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W J Gehring

Publications and source records attributed to W J Gehring.

At least 73 records · Page 4Linked to original sources
PubMed1994

spalt encodes an evolutionarily conserved zinc finger protein of novel structure which provides homeotic gene function in the head and tail region of the Drosophila embryo.

The region specific homeotic gene spalt (sal) of Drosophila melanogaster promotes the specification of terminal pattern elements as opposed to segments in the trunk. Our results show that the previously reported sal transcription unit was misidentified. Based on P-element mediated germ line transformation and DNA sequence analysis of sal mutant alleles, we identified the transcription unit that carries sal function. sal is located close to the misidentified transcription unit, and it is expressed in similar temporal and spatial patterns during embryogenesis. The sal gene encodes a zinc finger protein of novel structure composed of three widely spaced 'double zinc finger' motifs of internally conserved sequences and a single zinc finger motif of different sequence. Antibodies produced against the sal protein show that sal is first expressed at the blastoderm stage and later in restricted areas of the embryonic nervous system as well as in the developing trachea. The antibodies detect sal homologous proteins in corresponding spatial and temporal patterns in the embryos of related insect species. Sequence analysis of the sal gene of Drosophila virilis, a species which is phylogenetically separated by approximately 60 million years, suggests that the sal function is conserved during evolution, consistent with its proposed role in head formation during arthropod evolution.

Amino Acid Sequence↗
PubMed1994

Homeodomain proteins in development and therapy.

Homeobox genes encode transcriptional regulators found in all organisms ranging from yeast to humans. In Drosophila, a specific class of homeobox genes, the homeotic genes, specifies the identity of certain spatial units of development. Their genomic organization, in Drosophila, as well as in vertebrates, is uniquely connected with their expression which follows a 5'-posterior-3'-anterior rule along the longitudinal body axis. The 180-bp homeobox is part of the coding sequence of these genes, and the sequence of 60 amino acids it encodes is referred to as the homeodomain. Structural analyses have shown that homeodomains consist of a helix-turn-helix motif that binds the DNA by inserting the recognition helix into the major groove of the DNA and its amino-terminal arm into the adjacent minor groove. Developmental as well as gene regulatory functions of homeobox genes are discussed, with special emphasis on one group, the Antennapedia (Antp) class homeobox genes and a representative 60-amino acid Antennapedia peptide (pAntp). In cultured neuronal cells, pAntp translocates through the membrane specifically and efficiently and accumulates in the nucleus. The internalization process is followed by a strong induction of neuronal morphological differentiation, which raises the possibility that motoneuron growth is controlled by homeodomain proteins. It has been demonstrated that chimeric peptide molecules encompassing pAntp are also captured by cultured neurons and conveyed to their nuclei. This may be of enormous interest for the internalization of drugs.

Amino Acid Sequence↗
PubMed1994

Three maternal coordinate systems cooperate in the patterning of the Drosophila head.

In contrast to the segmentation of the embryonic trunk region which has been extensively studied, relatively little is known about the development and segmentation of the Drosophila head. Proper development of the cephalic region requires the informational input of three of the four maternal coordinate systems. Head-specific gene expression is set up in response to a complex interaction between the maternally provided gene products and zygotically expressed genes. Several zygotic genes involved in head development have recently been characterized. A genetic analysis suggests that the segmentation of the head may use a mechanism different from the one acting in the trunk. The two genes of the sloppy paired locus (slp1 and slp2) are also expressed in the embryonic head. slp1 plays a predominant role in head formation while slp2 is largely dispensible. A detailed analysis of the slp head phenotype suggests that slp is important for the development of the mandibular segment as well as two adjacent pregnathal segments (antennal and ocular). Our analysis of regulatory interactions of slp with maternal and zygotic genes suggests that it behaves like a gap gene. Thus, phenotype and regulation of slp support the view that slp acts as a head-specific gap gene in addition to its function as a pair-rule and segment polarity gene in the trunk. We show that all three maternal systems active in the cephalic region are required for proper slp expression and that the different systems cooperate in the patterning of the head. The terminal and anterior patterning system appear to be closely linked. This cooperation is likely to involve a direct interaction between the bcd morphogen and the terminal system. Low levels of terminal system activity seem to potentiate bcd as an activator of slp, whereas high levels down-regulate bcd rendering it inactive. Our analysis suggests that dorsal, the morphogen of the dorsoventral system, and the head-specific gap gene empty spiracles act as repressor and corepressor in the regulation of slp. We discuss how positional information established independently along two axes can act in concert to control gene regulation in two dimensions.

Animals↗
PubMed1994

The Drosophila SRF homolog is expressed in a subset of tracheal cells and maps within a genomic region required for tracheal development.

The Drosophila homolog of the vertebrate serum response factor (SRF) was isolated by low stringency hybridization. Nucleotide sequence analysis revealed that the Drosophila SRF homolog (DSRF) codes for a protein that displays 93% sequence identity with human SRF in the MADS domain, the region required for DNA binding, dimerization and interaction with accessory factors. The DSRF gene is expressed during several phases of embryonic development. In the egg, both the RNA and the protein are maternal in origin and slowly decrease in amount during gastrulation. After germ band retraction, high levels of zygotic expression are observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. Many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues. The DSRF gene was mapped to position 60C on the second chromosome, and overlapping deficiencies which remove the gene were identified. Analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. Although the initial migration of tracheal cells was not affected in those deficient embryos, many tracheal cells appeared not to maintain their correct position and continued to migrate. Thus, the DSRF gene might play a role in the proper formation and maintenance of the trachea.

Amino Acid Sequence↗
PubMed1993

Exploring the homeobox.

In Drosophila, homeotic mutations lead to the transformation of structures of one body segment into the corresponding structures of another segment. These mutations identify master regulator genes which specify segmental identity along the antero-posterior body axis. Dominant gain and recessive loss-of-function mutations generate to opposite segmental transformations. The cloning of the homeotic Antennapedia (Antp) gene led to the discovery of the homeobox, a 180-bp DNA segment characteristic for homeotic genes. It encodes the DNA-binding domain of the respective proteins which was designated as the homeodomain. Homeodomain proteins are transcriptional regulators which specify the body plan by controlling the transcription of their subordinate target genes. By inserting the Antp cDNA into a heat-inducible expression vector, the body plan can be altered in a predictable way. Using the homeobox as a probe, homologous Hox genes from vertebrates have been cloned. In the mouse, dominant gain and recessive loss-of-function mutations result in segmental transformations of opposite direction, as in Drosophila. Also, the mouse Hox genes can partially substitute the homologous Drosophila genes in transgenic flies. Therefore, the genetic control of the body plan is much more universal than anticipated. The three-dimensional structure of the Antp homeodomain and its complex with a consensus DNA-binding site was determined by nuclear magnetic resonance (NMR) spectroscopy. The homeodomain essentially consists of four alpha-helices, a helix-turn-helix motif, and a flexible N-terminal arm. Base-specific contacts are made by both the recognition helix and the N-terminal arm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Functional specificity of the Antennapedia homeodomain.

The segmental identity in animal development is determined by a set of homeotic selector genes clustered in the invertebrate HOM or vertebrate Hox homeo box complexes. These genes encode proteins with very similar homeodomains and highly diverged N- and C-terminal sequences. The Antennapedia (Antp) homeodomain, for instance, differs at only five amino acid positions from that of Sex combs reduced (Scr) protein. Using a heat shock assay in which chimeric Antp-Scr proteins are expressed ectopically in Drosophila, we have shown that the functional specificity of the Antp protein is determined by the four specific amino acids located in the flexible N-terminal arm of the homeodomain. The three-dimensional structure of the Antp homeodomain-DNA complex shows that this N-terminal arm is located in the minor groove of the DNA, suggesting that the functional specificity is determined either by slight differences in DNA binding and/or by selective interactions with other transcription factor(s).

Amino Acid Sequence↗
PubMed1993

Functional specificity of the homeodomain protein fushi tarazu: the role of DNA-binding specificity in vivo.

The mechanisms determining the functional specificity of Drosophila homeodomain proteins are largely unknown. Here, the role of DNA-binding specificity for the in vivo function of the homeodomain protein fushi tarazu (ftz) is analyzed. We find that specific DNA binding is an important but not sufficient determinant of the functional specificity of ftz in vivo: The ftz DNA-binding specificity mutant ftzQ50K retains partial ftz wild-type activity in gene activation and phenotypic rescue assays. Furthermore, specificity mutations in a ftz-in vivo binding site only partially reduce enhancer activity as compared to null mutations of this site. Despite bicoid-like DNA-binding specificity ftzQ50K does not activate natural or artificial bcd target genes in the realms of ftz. These results are discussed in the light of recent observations on the mechanism of action of the yeast homeodomain protein alpha 2.

Animals↗
PubMed1993

Analysis of a fushi tarazu autoregulatory element: multiple sequence elements contribute to enhancer activity.

Regulatory sequences or factors involved in the regulation of target genes of Drosophila homeodomain proteins are largely unknown. Here, we identify sequence elements that are involved in the function of the fushi tarazu (ftz) autoregulatory element AE, a direct in vivo target of the homeodomain protein ftz. A systematic deletion analysis of AE in transgenic embryos defines multiple elements that are redundantly involved in enhancer activity. Sequences juxtaposed to ftz binding sites are not strictly required for enhancer function. Several sequence motifs are conserved in other developmentally regulated genes of Drosophila melanogaster and in the AE homologue of Drosophila virilis. The D. virilis AE is functional in D. melanogaster. The sequence motifs identified here are candidate elements contributing to the target specificity of the homeodomain protein ftz.

Animals↗
PubMed1993

Regional repression of a Drosophila POU box gene in the endoderm involves inductive interactions between germ layers.

An induction process occurring between the mesodermal and the endodermal germ layers has recently been described in the regulation of the Drosophila homeotic gene labial (lab). We report here that proper spatial regulation of the Drosophila POU box gene pdm-1 products also involves interaction between these two germ layers. pdm-1 transcripts are initially present in both the anterior and the posterior endodermal midgut primordia. Upon fusion of the two primordia, transcripts disappear from two regions in the endoderm, a central domain and an anterior domain. The anterior repression domain of pdm-1 is independent of the expression of known homeotic genes and genes encoding secreted signalling molecules in the visceral mesoderm, both for its positioning and its repression. Repression in the central domain requires both the homeotic gene Ultrabithorax (Ubx) and the decapentaplegic (dpp) gene, which encodes a secreted protein. Both of these genes are also required for lab induction. However, the analysis of pdm-1 expression in various mutant backgrounds indicates that the regulation of lab and pdm-1 across germ layers is controlled by different genetic cascades. Our study indicates that dpp is not the signal that dictates central pdm-1 repression across germ layers and suggests that in the same midgut region, different signalling pathways result in the differential activation or repression of potential transcription factors.

Animals↗
PubMed1992

NMR structure determination reveals that the homeodomain is connected through a flexible linker to the main body in the Drosophila Antennapedia protein.

The secondary structure of an N-terminally elongated Antennapedia (Antp) homeodomain (HD) polypeptide containing residues -14 to 67, where residues 1-60 constitute the HD, has been determined by NMR in solution. This polypeptide contains the conserved motif -Tyr-Pro-Trp-Met- (YPWM) at positions -9 to -6. Despite the hydrophobic nature of this tetrapeptide motif, the N-terminal arm consisting of residues -14 to 6 is flexibly disordered, and the well-defined part of the HD structure with residues 7-59 is indistinguishable from that of the shorter Antp HD polypeptide (where positions 0, 1, and 67 are methionine, arginine, and glycine, respectively). In vitro biochemical studies showed that the stability and specificity of the DNA binding previously observed for the shorter Antp HD polypeptide is preserved in the elongated polypeptide. These results strongly support the view that the HD is connected through a flexible linker to the main body in the Antp protein and that the minor groove contacts by the N-terminal arm (residues 1-6) in the Antp HD-DNA complex are an intrinsic feature of the DNA-binding interactions of the intact Antp protein.

Amino Acid Sequence↗
PubMed1992

Developmentally regulated Drosophila gene family encoding the fork head domain.

We have isolated seven Drosophila genes by means of low-stringency hybridization to a DNA probe containing the coding sequence for the protein domain shared by the rodent hepatocyte-enriched nuclear transcription factor HNF3A (alpha) and the product of the Drosophila region-specific homeotic gene fork head (fkh). The previously unreported genes encode a 110-amino acid conserved sequence, which we call the fork head (fkh) domain. Two of these fkh-domain-encoding genes ("FD genes") map to the sloppy paired locus (slp), which exerts segmentation gene function. The expression patterns of the other FD genes suggest that their protein products are likely to be involved in gut formation, mesoderm specification, and some specific aspects of neural development. The FD gene products presumably represent a family of transcription factors that, like the previously identified DNA-binding proteins, contribute to early developmental decisions in cell fates during embryogenesis.

Amino Acid Sequence↗
PubMed1992

Occurrence of sialic acids in Drosophila melanogaster.

Sialylated oligosaccharides, which are cell type-specific and developmentally regulated, have been implicated in a variety of complex biological events. Their broad functional importance is reflected by their presence in a wide variety of phyla extending from Echinodermata through higher vertebrates. Here, sialic acids are detected throughout development in an insect, Drosophila. Homopolymers of alpha 2,8-linked sialic acid, polysialic acid, are developmentally regulated and only expressed during early Drosophila development.

Animals↗
PubMed1992

Direct homeodomain-DNA interaction in the autoregulation of the fushi tarazu gene.

A major problem in the elucidation of the molecular mechanisms governing development is the distinction between direct and indirect regulatory interactions among developmental control genes. In vivo studies have indicated that the Drosophila segmentation gene fushi tarazu (ftz) directly or indirectly autoregulates its expression. Here we describe a generally applicable experimental approach which establishes a direct in vivo interaction of the homeodomain protein ftz with the ftz cis-autoregulatory control region. In vitro studies have shown that the DNA-binding specificity of the ftz homeodomain can be changed by a single amino-acid substitution in the recognition helix (Gln 50----Lys). Whereas wild-type ftz homeodomain binds preferentially to a CCATTA motif, the mutant homeodomain (ftzQ50K) recognizes a GGATTA motif. We now find that the in vivo activity of an ftz autoregulatory enhancer element is reduced by mutations of putative ftz-binding sites to GGATTA. This down-regulatory effect is specifically suppressed in vivo by the DNA-binding specificity mutant ftzQ50K. These results establish a direct positive autoregulatory feedback mechanism in the regulation of this homeobox gene.

Animals↗
PubMed1992

Distamycin-induced inhibition of homeodomain-DNA complexes.

The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.

Animals↗
PubMed1992

Empty spiracles, a gap gene containing a homeobox involved in Drosophila head development.

The empty spiracles (ems) gene of Drosophila melanogaster is necessary for proper head formation and the development of the posterior spiracles. We have isolated a homeobox-containing gene, W13, by cross-homology using the Drosophila muscle segment homeobox gene (msh) as a probe. The W13 gene maps at 88A, where the ems locus has been previously localized genetically. The sequence alterations found in the W13 coding region from two mutant ems alleles show that W13 is the ems gene. A 2.4 kb RNA corresponding to the ems transcript is expressed from cellular blastoderm throughout all embryonic and larval stages. In situ hybridization to whole mount embryos reveals two domains of expression. During the cellular blastoderm stage ems is expressed in the developing head in a single anterior band. This is correlated with its possible function as an anterior gap gene that is expressed in the preantennal, antennal and intercalary segments and is required for the development of the antennal sense organ, the optic lobe and parts of the head skeleton. The early expression of the ems gene is controlled by the anterior morphogen bicoid (bcd). Using a gene fusion we identified a cis-acting element which is a target for the bcd gene product. Later during embryogenesis ems is expressed in lateral regions of each segment, where the tracheal pits form and lateral neuroblasts originate, as well as in the posterior spiracles. This late expression partially correlates with defects seen in the tracheal tree of ems embryos. In addition to a homeodomain, the N-terminal portion of the predicted protein sequence is very proline-rich, whereas the C-terminus has an acidic profile consistent with the role of the ems gene product as a transcription factor.

Amino Acid Sequence↗
PubMed1992

The homeobox in perspective.

The discovery of the homeobox marks the beginning of a new era in developmental biology in which a class of master control genes, which determine the body plan, have been identified. Their mechanism of action can now be studied at the molecular level and their occurrence seems to be much more universal than originally anticipated.

Amino Acid Sequence↗