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I E Scheffler

Publications and source records attributed to I E Scheffler.

At least 19 recordsLinked to original sources
PubMed1992

The C-terminus of the succinate dehydrogenase IP peptide of Saccharomyces cerevisiae is significant for assembly of complex II.

Site-directed mutagenesis was used to introduce mutations into the gene for the iron protein (IP) of succinate dehydrogenase (SDH) of Saccharomyces cerevisiae. Specifically, three mutations were examined which caused the synthesis of truncated IP peptides missing four, seven, or 17 amino acids from the C-terminus, respectively. The deletion of seven or more amino acids includes the loss of two lysine residues, which appear to have been highly conserved in evolution. While the deletion of four amino acids had no effect on the assembly of complex II and on its activity, the deletion including the two lysines abolished SDS activity completely and led to the failure of the imported IP peptide to be incorporated into a stable complex II or SDH complex. Replacement of one of the lysines by threonine had no effect, but replacement of both by threonine affected the specific activity of complex II but not its assembly and stability.

Amino Acid Sequence↗
PubMed1992

Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae.

We have examined the expression of the gene encoding the iron-protein subunit (Ip) of succinate dehydrogenase in Saccharomyces cerevisiae. The gene had been cloned by us and shown to be subject to glucose regulation (A. Lombardo, K. Carine, and I. E. Scheffler, J. Biol. Chem. 265:10419-10423, 1990). We discovered that a significant part of the regulation of the Ip mRNA levels by glucose involves the regulation of the turnover rate of this mRNA. In the presence of glucose, the half-life appears to be less than 5 min, while in glycerol medium, the half-life is greater than 60 min. The gene is also regulated transcriptionally by glucose. The upstream promoter sequence appeared to have four regulatory elements with consensus sequences shown to be responsible for the interaction with the HAP2/3/4 regulatory complex. A deletion analysis has shown that the two distal elements are redundant. These measurements were carried out by Northern (RNA) analyses of Ip mRNA transcripts as well as by assays of beta-galactosidase activity in cells carrying constructs of the Ip promoter linked to the lacZ coding sequence. These observations on the regulation of mRNA stability were also extended to the mRNA of the flavoprotein subunit of succinate dehydrogenase and in some experiments of iso-1-cytochrome c.

Cloning, Molecular↗
PubMed1990

The 5'- and 3'-untranslated regions of ornithine decarboxylase mRNA affect the translational efficiency.

We have determined the roles of the 5'- and 3'-untranslated regions (UTR) of ornithine decarboxylase (ODC) mRNA in the post-transcriptional regulation of this enzyme. A series of expression vectors were constructed in which portions of the ODC 5' and/or 3' UTRs were placed flanking a reporter gene coding sequence, either firefly luciferase or chloramphenicol acetyltransferase, so as to generate a hybrid transcript. Translation of these chimeric genes in transient expression assays in wild type and ODC-deficient hamster cells was examined in the presence of normal or depleted polyamine pools. The ODC 5' UTR suppresses translation of the coding sequence it precedes irrespective of polyamine levels, and this effect is shown to be due to the GC-rich 5' segment of the UTR. The same effect is observed in vivo and in a rabbit reticulocyte in vitro translation system. The GC-rich region has the potential to form a very stable hairpin structure and inhibits translation in a position-dependent but orientation-independent manner. Insertion of the 3' UTR of ODC downstream of the translation termination codon of the reporter gene but prior to the polyadenylation signal partially relieves the suppression of translation imposed by the 5' UTR; the overall translatability of the message improves 30-50-fold.

Animals↗
PubMed1990

Cloning and characterization of the iron-sulfur subunit gene of succinate dehydrogenase from Saccharomyces cerevisiae.

We describe the cloning and characterization of the complete gene for the iron-sulfur protein subunit of succinate dehydrogenase (EC 1.3.99.1) from Saccharomyces cerevisiae. The promoter and coding sequence have been cloned into an Escherichia coli-yeast shuttle vector. The cloned gene complements the defect in a succinate dehydrogenase-deficient yeast mutant isolated by us, and gene expression is fully responsive to induction by glucose deprivation, indicating that the promoter is intact.

Amino Acid Sequence↗
PubMed1990

Molecular and genetic characterization of an ornithine decarboxylase-deficient Chinese hamster cell line.

The ornithine decarboxylase (ODC)-deficient Chinese hamster ovary (CHO) cell line C55.7 has normal amounts of ODC mRNA with very low amounts of immunologically detectable ODC protein, suggesting a structural mutation; however, 5-azacytidine treatment leads to phenotypical reversion (Steglich, C., and Scheffler, I. E. (1985) Somat. Cell Mol. Genet. 11, 11-23). We have demonstrated by chemical cleavage a single base mismatch in DNA heteroduplexes composed of wild-type and mutant cDNA strands. DNA sequencing showed that the mutant phenotype results from an aspartate-glycine substitution at amino acid 381 of the protein. When 5-azacytidine-revertant cell lines were selected for resistance to alpha-difluoromethylornithine, the resulting amplified ODC gene was structurally indistinguishable from the wild type gene. These results suggested the existence of a single active ODC locus in CHO cells. Using the methylation-sensitive restriction endonucleases AvaI and HpaII, we found evidence for two differentially methylated alleles in wild type, ODC-deficient and alpha-difluoromethylornithine-resistant cells. One of the alleles appeared completely inactivated by hypermethylation but could be reactivated by demethylation in spontaneous or 5-azacytidine-induced revertants.

Animals↗
PubMed1989

Isolation and characterization of a Saccharomyces cerevisiae mutant with a disrupted gene for the IP subunit of succinate dehydrogenase.

A partial cDNA clone corresponding to the iron-sulfur protein of succinate dehydrogenase (EC 1.3.99.1) has been isolated by an application of the polymerase chain reaction (Gould, S. J., Subramani, S., and Scheffler, I. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1934-1938). We used this clone for targeted gene disruption to isolate clones of Saccharomyces cerevisiae which were totally defective in this enzyme. The disruption of the gene was verified by Southern analysis. A Northern analysis revealed the existence of a new transcript which includes the 5' end of the mRNA for the IP subunit, but extends into the vector sequence disrupting the gene. A Western analysis suggests that the large flavoprotein subunit (FP) is still made in the mutant and imported into the mitochondria, but at significantly lower levels.

Blotting, Northern↗
PubMed1989

Molecular characterization of human minichromosomes with centromere from chromosome 1 in human-hamster hybrid cells.

In this study we examine the amounts of four different human satellite DNA sequences in a series of human-hamster hybrid cells, which contain a human minichromosome including the centromere of human chromosome 1. Comparisons with the corresponding amounts in an intact human chromosome 1 suggest that the minichromosomes have lost satellite DNA sequences, and in one case a substantial fraction of several satellite DNAs is lost, without affecting the stability and normal mitotic segregation of the minichromosome. The smallest minichromosome appears to have lost all of the long arm and a significant portion of centromeric heterochromatin, while retaining 1000-2000 kb of the short arm of human chromosome 1. The satellite sequences examined include: a chromosome 1-specific satellite III probe, a chromosome 1-specific alpha satellite DNA, another alpha satellite DNA originally derived from the X chromosome, and an alphoid EcoRI dimer whose isolation from one of the minichromosomes and characterization is also described in this paper. One interpretation of these data indicates that an interspersion of blocks of satellite sequences occurs in the centromere region of chromosome 1. If these satellite sequences have functional significance, then there may be redundancy in the system that allows for a variation in the size of the kinetochore and the number of attachment sites for microtubules.

Base Sequence↗
PubMed1989

Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species.

The DNA polymerase chain reaction was developed for in vitro amplification of specific DNA sequences, and it has been used for a wide variety of purposes in several fields. We have developed an application of the polymerase chain reaction that is useful for the isolation of partial cDNA or genomic clones of conserved genes. We used this technique to clone the gene encoding the iron protein subunit (27 kDa) of succinate dehydrogenase (EC 1.3.5.1) from several species, including human, rat, Drosophila melanogaster, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Mixed oligonucleotide primers corresponding to two conserved regions of the protein were used in conjunction with genomic and cDNA templates in the reaction. The primers contained all possible nucleotide combinations that could encode the corresponding peptide sequences. These oligonucleotide mixtures contained 262,144 (2(18] and 8192 (2(13] unique sequences, respectively. Use of the polymerase chain reaction for homology probing allows one to utilize more complex mixtures of oligonucleotides as probes than is possible with filter hybridization screening techniques. In addition, the polymerase chain reaction offers the advantage of synthesizing the DNA product directly, in some cases obviating the need to construct cDNA or genomic libraries. This application of the polymerase chain reaction should be useful not only for the identification of conserved genes in a variety of species but also for the isolation of previously unknown members of gene families.

Amino Acid Sequence↗
PubMed1988

Temperature-sensitive Chinese hamster cell mutant with a defect in glycoprotein synthesis: accumulation of the EGF receptor in the endoplasmic reticulum and the role of the glucose-regulated protein GRP78.

A temperature-sensitive mutant of Chinese hamster fibroblasts with a defect in glycoprotein synthesis is investigated after transfection and amplification of the gene for the human EGF receptor. We demonstrate that at the nonpermissive temperature a partially glycosylated species of the receptor accumulates in the endoplasmic reticulum. The oligosaccharides present are the high mannose types, since they can be removed completely by treatment with endoglycosidase H. Pulse-chase experiments show that the abnormal species of the receptor cannot be chased to a form that is either resistant to endoglycosidase H, or altered in its mobility on SDS polyacrylamide gels. The abnormal species of the receptor appears within the first hour of a shift to the nonpermissive temperature, and no further changes are observed upon prolonged incubation of cells at 40 degrees C. However, after 3-4 hours immunoprecipitations of the receptor yield another protein, which has properties very similar, if not identical, to the glucose-regulated protein GRP78. The induction of this protein at 40 degrees C can be suppressed completely with an inhibitor of RNA synthesis, without any effect on the glycosylation defect, or on the accumulation of the EGF receptor in the endoplasmic reticulum.

Animals↗
PubMed1988

Characterization of single-copy probe from vicinity of centromere of human chromosome 1.

Human DNA sequences in the human-hamster somatic cell hybrid XJM12.1.3 exist in the form of a minichromosome including the centromere of human chromosome 1. We describe the cloning of XJM12.1.3 DNA into the lambda vector EMBL3, the identification of minichromosome DNA-containing recombinants by hybridization with human sequences, and the characterization of one recombinant as a specific and unique probe for a region close to the centromere of human chromosome 1. This probe and others isolated from the minichromosome DNA are being developed to permit molecular access to a human centromere and its functional sequences.

Animals↗
PubMed1988

Steady-state and nuclear run-on analyses of transcription in a temperature-sensitive Chinese hamster cell mutant with a defect in RNA metabolism.

We have further characterized a temperature-sensitive mutant of Chinese hamster lung fibroblasts in tissue culture with a defect in RNA metabolism. The mutant phenotype is reflected in transcription in crude extracts or in isolated nuclei, when these are made from cells shifted to the nonpermissive temperature; however, differential heat inactivation between mutant and wild-type extracts cannot be demonstrated with cell-free systems. We tentatively conclude that the mutation may affect initiation of transcription which cannot be observed in our in vitro systems. Partially purified RNA polymerase I, II, and III fractions are indistinguishable from wild type. A temperature shift does not affect transcription by RNA polymerase III measured with intact cells or by nuclear run-on experiments. The nuclear run-on and other experiments suggest that RNA polymerase II-dependent transcription is inhibited before RNA polymerase I-dependent transcription. This conclusion is also supported by Northern analyses of selected mRNAs in nonsynchronized and synchronized cells after a shift to the nonpermissive temperature.

Amanitins↗
PubMed1987

Analysis of the protein glycosylation defect of a temperature-sensitive cell cycle mutant by the use of mutant cells overexpressing the human epidermal growth factor receptor after transfection of the gene.

A temperature-sensitive mutant with a defect in glycoprotein synthesis and a cell cycle (G1)-specific arrest at the nonpermissive temperature (Tenner et al., J. Cell. Physiol., 90:145-160, 1977; Tenner and Scheffler, J. Cell. Physiol., 98:251-266, 1979) was investigated further after a human epidermal growth factor (EGF) receptor gene had been transfected and amplified in these cells. While a temperature shift-up lead to an immediate arrest in the biosynthesis of mature EGF receptor and its appearance on the plasma membrane, the observed turnover of the preexisting receptor was too slow to account for the arrest of DNA synthesis in these mutant cells. Tunicamycin could in fact mimic the effect of a temperature shift on the biosynthesis of EGF receptor, but it did not have the same rapid effect on DNA synthesis and cell cycle progression. These mutants have also been shown to induce a set of stress proteins or glucose-regulated proteins, GRPs (Lee et al., J. Cell. Physiol., 129:277-282, 1986). The question is addressed whether the defect in glycoprotein synthesis is the primary defect and a possible cause of the induction of the GRPs, or whether a more basic defect at the level of the endoplasmic reticulum (ER) is responsible for the complex phenotype of the mutant. Our results argue in favor of a primary defect which indirectly affects N-linked glycosylation of proteins, as well as several other functions associated with the ER. We hypothesize that the defect affects the calcium distribution between ER and cytosol, since the calcium ionophore A23187 has an effect similar to that of a temperature shift.

Animals↗
PubMed1986

Posttranscriptional regulation of ornithine decarboxylase activity.

We have used a Chinese hamster ovary cell line (DF3) that overproduces ornithine decarboxylase (ODC) to examine various parameters in the cell cycle-dependent regulation of this enzyme. Under a variety of conditions, alterations in the activity of ODC were accompanied by parallel changes in the levels of the protein, as measured by immunologically cross-reactive material (CRM). While putrescine has been known to suppress the induction of ODC, we have found that in DF3 cells 10(-4)M ornithine completely suppresses ODC activity. We also show that the levels of ODC mRNA are not modulated when the levels of ODC activity and CRM change drastically. The data can be interpreted in terms of models involving either an effect of putrescine on the translation of ODC mRNA, or on the activity of a relatively specific protease with ODC as its target.

Animals↗
PubMed1986

Enhanced synthesis of the glucose/calcium-regulated proteins in a hamster cell mutant deficient in transfer of oligosaccharide core to polypeptides.

The properties of two Chinese hamster temperature-sensitive mutants, K12 and H3.5, were examined. Both mutants originated from the same parental cell line, Wg1A, and were isolated as cell cycle mutants arrested in G1. Previously, we had been shown that the H3.5 ts mutation affected the transfer of the oligosaccharide from the lipid carrier to the nascent polypeptide and that the K12 ts mutation regulated the transcription of two glucose/calcium-regulated genes. We report here that these two mutants exhibit almost identical phenotypes at the biochemical level. Furthermore, a genetic complementation test demonstrates that the two ts lesions must be closely related, or even identical. Our results suggest that a specific defect in glycosylation may result in the overproduction of the glucose/calcium-regulated proteins and is capable of activating the promoter of the major glucose-regulated gene.

Animals↗
PubMed1986

Chinese hamster cells with a minichromosome containing the centromere region of human chromosome 1.

We describe a series of primary and secondary hamster-human hybrids which have selectively retained a small amount of human DNA. The hybrid XJM12.1.3 contains an estimated 4000-8000 kb of human DNA, and for a secondary hybrid derived from it, XEW8.2.3, our estimate is 1000-2000 kb. The hybridization of Southern blots of DNA from these hybrids with a variety of human satellite DNA probes reveals that these lines include centromere sequences of human chromosome 1. The identifiable human DNA is in the form of a minichromosome, as detected by in situ hybridization in the light microscope and in the electron microscope. At mitosis, the minichromosome can be observed to have kinetochores and to be associated with microtubules. Therefore, it can segregate in a stable fashion. It may be significant that in the selection of the hybrids we had selected for a human gene which has been mapped on human chromosome 1.

Animals↗
PubMed1985

Chinese hamster cells deficient in ornithine decarboxylase activity: reversion by gene amplification and by azacytidine treatment.

A group of Chinese hamster ovary (CHO) cell mutants deficient in ornithine decarboxylase (ODC) activity are described and compared to the prototype mutant reported previously (21). Although all mutants belong to the same complementation group, they can be divided into two classes: those with some residual enzyme activity and those with no activity. All mutants are putrescine auxotrophs, but they differ in their ability to utilize the enzyme's substrate, ornithine, a property which correlates with the amount of residual enzyme activity. The mutants also differ in their frequency of reversion to prototrophy. The leaky mutants revert at a high rate by overproducing a partially defective enzyme by a gene amplification mechanism similar to that leading to the ornithine analog-resistant mutants which have elevated enzyme levels. Spontaneous reversion in the null mutants is rare. However, one null mutant, which was induced with ethyl methane sulfonate and which makes ODC mRNA but no active enzyme, is nevertheless revertible with 5-azacytidine. We conclude that CHO cells are at least diploid at the ODC locus, but that only one allele is active. Further studies suggest the possibility that ethyl methane sulfonate is not just a classical mutagen but may also induce gene inactivations that are revertible by 5-azacytidine.

Animals↗