Biomedical subjects

P Schedl

Publications and source records attributed to P Schedl.

At least 55 records · Page 3Linked to original sources

PubMed1994

The Drosophila miti-mere gene, a member of the POU family, is required for the specification of the RP2/sibling lineage during neurogenesis.

The Drosophila POU gene miti-mere (previously known as pdm2) has a complex spatial and temporal pattern of expression during early development; initially it is expressed in gap-gene-like pattern, then in 14 stripes and finally in a subset of the cells in the developing CNS and PNS. To study the function of this gene during development, we generated a 'synthetic anti-morphic mutation' by expressing a truncated version of the miti protein from a constitutive hsp83 and an inducible hsp70 promoter. We show that these delta miti transgenes behave like classical antimorphic mutations. Using these dominant negative transgenes, together with deletions and a duplication for the gene, we show that miti is required during segmentation and neurogenesis. We have also used temperature-shift experiments with the hsp70 delta miti transgene to demonstrate that miti function in segmentation is distinct and separable from its function during neurogenesis. In segmentation, miti appears to be required in the specification of the segments A2 and A6. In the CNS, miti is required for the elaboration of the NB4-2-->GMC-1-->RP2/sib lineage. miti is initially required in this lineage to establish the identity of the parental ganglion mother cell, GMC-1. miti must then be down-regulated to allow the asymmetric division of GMC-1 into the RP2 and its sibling cell.

Animals↗

PubMed1993

The bluetail transposon: evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex.

An extremely large cis-regulatory region generates the parasegment-specific expression patterns of the homeotic genes in the bithorax complex. We present evidence supporting the idea that this cis-regulatory region is subdivided into independent cis-regulatory domains. We describe a Ubx-lacZ transposon which is inserted into one of these domains, iab-7. The PS12-specific pattern of LacZ expression from this reporter indicates that it is subject to the control of the iab-7 cis-regulatory domain, but is protected from the effects of adjacent regulatory domains. Protection on the proximal side appears to be provided by the Fab-7 boundary element. Deletion of this boundary results in the ectopic activation of iab-7 in PS11 (where the iab-6 cis-regulatory domain normally functions). We show that the Fab-7 boundary, like other boundaries, has an unusual chromatin structure.

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↗

PubMed1993

Sex-lethal autoregulation requires multiple cis-acting elements upstream and downstream of the male exon and appears to depend largely on controlling the use of the male exon 5' splice site.

The on/off state of the binary switch gene Sex-lethal (Sxl), which controls somatic sexual development in Drosophila melanogaster, is regulated at the level of alternative splicing. In males, in which the gene is off, the default splicing machinery produces nonfunctional mRNAs; in females, in which the gene is on, the autoregulatory activity of the Sxl proteins directs the splicing machinery to produce functional mRNAs. We have used germ line transformation to analyze the mechanism of default and regulated splicing. Our results demonstrate that a blockage mechanism is employed in Sxl autoregulation. However, in contrast to transformer, in which Sxl appears to function by preventing the interaction of splicing factors with the default 3' splice site, a different strategy is used in autoregulation. (i) Multiple cis-acting elements, both upstream and downstream of the male exon, are required. (ii) These cis-acting elements are distant from the splice sites they regulate, suggesting that the Sxl protein cannot function in autoregulation by directly competing with splicing factors for interaction with the regulated splice sites. (iii) The 5' splice site of the male exon appears to be dominant in regulation while the 3' splice site plays a subordinate role.

Animals↗

PubMed1993

Regulated splicing of the Drosophila sex-lethal male exon involves a blockage mechanism.

In Drosophila melanogaster, sex determination in somatic cells is controlled by a cascade of genes whose expression is regulated by alternative splicing [B. S. Baker, Nature (London) 340:521-524, 1989; J. Hodgkin, Cell 56:905-906, 1989]. The master switch gene in this hierarchy is Sex-lethal. Sex-lethal is turned on only in females, and an autoregulatory feedback loop which controls alternative splicing maintains this state (L. R. Bell, J. I. Horabin, P. Schedl, and T. W. Cline, Cell 65:229-239, 1991; L. N. Keyes, T. W. Cline, and P. Schedl, Cell 68:933-943, 1992). Sex-lethal also promotes female differentiation by controlling the splicing of RNA from the next gene in the hierarchy, transformer. Sosnowski et al. (B. A. Sosnowski, J. M. Belote, and M. McKeown, Cell 58:449-459, 1989) have shown that the mechanism for generating female transformer transcripts is not through the activation of the alternative splice site but by the blockage of the default splice site. We have tested whether an activation or a blockage mechanism is involved in Sex-lethal autoregulation. The male exon of Sex-lethal with flanking splice sites was placed into the introns of heterologous genes. Our results support the blockage mechanism. The poly(U) run at the male exon 3' splice site is required for sex-specific splicing. However, unlike transformer, default splicing to the male exon is sensitive to the sequence context within which the exon resides. This and the observation that the splice signals at the exon are suboptimal are discussed with regard to alternate splicing.

Alternative Splicing↗

PubMed1993

Expression of the Sex-lethal gene is controlled at multiple levels during Drosophila oogenesis.

In addition to controlling somatic sexual development in Drosophila melanogaster, the Sex-lethal (Sxl) gene is required for proper differentiation of female germ cells. To investigate its role in germ-line development, we have examined the expression of Sxl in wild-type ovaries and ovaries that are defective in early steps of germ cell differentiation. As in the soma, the basic mechanism for on/off regulation of Sxl relies on sex-specific processing of its transcripts in germ cells. One class of female-sterile mutations, which includes fs(1)1621 and the tumorous-ovary-producing allele of the ovarian tumor gene, otu1, is defective in the splicing process. These mutants have germ lines with high amounts of Sxl RNA spliced in the male mode and a severe reduction of protein levels in the germ cells. Another class of female-sterile mutations produces a phenotype similar to that seen in fs(1)1621 and otu1 but appears to express normal levels of Sxl protein in the germ cells. However, this second class does not show the changes in protein distribution normally observed in wild-type germ cells. In the wild-type germarium, the non-differentiated germ cells show a strong cytoplasmic accumulation of Sxl protein followed, as the germ cells differentiate, by a dramatic reduction and redistribution of the protein into nuclear foci. Interestingly, two female-sterile alleles of Sxl, Sxlf4 and Sxlf5 belong to the second class, which shows persistent cytoplasmic accumulation of Sxl protein. These Sxl female-sterile mutants encode an altered protein indicating that Sxl regulates processes that eventually lead to the changes in Sxl protein distribution. Lastly, we demonstrate that during the final stages of oogenesis several mechanisms must operate to prevent the progeny from inheriting Sxl protein. Conceivably, this regulation safeguards the inadvertent activation of the Sxl autoregulatory feedback loop in the male zygote.

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

The primary sex determination signal of Drosophila acts at the level of transcription.

For Drosophila, the choice between male and female development is made by the switch gene, Sxl, in response to the X:A ratio. Once Sxl is turned on in females, it actively maintains the determined state, independent of the X:A signal, by a positive autoregulatory feedback loop in which Sxl proteins direct the female-specific splicing of Sxl transcripts. In this paper we have investigated the mechanism controlling pathway initiation. Our results suggest a two-step model for the initial activation of Sxl in females. In the first step, a special class of Sxl mRNAs is expressed in female embryos from an early promoter that responds to the genes signaling the X:A ratio. The proteins produced from these early mRNAs then initiate the autoregulatory loop by directing the female-specific processing of transcripts from the late Sxl promoter.

Animals↗

PubMed1992

Two non-gypsy rudimentary mutations and their suppression by mutations of suppressor of Hairy-wing in Drosophila.

Two spontaneous mutations of rudimentary, the gene encoding the first steps of de novo pyrimidine biosynthesis in Drosophila, are suppressed by mutant alleles of the suppressor of Hairy-wing locus. This interaction differs from typical su(Hw) suppression in that neither rudimentary allele is associated with an insertion of the gypsy retrotransposon. One allele, rsP1, appears to be a point mutation. Adult rsP1 homozygous females accumulate substantially less 7.3 kb rudimentary transcript than do wild-type females. The other allele, rsP2, is an insertion of an mdg3 retrotransposon in the sixth exon of rudimentary and in the opposite transcriptional orientation. This insertion divides the rudimentary locus into two separate, yet functional, transcription units by truncating transcription from the rudimentary promoter and promoting transcription of downstream rudimentary sequences. Phenotypic suppression of both rsP1 and rsP2 by mutant alleles of the suppressor of Hairy-wing locus correlates with enhanced levels of the rsP1 and rsP2 transcripts.

Alleles↗

PubMed1992

A group of scs elements function as domain boundaries in an enhancer-blocking assay.

Chromosomes of higher eukaryotes are thought to be organized into a series of discrete and topologically independent higher-order domains. In addition to providing a mechanism for chromatin compaction, these higher-order domains are thought to define independent units of gene activity. Implicit in most models for the folding of the chromatin fiber are special nucleoprotein structures, the domain boundaries, which serve to delimit each higher-order chromosomal domain. We have used an "enhancer-blocking assay" to test putative domain boundaries for boundary function in vivo. This assay is based on the notion that in delimiting independent units of gene activity, domain boundaries should be able to restrict the scope of activity of enhancer elements to genes which reside within the same domain. In this case, interposing a boundary between an enhancer and a promoter should block the action of the enhancer. In the experiments reported here, we have used the yolk protein-1 enhancer element and an hsp70 promoter:lacZ fusion gene to test putative boundary DNA segments for enhancer-blocking activity. We have found that several scs-like elements are capable of blocking the action of the yp-1 enhancer when placed between it and the hsp70 promoter. In contrast, a MAR/SAR DNA segment and another spacer DNA segment had no apparent effect on enhancer activity.

Animals↗

PubMed1992

The Drosophila orb gene is predicted to encode sex-specific germline RNA-binding proteins and has localized transcripts in ovaries and early embryos.

We report the identification of a new gene, orb, which appears to be expressed only in the germline and encodes ovarian- and testis-specific transcripts. The predicted proteins contain two regions with similarity to the RRM family of RNA-binding proteins but differ at their amino termini. In testes, orb RNA accumulates in the primary spermatocytes and at the caudal ends of the spermatid bundles. In ovaries, orb transcripts display an unusual spatial pattern of accumulation in the oocyte. Preferential accumulation in the oocyte of orb RNA is first detected in region 2 of the germarium and is dependent on Bicaudal-D and egalitarian. While in stage 7 egg chambers orb RNA is localized posteriorly in the oocyte, during stages 8-10 it is localized at the anterior of the oocyte, asymmetrically along the dorsal-ventral axis. In embryos the transcripts accumulate at the posterior end and are included in the pole cells. This pattern of localization and the similarity to RNA-binding proteins suggest that the orb gene product may mediate the localization of maternal RNAs during oogenesis and early embryogenesis.

Amino Acid Sequence↗

PubMed1991

Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila.

Sex-lethal is a binary switch gene that controls all aspects of Drosophila sexual dimorphism. It must be active in females and inactive in males. The on/off regulation reflects alternative RNA splicing in which full-length proteins are produced only in females. Here we investigate the role of Sxl in maintaining sexual pathway commitments. By ectopic expression of a female Sxl cDNA in transgenic male flies, we show that Sxl protein induces a rapid switch from male- to female-specific splicing. The ectopically expressed Sxl protein wil trans-activate an endogenous wild-type Sxl gene. This establishes a feedback loop in which Sxl proteins induce their own synthesis by directing the female-specific splicing of Sxl transcripts. We conclude that the female determined state is maintained by Sxl through positive autoregulation, while the male determined state is maintained by default.

Alleles↗

PubMed1991

A position-effect assay for boundaries of higher order chromosomal domains.

Eukaryotic chromosomes are thought to be organized into a series of discrete higher order chromatin domains. This organization is believed to be important not only in the compaction of the chromatin fiber, but also in the utilization of genetic information. Each domain would define an independent unit of gene activity, insulated from the regulatory influences of adjacent domains. Critical to this model of chromosome organization and function are the domain boundaries: the special nucleoprotein structures that delimit each higher order domain and segregate the chromosome into units of independent gene activity. In the work reported here we have tested whether two putative domain boundaries, scs and scs', from the Drosophila 87A7 heat shock locus can establish a domain of independent gene activity in vivo and insulate against chromosomal position effects.

Animals↗

PubMed1991

Developmental distribution of female-specific Sex-lethal proteins in Drosophila melanogaster.

The binary switch gene Sex-lethal (Sxl) must be on in females and off in males to allow the proper elaboration of the appropriate sexual developmental pathway in Drosophila melanogaster. Previous studies suggested a mechanism in which the on/off regulation of Sxl occurs post-transcriptionally at the level of RNA splicing. A critical prediction of this model is that functional Sxl proteins are absent in males but present in females. In this report we show that the expected full-length proteins are only present in female animals. Multiple forms of Sxl protein are found in females, some of which are expressed in a stage- and tissue-specific pattern. Consistent with a role of Sxl proteins in regulating alternate splicing, the proteins are localized in the nucleus where they exhibit a punctate staining pattern. Surprisingly, several minor Sxl proteins appear to be present in specific tissues of both sexes of adults. The possible origin of these species is discussed. We also show that Sxl expression in the early embryo is sex specific and depends on maternal daughterless and zygotic sisterless-b activity in accordance with the established roles of these genes as positive regulators of Sxl. The onset of Sxl expression in the germ line occurs later than that in the soma.

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↗

PubMed1991

The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides.

Sex-lethal (Sxl), a key sex determination gene in Drosophila melanogaster, is known to express a set of three early transcripts arising during early embryogenesis and a set of seven late transcripts occurring from midembryogenesis through adulthood. Among the late transcripts, male-specific mRNAs were distinguished from their female counterparts by the presence of an extra exon interrupting an otherwise long open reading frame (ORF). We have now analyzed the structures of the late Sxl transcripts by cDNA sequencing, Northern (RNA) blotting, primer extension, and RNase protection. The late transcripts appear to use a common 5' end but differ at their 3' ends by the use of alternative polyadenylation sites. Two of these sites lack canonical AATAAA sequences, and their use correlates in females with the presence of a functional germ line, suggesting possible tissue-specific polyadenylation. Besides the presence of the male-specific exon, no additional sex-specific splicing events were detected, although a number of non-sex-specific splicing variants were observed. In females, the various forms of late Sxl transcript potentially encode up to six slightly different polypeptides. All of the protein-coding differences occur outside the previously defined ribonucleoprotein motifs. One class of Sxl mRNAs also includes a second long ORF in the same frame as the first ORF but separated from it by a single ochre codon. The function of this second ORF is unknown. Significant amounts of apparently partially processed Sxl RNAs were observed, consistent with the hypothesis that the regulated Sxl splices occur relatively slowly.

Amino Acid Sequence↗

PubMed1990

Spatial expression of the Drosophila segment polarity gene armadillo is posttranscriptionally regulated by wingless.

armadillo (arm) is one of a group of Drosophila segment polarity genes that are required for normal patterning within the embryonic segment. Although arm RNA is uniformly distributed in embryos, arm protein accumulates at higher levels in regions that contain wingless, another segment polarity gene which encodes a secreted protein that regulates patterning via cell-cell communication. These local increases in arm protein require wingless activity, and mutations that alter wingless distribution produce corresponding changes in the arm protein pattern. These results suggest that wingless regulates accumulation of arm protein by a posttranscriptional mechanism. Two other segment polarity genes, porcupine and dishevelled, are required for this effect. We also show that arm protein is closely associated with the plasma membrane in virtually all cell types and often colocalizes with F-actin.

Animals↗

PubMed1990

Transcription of adenovirus and HeLa cell genes in the presence of drugs that inhibit topoisomerase I and II function.

The requirements for topoisomerases in transcription of adenovirus and HeLa cell genes were analyzed using drugs that specifically inhibit either topoisomerases I or II. Cleavage of viral DNA by topoisomerases in the presence of either camptothecin or VM26 was used to determine drug concentrations that led to maximal inhibition of ligation in the cleavage and ligation step of topoisomerase I or II respectively. Inhibition of topoisomerase II with VM26 did not cause a direct reduction in transcription of adenoviral genes or HeLa cell heat shock genes. VM26 did, however, interfere with other cellular processes. It reduced nucleoside uptake into HeLa cells from the medium, and it altered the normal nuclear to cytoplasmic ratio of specific RNAs. Treatment of cells with camptothecin to inhibit topoisomerase I reduced but did not abolish transcription of viral and HeLa cell genes. Transcription mediated by both RNA polymerases I and II was reduced. Topoisomerase II did not appear to substitute for topoisomerase I in transcription since treatment of cells with VM26 and camptothecin did not reduce transcript accumulation relative to cells treated with camptothecin alone.

Adenovirus Early Proteins↗