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R Jaussi

Publications and source records attributed to R Jaussi.

At least 37 records · Page 2Linked to original sources
PubMed1993

The Gus-e locus regulates estrogen repression of androgen-induced beta-glucuronidase expression in mouse kidney.

Both enzyme activity and mRNA concentration of beta-glucuronidase were measured in kidneys of mice treated with testosterone and the synthetic estrogen, diethylstilbestrol. Six congenic strains, all having a C57BL6/J genetic background but each having a different haplotype of the beta-glucuronidase gene complex, were compared. In each strain the induction caused by androgen was partially repressed by estrogen. The extent of this antagonism varied among the six haplotypes and was not coordinate with the extent of induction by androgen alone. Antagonism appears to be regulated by at least two alleles of a new locus, Gus-e, within the beta-glucuronidase gene complex. Repression by estrogen, like induction by androgen, appears to take place primarily at the transcriptional level. Kinetic studies revealed that estrogen causes the androgen response curve to plateau earlier and at a lower level. This suggests that estrogen increases the rate of gene deactivation rather than decreasing the rate of gene activation. Isoelectric focusing of beta-glucuronidase from Gus-ea and Gus-eb mice and their F1 progeny revealed that the genes are regulated in cis. Together, these findings support a model in which both sex hormones exert their effects on separate DNA response elements located in close proximity to the gene or within the gene itself.

Androgens↗
PubMed1993

Staurosporine- and radiation-induced G2-phase cell cycle blocks are equally released by caffeine.

We show here that the arrests of cells in G2 phase of the cell cycle induced by either staurosporine or ionizing radiation are closely related phenomena governed by a common kinase signaling pathway. The protein kinase inhibitor staurosporine induces a complete G2-phase arrest in exponentially growing TK6 human lymphoblastoid and V79 Chinese hamster fibroblast cells. Both cell types are equally sensitive to the kinase inhibitor and the arrest is dependent on its continued presence. Caffeine completely abrogates this arrest at concentrations comparable to those which abrogate radiation-induced G2-phase arrest. The kinetics of caffeine-induced release of both kinds of arrest are essentially identical. The activity of p34cdc2 kinase was also found to increase in a parallel fashion after caffeine-induced release of both kinds of arrest. As opposed to those transformed cell types which arrest only in G2 phase in response to staurosporine, immortalized C3H 10T1/2 fibroblasts and Muntjak skin fibroblasts display both G1- and G2-phase arrests. The results suggest that staurosporine and radiation interact with regulatory pathways in the cell cycle, and specifically with a caffeine-sensitive signal transduction pathway which recognizes DNA damage, regulates the G2/M-phase transition, and attenuates the biological consequences of radiation exposure.

Alkaloids↗
PubMed1992

Precursor of mitochondrial aspartate aminotransferase synthesized in Escherichia coli is complexed with heat-shock protein DnaK.

On expression of the cDNA encoding the precursor of chicken mitochondrial aspartate aminotransferase (pmAspAT) in Escherichia coli, the bulk of pmAspAT was found to be associated with the 70-kDa heat-shock protein DnaK which is closely related to mitochondrial 70-kDa heat-shock protein (HSP70). Purification protocols for the DnaK/pmAspAT complex and its individual components were elaborated. The complex dissociated on treatment with MgATP or at pH 5.5. Like the mature enzyme, pmAspAT is a dimer (2 x 47 kDa) and exhibits about a third of its enzyme activity. In the DnaK/pmAspAT complex, one DnaK molecule is bound to each subunit of pmAspAT; this tetramer may further aggregate to an octamer. The complex is catalytically almost as active as free pmAspAT. It could be reconstituted from isolated DnaK and pmAspAT. No complex was formed with mAspAT. Apparently, DnaK binds to the solvent-exposed presequence of folded pmAspAT without significantly changing the structure and functional properties of its mature moiety.

Amino Acid Sequence↗
PubMed1992

Lack of adaptive response to low doses of ionizing radiation in human lymphocytes from five different donors.

Various investigators reported a reduced yield of chromosome and chromatid aberrations in short-term cultures of human lymphocytes if a 'challenge' exposure to ionizing radiation was preceded by an 'adaptive' exposure. In order to examine the cell cycle dependence of the 'adaptive response', chromosome and chromatid aberration yields were estimated after challenge doses in the G1, S or G2 phase of lymphocytes which had been adapted in the early G1 phase. On testing two donors no protective adaptive response was found. Blood samples of four donors were tested for their capability to evoke the adaptive response in a standard experiment with the adaptive dose in the S phase and the challenge dose in the G2 phase. A synergistic response occurred in one out of two similar experiments performed with the same blood sample. The three other blood samples tested did not respond. Apparently these data indicate a high frequency of human lymphocyte cultures that do not display an adaptive response.

Adaptation, Physiological↗
PubMed1992

Modulation of androgen-responsive gene expression by estrogen.

Studies on hormonal action frequently focus on a single hormone. In intact animals, however, genes may respond to the balance of multiple hormones. Therefore, we have studied the mutual influence of sex steroids on eight genes previously known to be testosterone-responsive in kidneys of mice. A variety of responses to estrogen were recorded. Effects occurred primarily at the transcriptional level although in several cases there was also evidence of decreased mRNA stability. Estrogen did not affect the nuclear location of the androgen receptor. Apparently each gene interacts with both androgen-receptor complex and estrogen-receptor complex, and the ultimate outcome depends on each gene's detailed regulatory structure.

Animals↗
PubMed1991

The precursor of mitochondrial aspartate aminotransferase is imported into mitochondria faster than the homologous cytosolic isoenzyme with the same presequence attached.

Mitochondrial and cytosolic aspartate aminotransferase (AspAT) are homologous proteins with identically folded polypeptide chains. The cDNAs of the two isoenzymes of chicken were used to express the following proteins in yeast: the precursor of mitochondrial AspAT, mature mitochondrial AspAT, and two chimeric proteins in one of which (pc) the presequence of the precursor was attached to the entire cytosolic isoenzyme and in the other one (pmc) the N-terminal segment (amino acid residues -22 to 23) of the precursor was linked to the slightly truncated cytosolic isoenzyme (residues 34 to 412). All presequence containing proteins were imported into the mitochondria and processed to the mature form whereas mature mitochondrial AspAT remained in the cytosol. The rate of import of the authentic precursor was four times faster than that of the chimeric proteins pc and pmc, t1/2 for importation at 29 degrees C being 3, 13 and 14 min, respectively. Apparently, the mature moiety of the precursor of mitochondrial AspAT promotes importation.

Aspartate Aminotransferases↗
PubMed1990

Structure of the genes of two homologous intracellularly heterotopic isoenzymes. Cytosolic and mitochondrial aspartate aminotransferase of chicken.

The genes of mitochondrial and cytosolic aspartate aminotransferase of chicken were cloned and sequenced. In both genes nine exons encode the mature enzyme. The additional exon for the N-terminal presequence that directs mitochondrial aspartate aminotransferase into the mitochondria is separated by the largest intron from the rest of the gene. A comparison of the two genes of chicken with the aspartate aminotransferase genes of mouse [Tsuzuki, T., Obaru, K., Setoyama, C. & Shimada, K. (1987) J. Mol. Biol. 198, 21-31; Obaru, K., Tsuzuki, T., Setoyama, C. & Shimada, K. (1988) J. Mol. Biol. 200, 13-22] reveals closely similar structures: in the gene of both the mitochondrial and the cytosolic isoenzyme all but one intron positions are conserved in the two species and five introns out of nine are placed at the same positions in all four genes indicating that the introns were in place before the genes of the two isoenzymes diverged. The variant consensus sequence (T/C)11 T(C/T)AG at the 3' splice site of the introns of the genes for nuclear-encoded mitochondrial proteins, which had been deduced from a total of 34 introns [Juretić, N., Jaussi, R., Mattes, U. & Christen, P. (1987) Nucleic Acids Res. 15, 10,083-10,086], was confirmed by including an additional 22 introns into the comparison. The position -4 at the 3' splice site is occupied by base T in 43% of the total 56 introns and appears to be subject to a special evolutionary constraint in this particular group of genes. The following course of evolution of the aspartate aminotransferase genes is proposed. Originating from a common ancestor, the genes of the two isoenzymes intermediarily evolved in separate lineages, i.e. the ancestor eukaryotic and ancestor endosymbiontic cells. When endosymbiosis was established, part of the endosymbiontic genome, including the aspartate aminotransferase gene, was transferred to the nucleus. This process probably led to the conservation of certain splicing factors specific for nuclear-encoded mitochondrial proteins. The presequence for the mitochondrial isoenzyme was acquired by DNA rearrangement. In the eukaryotic lineage, the mitochondrial isoenzyme evolved more slowly than its cytosolic counterpart.

Amino Acid Sequence↗
PubMed1990

Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence.

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.

Animals↗
PubMed1990

Evolutionary and biosynthetic aspects of aspartate aminotransferase isoenzymes and other aminotransferases.

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase are homologous proteins. Both are encoded by nuclear DNA and synthesized on free polysomes. The organization of their genes is very similar, five out of a total of eight introns are located at the same nucleotide position. A variant consensus sequence was observed at the 3' splice site of introns of genes of imported mitochondrial proteins which may reflect the existence of splicing factors specific for the genes of this particular group of nuclear-encoded proteins. To date the amino acid sequences of 22 aminotransferases are known. A rigorous analysis yielded clear evidence that aspartate, tyrosine, and histidinol-phosphate aminotransferases are homologous proteins despite their low degree of sequence identity. The evolutionary relationship among the vitamin B6-dependent enzymes in general appears less clear. Conceivably, their common structural and mechanistic features are dictated by the chemical properties of pyridoxal 5'-phosphate rather than being due to a common ancestor of their protein moieties. In agreement with this notion, the ubiquitous active-site lysine residue that forms a Schiff base with the coenzyme can be replaced in the case of aspartate aminotransferase by a histidine residue without complete loss of catalytic competence.

Animals↗
PubMed1989

Structure of cDNA of cytosolic aspartate aminotransferase of chicken and its expression in E. coli.

The structure of the mRNA of chicken cytosolic aspartate aminotransferase has been determined by analysis of cDNA and genomic clones. Two transcripts of different length were found that appear to arise from the alternate use of 2 polyadenylation signals in the 3' untranslated region. The expression product of the full-length construct in E. coli proved to be catalytically active and possessed the expected molecular weight.

Amino Acid Sequence↗
PubMed1987

Genes of nuclear encoded mitochondrial proteins: evidence for a variant of the 3' splice-site consensus sequence.

The introns of animal nuclear genes and of viral genes encoding protein possess at their 3' splice-site the consensus sequence (CT)11NTCAG (Mount, S.M. (1982) Nucl. Acids Res. 10, 459-472; Green, M.R. (1986) Ann. Rev. Genet. 20, 671-708). However, the total 39 introns of the 5 imported mitochondrial proteins of higher eucaryotes whose gene structure has been determined to date show a predominance of 44% for base T at position -4. Apparently, a variant consensus sequence, i.e. (CT)11TTCAG, characterizes the genes of nuclear encoded mitochondrial proteins.

5-Aminolevulinate Synthetase↗
PubMed1987

Expression of cDNAs encoding the precursor and the mature form of chicken mitochondrial aspartate aminotransferase in Escherichia coli.

Both the precursor and the mature form of chicken mitochondrial aspartate aminotransferase were synthesized in Escherichia coli. The precursor was found to sediment quantitatively together with insoluble cell material. In contrast, mature mitochondrial aspartate aminotransferase could be readily extracted from the cells and was indistinguishable from the enzyme isolated from chicken heart in all respects tested: specific activity 230 units mg-1; Mr 2 X 45,000; pI greater than 9; NH2-terminal sequence SSWWSHVEMG, the initiator methionine having been removed by the bacteria. Thus, the polypeptide chain representing mature mitochondrial aspartate aminotransferase is an autonomous folding unit which attains its functional spatial structure independently of the presence of the prepiece, trans-membrane passage, and proteolytic processing.

Amino Acid Sequence↗
PubMed1985

The primary structure of the precursor of chicken mitochondrial aspartate aminotransferase. Cloning and sequence analysis of cDNA.

The mitochondrial isoenzyme of aspartate amino-transferase (mAspAT; subunit Mr 45,000) is synthesized on free polysomes in the cytosol as a precursor of higher Mr (pre-mAspAT; Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345). We have isolated three overlapping cDNA clones that correspond almost to the full length of pre-mAspAT mRNA with 100 nucleotides at the 5' end missing. The mRNA is 2.1 kilobase pairs long and has a 3' noncoding region of 0.7 kilobase pairs. The cDNAs code for the 401 amino acid residues of mAspAT plus an NH2-terminal pre-piece. Deviations from the reported amino acid sequence were found at positions 154 and 202 where the cDNA specifies Gln instead of Glu. The pre-piece consists of 22 amino acid residues, among them 4 arginine and no acidic residues.

Amino Acid Sequence↗
PubMed1983

Vinblastine inhibits the maturation of the precursor of mitochondrial aspartate aminotransferase. Vincristine and six other cytoskeleton inhibitors do not show this effect.

Cytoskeleton inhibitors were tested in chicken embryo fibroblast cultures for possible effects on the import of the precursor of mitochondrial aspartate aminotransferase into mitochondria. Vinblastine (50 microM) increased the steady-state pool of the precursor 2.5-fold in pulse experiments with [35S]methionine. If the precursor was accumulated during a pulse in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and then chased under diluting CCCP, vinblastine (50 microM) prolonged the half-life of the precursor from 0.5 min in the control to 3 min. Other cytoskeleton inhibitors, i.e. vincristine (25 to 150 microM), colchicine (50 microM), nocodazole (50 microM), podophyllotoxin (50 microM), taxol (45 microM), cytochalasin D (20 microM) and phalloidin (25 microM) did not show this effect. The observed inhibition by vinblastine does not seem to relate to its action on microtubuli.

Alkaloids↗
PubMed1982

Biosynthesis and topogenesis of aspartate aminotransferase isoenzymes in chicken embryo fibroblasts. The precursor of the mitochondrial isoenzyme is either imported into mitochondria or degraded in the cytosol.

In chicken embryo fibroblasts pulsed wih [35S]methionine, a precursor of mitochondrial aspartate aminotransferase with higher molecular weight (delta Mr approximately 3000) was detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping of the precursor and the mature enzyme confirmed their precursor-product relationship. No precursor of the homologous cytosolic isoenzyme was found. The precursor of the mitochondrial isoenzyme is synthesized on membrane-free polysomes in the cytosol (Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345); its half-life is 30 to 60 s. The pronounced susceptibility of the precursor toward exogenous proteases contrasts the stability of the mature enzyme and thus indicates that the conformation or the quarternary structure of the protein must change concomitantly with its import into mitochondria. Administration of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to the cell cultures blocks the import of many matrix and inner membrane proteins into mitochondria. The precursor of mitochondrial aspartate aminotransferase is found to be accumulated in the cytosol. However, its steady state concentration in CCCP-treated cells exceeds the concentration in untreated cells by not more than 1 order of magnitude. During a chase, the radioactive precursor disappears with a half-life of approximately 5 with min without formation of mature enzyme. Thus, in CCCP-treated cells, a degradative process is limiting the accumulation of the precursor in the cytosol. When the chase is performed in the presence of cysteamine, an antagonist of CCCP, the precursor is processed to the mature enzyme. Newly synthesized cytosolic aspartate aminotransferase is not degraded.

Animals↗