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C F Beck

Publications and source records attributed to C F Beck.

At least 37 records · Page 2Linked to original sources
PubMed1993

Chlamydomonas mutants affected in the light-dependent step of sexual differentiation.

Sexual differentiation of Chlamydomonas reinhardtii is induced by the consecutive action of two extrinsic cues--nitrogen deprivation and blue light. The definition of a blue light-dependent step in gamete formation provided a basis for the isolation of mutants altered in the signal transduction pathway by which light controls sexual differentiation. In one mutant (lrg1), gamete formation has become light independent. In the other mutant (lrg2), perception or transduction of the light signal appears to be partially impaired. In both mutants, the expression of genes activated by light in the late phase of gamete formation is affected. Genetic analyses showed that genes LRG1 and LRG2 are linked. The recessive nature of the lrg1-1 mutation implies that the gene encodes a negative factor or a protein that controls the activity of a negative factor. In the case of lrg2-1, neither wild-type nor mutant allele was dominant. Rather, two copies of the lrg2-1 gene simulate a wild-type phenotype. The identification of genetic loci in the pathway for blue light-mediated differentiation provides a basis for the isolation of signal transduction genes in Chlamydomonas.

Animals↗
PubMed1993

Failure to make normal alpha ryanodine receptor is an early event associated with the crooked neck dwarf (cn) mutation in chicken.

We have investigated the molecular basis of the Crooked Neck Dwarf (cn) mutation in embryonic chickens. Using biochemical and pharmacological techniques we are unable to detect normal alpha ryanodine receptor (RyR) protein in intact cn/cn skeletal muscle. Extremely low levels of alpha RyR immunoreactivity can be observed in mutant muscles, but the distribution of this staining differs from that in normal muscle and colocalizes with the rough endoplasmic reticulum immunoglobulin binding protein, BiP. This suggests the existence of an abnormal alpha RyR protein in mutant muscle. In day E12 cn/cn muscle the levels of RyR mRNA are reduced by approximately 80%, while the levels of other muscle proteins, including the alpha 1 subunit of the dihydropyridine receptor, the SR Ca(2+)-ATPase, calsequestrin, and glyceraldehyde-3-phosphate dehydrogenase, and their associated mRNAs are essentially normal in cn/cn muscle. There is also a failure to express alpha RyR in cn/cn cerebellar Purkinje neurons. Expression of the beta RyR, a second RyR isoform, is not initiated in normal skeletal muscle until day E18. In cn/cn skeletal muscle significant muscle degeneration has occurred by this time and the beta RyR is found at low levels in only a subset of fibers suggesting the reduced levels of this isoform are a secondary consequence of the mutation. The cardiac RyR isoform is found in cn/cn cardiac muscle, which contracts in a vigorous manner. In summary, a failure to make normal alpha RyR receptor appears to be an event closely associated with the cn mutation and one which may be largely responsible for development of the cn/cn phenotype in embryonic skeletal muscle.

Animals↗
PubMed1993

Ryanodine receptor expression in embryonic avian cardiac muscle.

The cardiac ryanodine receptor serves as a sarcoplasmic reticulum calcium release channel and contributes to the rise in cytosolic calcium necessary for contraction of the heart. We have investigated the presence and oligomeric form of the ryanodine receptor during embryonic development of the avian heart. A single isoform of the ryanodine receptor was identified both in adult and in embryonic cardiac tissue from Days 4 to 20 of development, using anti-receptor monoclonal antibodies in conjunction with [3H]ryanodine binding. The results of sucrose density gradient sedimentation analysis and [3H]ryanodine binding indicated that the cardiac ryanodine receptor is present in a tetrameric form in both the adult and embryos at Day 6 of development. The observation of specific [3H]ryanodine binding in hearts from Days 4 and 5 of embryonic development also indicates the presence of a tetrameric receptor protein. Although the heart begins to beat at approximately 33-38 hr (Day 1.5) of embryonic development, we were unable to detect the cardiac ryanodine receptor, using biochemical or immunological techniques, prior to Embryonic Day 4. Ryanodine was found to alter the chronotropic state of intact hearts as early as Hamburger and Hamilton stages 15-19 (Embryonic Day 3) suggesting that receptor protein may be present in a limited subset of cells involved in pacemaker activity in the very early embryo. After Embryonic Day 4, ryanodine also exerted a negative inotropic effect on embryonic hearts. Our results suggest that a single isoform of the ryanodine receptor is present from Day 4 to Day 20 of embryonic development in avian cardiac muscle and that the ryanodine receptor assumes a tetrameric structure capable of forming a functional calcium release channel that participates excitation-contraction coupling as early as Embryonic Day 4. In addition, a ryanodine receptor-related function may serve as a determinant of chronotropic effects in the very early embryonic heart.

Animals↗
PubMed1993

Genes expressed during sexual differentiation of Chlamydomonas reinhardtii.

Four genes specifically expressed during gametogenesis of Chlamydomonas reinhardtii have been cloned and their expression patterns analyzed. mRNAs encoded by these gamete-specific genes (gas) were absent or present only at very low levels in vegetative cells and mature zygotes. In young zygotes 2 h after gamete fusion, the mRNAs of three gas genes still persisted. The gas mRNAs accumulated during gametic differentiation. The temporal patterns of accumulation of individual mRNAs differed; some started to increase early during gametogenesis, others accumulated in the late phase. The accumulation of one of the late mRNAs (gas28) was strictly light-dependent. To illustrate the utility of the genes cloned in the analysis of sexual differentiation in Chlamydomonas reinhardtii we show that in a gametogenesis-defective mutant, the expression of late genes is prevented while that of early genes is normal.

Animals↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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.

Journal Article↗
PubMed1991

Identification of novel genes specifically expressed in Chlamydomonas reinhardtii zygotes.

The maturation of zygotes formed by the fusion of two gametes is the essential part of the diploid phase of the Chlamydomonas reinhardtii sexual life cycle and results in mature zygotes competent to germinate. To understand the molecular mechanisms underlying zygote maturation and the attainment of competence for germination we isolated genomic clones representing three different genes that are specifically expressed in Chlamydomonas reinhardtii zygotes. Accumulation of the RNAs started more than 24 h after mating, setting these genes apart from genes expressed in young zygotes. Upon light-induced germination of zygotes, the mRNAs disappeared. The patterns of RNA accumulation and disappearance were gene-specific and suggested a function of these genes in maturation and/or in initial steps of germination.

Blotting, Northern↗
PubMed1991

Nonmammalian vertebrate skeletal muscles express two triad junctional foot protein isoforms.

Mammalian skeletal muscles express a single triad junctional foot protein, whereas avian muscles have two isoforms of this protein. We investigated whether either case is representative of muscles from other vertebrate classes. We identified two foot proteins in bullfrog and toadfish muscles on the basis of (a) copurification with [3H]epiryanodine binding; (b) similarity to avian muscle foot proteins in native and subunit molecular weights; (c) recognition by anti-foot protein antibodies. The bullfrog and toadfish proteins exist as homooligomers. The subunits of the bullfrog muscle foot protein isoforms are shown to be unique by peptide mapping. In addition, immunocytochemical localization established that the bullfrog muscle isoforms coexist in the same muscle cells. The isoforms in either bullfrog and chicken muscles have comparable [3H]epiryanodine binding capacities, whereas in toadfish muscle the isoforms differ in their levels of ligand binding. Additionally, chicken thigh and breast muscles differ in the relative amounts of the two isoforms they contain, the amounts being similar in breast muscle and markedly different in thigh muscle. In conclusion, in contrast to mammalian skeletal muscle, two foot protein isoforms are present in amphibian, avian, and piscine skeletal muscles. This may represent a general difference in the architecture and/or a functional specialization of the triad junction in mammalian and nonmammalian vertebrate muscles.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Action Spectrum for the Light-Dependent Step in Gametic Differentiation of Chlamydomonas reinhardtii.

Differentiation of Chlamydomonas reinhardtii vegetative cells to gametes requires two environmental signals: nitrogen starvation and light. Vegetative cells incubated without nitrogen differentiate into pregametes. Pregametes can be converted into sexually mature gametes by irradiation with light. The action spectrum for the light-dependent step in gamete formation showed two maxima at 370 and 450 nanometers. This is similar to the spectrum of other blue light/ultraviolet light-A-absorbing photoreceptors.

Journal Article↗
PubMed1990

Identification and localization of two triad junctional foot protein isoforms in mature avian fast twitch skeletal muscle.

We report evidence for two foot protein isoforms in chicken pectoral muscle. (i) Two polypeptides with molecular masses of approximately 500 kDa copurify with [3H]ryanodine binding. (ii) Both polypeptides are associated with oligomeric proteins similar in size to the mammalian skeletal muscle foot protein. (iii) The polypeptides are shown to be unique by limited proteolysis. (iv) By using isoform-specific antibodies, the polypeptides are shown to be subunits of different [3H]ryanodine-binding proteins. Using immunolabeling techniques, we have localized these proteins in chicken breast muscle by both light and electron microscopy. (v) From immunofluorescent light microscopy of longitudinal sections, it was determined that both ryanodine-binding protein isoforms exhibit identical repetitive punctate distributions near the Z-lines. (vi) In serial cross-sections both proteins have similar distributions in the same fibers. (vii) Both proteins were found to be associated with the terminal cisternae of the sarcoplasmic reticulum by immunoelectron microscopy. Based on their localization to the triadic junction, their large size and their ability to bind [3H]ryanodine, these proteins are identified as foot proteins. In conclusion, two distinct homo-oligomeric foot proteins coexist in avian fast twitch skeletal muscle. We have termed these proteins, alpha and beta foot proteins.

Alkaloids↗
PubMed1990

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.

Alkaloids↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

Overproduction of transposon Tn10-encoded tetracycline resistance protein results in cell death and loss of membrane potential.

High-level expression of the Tn10 tetracycline resistance protein TetA in Escherichia coli caused partial collapse of the membrane potential, arrest of growth, and killing of the cells. Since alpha-methylglucoside transport was not affected, the overproduced TetA protein may cause not destruction of membrane structure but rather unrestricted translocation of protons and/or ions across the membrane.

Bacterial Proteins↗