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Biomedical subjects

A Udvardy

Publications and source records attributed to A Udvardy.

At least 19 recordsLinked to original sources
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↗
PubMed1987

Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding.

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.

Animals↗
PubMed1986

Topoisomerase II cleavage in chromatin.

We have examined the effect of the anti-tumor drug VM-26 on purified Drosophila topoisomerase II, and used this drug to map (putative) topoisomerase II cleavage sites in chromatin. These studies indicate that VM-26 interferes with the strand breakage-rejoining catalytic cycle. VM-26 appears to stabilize the topoisomerase-II-cleavable complex and markedly enhances the formation of double-strand breaks in naked DNA. VM-26 also stimulates the formation of double-strand breaks in isolated Drosophila nuclei. Analysis of the parameters of the VM-26-stimulated cleavage reaction in nuclei strongly suggests that the double-strand scissions are generated by endogenous topoisomerase II. Finally, we have examined the distribution of (putative) cleavage sites for endogenous topoisomerase II in the chromatin of the 87A7 heat shock locus and the histone repeat unit. We have found that there are prominent VM-26-induced cleavage products from the 5' ends of the 87A7, the two heat shock protein 70 genes, and in the intergenic spacer separating these genes. Moreover, the pattern of VM-26-induced cleavage products is altered in nuclei prepared from heat-shocked cells. In the case of the histone repeat unit, only minor VM-26-induced cleavage products are observed in nuclei (in spite of the fact that experiments on naked DNA indicate that the histone repeat contains many major cleavage sites for purified topoisomerase II). These findings suggest that the nucleoprotein organization of different DNA segments may be important in determining whether specific sites are accessible to endogenous topoisomerase II in nuclei.

Adenosine Triphosphate↗
PubMed1985

The 87A7 chromomere. Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains.

The chromatin fiber of eukaryotic chromosomes is thought to be organized into a series of discrete domains or loops. To learn more about these large-scale structures, we have examined the sequence and chromatin organization of the DNA segments surrounding the two hsp 70 genes at the Drosophila melanogaster cytogenetic locus 87A7. These studies indicate that this heat shock locus is flanked on both the proximal and distal sides by novel chromatin structures, which we have called, respectively, scs and scs' (specialized chromatin structures). Each structure is defined by two sets of closely spaced nuclease-hypersensitive sites arranged around a central nuclease-resistant segment. Our findings suggest that these two structures define the proximal and distal boundaries of the 87A7 chromomere and, hence, may be one of the first examples of anchor points for the organization of eukaryotic chromosomes into a series of discrete higher order domains. Moreover, these structures may provide focal points both for the decondensation of the chromomere when the hsp 70 genes are induced by heat shock and for the subsequent rewinding and condensation of the chromomere during recovery from heat shock.

Animals↗
PubMed1985

Neurospora crassa and S1 nuclease cleavage in hsp 83 gene chromatin.

We have examined the distribution of Neurospora crassa and S1 nuclease cleavage products in the chromatin of the hsp 83 heat shock gene from the Drosophila melanogaster cytogenetic locus 63 BC. Both of these nucleases generate double strand breaks in chromatin at specific sites close to the 5' end of the hsp 83 gene. With N. crassa nuclease we observe one major 5' fragment which is derived from nuclease cleavage in a DNA segment mapping approximately 120 base-pairs from the beginning of the transcription unit. With S1 nuclease we observe one major fragment which overlaps the transcription start site. In addition to the major hypersensitive sites at the beginning of the gene, the hsp 83 transcription unit is also sensitive to attack by these nucleases both before and after heat shock; however, the yield of cleavage products from within the gene is considerably greater after heat induction.

Animals↗
PubMed1985

Chromatin structure of the 87A7 heat-shock locus during heat induction and recovery from heat shock.

We have examined the chromatin organization of the 87A7 heat-shock locus (which contains two hsp 70 genes transcribed in opposite orientation) as a function of the time of heat induction and during the course of recovery from heat shock. Our studies show that both induction and recovery from heat shock are accompanied by highly specific alterations in the nucleoprotein structure of this locus. Moreover, these changes parallel the transcriptional activity of the hsp 70 heat-shock genes. We have also examined the effect of inhibitors of transcription and translation. Cycloheximide, an inhibitor of translation, blocks both the attenuation of the heat-shock response (which occurs after a long-term incubation at elevated temperatures) and the re-establishment of the pre-induced chromatin organization of the locus during recovery from heat shock. Actinomycin D, an inhibitor of transcription, prevents some but not all of the alterations in chromatin structure which normally accompany heat induction.

Animals↗
PubMed1985

Novobiocin blocks the Drosophila heat shock response.

In the studies reported here we show that the antibiotic novobiocin, an in vitro inhibitor of topoisomerase II, blocks the Drosophila heat shock response. If novobiocin is added prior to induction, there is no detectable expression of the Drosophila heat shock genes. Moreover, analysis of the chromatin organization of the 87A7 heat shock locus indicates that the antibiotic prevents the structural alterations which normally accompany heat induction. When novobiocin is added after induction, transcription appears to be rapidly turned off, and the chromatin organization of the 87A7 locus is "fixed" in an "active" configuration. Novobiocin also prevents the re-establishment of the pre-induced 87A7 chromatin organization which occurs during recovery from heat shock. We have also presented data suggesting that this antibiotic blocks transcription at 25 degrees C. These findings raise the possibility that topoisomerase II may be required in eukaryotes for both gene activation and deactivation.

Autoradiography↗
PubMed1985

Novel partitioning of DNA cleavage sites for Drosophila topoisomerase II.

We have examined the long-range distribution of double-stranded DNA cleavage sites for Drosophila melanogaster topoisomerase II. These studies reveal a novel partitioning of preferred topoisomerase II cleavage sites. In the eukaryotic DNAs examined, major cleavage sites were typically found in nontranscribed spacer segments and close to the 5' and 3' boundaries of genes. In contrast, there were few if any prominent cleavage sites within genes. In addition, most of the major topoisomerase II cleavage sites closely corresponded to naked DNA hypersensitive sites for the prokaryotic enzyme, micrococcal nuclease.

Animals↗
PubMed1984

Transcriptionally active chromatin is sensitive to Neurospora crassa and S1 nucleases.

We have examined the distribution of Neurospora crassa and S1 nuclease cleavage products in the chromatin of the 87A7 heat shock locus of Drosophila melanogaster. Both of these nucleases generate single and double-strand breaks in chromatin at specific sites in the 87A7 locus. Before heat induction, we find that the 5' ends of the two 87A7 hsp 70 genes contain N. crassa and S1 nuclease hypersensitive sites, while there are only a few cleavage products from elsewhere in the locus. With N. crassa nuclease, we observe one major 5' fragment, and this is derived from cleavage in a DNA segment mapping about 90 to 115 base-pairs from the beginning of the transcription unit. With S1 nuclease, we find two 5' cleavage products. The first maps about 120 to 130 base-pairs from the beginning of the gene. Interestingly, this site is also sensitive to S1 nuclease in supercoiled but not linear naked DNA. The other fragment maps very close to the transcription start site (approximately 0 to -15 base-pairs). After heat induction, there is a transition in the chromatin architecture of 87A7. First, there is a marked reduction in the yield of the prominent 5' N. crassa and S1 nuclease fragments. Second, the entire hsp 70 gene, as well as the spacer DNA just downstream from the 3' end of the gene, becomes highly sensitive to both of these nucleases.

Animals↗
PubMed1984

Ribosomal RNA genes of Drosophila melanogaster have a novel chromatin structure.

We have examined the chromatin organization of the Drosophila melanogaster ribosomal RNA genes using both micrococcal nuclease and DNase I. Several findings are of interest. First, the transcribed DNA segments of the rRNA repeat unit appear to be packaged into an unstable or "multiphasic" nucleosome structure. Second, the 5' end of the transcription unit is preferentially exposed to nuclease attack. Third, the non-transcribed spacer immediately upstream from the transcription start site has a novel chromatin organization with micrococcal nuclease and DNase I cleavage sites spaced at intervals of about 240 base-pairs. This unusual fragment distribution appears to reflect the underlying sequence organization of the spacer DNA segment, which consists of a series of tandemly repeated 239 base-pair sequence blocks. We have also examined the chromatin structure of the rRNA repeat unit after extraction of nuclei with different concentrations of salt. Our results suggest that the higher order structures may be of importance in determining the novel chromatin organization of the rRNA repeat unit.

Animals↗
PubMed1984

Chromatin organization of the 87A7 heat shock locus of Drosophila melanogaster.

We have examined the chromatin structure of the hsp 70 gene complex at the 87A7 heat shock locus of Drosophila melanogaster. Our results indicate that this locus has a complex chromatin organization. Heat induction causes highly specific alterations in the chromatin throughout the locus. There are major changes within the heat shock gene transcription units, and in both the upstream and downstream flanking spacers.

Animals↗
PubMed1983

Structural polymorphism in DNA.

We have used the enzyme micrococcal nuclease and the methylating reagent dimethyl sulfate to examine the structural properties of eukaryotic DNAs. Our studies demonstrate extensive structural polymorphism in the DNA double helix. Moreover, we find that the distribution of helical variants is in some instances correlated with the functional organization of the DNA. These observations raise the possibility that eukaryotic DNAs may be organized into discrete functional units having characteristic structural properties. In addition, we find that boundaries between different functional units are typically marked by DNA segments having unusual conformational properties. Such structural perturbations could serve as signals in the utilization of genetic information in eukaryotes, and may be important in a variety of different protein-DNA interactions.

Animals↗