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A Udvardy

Publications and source records attributed to A Udvardy.

At least 19 recordsLinked to original sources
PubMed1996

The role of controlled proteolysis in cell-cycle regulation.

Cyclins and cyclin-dependent kinases are key regulators of the cell cycle. The binding of different cyclins, required to activate the catalytically inactive cyclin-dependent kinases, determines the substrate specificity of the enzymes. Cyclin-dependent-kinase inhibitors have an adverse effect, blocking the catalytic activity of cyclin-activated cyclin-dependent kinases. The cell cycle is a cyclic process of successive transient activation or inactivation of cyclin-dependent kinases by association with different cyclin regulatory subunits or cyclin-dependent kinase inhibitors. As the concentration of cyclin-dependent kinases is fairly constant during the cell cycle and exceeds the total amount of cyclins present in the cell, the exchange of regulatory subunits is determined by the availability of the different cyclins. Transcriptional control of cyclin gene expression is the most decisive factor determining the total amount of different cyclins synthesized. The actual concentration of a cyclin, however, is always the result of an equilibrium between the rates of its synthesis and degradation. While cyclin gene expression has long been known to be cell-cycle controlled, the idea of the rapid destruction of cyclins or cyclin-dependent-kinase inhibitors as an equally important factor contributing to the progress of the cell cycle is more recent. The role of controlled proteolysis in the regulation of cell cycle is discussed in this review. Two general features of this regulation are worth mentioning: cyclin-dependent kinases activated by different cyclin regulatory subunits have a central role both in the transcriptional regulation of their own genes and in the regulated, selective destruction of cyclins or cyclin-dependent kinase inhibitors; transcriptional regulation of cyclin gene expression ensures fine-tuned, continuous changes, and controlled proteolysis generates abrupt, irreversible transitions. The progress of the cell cycle is based on a delicate balance of the these mutual, but opposite regulations.

Animals↗
PubMed1996

Dissection of the regulator complex of the Drosophila 26S protease by limited proteolysis.

The 26S protease responsible for the selective degradation of ubiquitinated proteins is composed of a regulator complex and the 20S proteosome which is the catalytic core. In the absence of ATP the 26S protease dissociates to free regulator complex and 20S proteosome, and this process can be reversed in vitro in the presence of ATP. Trypsin, chymotrypsin or proteinase K digestion selectively removes several subunits of the free regulator complex of Drosophila 26S protease generating a well-defined new subparticle. Three subunits highly sensitive in the free regulator complex, however, were selectively protected within the in vitro reconstituted 26S protease, indicating that the ATP-dependent association of the 20S proteosome in the regulator complex selectively shields these subunits. In the same concentration range the 20S proteosome was completely resistant for proteolytic degradation.

Animals↗
PubMed1995

Cloning and sequencing a non-ATPase subunit of the regulatory complex of the Drosophila 26S protease.

We have cloned and sequenced a non-ATPase subunit of the regulatory complex of the Drosophila 26S protease. The gene is present in a single copy in the Drosophila genome. By comparing the nucleotide sequence of the genomic and cDNA clones three exons and two introns were localized. Two transcription start sites were identified 9 bp apart. The deduced protein sequence shows no significant similarity to any other protein in the database. In Drosophila embryos where the 26S protease is present in high concentration, the pool of free subunits of the regulatory complex is very low. Among the free subunits of the regulatory complex the cloned subunit is present in very large excess. This observation raises the possibility that this subunit is in a dynamic equilibrium, exchanging between a free and a particle-bound form, which may have important implications concerning its function.

Adenosine Triphosphatases↗
PubMed1995

The mechanism of nuclear transport of natural or artificial transport substrates in digitonin-permeabilized cells.

Characterization of nuclear protein transport in digitonin-permeabilized cells revealed that the number of the nuclear localization signal sequences (NLS) within the transport substrate basically influences the mechanism of the transport reaction. Phycoerythrine-NLS transport substrate carrying a maximum of 4-5 conjugated NLSs/subunit, or Bsp methyltransferase-NLS fusion protein were efficiently transported into the nuclei of digitonin-permeabilized cultured cells without any exogenously added cytosolic protein. All the characteristic properties of in vivo nuclear transport are faithfully reproduced with these transport substrates: (i) the transport requires a functional NLS in the transported protein, a transport-incompetent mutant NLS being ineffective; (ii) the transport is energy dependent; (iii) the wild type nuclear localization peptide efficiently competes for transport, while the transport-incompetent mutant peptide does not; and (iv) wheat germ agglutinin inhibits this transport reaction. Nuclear transport observed with these substrates was not due to any damage of the nuclear membrane or inefficient extraction of the cytosolic proteins during the permeabilization of the cells. The nuclear transport was proportional to the number of conjugated NLSs. Nuclear transport of phycoerythrine carrying 7-8 conjugated NLSs/subunit required the addition of exogenous cytosolic proteins. This transport also fulfilled all the characteristic properties of an authentic nuclear transport. Nuclear transport with different combinations of transport substrates further supported the assumption that distinct transport mechanisms operate for different substrates. From a mixture of PE-NLS7-8 and Bsp methyltransferase-NLS, the highly conjugated substrate was completely retained in the cytoplasm in the absence of exogenous cytosol, while Bsp methyltransferase-NLS was efficiently transported. Exogenous cytosol promoted the nuclear transport of the highly conjugated substrate.

Amino Acid Sequence↗
PubMed1993

S. cerevisiae 26S protease mutants arrest cell division in G2/metaphase.

We isolated two mutants from the yeast Saccharomyces cerevisiae, cim3-1 and cim5-1, that arrest cell division in G2/metaphase at 37 degrees C. CIM3 (identical to SUG1; ref. 1) and CIM5 are similar to each other and are members of a family of putative ATPases that have been proposed to be 26S protease subunits. We show here that CIM5 is the functional yeast homologue of the human MSS1 protein and that homologues of CIM3 and CIM5 are present in a highly purified preparation of the Drosophila 26S protease. The short-lived ubiquitin-proline-beta-galactosidase fusion protein is stabilized in cim mutants, but Leu-beta-galactosidase is not. The CLB2 and CLB3 cyclins also accumulate in the cim mutants. Thus the 26S protease is required in vivo for the degradation of ubiquitinated substrates and for anaphase chromosome separation.

Adenosine Triphosphatases↗
PubMed1993

Spermidine-induced alteration in the gene-spacer discrimination of nucleases in protonated DNA.

The sequence preference of a Drosophila lysosomal DNase was studied on the Drosophila hsp 70 heat-shock and histone recombinants, which carry six different genes, and the surrounding spacer sequences. The distribution of cleavage sites was random in respect of the locations of gene and spacer sequences. However, in the presence of 10 mM spermidine, a major transition was observed: the coding sequences became more susceptible than the spacer regions to nuclease attack. A similar transition was induced in the sequence preference of DNase I if the digestion was performed in the presence of spermidine at pH 5.2. At pH 7.5, spermidine does not influence the sequence preference of DNase I, which indicates the involvement of DNA protonation in this transition. In the presence of spermidine, the distributions of preferred and protected sequences were almost indistinguishable for these nucleases, suggesting that the protonated DNA, and not the enzymes, is the target of spermidine. A Drosophila embryonal protein was detected and partially purified which induced the same transition as observed in the presence of spermidine. The purified protein preferentially protected the spacer DNA sequences against acid DNase or DNase I cleavage in the hsp 70 heat-shock and histone gene recombinants. The protection was concentration dependent and occurred only at pH 5.2. The transition of nuclease specificity is probably due to a conformational change in the protonated DNA, induced by the binding of either the embryonal protein or spermidine.

Animals↗
PubMed1993

Z-DNA binding and inhibition by GTP of Drosophila topoisomerase II.

A Z-DNA binding protein has been isolated and characterized by biochemical means from Drosophila melanogaster tissue culture cells and embryos. This protein shares the following properties with the known, cloned Drosophila topoisomerase II: (1) expression of an ATP-dependent relaxation activity on supercoiled DNA; (2) a monomer mass of 165 kDa in SDS denaturing gels; (3) a sedimentation coefficient, S20,w, of approximately 10 S for the active enzyme; (4) cross-reactivity for the respective monoclonal and polyclonal antibodies; (5) generation of covalent enzyme-DNA intermediates at preferred cutting sites in the Drosophila HSP70 intergenic spacer region; (6) inhibition of DNA relaxation activity by antitumor drugs, e.g., the etoposide VM26, and by monospecific antibodies raised against the protein; and (7) in vitro phosphorylation by a casein kinase activity. However, we have identified new properties for our topoisomerase II preparation not previously reported for the conventionally isolated enzyme: (1) The enzyme binds to Z-DNA with an affinity 2 orders of magnitude greater than that for B-DNA. (2) The binding to Z-DNA is increased 5-10-fold by GTP or GTP-gamma-S. (3) GTP and GTP-gamma-S inhibit the catalytic activity of topoisomerase II through a proposed allosteric mechanism. (4) Z-DNA inhibits the relaxation of closed circular supercoiled DNA. (5) The preparation consists of a single polypeptide chain of 165 kDa on denaturing SDS gels with no evidence of proteolytic degradation. We postulate that the Z-DNA binding activity of undegraded topoisomerase II may be important in targeting the enzyme both to structural motifs required for chromatin organization and to sites of local supercoiling. Some of these features arise during processes such as replication and gene expression and may be more frequent during embryogenesis and early development.

Animals↗
PubMed1993

Purification and characterization of a multiprotein component of the Drosophila 26 S (1500 kDa) proteolytic complex.

A multiprotein complex, referred to as the mu particle, was purified to apparent homogeneity from Drosophila melanogaster embryos. This multiprotein complex has no protease activity, but it can be incorporated into an even larger multiprotein complex which exhibits strong and selective protease activity, i.e. it degrades only ubiquitin-conjugated proteins. Incorporation of the mu particle into the ubiquitin conjugate-degrading larger complex is absolutely ATP-dependent. On these criteria the larger complex corresponds to the 26 S (1500 kDa) proteolytic complex partially purified and characterized from reticulocytes. A procedure is described for the purification of the Drosophila 26 S (1500 kDa) proteolytic complex. It was found to be a stoichiometric complex of the mu particle and the 20 S proteosome. Although no other polypeptide was present in stoichiometric amount in the 26 S (1500 kDa) proteolytic complex besides the mu particle and the 20 S proteosome, an additional protein factor(s) is required for its assembly, the ubiquitin conjugate-degrading activity cannot be reconstituted from the purified mu particle and the 20 S proteosome. Synthesis of the mu particle is developmentally regulated; its concentration is highest in embryos. This is probably connected with massive degradation of yolk proteins during embryogenesis. In chicken, rabbit and human cells a high molecular weight multiprotein complex can be detected, which is immunologically related to the Drosophila mu particle.

Animals↗
PubMed1993

Mutations in the protein phosphatase 1 gene at 87B can differentially affect suppression of position-effect variegation and mitosis in Drosophila melanogaster.

The suppressor of position effect variegation (PEV) locus Su-var(3)6 maps to 87B5-10. The breakpoints of deficiencies that define this interval have been placed on a 250-kb molecular map of the region. The locus is allelic to the ck19 complementation group previously shown to encode a type 1 serine-threonine protein phosphatase (PP1) catalytic subunit. When introduced into flies by P element-mediated transformation, a 5.8-kb genomic fragment carrying this gene overcomes the suppressor phenotype of Su-var(3)6(01) and recessive lethality of all mutations of the locus. Four of the mutant alleles at the locus show a broad correlation between high levels of suppression of PEV, a high frequency of aberrant mitosis and low PP1 activity in larval extracts. However, some alleles with low PP1 activity show weak suppression of PEV with a high frequency of abnormal mitosis, whereas others show strong suppression of PEV with normal mitosis. The basis for these discussed.

Animals↗
PubMed1993

The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery.

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.

Animals↗
PubMed1992

Molecular characterization of the 5' end of the rudimentary gene in Drosophila and analysis of three P element insertions.

A detailed analysis of the 5' end of the rudimentary gene of Drosophila melanogaster is presented. Rudimentary transcripts are heterogeneous at their 5' ends indicating that transcription is initiated at multiple sites within a region of approximately 50 bp. These transcription initiation sites are within a region that is preferentially susceptible to nuclease cleavage in isolated nuclei. Additional nuclease hypersensitive regions were found within the first exon and the first intron. Within these internal nuclease hypersensitive regions are the insertion sites for previously identified P element transposons which disrupt rudimentary expression. One of these P element insertions, located in the first intron, is removed from the rudimentary transcript with the splicing of this intron. Another P element insertion, within the first exon, is removed from the rudimentary transcript by novel first intron splicing involving a cryptic splice donor site, located 5' to the insertion, and either the normal acceptor site or a cryptic splice acceptor site within the second exon.

Amino Acid Sequence↗
PubMed1992

Mouse microtubule-associated protein 2 expression in transgenic Drosophila.

Full-length mouse microtubule-associated protein 2 complementary DNA inserted between Drosophila heat-shock protein 70 promoter and trailer was introduced in the germ-line of Drosophila melanogaster by P element-mediated transformation. Three transformant lines contained microtubule-associated protein 2 complementary DNA in sense orientation and two in antisense orientation. All sense lines produced microtubule-associated protein 2 after heat-shock in several tissues at low level. In the adult brain, high-level microtubule associated protein 2 expression independent of heat induction occurred in specific neuron types in each sense transformant, suggesting the action of neuron-specific enhancer genes. High microtubule-associated protein 2 levels are expected to interfere with the cells' activity by forming unphysiological cross-links, which may help elucidate the function of the given neurons.

Animals↗
PubMed1991

Purification of a lysosomal DNase from Drosophila melanogaster.

An acid DNase was purified from Drosophila melanogaster till apparent homogeneity by six consecutive chromatographic steps. The enzyme is a lysosomal DNase, because it is glycosylated and carries 1.8-2.4 mol of mannose-6-phosphate/mol of enzyme. The enzyme is fully active without any divalent cation and introduces single stranded nicks into a supercoiled DNA.

Animals↗
PubMed1991

Chromatin structure, not DNA sequence specificity, is the primary determinant of topoisomerase II sites of action in vivo.

In the studies reported here we have used topoisomerase II as a model system for analyzing the factors that determine the sites of action for DNA-binding proteins in vivo. To localize topoisomerase II sites in vivo we used an inhibitor of the purified enzyme, the antitumor drug VM-26. This drug stabilizes an intermediate in the catalytic cycle, the cleavable complex, and substantially stimulates DNA cleavage by topoisomerase II. We show that lysis of VM-26 treated tissue culture cells with sodium dodecyl sulfate induces highly specific double-strand breaks in genomic DNA, and we present evidence indicating that these double-strand breaks are generated by topoisomerase II. Using indirect end labeling to map the cleavage products, we have examined the in vivo sites of action of topoisomerase II in the 87A7 heat shock locus, the histone repeat, and a tRNA gene cluster at 90BC. Our analysis reveals that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase II site selection in vivo. We suggest that chromatin organization may provide a general mechanism for generating specificity in a wide range of DNA-protein interactions in vivo.

Animals↗
PubMed1988

Intracellular forms of Drosophila topoisomerase II detected with monoclonal antibodies.

We developed monoclonal antibodies against Drosophila topoisomerase II and studied the intracellular forms and the in vivo and in vitro proteolytic degradation of the enzyme. In purified enzyme preparations polyclonal sera and monoclonal antibodies recognized several polypeptides in the 170-132 kD molecular weight range. In vivo, however, the pattern was much simpler. In Drosophila embryos, pupae, fly heads and Schneider S3 tissue culture cells topoisomerase II appeared as a single 166 kD polypeptide. In Drosophila embryos, with two monoclonal antibodies topoisomerase II appeared as a doublet composed of the 166 kD canonical form and a slightly higher molecular weight polypeptide. Topoisomerase II was shown to be present also in fly heads which are composed entirely of nonproliferative tissues.

Animals↗
PubMed1988

Drosophila homolog of the murine Int-1 protooncogene.

We have isolated phage clones from Drosophila melanogaster genomic and cDNA libraries containing a sequence homologous to the murine Int-1 protooncogene. The Drosophila gene is represented by a single locus at position 28A1-2 on chromosome 2. The gene is expressed as a 2.9-kilobase-long polyadenylylated mRNA in embryo, larval, and pupal stages. It is hardly detectable in adult flies. The longest open reading frame of the cDNA clone corresponds to a protein 469 amino acids long. Alignment of the predicted amino acid sequences shows that the Drosophila protein is 86 amino acids longer than its murine counterpart. In spite of the difference in length, the two proteins are highly conserved with an overall sequence homology of 54%. Both Drosophila and murine Int-1 proteins begin with a hydrophobic leader sequence and contain cysteine residues and sites for glycosylation (four in the murine protein and one in the Drosophila protein) in conserved positions, suggesting that they play important functional roles.

Amino Acid Sequence↗