Biomedical subjects

U Heberlein

Publications and source records attributed to U Heberlein.

31 records · Page 2Linked to original sources

PubMed1994

The C-terminus of the homeodomain is required for functional specificity of the Drosophila rough gene.

In contrast to most Drosophila homeobox genes, which are required during embryogenesis, the rough gene is involved in photoreceptor cell specification in the compound eye. Taking advantage of the viability of null rough alleles and the small size of the rough gene, we have combined in vivo and in vitro mutagenesis to define important functional domains in the rough protein. All missense mutations found to disrupt rough function mapped to highly conserved amino acids in the homeodomain (HD), suggesting that the nature of few, if any, single amino acids outside the HD is critical for rough activity. The analysis of chimeric proteins, in which the whole HD or parts of it were swapped between the rough and Antennapedia (Antp) proteins, revealed that the C-terminus of the rough HD is important for rough activity in vivo. This C-terminal region was also found to be required for the recognition of rough binding sites in vitro. Our data suggest that amino acids located in the C-terminus of the homeodomain may play important roles in selective binding site recognition.

Alleles↗

PubMed1993

The TGF beta homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophila retina.

Development of the Drosophila retina occurs asynchronously; differentiation, its front marked by the morphogenetic furrow, progresses across the eye disc epithelium over a 2 day period. We have investigated the mechanism by which this front advances, and our results suggest that developing retinal cells drive the progression of morphogenesis utilizing the products of the hedgehog (hh) and decapentaplegic (dpp) genes. Analysis of hh and dpp genetic mosaics indicates that the products of these genes act as diffusible signals in this process. Expression of dpp in the morphogenetic furrow is closely correlated with the progression of the furrow under a variety of conditions. We show that hh, synthesized by differentiating cells, induces the expression of dpp, which appears to be a primary mediator of furrow movement.

Animals↗

PubMed1993

Star is required for neuronal differentiation in the Drosophila retina and displays dosage-sensitive interactions with Ras1.

The photoreceptor cells R8, R2, and R5 are the first cells to initiate neuronal differentiation in the Drosophila eye imaginal disc. We have shown previously that these three cells require Star gene function for proper ommatidial assembly. Due to the embryonic lethality associated with mutations in Star, we have analyzed the consequence of loss of Star function in mosaic eye imaginal discs. Presumptive R8, R2, and R5 cells that lack Star function fail to differentiate neuronally and die a few hours later. Enhancer trap insertions reveal that Star expression in the eye disc is restricted to the developing R8, R2, and R5 cells. Taken together, these data suggest that Star is required for the reception of a signal and/or the execution of a developmental program that leads to the neuronal differentiation of R8, R2, and R5. Star is also required for the formation of wing veins and is expressed in developing veins, suggesting that at least partially overlapping pathways may operate during photoreceptor cell differentiation and wing vein formation. The role of Star in cell-cell signaling is supported by the observation of genetic interactions between Star and mutations that reduce signaling through both sevenless and the Drosophila EGF-receptor homologue, including Ras1 and Son of sevenless.

Animals↗

PubMed1991

Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough.

We report that mutations at the Star locus act as dominant enhancers of the eye phenotype displayed by flies carrying a null allele of rough. Our analysis of double mutants at different stages of eye development suggests that this phenotype results from defects in the early stages of photoreceptor cell differentiation in the eye imaginal disc. Complete loss of Star function during retinal development, analyzed in mosaic animals, results in cell death, visible as scars in the adult eye. The requirement for wild-type Star function, however, is confined to only a subset of photoreceptor cells, R8, R2, and R5, which are the first three cells to differentiate neurally in the developing retina. These results suggest an essential role for the Star gene in the initial events of ommatidial cluster formation during the development of the Drosophila compound eye.

Alleles↗

PubMed1991

Cell-fate determination in the developing Drosophila eye: role of the rough gene.

The homeobox-gene rough is required in photoreceptor cells R2 and R5 for normal ommatidial assembly in the developing Drosophila eye. We have used several cell-type-specific markers and double mutant combinations to analyze cell-fate determination in rough. We show that the cells that would normally become R2 and/or R5 express a marker, a lacZ insertion in the seven-up (svp) gene, which is indicative of the R1/3/4/6 cell fate. In addition, the analysis of mitotically induced svp,ro double mutant clones in the eye indicates that in rough all outer photoreceptors are under the genetic control of the svp gene. These results show that, in the absence of rough function, R2 and R5 fail to be correctly determined and appear to be transformed into cells of the R3/4/1/6 subtype. This transformation and the subsequent developmental defects do not preclude the recruitment of R7 cells. However, the presence of ommatidia containing more than one R7 and/or R8 cell in rough implies a complex network of cellular interactions underlying cell-fate determination in the Drosophila retina.

Animals↗

PubMed1990

Purified Drosophila transcription factor, Adh distal factor-1 (Adf-1), binds to sites in several Drosophila promoters and activates transcription.

Adh distal factor-1 (Adf-1) is a sequence-specific DNA-binding activity originally identified in Drosophila tissue culture cells and embryos. Adf-1 binds to upstream recognition elements in each of the two promoters of the Drosophila alcohol dehydrogenase gene (Adh), and binding of Adf-1 to the Adh distal promoter site activates transcription. We have carried out a mutational analysis of the Adh distal promoter using both an in vitro transcription assay and a transient transfection assay in Drosophila tissue culture cells, and in both cases find that deletion of sequences required for Adf-1 binding leads to a 3-4-fold drop in transcription. We have purified Adf-1 and demonstrate by a sodium dodecyl sulfate-gel renaturation assay that it is a 34-kDa protein. Purified Adf-1 activates Adh distal promoter transcription in vitro in a binding site-dependent manner. DNase I footprint analysis shows that the purified protein binds not only to the two previously characterized sites in Adh but also to transcriptional regulatory elements in the dopa decarboxylase (Ddc) and Antennapedia (Antp) P1 promoters. Thus, it appears that Adf-1 may play an important role not only in the regulation of Adh expression but also in the transcription of other Drosophila genes as well.

Alcohol Dehydrogenase↗

PubMed1990

Structural and functional comparisons of the Drosophila virilis and Drosophila melanogaster rough genes.

We have isolated the homeobox gene rough (ro) from Drosophila virilis. Comparison of the predicted amino acid sequences of the D. melanogaster and D. virilis rough proteins reveals that domains of high conservation, including the homeodomain, are interspersed with highly diverged regions. Stretches of significant sequence conservation are also observed in the 5' promoter region and in the introns. The D. virilis rough gene rescues the rough mutant phenotype and is properly regulated when introduced into the D. melanogaster genome. Thus the rough protein as well as the cis-regulatory elements that ensure proper temporal and spatial regulation are functionally conserved between these Drosophila species.

Amino Acid Sequence↗

PubMed1990

The homeo domain protein rough is expressed in a subset of cells in the developing Drosophila eye where it can specify photoreceptor cell subtype.

The Drosophila homeo box gene rough is required in photoreceptor cells R2 and R5 for normal eye development. We show here that rough protein expression is limited to a subset of cells in the developing retina where it is transiently expressed for 30-60 hr. The rough protein is first expressed broadly in the morphogenetic furrow but is rapidly restricted to the R2, R3, R4, and R5 precursor cells. Ubiquitous expression of rough under the control of the hsp70 promoter in third-instar larvae suppresses the initial steps of ommatidial assembly. Structures derived from other imaginal discs are not affected. Ectopic expression of rough in the R7 precursor, through the use of the sevenless promoter, causes this cell to develop into an R1-6 photoreceptor subtype; however, this cell still requires sevenless function for its neural differentiation. Taken together with previous analyses of the rough mutant phenotype, these results suggest that the normal role of rough is to establish the unique cell identity of photoreceptors R2 and R5.

Animals↗

PubMed1990

The Adh gene promoters of Drosophila melanogaster and Drosophila orena are functionally conserved and share features of sequence structure and nuclease-protected sites.

The sibling species Drosophila melanogaster and D. orena show similar patterns of alcohol dehydrogenase expression, both spatially and temporally. These two species diverged from a common ancestor 6 million to 15 million years ago, and the DNA sequences of the promoter regions of their Adh genes show a mosaic pattern of conservation and change. By interspecific transformation of D. orena sequences into D. melanogaster, we demonstrate a functional equivalence between these sequences. Using both D. melanogaster embryo extracts and purified transcription factor Adf-1, we compare the protection of these promoter sequences from nuclease, demonstrating considerable conservation.

Alcohol Dehydrogenase↗

PubMed1988

Temporal pattern of alcohol dehydrogenase gene transcription reproduced by Drosophila stage-specific embryonic extracts.

Regulation of transcription during development has been analysed in vitro using extracts derived from Drosophila embryos. Transcription of mutant templates reveals cis-control regions of the alcohol dehydrogenase promoter responsible for temporal regulation. The activity of a trans-acting protein correlates with the profile of alcohol dehydrogenase expression during embryogenesis.

Alcohol Dehydrogenase↗

PubMed1988

A cis-acting element and associated binding factor required for CNS expression of the Drosophila melanogaster dopa decarboxylase gene.

A cis-acting sequence from the Drosophila melanogaster dopa decarboxylase (Ddc) gene is selectively required for Ddc expression in the central nervous system. We analyze several parameters influencing the function of the sequence element and describe a factor which interacts with it and mediates CNS expression of Ddc. The element, element I, can function in vivo when included on a synthetic oligonucleotide inserted near its normal location, or closer to the RNA startpoint. It displays partial activity when inverted. Two different 2-bp mutations in element I abolish its ability to stimulate neuronal Ddc expression in the CNS. A factor present in embryonic nuclear extracts specifically protects element I in DNase I footprinting assays. The binding affinity of this factor is reduced by each alteration of element I that inhibits neuronal expression, indicating a role in mediating CNS expression of Ddc. Element I alone has no detectable activity when placed adjacent to a heterologous promoter, although 2.2 kb of 5' Ddc sequences direct correct cell-specific expression of a heterologous promoter.

Animals↗

PubMed1985

Characterization of Drosophila transcription factors that activate the tandem promoters of the alcohol dehydrogenase gene.

Fractionation of a nuclear extract derived from Drosophila tissue culture cells reveals the presence of multiple components involved in accurate transcription of both distal and proximal promoters of the alcohol dehydrogenase (Adh) gene. Transcription of deletion mutants indicates that a region between -24 and -85 upstream of the distal start site contains sequences required for RNA synthesis in vitro. Moreover, sequences that overlap this same upstream control region are specifically bound and protected from DNAase digestion by a promoter-specific transcription factor, Adf-1. Analysis of proximal promoter mutants identified multiple upstream elements that influence transcription, and DNAase footprint analysis detected three specific binding regions. Adf-1 binds at least one of these proximal promoter regions but interaction at this site is not specifically required for transcription. Our results suggest that multiple sequence-specific DNA binding proteins interact differentially with the proximal and distal promoters of Adh to activate transcription.

Alcohol Dehydrogenase↗

PubMed1983

Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast.

We have chemically synthesized and expressed in yeast a gene coding for human epidermal growth factor (urogastrone), a 53-amino-acid polypeptide that has been shown to promote epithelial cell proliferation and to inhibit gastric acid secretion. The synthetic gene, consisting of 170 base pairs, was designed with yeast-preferred codons and assembled by enzymatic ligation of synthetic fragments produced by phosphoramidite chemistry. The DNA synthesis protocol used allows for facile synthesis of oligonucleotides larger than 50 bases. Yeast cells were transformed with plasmids containing the synthetic gene under control of a yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter and were shown to synthesize a biologically active human epidermal growth factor.

Amino Acid Sequence↗