(1/933) The tyrosine kinase Abl and its substrate enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance.

Genetic analysis of growth cone guidance choice points in Drosophila identified neuronal receptor protein tyrosine phosphatases (RPTPs) as key determinants of axon pathfinding behavior. We now demonstrate that the Drosophila Abl tyrosine kinase functions in the intersegmental nerve b (ISNb) motor choice point pathway as an antagonist of the RPTP Dlar. The function of Abl in this pathway is dependent on an intact catalytic domain. We also show that the Abl phosphoprotein substrate Enabled (Ena) is required for choice point navigation. Both Abl and Ena proteins associate with the Dlar cytoplasmic domain and serve as substrates for Dlar in vitro, suggesting that they play a direct role in the Dlar pathway. These data suggest that Dlar, Abl, and Ena define a phosphorylation state-dependent switch that controls growth cone behavior by transmitting signals at the cell surface to the actin cytoskeleton.  (+info)

(2/933) Specific myosin heavy chain mutations suppress troponin I defects in Drosophila muscles.

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.  (+info)

(3/933) Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMP-II homologue.

Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme-mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type-like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin.  (+info)

(4/933) An improved system for selection of forward mutations in an Escherichia coli supF gene carried by plasmids.

An improved system to examine forward mutations that occurred in the supF gene of Escherichia coli carried on a multicopy plasmid is described. The system was validated by measuring spontaneous mutations of supF plasmids propagated in wild-type, recA- and mutM- mutY- E. coli strains, the mutation frequencies of which were 1.3 x 10(-7), 6.3 x 10(-7) and 1.5 x 10(-6), respectively. Sequence analysis of the supF mutant plasmids revealed that G:C-->T:A and G:C-->C:G transversions dominated. This improved system allows rapid scoring and sequencing forward mutations in the supF gene, thus permitting its use as a genetic target for repair and mutagenesis studies in bacteria and mammalian cells.  (+info)

(5/933) Extragenic suppressors of the Arabidopsis gai mutation alter the dose-response relationship of diverse gibberellin responses.

Active gibberellins (GAs) are endogenous factors that regulate plant growth and development in a dose-dependent fashion. Mutant plants that are GA deficient, or exhibit reduced GA responses, display a characteristic dwarf phenotype. Extragenic suppressor analysis has resulted in the isolation of Arabidopsis mutations, which partially suppress the dwarf phenotype conferred by GA deficiency and reduced GA-response mutations. Here we describe detailed studies of the effects of two of these suppressors, spy-7 and gar2-1, on several different GA-responsive growth processes (seed germination, vegetative growth, stem elongation, chlorophyll accumulation, and flowering) and on the in planta amounts of active and inactive GA species. The results of these experiments show that spy-7 and gar2-1 affect the GA dose-response relationship for a wide range of GA responses and suggest that all GA-regulated processes are controlled through a negatively acting GA-signaling pathway.  (+info)

(6/933) Cobaltous chloride-induced mutagenesis in the supF tRNA gene of Escherichia coli.

The spectrum of mutations induced by cobalt(II) chloride (CoCl2) was examined using plasmid pUB3 DNA, which was propagated after transfection into Escherichia coli SY1032/pKY241 host cells. The vector plasmid carried an E.coli supF suppressor tRNA gene as a target for mutations. After CoCl2 treatment, 64 independent nalidixic acid-resistant, ampicillin-resistant and Lac+ (SupF-) clones were obtained and the altered sequences of the mutated supF genes were determined. Deletions and frameshifts were the predominant mutational event (61%) induced by CoCl2 and base substitutions were induced to a lesser degree (29%). Analysis of sequence alterations at all the sites of mutation revealed that: (i) 18 of 19 base substitutions and eight of 10 frameshifts occurred at G:C sites, suggesting that the formation of N7G-Co(II) adducts may be responsible for premutagenic lesions of these mutations; (ii) short sequence repeats were mostly found at the sites of deletions and frameshifts. Slippage-misalignment is also suggested to be a mechanism for the induction of mutations at these sites.  (+info)

(7/933) Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity.

In the yeast, Saccharomyces cerevisiae, pyruvate decarboxylase (Pdc) is encoded by the two isogenes PDC1 and PDC5. Deletion of the more strongly expressed PDC1 gene stimulates the promoter activity of both PDC1 and PDC5, a phenomenon called Pdc autoregulation. Hence, pdc1Delta strains have high Pdc specific activity and can grow on glucose medium. In this work we have characterized the mutant alleles pdc1-8 and pdc1-14, which cause strongly diminished Pdc activity and an inability to grow on glucose. Both mutant alleles are expressed as detectable proteins, each of which differs from the wild-type by a single amino acid. The cloned pdc1-8 and pdc1-14 alleles, as well as the in-vitro-generated pdc1-51 (Glu51Ala) allele, repressed expression of PDC5 and diminished Pdc specific activity. Thus, the repressive effect of Pdc1p on PDC5 expression seems to be independent of its catalytic activity. A pdc1-8 mutant was used to isolate spontaneous suppressor mutations, which allowed expression of PDC5. All three mutants characterized had additional mutations within the pdc1-8 allele. Two of these mutations resulted in a premature translational stop conferring phenotypes virtually indistinguishable from those of a pdc1Delta mutation. The third mutation, pdc1-803, led to a deletion of two amino acids adjacent to the pdc1-8 mutation. The alleles pdc1-8 and pdc1-803 were expressed in Escherichia coli and purified to homogeneity. In the crude extract, both proteins had 10% residual activity, which was lost during purification, probably due to dissociation of the cofactor thiamin diphosphate (ThDP). The defect in pdc1-8 (Asp291Asn) and the two amino acids deleted in pdc1-803 (Ser296 and Phe297) are located within a flexible loop in the beta domain. This domain appears to determine the relative orientation of the alpha and gamma domains, which bind ThDP. Alterations in this loop may also affect the conformational change upon substrate binding. The mutation in pdc1-14 (Ser455Phe) is located within the ThDP fold and is likely to affect binding and/or orientation of the cofactor in the protein. We suggest that autoregulation is triggered by a certain conformation of Pdc1p and that the mutations in pdc1-8 and pdc1-14 may lock Pdc1p in vivo in a conformational state which leads to repression of PDC5.  (+info)

(8/933) Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP pathways during invasive growth of Saccharomyces cerevisiae.

The two highly conserved RAS genes of the budding yeast Saccharomyces cerevisiae are redundant for viability. Here we show that haploid invasive growth development depends on RAS2 but not RAS1. Ras1p is not sufficiently expressed to induce invasive growth. Ras2p activates invasive growth using either of two downstream signaling pathways, the filamentation MAPK (Cdc42p/Ste20p/MAPK) cascade or the cAMP-dependent protein kinase (Cyr1p/cAMP/PKA) pathway. This signal branch point can be uncoupled in cells expressing Ras2p mutant proteins that carry amino acid substitutions in the adenylyl cyclase interaction domain and therefore activate invasive growth solely dependent on the MAPK cascade. Both Ras2p-controlled signaling pathways stimulate expression of the filamentation response element-driven reporter gene depending on the transcription factors Ste12p and Tec1p, indicating a crosstalk between the MAPK and the cAMP signaling pathways in haploid cells during invasive growth.  (+info)