Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons.

Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.

Identification and localization of ryanodine binding proteins in the avian central nervous system.

Ryanodine binding proteins of the CNS have been identified using monoclonal antibodies against avian skeletal muscle ryanodine binding proteins. These proteins were localized to intracellular membranes of the dendrites, perikarya, and axons of cerebellar Purkinje neurons using laser confocal microscopy and immunoelectron microscopy. Ryanodine binding proteins were not found in dendritic spines. Immunoprecipitation and [3H]epiryanodine binding experiments revealed that the cerebellar ryanodine binding proteins have a native molecular weight of approximately 2000 kd and are composed of two high molecular weight (approximately 500 kd) polypeptide subunits. A comparable protein having a single high molecular weight polypeptide subunit was observed in the remainder of the brain. If the ryanodine binding proteins in muscle and nerve are similar in function, then the neuronal proteins may participate in the release of calcium from intracellular stores that are mechanistically and spatially distinct from those gated by inositol trisphosphate receptors.

Three light-inducible heat shock genes of Chlamydomonas reinhardtii.

Genomic clones representing three Chlamydomonas reinhardtii genes homologous to the Drosophila hsp70 heat shock gene were isolated. The mRNAs of genes hsp68, hsp70, and hsp80 could be translated in vitro into proteins of Mr 68,000, 70,000, and 80,000, respectively. Transcription of these genes increased dramatically upon heat shock, and the corresponding mRNAs rapidly accumulated, reaching a peak at around 30 min after a shift to the elevated temperature. Light also induced the accumulation of the mRNAs encoded by these heat shock genes. A shift of dark-grown cells to light resulted in a drastic increase in mRNA levels, which reached a maximum at around 1 h after the shift. Thus, in Chlamydomonas, expression of hsp70-homologous heat shock genes appears to be regulated by thermal stress and light.

Genes involved in light control of sexual differentiation in Chlamydomonas reinhardtii.

Gamete formation requires the sequential action of two extrinsic cues, nitrogen deprivation and blue light. The mutants described here are specifically altered in the light-dependent step. Mutations lrg1, lrg3, and lrg4 overcome this light dependence while mutation lrg2 results in a delayed execution of the light-mediated step. The four mutations are linked. The recessive nature of the lrg1, lrg3, and lrg4 mutations implies that they encode elements of negative control in this light response pathway. Analyses of diploids suggest an interaction between the gene products of the mutated loci with a central role for lrg4. The lrg4 mutation is unique also because it overcomes the light dependence of Chlamydomonas zygote germination when present in homozygous form. These data indicate that there are common components in the signal chains that control gametogenesis and zygote germination.

Characterization of Blue Light Signal Transduction Chains That Control Development and Maintenance of Sexual Competence in Chlamydomonas reinhardtii.

Blue light induces the differentiation of Chlamydomonas reinhardtii pregametes to gametes. The light-induced conversion of pregametes to gametes is protein synthesis dependent and proceeds only after a lag phase. Upon incubation in the dark, gametes lost their mating ability, resulting in dark-inactivated gametes. Reillumination rapidly restored mating competence and this was shown to be independent of protein synthesis. Apparently, differentiation and maintenance of gametic competence are both regulated by light. Whether one or two light-activated signal pathways are involved was investigated using pharmacological compounds that affect signal transduction. Compounds that affected pregamete-to-gamete conversion affected the expression of a gamete-specific gene in a similar fashion. Other drugs affected only dark-inactivated gametes, suggesting that reactivating gametes requires a separate signaling pathway. Combined treatments provided evidence for the consecutive action of a phosphatase and a protein kinase C-like kinase in the light-induced reactivation process.

Dissection of the Blue-Light-Dependent Signal-Transduction Pathway Involved in Gametic Differentiation of Chlamydomonas reinhardtii.

Gametogenesis of the green alga Chlamydomonas reinhardtii may be viewed as a two-step process that is controlled by the environmental cues of nitrogen deprivation and blue light. Initiation of gametogenesis is induced by nitrogen deprivation, resulting in mating-incompetent pregametes, when cells are kept in the dark. For the completion of gametic differentiation light is required. Pregametes were treated with pharmacological compounds to influence the light-dependent conversion to mature gametes. Dibutyryl-cyclic 3[prime]5[prime] adenosinemonophosphate, papaverine, and genistein were found to inhibit the progression of gametogenesis in the light. Treatment of pregametes in the dark with either staurosporine or papaverine resulted in their conversion to mature gametes. Apparently, papaverine has different effects in the dark and in the light; the effect of staurosporine suggested that a protein kinase C-like component inhibits the conversion of pregametes to gametes, a block that normally is relieved by illumination. This hypothesis was corroborated by the observation that activators of protein kinase C, N-heptyl-5-chloro-1-naphthalenesulfonamide, N- (6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, and the phorbolester phorbol-12-myristate 13-acetate inhibited gametogenesis in the light. Genistein and dibutyryl-cyclic 3[prime]5[prime] adenosinemonophosphate were able to inhibit the dark activation caused by staurosporine treatment, suggesting that their targets work downstream from the "protein kinase C-like" kinase. Surprisingly, staurosporine and papaverine worked synergystically on the activation of pregametes in the dark.

Point mutations that affect translation initiation of the transposon Tn10 tetA gene.

Single base-pair changes affecting the 5' untranslated leader of the tetracycline-resistance gene (tetA) mRNA resulted in increased or decreased levels in expression of tetA-lacZ protein fusions. The base changes affected the rate of initiation of translation of tetA mRNA because operon fusions revealed that the mutations had little or no effect on transcription. The translational efficiency of wild-type and mutant tetA mRNAs varied by more than a factor of 2000. The observed variations could be correlated with stabilization and destabilization of RNA secondary structures. These structures, located 5' from the translation start codon, sequester the Shine-Dalgarno sequence.

Promoter-probe vectors for the analysis of divergently arranged promoters.

A series of plasmid-based promoter-probe vectors has been constructed which are particularly useful for the analysis of divergent control regions. Each vector contains a pair of divergently oriented indicator genes whose expression can be monitored over a wide range by simple assay methods. These genes are separated by different polylinkers. Specifically, the beta-galactosidase gene (lacZ) was employed in combination with either the galactokinase gene (galK) or the alkaline phosphatase gene (phoA). In all cases translational stop codons are present in all three reading frames upstream from the initiation codon. The vectors permit direct detection of promoters--independent of insert orientation--on indicator plates after transformation. Using this vector system, we further characterized the divergent tet control regions of transposon Tn10 and plasmid pBR322.

Structure of a gene encoding heat-shock protein HSP70 from the unicellular alga Chlamydomonas reinhardtii.

The structure of a gene encoding a 70-kDa heat-shock protein (HSP70) from the unicellular alga, Chlamydomonas reinhardtii, is described. This gene shows a remarkable expression pattern, because it is inducible by light as well as by elevated temperature [von Gromoff et al., Mol. Cell. Biol. 9 (1989) 3911-3918]. As a first step in the investigation of trans-acting factors involved in environmentally controlled expression of this hsp70 gene, the nucleotide sequence of the entire gene, including its 5'- and 3'-flanking regions was determined. Although the deduced amino acid sequence exhibits a high degree of conservation to the HSP70 from higher plants, the C. reinhardtii gene has a unique structure among the members of the hsp70 gene family. While most hsp70 genes have only one or no intron, the coding region of the C. reinhardtii gene is interrupted by six introns. Besides putative TATA and CCAAT boxes, two heat-shock elements (HSE) were found in the promoter region, and a third HSE motif was located within the fourth intron. A computer search for regulatory cis-acting elements revealed a noted similarity of a 5'-upstream sequence motif to the G-box motif conserved in higher plants. A polyadenylation recognition sequence canonical for nuclear genes of C. reinhardtii is located downstream from the coding sequence.

An ISFET-algal (Chlamydomonas) hybrid provides a system for eco-toxicological tests.

A cellular sensoring system was designed in which metabolism-dedicated pH-ISFETs and the unicellular green alga Chlamydomonas reinhardtii as a biological component, were combined. The system permits on-line detection of pH changes caused by the metabolic and photosynthetic activities of the cells. Photosynthetic activity results in a basification of the medium caused by uptake of CO2. In darkness, an acidification of the medium, resulting from the production of CO2 by degradation of starch was observed. Both, acidification and basification, are sensitive indicators for the physiological activity of the alga. Experiments using inhibitors of energy metabolism or photosynthesis illustrate the utility of this system for an on-line monitoring of substances of eco-toxicological importance.

Monoclonal antibodies specific for Corynebacterium sepedonicum, the causative agent of potato ring rot.

BALB/c mice were immunized with Corynebacterium sepedonicum, and spleen cells from the immunized animals were fused with cells of the mouse myeloma line P3-X63-Ag8.653. Several hybridoma cell cultures were selected for further study. Monoclonal CS-B-5 was specific for C. sepedonicum and did not react significantly with other closely related phytopathogenic corynebacteria in an enzyme-linked immunosorbent assay (ELISA). As few as 10(3) organisms could be detected. This approach should prove useful for developing improved diagnostic procedures for a number of bacterial plant pathogens.

A genetic approach to analysis of transposons.

Integration of the tetracycline resistance transposon Tn10 into lacI of a lacI-lacZ gene fusion permits the isolation of deletions that excise DNA from one end of Tn10 and fuse Tn10 genes with lacZ in such a manner that chimeric proteins with beta-galactosidase activity are produced. The synthesis of the chimeric proteins is under the same control as the transposon genes. Thus, regulation of expression of Tn10 genes can be investigated by measuring beta-galactosidase activity. Analysis of Tn10-lacZ fusions revealed different deletion endpoints within Tn10; lacZ has been fused to at least three different Tn10 genes or operons. Two of these genes are under the control of a tetracycline repressor.

Tetracycline diffusion through phospholipid bilayers and binding to phospholipids.

The ability of tetracycline to pass through phospholipid bilayers by diffusion was investigated. Liposomes did not retain enclosed tetracycline. Accumulation of tetracycline was observed with liposomes containing entrapped Tet repressor protein. These results indicate that the drug can pass through lipid bilayers. The antibiotic was also shown to bind to liposomes and isolated phospholipids.

Topology of the transposon Tn10-encoded tetracycline resistance protein within the inner membrane of Escherichia coli.

The transposon Tn10-encoded tetracycline resistance protein TetA is an integral membrane protein responsible for the export of tetracycline from the cytoplasmic to the periplasmic side of the inner membrane of Gram-negative bacteria. From a plot of the average hydrophobicity along the sequence of this protein, a two-dimensional membrane topology with 12 transmembrane domains may be predicted. Using plasmid-bearing Escherichia coli maxicells we specifically radiolabeled the TetA protein. The amino terminus of this membrane protein was shown not to be processed, and its location on the inner side of the cytoplasmic membrane was demonstrated by a newly developed use of a chemical method. Spheroplasts and inside-out vesicles of the TetA protein synthesizing maxicells were subjected to limited digestion by proteases of different specificities. The TetA protein was not accessible to proteases from the periplasmic side. On the inner side of the cytoplasmic membrane, the carboxyl terminus and four sites accessible to endoproteases could be identified. The cleavage sites are proposed to be localized between amino acid residues 60-70, 110-130, 180-200, and at amino acid 327. These results allow the definition of a model for the two-dimensional topology of the TetA protein.

Gametic Differentiation of Chlamydomonas reinhardtii: Control by Nitrogen and Light.

Gametic differentiation of the unicellular green alga Chlamydomonas reinhardtii proceeds in two steps controlled by the extrinsic signals nitrogen deficiency and light. Nitrogen deprivation induces the differentiation of vegetative cells to sexually immature pregametes. A light signal is required to convert the pregametes to gametes. Both signals are also required for the maintenance of mating competence. Two converging signal transduction chains are proposed to control gamete formation. For the differentiation of pregametes to gametes, a fluence rate-dependent reaction, requiring continuous irradiation, is suggested by photobiological experiments.