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R Klemenz

Publications and source records attributed to R Klemenz.

54 records · Page 3Linked to original sources
PubMed1991

Molecular and functional analysis of tumor-suppressor genes by transfection.

The transformed and tumorigenic phenotype of H-ras transfected rat FE-8 cells can be suppressed by cell fusion with normal rat embryo fibroblasts. Transfection into FE-8 cells of DNA prepared from normal human placenta followed by selective elimination of tumorigenic transfected cell clones resulted in the isolation of phenotypically normal revertants. These cells exhibited a fibroblastlike, normal morphology; were anchorage-dependent; and were unable to proliferate in medium with reduced serum concentrations. Their tumorigenicity was also reduced. The suppressed phenotype has been transferred in a second transfection cycle. Human repetitive DNA sequences were detected in secondary transfectant DNA. A putative human suppressor gene, designated NTS-1, has been molecularly cloned. Reintroduction of cloned NTS-1 sequences into FE-8 cells resulted in suppression of the neoplastic phenotype in spite of a high ras expression.

Animals↗
PubMed1990

[Tumor suppressor genes: identification and role in malignant transformation].

Two lines of evidence have suggested that the loss of tumor suppressor gene function contributes to the multi-step process of tumorigenesis. Tumorigenicity and transformed phenotypes are frequently suppressed in somatic cell hybrids of tumor and normal cells. Consistent chromosomal aberrations indicating gene losses and mutations have been found in hereditary and sporadic human tumors. Tumor suppressor genes can be molecularly cloned, even if the encoded protein products have not been characterized biochemically. Here we describe a functional assay based on DNA transfection permitting the molecular identification of candidate human tumor suppressor genes (R. SCHAFER et al., Proc. Natl. Acad. Sci. USA 85, 1590-1594, 1988). A molecular clone has been identified capable of suppressing the neoplastic phenotype induced by an oncogene belonging to the family of ras genes, whose activation is frequently found in tumors.

Animals↗
PubMed1989

Oncogene mediated repression of glucocorticoid hormone response elements and glucocorticoid receptor levels.

We have previously described the inhibition of glucocorticoid-dependent transcription from the mouse mammary tumor virus long terminal repeat promoter by products of the H-ras and v-mos oncogenes. We have studied the effects of conditional oncogenes on expression of glucocorticoid-dependent indicator genes. Expression of the glucocorticoid-dependent transcription of the tyrosine aminotransferase gene was monitored in FTO-2B rat hepatoma cells during Mr 21,000 protein (p21) H-ras induction. A strong transcriptional repression of the tyrosine aminotransferase gene followed p21 H-ras expression. The sequences in a glucocorticoid-dependent promoter which are responsible for the oncogene-mediated repression could be localized to the glucocorticoid response element; a construct in which a 15-base pair glucocorticoid response element was inserted 5' of the thymidine kinase promoter exhibited the oncogene-mediated repression of transcription. We observed a strong repression of glucocorticoid-dependent promoters and promoter constructs not only in the presence of p21 H-ras and p37 v-mos but also with p60 v-src. p57 v-myc, however, had no effect. Oncogene expression is not a sufficient prerequisite for an initial repression of glucocorticoid hormone-dependent gene transcription, since even in the presence of constitutively high levels of oncogene product a transient stimulation of glucocorticoid-dependent gene expression was found. Protein synthesis inhibition experiments revealed that no hormonally induced cellular protein is needed for the oncogene-mediated repression. It seemed reasonable that this phenomenon might reflect oncogene effects on the glucocorticoid receptor. We, therefore, made measurements of the glucocorticoid receptor protein. In the presence of glucocorticoid hormone the receptor translocated rapidly from the cytoplasm to the nucleus. In normal NIH 3T3 cells, after 24-h treatments the nuclear receptor levels had declined to about 50% of those determined at 2 h and in the presence of p21 H-ras they declined to 15%. The levels of cytoplasmic receptor were not affected by p21 H-ras expression.

Animals↗
PubMed1989

Serum- and oncoprotein-mediated induction of a gene with sequence similarity to the gene encoding carcinoembryonic antigen.

We describe the molecular identification of a gene, designated T1, whose expression in mouse NIH 3T3 cells is strongly induced by the Ha-ras(EJ) and v-mos oncogenes and by serum. The T1 gene encodes a 38.5-kDa protein, as predicted from its primary sequence and shown by in vitro translation. The protein was processed at its amino terminus and extensively modified by N-linked glycosylation in vitro in the presence of microsomal vesicles. Sequence comparison of T1 with the MIPSX data base (Max-Planck-Institut für Biochemie, Martinsried/Munich) revealed similarity to the human carcinoembryonic antigen, a tumor marker which is overexpressed in colon adenocarcinomas and in fetal tissues. Considerable sequence similarity has also been observed to the short conserved region of other proteins which, like carcinoembryonic antigen, are encoded by members of the immunoglobulin gene superfamily.

Amino Acid Sequence↗
PubMed1989

Induction of a mitogen-responsive gene after expression of the Ha-ras oncogene in NIH 3T3 fibroblasts.

A cDNA clone, T1, has been isolated whose corresponding mRNA was transiently expressed at highly elevated levels after conditional expression of the Ha-ras(EJ) gene and after mitogenic activation of quiescent NIH 3T3 cells. Glucocorticoid hormone stimulated substantial T1 expression as well but only in proliferating cells. At least two different signaling pathways participate in the regulation of the T1 gene: a protein kinase C-dependent signal is involved in the response of proliferating NIH 3T3 cells to glucocorticoid in the absence but not the presence of p21ras, whereas a protein kinase C-independent mechanism mediates the response to serum factors. Treatment of cells with the protein kinase inhibitor 2-aminopurine blocked induction of expression of the T1 gene. T1 mRNA accumulation is regulated at the transcriptional level.

2-Aminopurine↗
PubMed1989

The v-mos and c-Ha-ras oncoproteins exert similar effects on the pattern of protein synthesis.

The effects of the ras and the mos oncogene products on the pattern of newly synthesized proteins was investigated in NIH3T3 cell lines. A conditional expression system which allowed hormonal induction of the oncogenes was utilized to detect effects on the accumulation of oncoproteins by two dimensional gel electrophoresis of crude cell extracts. Strong and reproducible changes of protein synthesis following the expression of the ras and mos oncogenes were detected. Transiently induced synthesis of four proteins with a molecular mass of 23 kDa, 32 kDa, 35 kDa, and 47 kDa was observed. These changes were qualitatively indistinguishable in both, ras and mos oncogene expressing cells. This is in agreement with the notion that the two oncogene products act on a common signal transduction pathway. Serum mediated growth induction of quiescent NIH3T3 cells led to a different pattern of altered protein synthesis. We observed the transient alteration in the synthesis rates of three proteins with a molecular mass of 27 kDa, 47 kDa and 52 kDa. Only the 47 kDa protein was also subject to regulation by the oncoproteins. One of the proteins whose synthesis was strongly induced by the ras and mos oncogene products is also expressed by heat shock.

Animals↗
PubMed1988

Expression, modification, and localization of the fushi tarazu protein in Drosophila embryos.

The fushi tarazu (ftz) protein of Drosophila is required during embryogenesis for the process of body segmentation. To study the biochemical properties of the ftz protein, ftz cDNA was expressed in Escherichia coli and the protein was purified to homogeneity. Polyclonal antibodies raised against the purified protein were used to localize and quantitate the protein during embryogenesis. Three temporally and spatially distinct phases of expression were observed, which include a previously undetected period later in embryogenesis. During this last phase, the protein is localized predominantly in the developing hindgut. Analysis of embryonic ftz protein on Western blots permitted us to approximate the number of protein molecules per nucleus. During the blastoderm phase of development, when ftz protein is most abundant, we estimate that there are approximately 20,000 molecules of protein per ftz-expressing nucleus. The embryonic ftz protein migrates more slowly on SDS-polyacrylamide gels than protein made either in E. coli or in a reticulocyte lysate system in vitro, indicating that it is modified in the embryo. To facilitate characterization of ftz protein made in embryos, an ftz overexpression system functional in Drosophila was developed. When fused to an hsp70 heat shock promoter and introduced into the germ line by P-element-mediated transformation, ftz could be overexpressed at all stages of development by heat shock. This protein is localized in the nucleus comigrates on SDS-polyacrylamide gels with endogenous ftz protein. Two-dimensional gel electrophoresis followed by Western blotting resolves the overexpressed protein into a series of isoforms that differ in charge and electrophoretic mobility. Post-translational modification may influence the biochemical properties and functions of the ftz protein during embryogenesis.

Animals↗
PubMed1987

The white gene as a marker in a new P-element vector for gene transfer in Drosophila.

We describe new vectors suitable for P-element mediated germ line transformation of Drosophila melanogaster using passenger genes whose expression does not result in a readily detectable phenotypic change of the transformed flies. The P-element vectors contain the white gene fused to the heat shock protein 70 (hsp70) gene promoter. Expression of the white gene rescues the white phenotype of recipient flies partly or completely even without heat treatment. Transformed descendents of most founder animals (GO) fall into two classes which are distinguishable by their orange and red eye colours. The different levels of white expression are presumably due to position effects associated with different chromosomal sites of insertion. Doubling of the gene dose in orange eyed fly stocks results in an easily visible darkening of the eye colour. Consequently, the generation of homozygous transformants is easily possible by simple inbreeding due to the phenotypic distinction of homo- and heterozygous transformants. Cloning into these P-element vectors is facilitated by the presence of polylinkers with 8 and 12 unique restriction sites.

Animals↗
PubMed1986

Translational and transcriptional control elements in the untranslated leader of the heat-shock gene hsp22.

Downstream of the transcription start site in the Drosophila heat-shock gene hsp22, we have identified a region that is necessary for efficient transcription, and also for selective translation during heat shock. We assayed the expression of mutated genes after P-factor-mediated insertion into the genome. Deletions within the first 26 nucleotides block the preferential translation of hsp22 mRNA at high temperature. The rate of transcription is also decreased, though transcription is still heat-inducible. Leaving this region intact, up to 86% of the leader can be deleted without affecting translation or transcription. The functional region coincides with a region of sequence homology between the heat-shock genes. Only partial homology is found to a conserved sequence, ATCAGTTCT, found at the very 5' end of other insect genes.

Animals↗
PubMed1986

Sequence requirement for expression of the Drosophila melanogaster heat shock protein hsp22 gene during heat shock and normal development.

A 14-base-pair sequence element present in almost all Drosophila melanogaster heat shock genes has been implicated in the heat inducibility of transcription. The D. melanogaster gene encoding the smallest heat shock protein, hsp22, contains within its 5' flanking sequences three such repeats, two close to the transcription start site and a distally located third one 101 base pairs further upstream. Deletion analyses reveal that the 5' flanking sequences required for full expression of the hsp22 gene extend beyond the distal repeat. Deletion of the furthest upstream repeat results in a five to sixfold reduction of gene expression. The small heat shock genes are transiently expressed in the late third instar larval and early pupal stages without external stimulation. A deletion of 5' flanking sequences to position -194, which includes two nucleotides of the distal heat shock element, has no effect on the developmental expression, whereas removal of an additional 18 nucleotides, including 12 nucleotides of the distal heat shock element, severely reduces developmental expression.

Animals↗
PubMed1985

Selective translation of heat shock mRNA in Drosophila melanogaster depends on sequence information in the leader.

One of the effects of a temperature increase above 35 degrees C on Drosophila melanogaster is a rapid switch in selectivity of the translational apparatus. Protein synthesis from normal, but not from heat shock, mRNA is much reduced. Efficient translation at high temperature might be a result of the primary sequence of heat shock genes. Alternatively a mRNA modification mechanism, altered as a consequence of heat shock, might allow for efficient high temperature translation of any mRNA synthesized during a heat shock. The gene for alcohol dehydrogenase (Adh) was fused to the controlling elements of a heat shock protein 70 (hsp70) gene. Authentic Adh mRNA, synthesized from this fusion gene at elevated temperatures was not translated during heat shock. A second Adh fusion gene in which the mRNA synthesized contained the first 95 nucleotides of the Hsp70 non-translated leader sequence gave rise, at high temperature, to mRNA which was translated during the heat shock. Thus, the signal(s) in the mRNAs controlling translation efficiency at heat shock temperatures is encoded within the heat shock genes.

Alcohol Dehydrogenase↗
PubMed1984

Functional analysis of the white gene of Drosophila by P-factor-mediated transformation.

A 12-kb DNA segment spanning the white (w) locus of Drosophila has been inserted into a P-transposon vector and used for P-factor-mediated germ-line transformation. Several red-eyed transformants were recovered which complement the white mutant phenotype. Analysis of the eye pigments and the interaction with the zeste mutation indicates that the w gene inserted at several new chromosomal sites is expressed normally. The tissue-specific accumulation of w transcripts, as studied by in situ hybridization to tissue sections, is the same in transformant and wild-type larvae. This indicates that all the genetic information specified by the w locus is contained within this 12-kb segment of DNA. By secondary mobilization it was shown that the w sequences have been inserted as a functional P(w) transposon which is capable of further transposition.

Journal Article↗
PubMed1982

Specific interactions of Saccharomyces cerevisiae proteins with a promoter region of eukaryotic tRNA genes.

The specific binding of one or several Saccharomyces cerevisiae proteins to a segment of genes that code for different yeast tRNAs has been demonstrated with the use of the DNase I-protection "footprint" assay of Galas and Schmitz. The analyzed binding occurs near the 3' ends of the genes and is centered on an 11-base-pair DNA sequence that has been well conserved among eukaryotic tRNA genes. Others have shown the involvement of this sequence in initiating the transcription of tRNA genes by RNA polymerase III. The adenovirus gene that codes for VAI RNA also contains this conserved sequence element, and we detect binding of yeast protein(s) to this gene. Competition experiments show that a common set of proteins binds to different tRNA genes. The DNA-protein complex is quite stable at 20 degrees C and low ionic strength.

Carrier Proteins↗
PubMed1981

Sequence determination of the 3' end of mouse mammary tumor virus RNA.

70S RNA has been prepared from mouse mammary tumor virus (MMTV) produced by the GR tumor cell line. After denaturation for 3 min at 60 degrees C the RNA was applied to a sucrose gradient and molecules sedimenting between 10-16S were selected and passed over an oligo-dT cellulose column. The poly A containing RNA fraction was used as a template for the synthesis of complementary DNA with reverse transcriptase in the presence of dideoxynucleoside triphosphates. Oligo-nucleotides p(dT)7rG, p(dT)7rA and (p(dT)7rC were tested for their primer activity. Two of the three primers gave readable sequences. This suggests a heterogeneity in the 3' end of the viral RNA, a phenomenon also observed with avian retroviruses. Nucleotides 37-45 from the 3' end are made up of only A's and T's and resemble a Hogness box. This finding could have biological consequences for the transcription of proviral DNA in the unintegrated and integrated state. Instead of the usual AAUAAA sequence present in mRNA's close and in front of the site of polyadenylation we find a sequence AGUAAA.

Animals↗
PubMed1980

The 5' terminus of the precursor ribosomal RNA of Saccharomyces cerevisiae.

The 5' terminus of Saccharomyces cereviasiae 35S pre rRNA was mapped on the rDNA using two methods: 1) Suitable restriction endonuclease fragments were hybridized to total high molecular weight RNA and extended with reverse transcriptase to the 5' end of the RNA template. 2) Other restriction fragments spanning the 5' terminus of 35S pre rRNA and radioactively labeled at their ends were hybridized to high molecular weight RNA and the non hybridized nucleic acids were digested with S1 nuclease. On the basis of these experiments, the 5' terminus of 35S pre rRNA was placed approximately 670 nucleotides upstream from the 17S rRNA coding region. The exact position was determined by reverse transcription as above, but in the presence of dideoxyribonucleoside triphosphates, which served as a way of sequencing the 5' terminal region. 35S pre rRNA synthesis is initiated at a site in EcoRI restriction fragment B which is 48 nucleotides upstream from the EcoRI cleavage site in the coding strand.

Base Sequence↗
PubMed1979

Extracellular cleavage of the glycoprotein precursor of Rous sarcoma virus.

The kinetics of cleavage of pr92gp, the precursor of the two glycoproteins of Rous sarcoma virus gp85 and gp35, were followed. Viral glycoproteins were detected by immunoprecipitation with anti-gp85 and anti-gp35 serum. It could be shown in pulse-chase experiments that little or no intracellular cleavage of the precursor took place during the time in which the majority of newly synthesized viral glycoprotein was exported from the cells. Soon after its synthesis, however, pr92gp underwent some modification that made it migrate slightly faster on sodium dodecyl sulfate-polyacrylamide gels. Under steady state conditions the precursor was shown to be the predominant form of intracellular viral glycoprotein. Virus which was harvested every 2 min from infected cells prelabeled for 90 min with [3H]mannose contained mostly uncleaved and only a little mature glycoprotein. By incubation of this freshly released virus in serum-free buffer, the majority of the glycoprotein precursor could be cleaved into mature gp85 and gp35. Virus harvested every 10 min contained only mature glycoproteins.

Avian Sarcoma Viruses↗
PubMedDate not supplied

Redesigning the body plan of Drosophila by ectopic expression of the homoeotic gene Antennapedia.

Genetic and molecular studies on the expression of Antennapedia (Antp) have suggested that this gene specifies mainly the second thoracic segment. On the basis of our molecular analysis of dominant gain-of-function mutants we have postulated that the transformation of antennae into second legs is due to the ectopic overexpression of the Antp+ protein. This hypothesis was tested by inserting the complementary DNA encoding the normal Antp protein into a heat-shock expression vector and subsequent germ-line transformation. As predicted, heat induction at defined larval stages leads to the transformation of antennae into second legs. The dorsal part of the head can also be transformed into second thoracic structures (scutum) indicating that Antp indeed specifies the second thoracic segment. By ectopic overexpression of the Antp protein the body plan of the fruit fly can be altered in a predictable way.

Animals↗