The assembly of tetrameric prolyl hydroxylase in tendon fibroblasts from newly synthesized α-subunits and from preformed cross-reacting protein

Embryonic-chick tendon cells were incubated in suspension for 4h with (14)C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the beta-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition alpha(2)beta(2) where alpha and beta are non-identical subunits. Only the alpha-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The beta-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the beta-subunit of prolyl hydroxylase. Pulse-chase experiments were performed on cultured chick tendon cells to demonstrate that alpha-subunits and cross-reacting protein had half-lives of about 60h. The half-life of beta-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from alpha-subunits which are newly synthesized, and from beta-subunits which are derived from cross-reacting protein.     

Genetics of cell-surface antigens: regional mapping of three components of the human cell-surface antigen complex, AL, on chromosome 11

Cytogenetic analysis has been performed on a series of deletion mutations on human chromosome 11 of AL hybrid clones in which specific markers have been lost as a result of treatment with mutagenic agents. Such analysis has localized the three previously identified components of the AL cell-surface antigen complex to the indicated regions of chromosome 11: a1 and a3:11p13 leads to 11pter; a2:11q13 leads to 11qter. Using these methodologies human lactic dehydrogenase A localization on the short arm as reported by others has been confirmed. Evidence is presented provisionally assigning this gene to 11p13 leads to 11pter.     

Ligand-induced internalization and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor.

Signals that can mediate ligand-induced receptor internalization and calcium regulation are present in a 48-amino acid "calcium-internalization" domain in the C' terminus of the epidermal growth factor (EGF) receptor. The basis of calcium and internalization regulation signalled by this 48-amino acid sequence was analyzed using deletion and substitution mutant receptors. Cells expressing truncated receptors containing either the NH2- or COOH-terminal portion of the 48-residue domain displayed high affinity EGF-dependent endocytosis and receptor down-regulation. These endocytosis-competent EGF receptor mutants that lacked any autophosphorylation site were unable to increase the concentration of intracellular calcium. To investigate the role of self-phosphorylation in EGF-induced calcium mobilization, phenylalanine was substituted for the single autophosphorylated tyrosine residue in this region of an internalization-competent truncated receptor. The receptor-mediated calcium response was abolished, while ligand-dependent receptor internalization was unimpaired. These results demonstrate that EGF-dependent receptor endocytosis and calcium mobilization are separate events. Tyrosine self-phosphorylation is required for increased [Ca2+]i, while structural features distinct from autophosphorylation are required for receptor internalization.     

A gene cluster required for coordinated biosynthesis of lipopolysaccharide and extracellular polysaccharide also affects virulence of Pseudomonas solanacearum.

Bacterial cell surface components can be important determinants of virulence. At least three gene clusters important for extracellular polysaccharide (EPS) biosynthesis have been previously identified in the plant pathogen Pseudomonas solanacearum. We have found that one of these gene clusters, named ops, is also required for lipopolysaccharide (LPS) biosynthesis. Mutations in any complementation unit of this cluster decreased EPS production, prevented the binding of an LPS-specific phage, and altered the mobility of purified LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, restoration of LPS biosynthesis alone was not sufficient to restore virulence to the wild-type level, suggesting that EPS is important for pathogenesis.     

Brome mosaic virus RNA replication proteins 1a and 2a from a complex in vitro.

Brome mosaic virus (BMV) is a positive-strand RNA virus that encodes two RNA replication proteins, the helicaselike 1a and the polymeraselike 2a. 1a and 2a share extensive sequence similarities with proteins encoded by many other members of the alphaviruslike superfamily. While further purifying enzymatically active RNA-dependent RNA polymerase from plants infected by BMV, we observed that 1a, 2a, and the polymerase activity all cofractionated through multiple independent purification steps. Moreover, using immunoprecipitation, we found that BMV 1a and 2a proteins synthesized in rabbit reticulocyte lysates or insect cells can form a specific complex in vitro. Complex formation was more efficient when 1a and 2a were cotranslated than when they were mixed after independent synthesis. In an antibody-independent assay, in vitro-translated 1a protein was also found to bind to 2a protein fixed on a nylon membrane. A three-amino-acid insertion in 1a that blocks BMV RNA replication in vivo also blocked in vitro interaction with 2a, while another two-amino-acid insertion that renders the 1a protein temperature sensitive for RNA replication interacted in vitro with 2a at 24 degrees C but not at 32 degrees C. These results and previous genetic data suggest that the 1a-2a interaction observed in vitro is required for BMV RNA replication and may have direct implications for other members of the alphaviruslike superfamily.     

Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus.

Brome mosaic virus is a positive-strand RNA virus whose RNA replication requires viral protein 1a, which has putative helicase and capping functions, and 2a, which has putative polymerase function. Since domains of related sequence are conserved in a wide range of plus-strand RNA viruses, analysis of 1a and 2a function should have applicability to many other viruses. We have recently demonstrated that 1a and 2a form a complex in vivo and in vitro. Using immune coprecipitation and mutant polypeptides made in reticulocyte lysates, we have now mapped both the 1a and 2a domains necessary for complex formation. The sequences needed to bind 2a map to the carboxy-terminal helicase-like domain of 1a. Truncated polypeptides containing this domain were able to bind to 2a, while several small insertions in the helicase-like domain disrupted binding. The sequence required for binding 1a lies within a 115-residue subset of the 2a N-terminal segment preceding the polymerase-like domain. Truncations or fusion polypeptides containing this segment can bind 1a. We also determined that highly purified 2a protein made in insect cells can form a complex with highly purified 1a helicase-like domain made in Escherichia coli, suggesting that no other factor is required to mediate 1a-2a interaction. Previous genetic analyses of 1a and 2a are consistent with this mapping and show that the newly defined 1a and 2a binding regions are required for RNA synthesis. The locations of these interacting regions are discussed with regard to models of viral replication and the evolution of positive-strand RNA virus genomes.     

Culture conditions for induction of green plants from barley microspores by anther culture methods

Summary With barley a large variation in frequency of plant formation from microspores of spikes from the same plant has been observed. The highest frequency of plant formation was obtained when culturing anthers in the dark on a high Ficoll medium containing 2,4-D and kinetin to induce proembryo (or callus) formation. Subsequently the proembryos or calli were cultured in dim light on a high Ficoll-high sugar medium containing IBA and kinetin. Finally the embryos were transferred to a starch agar medium. A maximum of 13 green plants were obtained from microspores of a single anther.The ratios of green to albino microspore derived plants varied from 91 to 19 depending on culture conditions. Under anaerobic conditions, lactic acid and other organic acids may have damaged the organelles in the cells resulting in the formation of albino plants. Thus, direct embryogenesis by using a well-buffered, high Ficoll-high sugar medium and proper aeration are essential for obtaining high frequency of green plants from microspores.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA 3 indolylbutyric acid     

Cyclic AMP induces activity increase of kinase FA (a transmembrane signal of insulin) during NG108-15 hybrid cell differentiation

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) has been identified to exist in neuroblastoma x glioma hybrid 108-15 cells (NG108-15 cells). More importantly, when NG cells were induced to differentiate with N6, O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), the cellular activity of kinase FA was found to increase dramatically. Time course study further revealed that induction of differentiation in NG cells by dibutyryl cAMP treatment increased the FA activity to over 3 times the levels found in undifferentiated cells and in a linear day-dependent manner, indicating that the FA activity level is correlated with the state of differentiation of NG108-15 cells. This is the first report providing initial evidence that protein kinase FA (a transmembrane signal of insulin) is involved in the induction of neuronal cell differentiation.     

Time-resolved fluorometric and differential scanning calorimetric investigation of dehydroergosterol in 1-stearoyl-2-caprylphosphatidylcholine bilayers

Thermal and dynamic properties of dehydroergosterol (DHE) in 1-stearoyl-2-capryl-sn-glycero-3-phosphocholine [C(18):C(10)PC] have been studied by differential scanning calorimetry (DSC) and multifrequency phase-modulation fluorometry. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form highly ordered mixed interdigitated bilayers below the maximal transition temperature, Tm, and partially interdigitated bilayers above Tm. This lipid system is thus unique in assessing the interactions between sterols and interdigitated lipid bilayers. DHE is a fluorescent analogue of cholesterol shown in previous studies to behave like cholesterol in noninterdigitated symmetric diacylphosphatidylcholines. DSC data show that DHE exhibits similar characteristics to cholesterol [Chong & Choate (1989) Biophys. J. 55, 551-556] in C(18):C(10)PC bilayers. DHE abolishes the phase transition of C(18):C(10)PC at 27 mol % compared to 25 mol % for cholesterol and decreases Tm, the onset temperature (To), and the completion temperature (Tc), at a similar rate to cholesterol at about -0.25 degrees C per mole percent DHE. Fluorescence data show that the rotational motion of DHE can be described by a hindered anisotropic model. In the gel state of C(18):C(10)PC, the rotational correlation of DHE decreases monotonically with increasing DHE content up to 24 mol %, suggesting that DHE causes a disordering/spacing effect on the packing of mixed interdigitated C(18):C(10)PC bilayers. The rotational correlation time undergoes an abrupt increase from 24 to 27 mol % DHE. Abrupt changes in the DSC parameters were also observed in the neighborhood of 27 mol %, suggesting that major reorganization takes place around this concentration.(ABSTRACT TRUNCATED AT 250 WORDS)