies of AST IV gene transcript levels showed that the livers of rats fed 2AAF for 3 wk (early stage hepatocarcinogenesis) and hyperplastic nodules from the livers of rats fed 2AAF for 19 wk (intermediate stage hepatocarcinogenesis) displayed transcript levels similar to those of livers from normal rats. This contrasted with the 60 to 70% lower than normal capacity of the mRNA fractions to express AST IV observed during in vitro translation. These results indicated that modulation of AST IV expression at early and intermediate stages of hepatocarcinogenesis involved regulatory mechanisms at the translational level. In contrast, mRNA fractions isolated from some 2AAF-induced liver tumors or from known chemical carcinogen-derived rat hepatoma cell lines showed losses of both AST IV transcript level and in vitro translation capacity, suggesting that regulation at the transcriptional level may become important at late stages of 2AAF-induced hepatocarcinogenesis. These results indicated that the molecular mechanisms for the 2AAF-mediated down regulation of AST IV expression during 2AAF-induced hepatocarcinogenesis involved alterations in regulation at both translational and transcriptional levels.

2-Acetylaminofluorene↗

PubMed1991

Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin-inhibited DNA synthesis.

Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.

Amides↗

PubMed1990

Fluorescence of phosphotyrosine--terbium(III) complexes.

Phosphotyrosine, a biologically important protein residue, was investigated for the ability to enhance terbium (Tb3+) fluorescence. Spectroscopic analysis of the Tb3+: phosphotyrosine interaction indicated the development of a new excitation peak at 275 nm and strong Tb+ fluorescence enhancement at 488 and 540 nm that was linear over a range from 0.5 to 100 microM amino acid. Subsequent experiments comparing the ability of phosphotyrosine, phosphothreonine, phosphoserine and 20 other common non-phosphorylated amino acids showed that only phosphotyrosine produced significant Tb3+ fluorescence enhancement. Analysis of various phospho-sugars and nucleotides showed (with the expected exception of GMP) that they produced little or no significant fluorescence enhancement, indicating a further selectiveness for the phosphotyrosine: Tb3+ fluorescence enhancement event. These results establish a basis for the future use of Tb3+ fluorescence enhancement as a unique probe for the investigation of phosphotyrosine residues.

In Vitro Techniques↗

PubMed1987

Evidence for nucleoside channeling in vivo: deoxythymidine incorporation into rat liver dTTP and nuclear matrix DNA.

Previous studies in prokaryotes and in eukaryotic cell lines have indicated the possible existence of more than one dTTP pool accessible to DNA synthesis. To investigate this possibility in eukaryotes in vivo, the incorporation of [3H] deoxythymidine into nuclear matrix-attached DNA and intracellular dTTP was examined in regenerating rat liver. The labeling of matrix DNA reached a maximum after a 5 min pulse and then began to rapidly decrease. Conversely, [3H] deoxythymidine incorporation into dTTP began to increase after 5 min and peaked 10 min after injection. Since the peak specific activity for [3H] deoxythymidine incorporation into matrix DNA precedes that into dTTP, there seems to be channeling of exogenous thymidine directly to sites of DNA replication, bypassing existing nucleotide pools.

Animals↗

PubMed1987

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A.

The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A-stimulated guinea pig lymphocytes incorporate [3H]dThd, [3H]dCyd, [3H]dUrd, [3H]Cyd and [3H]Urd into DNA-thymines and DNA-cytosines in a highly conserved distribution pattern. DNA-thymines were labeled only by dThd and dUrd, while DNA-cytosines were labeled only by dCyd, Cyd and Urd. The kinetics for the incorporation of the [3H]nucleosides were essentially identical, indicating equivalent abilities to measure DNA synthesis. Pyrazofurin inhibition of the pyrimidine de novo synthetic pathway inhibited cell proliferation and the levels of [3H]nucleoside incorporation by approx. 50%, but did not alter restricted distribution of the [3H]nucleosides among DNA-thymines and DNA-cytosines. These findings indicate the absence of Cyd and dCMP deaminase salvage pathways and suggest either subcellular compartmentalization or differential regulation of ribonucleoside diphosphoreductase which permits reduction of CDP but not UDP.

Amides↗

PubMed1985

The influence of DNA sequence on terbium (III) fluorescence enhancement by DNA.

The synthetic DNA duplexes, poly(dA-dC):poly(dG-dT), poly(dG):poly(dC), poly(dG-dC):poly(dG-dC), and poly(dG-m5dC):poly(dG-m5dC), were analyzed as double- and single-strand polymers for the ability to enhance terbium fluorescence. Using conditions which limited the enhancement of Tb3+ fluorescence to that from DNA-guanosines, our results showed that (a) guanosines in single-strand DNA enhanced terbium fluorescence equally well irrespective of the primary sequence surrounding them, and (b) guanosines in either left- (Z-form) or right- (B-form) handed double helixes failed to enhance terbium fluorescence.

DNA↗