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J D Gross

Publications and source records attributed to J D Gross.

At least 19 recordsLinked to original sources
PubMed2000

Identification of STKA-dependent genes in Dictyostelium discoideum.

During culmination of Dictyostelium aggregates, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. Disruption of the cell-fate gene stkA leads to a phenotype in which all the cells destined to become spores end up as stalk cells. 'Stalky' mutants express normal levels of prespore cell transcripts but fail to produce the culmination-stage spore transcript spiA. The stkA gene encodes a putative GATA-type transcription factor (STKA). In order to identify possible downstream targets of STKA we used the technique of mRNA differential display and isolated four cDNA fragments that hybridise to mRNAs present during the later stages of development. All four gene tags were cloned and sequenced. mRNAs represented by these four sequence tags do not accumulate during culmination of 'stalky' cells and therefore must be specific to the spore pathway. By screening a cDNA library, longer cDNAs for all four were cloned and sequenced. Three of these contained complete protein-coding regions while only a partial cDNA was recovered for the fourth. One of the corresponding proteins has significant homology to a surface zinc metalloproteinase (GP63) of the protozoan parasite Leishmania, while another is closely related to a human pre-RNA binding protein (hnRNP R).

Amino Acid Sequence↗
PubMed1999

A negative regulatory element in a prespore-specific promoter of dictyostelium discoideum(1).

We previously isolated several 'promoter-trap' transformants in which insertion of a promoterless beta-galactosidase gene into the genome caused expression of beta-galactosidase in specific cell types. The upstream flanking region was rescued from one transformant specifically expressing beta-galactosidase in prespore cells. We sequenced the promoter of the gene that is fused in-frame with lacZ and characterised a negative element that inhibits expression in pstO cells (a subtype of prestalk cells). Gel-retardation assays show that a developmentally regulated factor(s) recognises and binds to this element.

Amino Acid Sequence↗
PubMed1999

The Cap-binding protein eIF4E promotes folding of a functional domain of yeast translation initiation factor eIF4G1.

The association of eucaryotic translation initiation factor eIF4G with the cap-binding protein eIF4E establishes a critical link between the mRNA and the ribosome during translation initiation. This association requires a conserved seven amino acid peptide within eIF4G that binds to eIF4E. Here we report that a 98-amino acid fragment of S. cerevisiae eIF4G1 that contains this eIF4E binding peptide undergoes an unfolded to folded transition upon binding to eIF4E. The folding of the eIF4G1 domain was evidenced by the eIF4E-dependent changes in its protease sensitivity and (1)H-(15)N HSQC NMR spectrum. Analysis of a series of charge-to-alanine mutations throughout the essential 55.4-kDa core of yeast eIF4G1 also revealed substitutions within this 98-amino acid region that led to reduced eIF4E binding in vivo and in vitro. These data suggest that the association of yeast eIF4E with eIF4G1 leads to the formation of a structured domain within eIF4G1 that could serve as a specific site for interactions with other components of the translational apparatus. They also suggest that the stability of the native eIF4E-eIF4G complex is determined by amino acid residues outside of the conserved seven-residue consensus sequence.

Amino Acid Sequence↗
PubMed1999

A myb-related protein required for culmination in Dictyostelium.

The avian retroviral v-myb gene and its cellular homologues throughout the animal and plant kingdoms contain a conserved DNA binding domain. We have isolated an insertional mutant of Dictyostelium unable to switch from slug migration to fruiting body formation i.e. unable to culminate. The gene that is disrupted, mybC, codes for a protein with a myb-like domain that is recognized by an antibody against the v-myb repeat domain. During development of myb+ cells, mybC is expressed only in prestalk cells. When developed together with wild-type cells mybC- cells are able to form both spores and stalk cells very efficiently. Their developmental defect is also bypassed by overexpressing cAMP-dependent protein kinase. However even when their defect is bypassed, mybC null slugs and culminates produce little if any of the intercellular signalling peptides SDF-1 and SDF-2 that are believed to be released by prestalk cells at culmination. We propose that the mybC gene product is required for an intercellular signaling process controlling maturation of stalk cells and spores and that SDF-1 and/or SDF-2 may be implicated in this process.

Amino Acid Sequence↗
PubMed1998

A novel adenylyl cyclase detected in rapidly developing mutants of Dictyostelium.

Disruption of either the RDEA or REGA genes leads to rapid development in Dictyostelium. The RDEA gene product displays homology to certain H2-type phosphotransferases, while REGA encodes a cAMP phosphodiesterase with an associated response regulator. It has been proposed that RDEA activates REGA in a multistep phosphorelay. To test this proposal, we examined cAMP accumulation in rdeA and regA null mutants and found that these mutants show a pronounced accumulation of cAMP at the vegetative stage that is not observed in wild-type cells. This accumulation was due to a novel adenylyl cyclase and not to the known Dictyostelium adenylyl cyclases, aggregation stage adenylyl cyclase (ACA) or germination stage adenylyl cyclase (ACG), since it occurred in an acaA/rdeA double mutant and, unlike ACG, was inhibited by high osmolarity. The novel adenylyl cyclase was not regulated by G-proteins and was relatively insensitive to stimulation by Mn2+ ions. Addition of the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) permitted detection of the novel adenylyl cyclase activity in lysates of an acaA/acgA double mutant. The fact that disruption of the RDEA gene as well as inhibition of the REGA-phosphodiesterase by IBMX permitted detection of the novel AC activity supports the hypothesis that RDEA activates REGA.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Evidence that the RdeA protein is a component of a multistep phosphorelay modulating rate of development in Dictyostelium.

We have isolated an insertional mutant of Dictyostelium discoideum that aggregated rapidly and formed spores and stalk cells within 14 h of development instead of the normal 24 h. We have shown by parasexual genetics that the insertion is in the rdeA locus and have cloned the gene. It encodes a predicted 28 kDa protein (RdeA) that is enriched in charged residues and is very hydrophilic. Constructs with the DNA for the c-Myc epitope or for the green fluorescent protein indicate that RdeA is not compartmentalized. RdeA displays homology around a histidine residue at amino acid 65 with members of the H2 module family of phosphotransferases that participate in multistep phosphoryl relays. Replacement of this histidine rendered the protein inactive. The mutant is complemented by transformation with the Ypd1 gene of Saccharomyces cerevisiae, itself an H2 module protein. We propose that RdeA is part of a multistep phosphorelay system that modulates the rate of development.

Adenylyl Cyclases↗
PubMed1998

Determination of the peptide torsion angle phi by 15N chemical shift and 13Calpha-1Halpha dipolar tensor correlation in solid-state MAS NMR.

We demonstrate a dipolar-chemical shift correlation technique for sign-sensitive determination of the torsion angle phi in solid peptides and proteins under magic-angle spinning. The indirect dimension of the experiment is obtained by separate but synchronous evolution of the magnetization under the 15N chemical shift and the C-H dipolar coupling. The resulting sum and difference spectrum of the two frequencies, with more than ten independent sidebands, depends strongly on the relative orientation of the 15N chemical shift tensor and the Calpha-Halpha bond. This relative orientation reflects the C(O)i-1-N-Calpha-C(O)i torsion angle. The technique can distinguish phi angles over the full range of 360 degrees when the amide 15N chemical shift tensor does not possess reflection symmetry with respect to the peptide plane. Thus it complements our previous HNCH experiment, in which two mirror-symmetric conformers of the HN-N bond relative to the Calpha-Halpha bond around the N-Calpha axis cannot be distinguished.

Magnetic Resonance Spectroscopy↗
PubMed1997

Non-invasive measurements of breast tissue optical properties using frequency-domain photon migration.

A multiwavelength, high bandwidth (1 GHz) frequency-domain photon migration (FDPM) instrument has been developed for quantitative, non-invasive measurements of tissue optical and physiological properties. The instrument produces 300 kHz to 1 GHz photon density waves (PDWs) in optically turbid media using a network analyser, an avalanche photodiode detector and four amplitude-modulated diode lasers (674 nm, 811 nm, 849 nm, and 956 nm). The frequency of PDW phase and amplitude is measured and compared to analytically derived model functions in order to calculate absorption, mu a, and reduced scattering, mu s, parameters. The wavelength-dependence of absorption is used to determine tissue haemoglobin concentration (total, oxy- and deoxy- forms), oxygen saturation and water concentration. We present preliminary results of non-invasive FDPM measurements obtained from normal and tumour-containing human breast tissue. Our data clearly demonstrate that physiological changes caused by the presence of small (about 1 cm diameter) palpable lesions can be detected using a handheld FDPM probe.

Adult↗
PubMed1997

Coupling amplification in 2D MAS NMR and its application to torsion angle determination in peptides.

A technique for amplifying the apparent magnitudes of 13C-1H and 15N-1H dipolar interactions in magic-angle spinning experiments is described. By inserting rotor-synchronized 180 degrees pulses in the evolution period of a 2D dipolar-chemical shift experiment, heteronuclear dipolar couplings are doubled or quadrupled relative to the spinning speed. The increased number of dipolar sidebands is desirable for retaining structural information in the indirectly detected dipolar dimension while resolving inequivalent sites in the isotropic chemical shift dimension at relatively high spinning speeds. This coupling amplification method is incorporated into an experiment that determines the peptide torsion angle phi through the relative orientation of the Calpha-Halpha and N-HN bonds. It is shown both experimentally and theoretically that the angular resolution of the measurement is enhanced significantly by the selective doubling of the N-HN coupling.

Chemical Phenomena↗
PubMed1996

Identification of the cell fate gene stalky in Dictyostelium.

Using insertional mutagenesis, we have isolated a "stalky" mutant in which cells destined to become spores end up as stalk cells. Similar mutants were previously observed after chemical mutagenesis, but the affected gene could not be isolated. Our mutant, like the previous ones, is in stkA. Its defect is cell-autonomous and not overcome by overexpressing cAMP-dependent protein kinase. stkA is strongly expressed in the prespore region of aggregates but not in the anterior prestalk zone. The mutant expresses normal levels of prespore-cell transcripts but fails to produce the spore transcript spiA. stkA encodes a predicted 99 kDa protein (STKA) with two putative C4 zinc fingers, one of which is a GATA-type finger, indicating that it may be a transcription factor. This conclusion is supported by localization of STKA in the nucleus.

Amino Acid Sequence↗
PubMed1996

Vacuolar H(+)-ATPase and weak base action in Dictyostelium.

Amoebae of Dictyostelium discoideum release ammonia during development, and the accumulation of this weak base is believed to be responsible for inhibiting fruiting-body formation and switching aggregates into migrating slugs. Exposure to weak bases can also inhibit aggregation and cell-type specific gene expression. The pathway by which weak bases influence development is not understood. We show here that the development of a set of mutants defective in acidification of intracellular acidic compartments is abnormally sensitive to inhibition by weak bases. Moreover even in the absence of added weak bases these mutants are delayed in aggregation and have a protracted migratory phase. The same behaviour is observed in transformants harbouring an antisense construct for one of the vacuolar H(+)-ATPase subunits. These results support the idea that weak bases exert their effects by inhibiting acidification of an intracellular acidic compartment.

Acids↗
PubMed1995

The role of calcium in aggregation and development of Dictyostelium.

Changes in cytosolic Ca2+ play an important role in a wide array of cell types and the control of its concentration depends upon the interplay of many cellular constituents. Resting cells maintain cytosolic calcium ([Ca2+]i) at a low level in the face of steep gradients of extracellular and sequestered Ca2+. Many different signals can provoke the opening of calcium channels in the plasma membrane or in intracellular compartments and cause rapid influx of Ca2+ into the cytosol and elevation of [Ca2+]i. After such stimulation Ca2+ ATPases located in the plasma membrane and in the membranes of intracellular stores rapidly return [Ca2+]i to its basal level. Such responses to elevation of [Ca2+]i are a part of an important signal transduction mechanism that uses calcium (often via the binding protein calmodulin) to mediate a variety of cellular actions responsive to outside influences.

Animals↗
PubMed1995

Trapping developmental promoters in Dictyostelium.

Recently an insertional mutagenesis procedure has been developed to permit cloning of genes affected in developmental mutants of Dictyostelium discoideum (Kuspa and Loomis, Proc. Natl. Acad. Sci. USA 89, 8803-8807, 1992). In this procedure a plasmid bearing the URA (pyr5-6) gene is linearized with a restriction enzyme and electroporated into URA- amoebae (auxotrophic for uracil) together with the corresponding restriction enzyme. Transformants that can grow without uracil are screened for developmental defects resulting from insertion of the plasmid into a gene of developmental importance. We have modified this procedure to permit characterization of the promoters and structural sequences of genes that would be missed by the standard procedure because their disruption produces no obvious phenotype. Constructs carrying a promoter-less Escherichia coli lacZ gene were designed so that expression of lacZ requires insertion into an active host transcription unit. By screening restriction enzyme-generated transformants we have isolated several strains in which lacZ is under the control of a developmentally activated promoter and have cloned the 5' flanking DNA adjacent to the insertion site. Sequencing the junction between plasmid and host genome has confirmed in-frame fusion with the lacZ gene, and reintroduction of the cloned plasmids into parental cells has shown that the cloned sequences do actually contain the relevant promoters. This procedure should give ready access to a wide range of developmental promoters without the need for prior identification of the developmental genes involved.

Animals↗
PubMed1995

Anion effects on vesicle acidification in Dictyostelium.

Chloride ions stimulated the ATP-dependent formation of a proton gradient in vesicles derived from amoebae of the cellular slime mould, D. discoideum, and reduced the formation of a membrane potential, inhibited rather than stimulated the formation of the proton gradient. Since bicarbonate ions did not inhibit H(+)-ATPase activity we conclude that they enter the vesicles and combine with translocated protons. This finding is consistent with the suggestion that the membranes of the light vesicle fraction are fragments of contractile vacuole complexes, and that these organelles increase their osmotic activity by taking up bicarbonate ions and protons from the cytoplasm, and then release water and carbonic acid into the extracellular milieu.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Characterisation of an intracellular Ca2+ pump in Dictyostelium.

Calcium uptake by microsomal membranes from the cellular slime mould Dictyostelium discoideum was measured using Calcium Green-2 as a fluorescent probe of external free Ca2+ concentration. High-affinity Ca2+ uptake was found to be completely inhibited by low concentrations of vanadate, but not by thapsigargin, suggesting that the activity is mediated by a Ca(2+)-ATPase distinct from sarco(endo)plasmic reticulum type of higher animal cells. On sucrose density gradients, Ca2+ uptake distributes with vacuolar proton pump activity and part of the observed Ca2+ uptake is dependent on the pH gradient generated by the vacuolar-type H(+)-ATPase, indicating that the Ca2+ pump is located on both acidic and non-acidic vesicles, possibly derived from the H(+)-ATPase-rich contractile vacuole complex.

Animals↗