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H M Krause

Publications and source records attributed to H M Krause.

29 records · Page 2Linked to original sources
PubMed1993

Control of segmental asymmetry in Drosophila embryos.

During Drosophila development, an important aspect of body patterning is the division of the embryo into repeating morphological units referred to as parasegments. The parasegmental domains are first defined at the blastoderm stage by alternating stripes of transcripts encoded by the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) and later by stripes encoded by the segment polarity genes engrailed (en) and wingless. Here, we show that the runt gene (run) is required to generate asymmetries within these parasegmental domains. Using a heat-shock-inducible run transgene, we found that ectopic run expression leads to rapid repression of eve stripes and a somewhat delayed expansion of ftz stripes. Unexpectedly, we also found that ectopic run was a rapid and potent repressor of odd-numbered en stripes. Two remarkably different segmental phenotypes were generated as a consequence of these effects. In solving the mechanisms underlying these phenotypes, we discovered that the positioning of en stripes is largely determined by the actions of negative regulators. Our data indicate that run is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of run and odd by the product of the eve gene. The spatial restriction of gene expression via negative and double negative pathways such as these is likely to be a common theme during development.

Animals↗
PubMed1992

Homeodomain-independent activity of the fushi tarazu polypeptide in Drosophila embryos.

The Drosophila segmentation gene fushi tarazu (ftz) encodes a homeodomain-containing protein, ftz, that can act as a DNA-binding activator of transcription. In the developing embryo, ftz is expressed in seven stripes which correspond to the even-numbered parasegments. These parasegments are missing in ftz- embryos. When ftz is expressed throughout blastoderm embryos under the control of a heat-shock promoter, the odd-numbered parasegments are lost. This 'anti-ftz' phenotype has been attributed to autoactivation of the endogenous ftz gene by the ectopically expressed protein. Here we show that the same phenotype is induced by ectopic expression of a ftz polypeptide containing a deletion in the homeodomain. Thus, ftz can alter gene expression without binding directly to DNA.

Amino Acid Sequence↗
PubMed1992

Modifiers of bx1 alter the distribution of Ubx proteins in haltere imaginal discs of Drosophila.

The bithorax (bx) mutations in the Ultrabithorax (Ubx) gene of Drosophila melanogaster cause homeotic transformations of anterior third thoracic structures (T3a) toward anterior second thoracic structures (T2a) in the adult fly. A corresponding loss of Ubx protein expression in T3a of bx imaginal discs has been observed (White and Wilcox, 1985). We describe two genetic loci which modify the bx-induced transformation. A locus which we map very close to the pink peach (pp) gene suppresses the bx1 phenotype. In contrast, mutations in the suppressor of sable (su(s)) gene enhance the bx1 phenotype. A correlation was observed between patterns of Ubx protein expression and the phenotypic transformations observed.

Animals↗
PubMed1992

Concentration-dependent activities of the even-skipped protein in Drosophila embryos.

The Drosophila pair-rule gene even-skipped (eve) encodes a homeo-domain-containing protein (Eve) that is required for the development of both odd- and even-numbered parasegments. We have used a heat shock-inducible eve transgene to study the regulatory functions of Eve in vivo. Transcripts encoded by eight other segmentation genes were monitored for changes in distribution and abundance following short pulses of ectopic Eve expression. Two tiers of response times appeared to distinguish between genes that were direct [fushi tarazu (ftz), odd-skipped (odd), runt (run), paired, and wingless] and indirect [eve, hairy, and engrailed (en)] targets of Eve. Genes that appeared to be directly regulated by Eve were differentially repressed in a concentration-dependent fashion. Interestingly, the run and ftz genes could also be activated by Eve during a brief 20- to 30-min stage in development. The delayed actions upon the eve and en genes appeared to be mediated by run and odd. As in eve- embryos, these effects on segmentation gene expression patterns caused defects in both odd- and even-numbered parasegments. Four sequential phenotypes could be induced, each of which was attributable to the altered expression of a unique subset of target genes.

Animals↗
PubMed1989

Supercoiling and integration host factor change the DNA conformation and alter the flow of convergent transcription in phage Mu.

Transcription of bacteriophage Mu is modulated by its repressor, by negative supercoiling, and by the Escherichia coli protein integration host factor (IHF). Two converging Mu promoters regulate lytic and lysogenic development. The influence of IHF on these convergent promoters depended on the DNA conformation. When Mu operator DNA changed from the relaxed to the negative supercoiled form, IHF changed from a stimulatory factor to an inhibitor of the repressor promoter, and the ratio of the lytic transcript relative to the repressor transcript increased by 40-fold. Flexibility in Mu operator DNA was demonstrated by an unusual supercoil-induced DNA conformation, which was detectable by chemical modification with bromoacetaldehyde or digestion with P1 nuclease. IHF binding adjacent to this site induced dramatic bending of Mu DNA. A topological model we call a superloop is proposed to explain the effect of IHF on Mu transcription in vitro and on the lytic-lysogeny decision of the virus grown in IHF and gyrase mutants.

Bacterial Proteins↗
PubMed1989

Stage-specific phosphorylation of the fushi tarazu protein during Drosophila development.

The regulatory protein encoded by the fushi tarazu (ftz) gene is expressed during three different stages of Drosophila embryogenesis in three different developing tissues. Previously, we demonstrated that ftz protein ectopically expressed throughout developing embryos under the control of an hsp70 heat shock promoter is heavily modified. Here we show that these negatively charged isoforms of the protein are the result of phosphorylation at as many as 16 sites. Phosphate groups could be removed in vitro by treatment with various phosphatases and could be added in vivo by incubating embryo-derived cells or nuclei in the presence of [32P]-orthophosphate. Phosphoamino acid analysis of immunoprecipitated ftz protein yielded both phosphoserines and phosphothreonines at a ratio of approximately 1:1. Interestingly we find that the endogenous ftz protein is also phosphorylated at multiple sites and that different subsets of the phosphoisoforms occur during different stages of development.

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↗
PubMed1988

The location, modification, and function of the fushi tarazu protein during Drosophila embryogenesis.

The fushi tarazu (ftz) protein of Drosophila is required during embryogenesis for the process of body segmentation. In order to study the biochemical properties of the ftz protein, ftz cDNA was expressed in E. coli, and the protein 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 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 calculate that there are 15,000 molecules of protein per ftz-expressing nucleus. Since embryonic ftz protein migrates more slowly on SDS polyacrylamide gels than protein expressed either in E. coli, or in vitro in a reticulocyte lysate system, it is apparently modified in the embryo. Two-dimensional gel electrophoresis followed by Western blotting resolves the protein into a series of isoforms which have variable charge and electrophoretic mobility. When compared in its ability to bind DNA in a sequence-specific manner, it was also found that ftz protein partially purified from embryos binds with greater specificity than its bacterially expressed counterpart. In this paper, we demonstrate that embryonic ftz protein binds to a specific region within the ftz enhancer element. The potential relationship between these observations is discussed.

Animals↗
PubMed1986

Positive and negative regulation of the Mu operator by Mu repressor and Escherichia coli integration host factor.

Bacteriophage Mu utilizes two converging promoters to regulate the lytic and lysogenic pathways. Messenger RNA encoding the repressor gene is synthesized leftward from a promoter (PcM) located 1066 base pairs from the Mu left end. This transcript overlaps and is complementary to RNA synthesized rightward from the early promoter (PE) for transposase and replication proteins which initiates transcription at base pair 1028. Purified Mu repressor binds to three distinct operator sites (O1, O2, and O3); repressor binding at O2 blocks RNA polymerase binding at PE and repressor binding at O3 blocks RNA polymerase binding at PcM. O1 and O2 have higher affinity for repressor than O3, and transcription from PE is blocked at repressor concentrations that do not affect PcM. Thus, maintenance of the lysogenic state and autoregulation of the repressor gene is achieved by RNA polymerase transcription through DNA-repressor ensembles at O1 and O2. Integration host factor (IHF) encoded by the Escherichia coli him A and him D genes binds to Mu operator DNA between sites O1 and O2. IHF enhances transcription from PE 3-5-fold on supercoiled plasmid substrates in vitro and in Mu monolysogens in vivo. In vitro, IHF simultaneously decreases transcription from PcM 5-10-fold which results in a 25-fold change in lytic transcription relative to repressor transcription. A model for regulating the Mu lysis-lysogeny decision is presented.

Bacterial Proteins↗
PubMed1983

The early promoter of bacteriophage Mu: definition of the site of transcript initiation.

The early promoter of bacteriophage Mu has been identified and characterized by a nitrocellulose filter binding assay and analysis of RNA products transcribed in vitro and in vivo. A tight, heparin resistant, RNA polymerase-DNA binding site overlaps the left Mu Hind III site. In vitro [gamma 32P] ATP initiated transcription begins approximately 25 nucleotides to the right of the Hind III restriction site. Dinucleotide initiation with ApA and S1 mapping of in vitro and in vivo transcripts shows that transcription is initiated 1028 base pairs from the Mu genetic left end.

Base Sequence↗