Biomedical subjects

P Schedl

Publications and source records attributed to P Schedl.

At least 73 records · Page 4Linked to original sources

PubMed1990

Molecular analysis of odd-skipped, a zinc finger encoding segmentation gene with a novel pair-rule expression pattern.

odd-skipped (odd) is one of eight known pair-rule genes that establish portions of alternating segments during Drosophila embryogenesis; odd mutant embryos exhibit pattern defects in anterior regions of odd-numbered segments. P element transposon tagging was used to clone 25 kb of DNA from the odd genomic region. Molecular analysis of phenotypic revertants confirmed that the P element used to tag the locus was responsible for the corresponding odd mutation, and significant structural changes were identified in two additional odd mutants. Several cDNA clones derived from a 2.2 kb embryonic transcript were isolated and the longest was sequenced. The predicted odd protein of 392 amino acids is highly basic and contains four tandem Cys-Cys/His-His zinc finger repeats, consistent with a presumed function for odd as a DNA binding protein and transcriptional regulator. In situ hybridization analysis indicated that odd transcripts accumulate in a dynamic pattern during early embryogenesis, with two temporally distinct modes of expression. The first mode results in a 'pair-rule' pattern of seven stripes at the blastoderm stage, representing the expected double segment periodicity. During gastrulation, the seven primary stripes are supplemented by secondary stripes which appear in alternate segments, resulting in the equivalent labeling of every segment in the extended germ band. Similar double to single segment transitions have now been reported for four of the six pair-rule genes analyzed.

Amino Acid Sequence↗

PubMed1990

Topoisomerase I and II cleavage of adenovirus DNA in vivo: both topoisomerase activities appear to be required for adenovirus DNA replication.

Sites of topoisomerase I and II cleavage across large portions of the adenovirus type 5 genome were mapped by using the drugs camptothecin and VM26, respectively. These drugs prolong the half-lives of the covalent DNA-protein intermediates in which the DNA is transiently cleaved, and so treatment with protein denaturants after exposure to the drugs leads to DNA strand scission at the site of topoisomerase cleavage. Strong topoisomerase II cleavage sites occurred in clusters throughout the regions examined, including both transcribed regions and transcriptional control regions. The efficiency of topoisomerase II cleavage increased as the rate of adenovirus DNA replication increased and then decreased with the decreasing rate of replication late in the infection cycle. The increase was not dependent on expression of the E1A gene, whose products activate transcription of the early viral genes. Positions of topoisomerase II cleavage sites did not vary during the infection. Topoisomerase I cleavage sites were also found throughout the examined regions, with the strongest sites occurring near the ends of the transcription units. Topoisomerase I cleavage in the E1 region occurred much more frequently than topoisomerase II cleavage, was not dependent on E1A gene expression, and remained at a similar level from the early viral phase into the late viral phase. Treatment of infected cells with either drug prevented efficient replication of adenovirus DNA. Inhibition of topoisomerase I activity led to an immediate cessation of adenovirus DNA replication, while inhibition of topoisomerase II blocked replication only after completion of approximately one additional round.

Adenovirus Early Proteins↗

PubMed1989

The cloning of the Bar region and the B breakpoint in Drosophila melanogaster: evidence for a transposon-induced rearrangement.

We have cloned the B breakpoint in Drosophila melanogaster using DNA from a P-M-induced revertant of B, which has a P element inserted at the B breakpoint. The analysis of the B DNA reveals that there is a transposable element, B104, right at the breakpoint. This suggests that this element may have been involved in the generation of the B breakpoint and the associated tandem duplication. One possible mechanism to generate the B duplication is a recombination event between two B104 elements, one at 16A1 and the other at 16A7. DNA sequencing data of the junctions of the B104 element support this model. Four partial revertants of B are the result of insertions of transposable elements very close to the B breakpoint. This supports the hypothesis that the breakpoint is the cause of the B mutation. The clones from B were used to isolate wild-type clones from 16A1, the location of the Bar gene. Four rearrangement breakpoints associated with various Bar mutations map within a 37-kb region, suggesting that the Bar gene is very large.

Animals↗

PubMed1989

Molecular analysis of the armadillo locus: uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene.

During Drosophila embryogenesis, the segment polarity genes are required for the formation of specific pattern domains within each segment. Mutations in the armadillo (arm) gene primarily affect the posterior part of the segment and lead to the production of anterior structures within this region. To examine the molecular basis for these effects, we have cloned the arm region and identified the gene by germ-line transformation. The arm gene produces two types of very abundant 3.2-kb transcripts that differ only in their first exons. These RNAs appear to be formed by independent transcriptional initiation but have similar patterns of expression throughout development. Both arm transcripts are present in virtually all of the cell types contained in embryos, third-instar larvae, and adult ovaries, suggesting that arm may be required in all cells. In addition, the arm transcripts are uniformly distributed in embryonic segments, so the regional pattern defects associated with its embryonic phenotype may result from interactions between arm and other localized factors. Both arm RNAs encode the same 91-kD polypeptide. This protein has no probable secretory or membrane-spanning regions and contains a series of novel internal repeats that are conserved in sequence, length, and spacing. Considering these results and previous genetic observations, we discuss potential roles for the arm gene in pattern formation processes.

Amino Acid Sequence↗

PubMed1989

The Drosophila female-specific sex-determination gene, Sex-lethal, has stage-, tissue-, and sex-specific RNAs suggesting multiple modes of regulation.

For proper sexual development of females, the Sex-lethal (Sxl) gene must be activated early in development and remain on during the rest of the life cycle. Conversely, in males, Sxl must remain functionally off through development. Here, we show that the Sxl transcription unit spans a DNA segment of greater than 20 kb and encodes at least 10 distinct, but overlapping, RNA species. These RNAs range in size from 4.4 to 1.7 kb and exhibit sex, stage, and tissue specificity. Six RNAs, three female specific and three male specific, are first detected by midembryogenesis and persist through the adult stage: Their expression reflects the on/off regulation of Sxl's activity at the level of sex-specific alternate splicing. Four Sxl RNAs are found in adult females. Two of these RNAs are dependent on the presence of a functional germ line and may be relevant to Sxl's role in adult germ-line development. All four are present in unfertilized eggs. Finally, three Sxl RNAs are found only transiently during very early embryogenesis; we suggest that the expression of these RNAs may reflect an early regulation of Sxl at the level of transcription and that these transcripts are involved in the initial selection of the Sxl activity state in response to the primary sex-determination signal, the X/A ratio.

Animals↗

PubMed1988

Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins.

The switch gene, Sex-lethal (Sxl), controls sexual development and dosage compensation. It must be active in females and inactive in males throughout development. Analysis of Sxl cDNAs shows that this on/off regulation may be explained by differential RNA splicing; only female transcripts appear to encode functional products, whereas all male transcripts contain an exon that truncates the open reading frame. The functional female product shows sequence similarities with ribonucleoproteins, suggesting that it is an RNA binding protein. Thus, we propose that Sxl encodes a factor that interacts with both its own pre-mRNA (accounting for positive autoregulation) and that of downstream genes to confer female-specific splicing. In this way, a single, simple mechanism could account for both the maintenance and expression of the sexually determined state.

Amino Acid Sequence↗

PubMed1988

The dorsal protein is distributed in a gradient in early Drosophila embryos.

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is closely related to the vertebrate proto-oncogene c-rel. Genetic experiments suggest that dorsal represents one of the last (if not the last) steps in the maternal pathway involved in establishing dorsal-ventral polarity in the early embryo. Even though the dorsal RNA is uniformly distributed in the embryo, we have found that the dorsal protein is specifically localized in peripheral nuclei of syncytial and cellular blastoderm stage embryos, and it is distributed in a ventral-to-dorsal gradient. These findings suggest possible mechanisms for how the dorsal protein may communicate maternal positional information to the zygotic genome.

Animals↗

PubMed1988

Molecular characterization of daughterless, a Drosophila sex determination gene with multiple roles in development.

The daughterless (da) gene in Drosophila acts both maternally and zygotically to provide essential functions during development. Maternal da+ expression is required by embryos for the regulation of sex determination and dosage compensation. Zygotic da+ expression is required throughout development--early for the formation of the peripheral nervous system and perhaps for the proper functioning of genes in heterochromatic regions of the genome; during larval stages for growth and differentiation of the future adult epidermis; and in the somatic part of the gonad of adult females for proper egg membrane synthesis. Here we describe the cloning of da by the transposon tagging approach as well as some aspects of the molecular characterization of wild-type and mutant alleles. Despite the multiple developmental roles of da, the organization and expression of this gene appear relatively simple at this level of analysis. The gene codes for two transcripts, present in both sexes and at all stages of development. The nucleotide sequence of a nearly full-length cDNA predicts a protein product of 710 amino acids that shares sequence similarity with the His-Pro repeat of the Drosophila genes bicoid and paired. Two partial-loss-of-function da mutations (one of which is temperature sensitive) appear to be caused by DNA insertions in the 5'-untranslated region of the gene.

Alleles↗

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↗

PubMed1987

Functional changes associated with structural alterations induced by mobilization of a P element inserted in the Sex-lethal gene of Drosophila.

Genetic analysis of rearrangements within the multifunctional sex determining gene Sex-lethal has allowed correlation of changes in specific functions with DNA alterations. Rearrangements were isolated by mobilization of a P element which is on the 5' side of the gene, at coordinate 0. Previous work has shown that rearrangements associated with alterations in Sxl gene function are found within an 11-kb region between coordinates-11 and 0. Here it is shown that insertion of foreign DNA, per se, at coordinate 0 is compatible with wild-type gene function. However, deletion of sequences on either side of this point generates a mutant phenotype. Deletions extending distally beyond coordinate -6.5 kb result in a null phenotype, whereas smaller distal deletions or proximal deletions eliminate only some Sxl functions.

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↗

PubMed1986

Molecular organization of the rudimentary gene of Drosophila melanogaster.

We have determined the molecular structure of the rudimentary gene of Drosophila melanogaster. The transcription unit, which extends over 14,000 bases, is split into seven exons and six introns. Sequences presenting homologies with a TATA box and a CAT box are located upstream from the transcription initiation site, and there is a consensus polyadenylation signal in the vicinity of the 3' end of the messenger RNA coding sequence. Short intronic sequences fit well with consensus signals found in other eukaryotic genes and involved in the splicing of the precursor messenger RNA. Partial genomic and complementary DNA sequencing has revealed stretches of amino acid sequence homologies with the corresponding enzymes in Saccharomyces cerevisiae and Escherichia coli. This is in good agreement with the genetic map of the locus, which indicates that the enzymic activities encoded by the gene are organized linearly along the DNA.

Amino Acid Sequence↗

PubMed1986

Organization of transcription units around the Drosophila melanogaster rudimentary locus and temporal pattern of expression.

The molecular organization of 90 kb of DNA derived from a region of the X chromosome that encompasses the rudimentary locus of D. melanogaster is presented. This segment spans the cytogenetic region 14F2-3 to 15A1-2, and there are, in addition to the rudimentary gene several transcription units present, whose functions are still unknown. We have determined the pattern of expression of all these genes at several stages of development, and found that they all show a different temporal modulation of their activity. The accumulation of the r product correlates well with the enzymatic activity determined for the protein product of the gene, being highest in very early embryos and adult females.

Animals↗

PubMed1986

Hybrid dysgenesis-induced revertants of insertions at the 5' end of the rudimentary gene in Drosophila melanogaster: transposon-induced control mutations.

Mutations in the 5' control region of the rudimentary (r) gene of Drosophila melanogaster were generated by using hybrid dysgenesis to mobilize P elements that were already inserted within this region. Eighteen new mutations out of 7793 chromosomes were isolated. Among the mutations were small insertions, deletions and inversions. All five of the deletions deleted into the 5' coding region of the r gene resulting in a severe mutant phenotype. The inversions and small insertions left the coding region intact, but altered the 5' control region. Northern analyses of the message levels in adult females indicated that these mutations alter the amount of the wild-type rudimentary message. These data also indicated that there is a region about 800 bp upstream of the start of transcription which is necessary for normal r expression. This region is not involved with the temporal and spatial regulation of r gene expression, but with the quantitative expression. An analysis of the DNA changes associated with each of the mutations demonstrates that P elements usually mobilize in an imprecise manner. Of the 22 mutations that have been isolated to date, only three are precise excisions. This means that P elements are potent mutators of the genome.

Animals↗

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↗