Biomedical subjects

P Barthmaier

Publications and source records attributed to P Barthmaier.

6 recordsLinked to original sources

PubMed2001

Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2 identified using a cell-based screen in yeast.

Multicellular organisms must have means of preserving their genomic integrity or face catastrophic consequences such as uncontrolled cell proliferation or massive cell death. One response is a modification of nuclear proteins by the addition and removal of polymers of ADP-ribose that modulate the properties of DNA-binding proteins involved in DNA repair and metabolism. These ADP-ribose units are added by poly(ADP-ribose) polymerase (PARP) and removed by poly(ADP-ribose) glycohydrolase. Although budding yeast Saccharomyces cerevisiae does not possess proteins with significant sequence similarity to the human PARP family of proteins, we identified novel small molecule inhibitors against two family members, PARP1 and PARP2, using a cell-based assay in yeast. The assay was based on the reversal of growth inhibition caused by the heterologous expression of either PARP1 or PARP2. Validation of the assay was achieved by showing that the growth inhibition was relieved by a mutation in a single residue in the catalytic site of PARP1 or PARP2 or exposure of yeast to a known PARP1 inhibitor, 6(5H)-phenanthridinone. In separate experiments, when a putative protein regulator of PARP activity, human poly(ADP-ribose) glycohydrolase, was coexpressed with PARP1 or PARP2, yeast growth was restored. Finally, the inhibitors identified by screening the yeast assay are active in a mammalian PARP biochemical assay and inhibit PARP1 and PARP2 activity in yeast cell extracts. Thus, our data reflect the strength of using yeast to identify small molecule inhibitors of therapeutically relevant gene families, including those that are not found in yeast, such as PARP. The resultant inhibitors have two critical uses (a) as leads for drug development and (b) as tools to dissect cellular function.

ATP-Binding Cassette Transporters↗

PubMed2001

A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays.

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.

Amino Acid Sequence↗

PubMed1998

A family of Drosophila genes encoding quaking-related maxi-KH domains.

We recently identified a Drosophila gene, wings held out (who), that specifies a STAR (signal transduction and RNA activation) protein expressed within mesoderm and muscles. Genetic evidence suggests that WHO regulates muscle development and function in response to steroid hormone titer. who is related to the mouse quacking gene, essential for embryogenesis and neural myelination, and gld-1, a nematode tumor suppressor gene necessary for oocyte differentiation, both of which contain RNA binding "maxi-KH" domains presumed to link RNA metabolism to cell signaling. To initiate a broader study of Drosophila WHO-related proteins we used degenerate primers encoding peptides unique to maxi-KH domains to amplify the corresponding genes. We recovered nine genes, all specifying single maxi-KH domain proteins having tripartite regions of similarity that extend over 200 amino acids. One is located within the 54D chromosome subdivision, and one within 58C, while the remaining seven are within the 58E subdivision. At least four of these STAR proteins are expressed in a general manner, suggesting that maxi-KH domains are employed widely in Drosophila.

Amino Acid Sequence↗

PubMed1997

A Drosophila muscle-specific gene related to the mouse quaking locus.

We have characterized a novel muscle-specific gene of Drosophila melanogaster, defined by enhancer trap strain 24B of Brand and Perrimon (1993). We show that transcripts of the gene accumulate within presumptive mesoderm and persist within developing muscles, strongly suggesting that the encoded protein is involved in muscle cell determination and differentiation. cDNA sequences reveal that the Drosophila protein is similar to quaking (64% identity over 210 amino acids), a protein essential for mouse embryogenesis, and gld-1 (53% identity over 162 amino acids) a germ-line-specific tumor suppressing protein of the nematode, Caenorhabditis elegans. We demonstrate that the Drosophila gene resides within the 93F chromosome subdivision, and describe its physical map. Finally, we have used the gene, which we have named quaking-related 93F (qkr93F), to identify a family of closely related KH domains.

Amino Acid Sequence↗

PubMed1997

Green fluorescent protein/beta-galactosidase double reporters for visualizing Drosophila gene expression patterns.

We characterized 120 novel yeast Ga14-targeted enhancer trap lines in Drosophila using upstream activating sequence (UAS) reporter plasmids incorporating newly constructed fusions of Aequorea victoria green fluorescent protein (GFP) and Escherichia coli beta-galactosidase genes. Direct comparisons of GFP epifluorescence and beta-galactosidase staining revealed that both proteins function comparably to their unconjugated counterparts within a wide variety of Drosophila tissues. Generally, both reporters accumulated in similar patterns within individual lines, but in some tissues, e.g., brain, GFP staining was more reliable than that of beta-galactosidase, whereas in other tissues, most notably tests and ovaries, the converse was true. In cases of weak enhancers, we occasionally could detect beta-galactosidase staining in the absence of discernible GFP fluorescence. This shortcoming of GFP can, in most cases, be alleviated by using the more efficient S65T GFP derivative. The GFP/beta-gal reporter fusion protein facilitated monitoring several aspects of protein accumulation. In particular, the ability to visualize GFP fluorescence enhances recognition of global static and dynamic patterns in live animals, whereas beta-galactosidase histochemistry affords sensitive high resolution protein localization. We present a catalog of Ga 14-expressing strains that will be useful for investigating several aspects of Drosophila melanogaster cell and developmental biology.

Animals↗

PubMed1995

Monitoring development and pathology of Drosophila indirect flight muscles using green fluorescent protein.

We describe the use of a green fluorescent protein (GFP) reporter construct to monitor indirect flight muscle development in normal and mutant Drosophila melanogaster strains. We used polymerase chain reaction to amplify a portion of the Act88F actin gene that includes 1420 nucleotides of flanking DNA, the transcription start, first intron, and initiator codon, incorporating the fragment into the Drosophila germ line transformation vector pCaSpeR. We fused the fragment to the gene encoding green fluorescent protein of the bioluminescent jellyfish Aequorea victoria. We could detect GFP protein in transgenic strains and found that its accumulation, conveniently visualized in living flies using epifluorescence microscopy, was limited to the indirect flight muscles. GFP fluorescence can be used to visualize all stages of flight muscle development subsequent to myoblast fusion nad facilitates the detection of morphological changes in fibers caused by particular mutations.

Actins↗