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D S Hogness

Publications and source records attributed to D S Hogness.

At least 19 recordsLinked to original sources
PubMed1995

Identification of homeotic target genes in Drosophila melanogaster including nervy, a proto-oncogene homologue.

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.

Amino Acid Sequence↗
PubMed1995

The Drosophila E74 gene is required for metamorphosis and plays a role in the polytene chromosome puffing response to ecdysone.

The steroid hormone ecdysone initiates Drosophila metamorphosis by reprogramming gene expression during late larval and prepupal development. The ecdysone-inducible gene E74, a member of the ets proto-oncogene family, has been proposed to play a key role in this process. E74 is encoded within the 74EF early puff and consists of two overlapping transcription units, E74A and E74B. To assess the function(s) of E74 during metamorphosis, we have isolated and characterized recessive loss-of-function mutations specific to each transcription unit. We find that mutations in E74A and E74B are predominantly lethal during prepupal and pupal development, consistent with a critical role for their gene products in metamorphosis. Phenotypic analysis reveals that E74 function is required for both pupariation and pupation, and for the metamorphosis of both larval and imaginal tissues. E74B mutants are defective in puparium formation and head eversion and die as prepupae or cryptocephalic pupae, while E74A mutants pupariate normally and die either as prepupae or pharate adults. We have also investigated the effects of the E74 mutations on gene expression by examining the puffing pattern of the salivary gland polytene chromosomes in newly formed mutant prepupae. Most puffs are only modestly affected by the E74B mutation, whereas a subset of late puffs are sub-maximally induced in E74A mutant prepupae. These observations are consistent with Ashburner's proposal that early puff proteins induce the formation of late puffs, and define E74A as a regulator of late puff activity. They also demonstrate that E74 plays a wide role in reshaping the insect during metamorphosis, affecting tissues other than the salivary gland in which it was originally identified.

Animals↗
PubMed1992

DHR3: a Drosophila steroid receptor homolog.

In Drosophila the steroid hormone ecdysone triggers a genetic regulatory hierarchy in which ecdysone combines with a receptor protein to form a complex that induces the transcription of a small class of "early" genes, which encode transcription factors that regulate other genes. We previously reported that one of the early genes, E75, encodes members of the steroid receptor superfamily. Using an E75 hybridization probe, we have identified two additional Drosophila genes that encode members of this superfamily. One of these is the ecdysone receptor gene, EcR, as previously reported. In this work, we examine the sequence, genomic organization, and developmental expression of the other gene, DHR3, which, like E75, encodes one of a growing number of "orphan" receptors for which ligands have not yet been identified. The structure of the DHR3 protein is strikingly similar to that of the MHR3 protein (e.g., 97% amino acid identity for the DNA binding domains), another orphan receptor encoded by an ecdysone-inducible early gene of another insect, Manduca sexta. The temporal developmental profile for DHR3 expression closely parallels that for the ecdysone titer and for the ecdysone-inducible E75 and E74 Drosophila early genes. The structural similarity to a Manduca early gene and the expression similarities to Drosophila early genes suggest that the DHR3 gene may also belong to the early gene class.

Age Factors↗
PubMed1991

Immunochemical dissection of the Ultrabithorax homeoprotein family in Drosophila melanogaster.

The homeotic gene Ultrabithorax (Ubx) specifies metameric identities in multiple tissues of the thorax and abdomen in Drosophila melanogaster. Alternatively spliced Ultrabithorax mRNAs encode five protein isoforms that differ in internal sequences immediately adjacent to a homeodomain DNA-binding motif. Each of these proteins is phosphorylated in vivo at multiple serine and threonine residues. An extensive panel of monoclonal antibodies was raised against the Ultrabithorax proteins, including antibodies specific for individual isoforms and antibodies that discriminated between different phosphorylation states. Characterization of these antibodies provided insights into shared and isoform-specific features of Ultrabithorax protein structure that may be functionally important. Immunohistochemical staining experiments demonstrated that each isoform is expressed in a different stage- and tissue-specific pattern and suggested that Ultrabithorax protein phosphorylation is also developmentally regulated. These results support the hypothesis that alternative splicing and phosphorylation modulate developmentally specific functions of the Ubx gene.

Amino Acid Sequence↗
PubMed1991

The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily.

The steroid hormone ecdysone triggers coordinate changes in Drosophila tissue development that result in metamorphosis. To advance our understanding of the genetic regulatory hierarchies controlling this tissue response, we have isolated and characterized a gene, EcR, for a new steroid receptor homolog and have shown that it encodes an ecdysone receptor. First, EcR protein binds active ecdysteroids and is antigenically indistinguishable from the ecdysone-binding protein previously observed in extracts of Drosophila cell lines and tissues. Second, EcR protein binds DNA with high specificity at ecdysone response elements. Third, ecdysone-responsive cultured cells express EcR, whereas ecdysone-resistant cells derived from them are deficient in EcR. Expression of EcR in such resistant cells by transfection restores their ability to respond to the hormone. As expected, EcR is nuclear and found in all ecdysone target tissues examined. Furthermore, the EcR gene is expressed at each developmental stage marked by a pulse of ecdysone.

Amino Acid Sequence↗
PubMed1991

The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila.

The Polycomb (Pc) gene of Drosophila melanogaster is a member of a large class of genes (Pc group) required for the segment-specific repression of homeotic selector genes. Mutations in Pc-group genes show strong posterior transformations in homozygous embryos resulting from an ectopic expression of homeotic genes in segments where they are not supposed to be active. Genetic evidence suggests that Pc is part of a cellular memory mechanism responsible for the transmission of the homeotic expression pattern through developmental time. We have determined the nucleotide sequence for the genomic DNA of the Pc gene and for cDNAs corresponding to the 2.5-kilobase Pc mRNA. The deduced sequence of the Pc protein exhibits a homology to the heterochromatin-associated protein HP1 encoded by the suppressor of position effect variegation gene Su(var) 205. The homology is confined to a 37-amino acid domain in the N-terminal part of the two proteins. Our findings extend to the molecule level the genetically identified parallels between the Pc-group genes and the modifiers of position effect variegation. This suggests that Pc could use analogous mechanisms at the level of the higher order chromatin structure for the stable transmission of a determined state, as has been proposed for the clonal propagation of heterochromatin domains.

Amino Acid Sequence↗
PubMed1991

The large upstream control region of the Drosophila homeotic gene Ultrabithorax.

Ultrabithorax (Ubx) is a Drosophila homeotic gene that determines the segmental identities of parts of the thorax and abdomen. Appropriate Ubx transcription requires a long upstream control region (UCR) that is defined genetically by the bithoraxoid (bxd) and postbithorax (pbx) subfunction mutations. We have directly analyzed UCR functions by the examination of beta-galactosidase expression in flies containing Ubx-lacZ fusion genes. 35 kb of UCR DNA confers upon beta-galactosidase an expression pattern that closely parallels normal Ubx expression throughout development. In contrast, 22 kb of UCR DNA confers fewer features of normal Ubx expression, and with 5 kb of UCR DNA the expression pattern has no resemblance to Ubx expression except in the visceral mesoderm. We have also shown that bxd chromosome breakpoint mutants form a comparable 5' deletion series in which the severity of the effect on Ubx expression correlates with the amount of upstream DNA remaining in the mutant. In Ubx-lacZ fusions containing 22 kb of UCR DNA, and in comparable bxd mutants, there is a persistent pair-rule pattern of metameric expression in early development, demonstrating that there are distinct mechanisms with different sequence requirements for the initial activation of Ubx in different metameres. The correction of this pair-rule pattern later in embryogenesis shows that there are also distinct mechanisms for the activation of Ubx at different times during development.

Animals↗
PubMed1991

Phosphorylation, expression and function of the Ultrabithorax protein family in Drosophila melanogaster.

Alternative splicing of the Ultrabithorax homeotic gene transcript generates a family of five proteins (UBX isoforms) that function as transcription factors. All isoforms contain a homeodomain within a common 99 aa C-terminal region (C-constant region) which is jointed to a common 247 aa N-terminal (N-constant) region by different combinations of three small optional elements. Unlike the UBX proteins expressed in E. coli, UBX isoforms expressed in D. melanogaster cells are phosphorylated on serine and threonine residues, located primarily within a 53 aa region near the middle of the N-constant region, to form at least five phosphorylated states per isoform. Similar, if not identical states can be generated in vitro from purified E. coli UBX protein by a kinase activity in nuclear extracts from D. melanogaster cells. Temporal developmental profiles of UBX isoforms parallel those for the respective mRNAs, and all isoforms are similarly phosphorylated throughout embryogenesis. Analysis by cotransfection assays of the promoter activation and repression functions of mutant UBX proteins with various deletions in the N-constant region shows that repression is generally insensitive to deletion and, hence, presumably to phosphorylation. By contrast, the activation function is differentially sensitive to the different deletions in a manner indicating the absence of a discrete activating domain and instead, the presence of multiple activating sequences spread throughout the region.

Animals↗
PubMed1990

The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets-related proteins.

We have isolated an ecdysone-inducible gene, E74, from the early puff at position 74EF in the Drosophila polytene chromosomes. We show that E74 consists of three nested transcription units that derive from unique promoters but share a single polyadenylation site. The 60 kb E74A unit is directly induced by ecdysone and leads to the synthesis of a 6.0 kb mRNA that contains an unusually long 5' leader (1891 nucleotides) with 17 short ORFs. Within the fifth of the seven E74A introns are two E74B promoters that direct the synthesis of 4.8 and 5.1 kb mRNAs. The nested arrangement of these transcription units leads to the formation of two E74 proteins, each with a unique N-terminal domain joined to a common C-terminal domain. The unique N-terminal domains contain regions rich in acidic amino acids while the C-terminal domain is rich in basic amino acids and is very similar to proteins encoded by the ets proto-oncogene superfamily.

Amino Acid Sequence↗
PubMed1990

Spatial and temporal patterns of E74 transcription during Drosophila development.

The E74 gene occupies one of the early puff loci (74EF) central to the Ashburner model for the ecdysone-induced puffing pattern in Drosophila. In support of this model, we show that the E74A promoter is directly activated by ecdysone and is subsequently repressed by ecdysone-induced proteins. Further support derives from the correspondence observed between 74EF puff size and the accumulation of nascent transcripts on the E74A unit. These transcripts elongate at 1.1 kb/min so that this 60 kb unit acts as a timer, delaying the appearance of its mRNA by 1 hr. E74A transcription is induced in a variety of ecdysone target tissues in late third instar larvae and during each of the ecdysone pulses that mark the six stages of Drosophila development. These results support an extension of the Ashburner model in which ecdysone pulses coordinate tissue development. The temporal pattern of E74B transcription overlaps but is distinct from that of E74A.

Aging↗
PubMed1990

Functional dissection of Ultrabithorax proteins in D. melanogaster.

Expression of Ultrabithorax (UBX) proteins via a heat-inducible promoter generated homeotic transformations of segmental identities in the embryonic cuticle and peripheral nervous system (PNS) of Drosophila and transformed antennae into legs in the adult. The embryonic transformations were used to determine the identity functions of members of the UBX family and UBX mutant forms. Whereas UBX forms I and IV each induced the cuticle transformations, only form I induced the PNS transformations. Analysis of the transformations generated by UBX deletions and by a chimeric Ultrabithorax-Antennapedia protein demonstrated that the majority of the UBX identity information is contained within the C-terminal, homeodomain-containing portion of the protein. Implications of these results for how homeotic proteins select particular metameric identities are discussed.

Amino Acid Sequence↗
PubMed1990

The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily.

A pulse of the steroid hormone ecdysone at the end of Drosophila larval development triggers coordinate changes in both larval and imaginal tissues that result in metamorphosis to the adult fly. In larval salivary glands, this pulse activates a genetic regulatory hierarchy manifested by the induction of two kinds of transcription puffs in the polytene chromosomes: a small set of "early" puffs representing a primary response to the hormone, and a complex set of "late" puffs whose delayed appearance is dependent on proteins synthesized during the primary response. We isolated a 50-kb ecdysone-inducible gene, E75, that occupies the early puff locus at 75B. E75 contains two overlapping transcription units. The E75 A unit is a coextensive with the E75 gene and contains six exons: two 5'-proximal exons, A0 and A1, which are specific to this unit, and exons 2-5, which are common to both units. The E75 B unit is 20 kb long and contains five exons, a 5'-terminal exon, B1, located within the second intron of E75 A, and the common exons 2-5. Large open reading frames start within the first exon of each unit and continue into the last exon and therefore encode two different proteins. Both proteins exhibit sequence similarity to the conserved DNA-binding and hormone-binding domains of proteins in the steroid receptor superfamily. The two putative zinc fingers that characterize the DNA-binding domain are encoded by exon A1 and exon 2, so that the E75 A protein contains both fingers, whereas the E75 B protein contains only the second. Both proteins contain the same putative hormone-binding domain encoded by exon 4.

Amino Acid Sequence↗
PubMed1989

Transcriptional activation by the Antennapedia and fushi tarazu proteins in cultured Drosophila cells.

Drosophila homeodomain proteins bind to specific DNA sequences in vitro and are hypothesized to regulate the transcription of other genes during development. Using a cotransfection assay, we have shown that homeodomain proteins encoded by the homeotic gene Antennapedia (Antp) and the segmentation gene fushi tarazu, as well as a hybrid homeodomain protein, are activators of transcription from specific promoters in cultured Drosophila cells. Sequences downstream of the Antp P1 and Ultrabithorax transcription start sites mediate the observed activation. A TAA-rich DNA sequence to which the Antp protein binds in vitro is sufficient to confer regulation on a heterologous promoter. The results demonstrate that homeodomain proteins are transcriptional regulators in vivo and that in cultured cells, different homeodomain-containing proteins can act upon a common sequence to modulate gene transcription.

Animals↗
PubMed1989

Transcriptional activation and repression by Ultrabithorax proteins in cultured Drosophila cells.

Homeotic genes of Drosophila melanogaster such as Ultrabithorax (Ubx) and Antennapedia (Antp) have long been thought to select metameric identity during development by controlling the expression of various target genes. Here we describe a cotransfection assay in cultured D. melanogaster cells that is used to demonstrate that Ubx proteins (UBX) can repress an Antp promoter fusion and activate a Ubx promoter fusion, activities predicted from genetic studies. We show (a) that UBX proteins regulated the level of accurately initiated Antp P1 and Ubx transcripts, (b) that activation of the Ubx promoter required a downstream cluster of UBX binding sites, and (c) that binding site sequences were sufficient to confer regulation on a heterologous promoter, regardless of their orientation or precise position. We conclude that UBX proteins are transcriptional repressors and activators, and that their actions are mediated by binding to promoter region sequences. Each member of the UBX protein family has similar regulatory abilities, but the properties of synthetic mutant forms suggest that UBX proteins may have a modular design similar to other transcriptional regulators.

Animals↗
PubMed1989

Structure and expression of a family of Ultrabithorax mRNAs generated by alternative splicing and polyadenylation in Drosophila.

The 77-kb primary transcript of the homeotic Ultrabithorax (Ubx) gene is alternatively spliced to yield at least five different coding regions. Each is restricted to either a 3.2- or a 4.3-kb size class generated by alternative polyadenylation. The pathways for splicing and polyadenylation are therefore coordinately regulated, and because the relative abundance of the respective mRNAs varies throughout development, these pathways also appear to be developmentally regulated. Translation of these mRNAs yields a family of Ubx proteins characterized by constant amino- and carboxy-proximal regions of 247 and 99 amino acid residues, respectively. Members of this family are distinguished by a short variable region that links the constant regions and consists of different combinations of three optional elements of 9, 17, and 17 residues. Only four amino acid residues separate this variable region from the 60-residue homeo domain of the carboxy-terminal constant region. This proximity suggests that functional differences among the Ubx proteins derive from the differential effects of their variable regions on the DNA-binding capacity of the homeo domain. An argument is made that these functional differences are tissue specific.

Amino Acid Sequence↗
PubMed1988

An Ultrabithorax protein binds sequences near its own and the Antennapedia P1 promoters.

The homeotic gene Ultrabithorax (Ubx), located in the bithorax complex of Drosophila, encodes a family of closely related proteins that direct the developmental fates of posterior thoracic and anterior abdominal metameres. We have purified a member of the Ubx protein family from an overproducing E. coli strain and have shown that it is sequence-specific DNA binding protein. The protein binds tightly to sequences near its own promoter and near the P1 promoter of Antenna-pedia (Antp), a homeotic gene Ubx is known to repress from genetic studies. The binding sites occur in clusters downstream of the transcription start sites, and far upstream at Antp P1. They range in size from 40 to 90 bp, and contain tandem repeats of the trinucleotide TAA or the related hexanucleotide TAA-TCG. These results suggest that the regulatory activities of Ubx are direct and are mediated by binding of Ubx proteins to promoter region sequences.

Animals↗