Biomedical subjects

R A Holmgren

Publications and source records attributed to R A Holmgren.

5 recordsLinked to original sources

PubMed1999

The subcellular localization and activity of Drosophila cubitus interruptus are regulated at multiple levels.

Cubitus interruptus (Ci), a Drosophila transcription factor, mediates Hedgehog (Hh) signaling during the patterning of embryonic epidermis and larval imaginal discs. In the absence of Hh signal, Ci is cleaved to generate a truncated nuclear form capable of transcriptional repression. Hh signaling stabilizes and activates the full-length Ci protein leading to strong activation of downstream target genes including patched and decapentaplegic. A number of molecules have been implicated in the regulation of Ci. Mutations in these molecules lead to changes in Ci protein level, the extent of Ci proteolysis and the expression of Ci target genes. This paper examines the regulation of Ci subcellular localization and activity. We first characterize a bipartite nuclear localization signal (NLS) within Ci. We propose that the subcellular distribution of Ci is affected by two opposing forces, the action of the NLS and that of at least two regions targeting Ci to the cytoplasm. Further our data show that loss of PKA or Costal-2 activity does not fully mimic Hh signaling, demonstrating that Ci proteolysis and Ci activation are two distinct events which are regulated through different paths. Finally, we propose that there are three levels of apparent Ci activity, corresponding to three zones along the AP axis with different sets of gene expression and different levels of Hh signaling.

Amino Acid Sequence↗

PubMed1994

Invariant tryptophan at a shielded site promotes folding of the conformational unit of spectrin.

The tryptophan that is highly conserved among repeating structural units of spectrin is reported to promote the conformational stability of one such unit of chicken brain alpha-spectrin. Four constructs were inserted into pET vectors for overexpression in Escherichia coli of the following spectrin peptides: (i) two adjacent but separately expressed "conformationally phased" repeating units, R16 and R17, one of which (R17) contains a single tryptophan; (ii) a mutant, M17, of the single tryptophan-containing unit with alanine substituted for the tryptophan; and (iii) a conformationally unphased unit, 1617, composed of half of each of the phased units. Both the mutant unit and the unphased unit were much more readily digested by chymotrypsin and by elastase than the phased units and exhibited only 38% and 54% as much alpha-helical structure, respectively, as the phased units by their far UV CD spectra; 90 degrees light scattering measurements revealed the folded peptides to be predominantly monomeric in solution, whereas the unfolded, protease-sensitive peptides consisted of dimers and/or trimers. This trend was corroborated by their dynamic light scattering. Both the blue-shifted wavelength of maximal emission and the relative inaccessibility to acrylamide of the single tryptophan in the folded unit indicate that the invariant tryptophan occupies a site that is shielded from the aqueous phase.

Amino Acid Sequence↗

PubMed1993

A GATA family transcription factor is expressed along the embryonic dorsoventral axis in Drosophila melanogaster.

The GATA transcription factors are a family of C4 zinc finger-motif DNA-binding proteins that play defined roles in hematopoiesis as well as presumptive roles in other tissues where they are expressed (e.g., testis, neuronal and placental trophoblast cells) during vertebrate development. To investigate the possibility that GATA proteins may also be involved in Drosophila development, we have isolated and characterized a gene (dGATAa) encoding a factor that is quite similar to mammalian GATA factors. The dGATAa protein sequence contains the two zinc finger DNA-binding domain of the GATA class but bears no additional sequence similarity to any of the vertebrate GATA factors. Analysis of dGATAa gene transcription during Drosophila development revealed that its mRNA is expressed at high levels during early embryogenesis, with transcripts first appearing in the dorsal portion of the embryo just after cellularization. As development progresses, dGATAa mRNA is present at high levels in the dorsal epidermis, suggesting that dGATAa may be involved in determining dorsal cell fate. The pattern of expression in a variety of dorsoventral polarity mutants indicates that dGATAa lies downstream of the zygotic patterning genes decapentaplegic and zerknüllt.

Amino Acid Sequence↗

PubMed1992

Developmental potential.

In summary (and probably to no one's genuine surprise), it seems clear that some of the key themes in the mechanisms employed during development reiterate themselves throughout the animal kingdom. Yet, as our understanding becomes more refined, new and beguiling observations point to unique aspects of each developmental program. The concentration and absolute position of a variety of positional signaling molecules is likely to be very important in determinative events (establishment of the anteroposterior positioning in a field as in retinal development, establishment or enactment of a hox code, and selector gene regulation through gradients in Drosophila). Appropriate signalling responses are virtually certain to depend critically on the appropriate expression of each component of cellular signal transduction pathways (initiated by the activation of cell-surface receptor protein kinases to finally eliciting gene expression changes through the differential activity of specific transcription factors). The important biochemical details of transcription factor activation of specific respondent genes may be either simpler (as indicated from the murine/Drosophila domain swap experiments) or more complicated (from the responses of mim-1 to cellular versus viral myb proteins) than we had heretofore anticipated.

Animals↗

PubMed1990

Cloning and characterization of the segment polarity gene cubitus interruptus Dominant of Drosophila.

The segment polarity mutation, cubitus interruptus Dominant (ciD), of Drosophila melanogaster causes defects in the posterior half of every embryonic segment. We cloned sequences from the ciD region on the proximal fourth chromosome by "tagging" the gene with the transposable element P. Genetic and molecular evidence indicates that the P-element insertions, which all occurred within the same restriction fragment, are in 5'-regulatory regions of the ciD gene within 3 kb of the first exon of its transcript. The putative ciD transcript was identified on the basis of its absence in homozygous ciD embryos. Its spatial pattern of expression during development is unusual in that, unlike most other segmentation genes, it exhibits uniform expression throughout cellular blastoderm and gastrulation and does not resolve into a periodic pattern until the end of the fast phase of germ-band elongation when it is present in 15 broad segmentally repeating stripes along the anterior-posterior axis of the embryo. Registration of the ciD stripes of expression relative to the stripes of other segment polarity genes shows that ciD is expressed in the anterior three-quarters of every segment. This registration does not correlate with the pattern defects observed in ciD mutants. Sequence analysis indicates that the protein encoded by the ciD transcript contains a domain of five tandem amino acid repeats that have sequence similarity to the zinc-finger repeats of the Xenopus transcription factor TFIIIA and that share the highest degree of identity with the human zinc-finger protein GLI, which has been found to be amplified in several human glioblastomas.

Amino Acid Sequence↗