Changes in Protein Kinase A Activity Accompany Sclerotial Development in Sclerotinia sclerotiorum.

ABSTRACT Sclerotia of Sclerotinia sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Sclerotial formation has been shown to be affected by increased intracellular cAMP levels. Cyclic AMP (cAMP) is a key modulator of cAMP-dependent protein kinase A (PKA) and the latter may prove to play a significant role in sclerotial development. Therefore, we monitored changes in relative PKA activity levels during sclerotial development. To do so, we first developed conditions for near-synchronous sclerotial development in culture, based on hyphal maceration and filtering. Relative PKA activity levels increased during the white-sclerotium stage in the wild-type strain, while low levels were maintained in nonsclerotium-producing mutants. Furthermore, applying caffeine, an inducer of PKA activity, resulted in increased relative PKA activity levels and was correlated with the formation of sclerotial initial-like aggregates in cultures of the non-sclerotium-producing mutants. In addition, low PKA activities were found in an antisense smk1 strain, which exhibits low extracellular-signal-regulated kinase (ERK)-type mitogen-activated protein kinase (MAPK) activity, and does not produce sclerotia. The changes in PKA activity, as well as the abundance of phosphorylated MAPKs (ERK-like as well as p38-like) that accompany sclerotial development in a distinct developmental phase manner represent a potential target for antifungal intervention.

Hormonal and non-hormonal regulation of glutamine synthetase in the developing neural retina.

Two isoforms of the glucocorticoid receptor, with apparent molecular mass of 90 and 95 kDa, are expressed in embryonic chicken neural retina. The 95-kDa receptor represents a hyperphosphorylated form of the 90-kDa receptor. Activation of the glucocorticoid receptor by cortisol results in a dose-dependent increase in receptor phosphorylation, translocation of receptor molecules into the nucleus and a decline in the total amount of the receptor. Activation of the glucocorticoid receptor can also be observed in the developing retinal tissue in ovo. At late embryonic ages, when the systemic level of glucocorticoids increases, a substantial quantity of receptor molecules becomes translocated into the nucleus, the relative level of the 95-kDa isoform increases, and the total amount of receptor declines. Activation of the receptor molecules in ovo correlates directly with an increase in transcription of the glucocorticoid-inducible gene, glutamine synthetase. The close correlation between the increase in systemic glucocorticoids, activation of glucocorticoid receptor molecules and induction of glutamine synthetase gene transcription suggests that glucocorticoids are directly involved in the developmental control of glutamine synthetase expression. Long-term organ culturing of embryonic retinal tissue in the absence of hormone results in an increase in glutamine synthetase expression. This increase, which is only 5 to 10% of that observed in ovo, is not mediated by activated receptor molecules and represents a mechanism for non-hormonal regulation of glutamine synthetase.

Molecular basis for differential expression of glutamine synthetase in retina glia and neurons.

Glutamine synthetase (GS) is a differentiation marker of retina glial cell. It is expressed in the chicken neural retina at a particularly high level, is inducible by glucocorticoids and is always confined to Müller glia. This study investigated the molecular basis for tissue and cell-type specific expression of the GS gene. A high level of GS expression in the retina was found to coincide with the accumulation of a relatively high level of GS mRNA in this tissue. The gliatoxic agent alpha-aminoadipic acid, which can selectively destroy glia cells, was used to demonstrate that restriction of GS induction to Müller glia is controlled at a transcriptional level. Cortisol could induce accumulation of GS mRNA and transcription of the GS gene in Müller glia but not in retina neurons. Glia and neurons were also found to differ in their ability to express the glucocorticoid inducible CAT construct, p delta G46TCO, which is controlled by a 'simple GRE' promoter. When introduced into cells of retina tissue, this construct was cortisol-inducible in glia whereas in neurons it was only slightly inducible or not at all. Introduction of a glucocorticoid receptor expression vector into the cells facilitated induction of the CAT construct in neurons. Analysis by immunoblotting revealed that expression of the glucocorticoid receptor protein is predominantly restricted to Müller glia. These results suggest that differential levels of glucocorticoid receptor expression in glia and neurons might be the basis for cell-type specific induction of GS.

[Effectiveness of expression of chloramphenicol acetyltransferase gene controlled by foreign regulatory regions in Escherichia coli. III. Relative effectiveness of cloned promoters of Escherichia coli and coliphages]. / Issledovanie éffektivnosti ékspressii gena khloramfenikolatsetiltransferazy pod kontrolem chuzherodnykh reguliatornykh oblastei v kletkakh Escherichia coli. III. Izuchenie otnositel'noi éffektivnosti klonirovannykh promotorov Escherichia coli i kolifagov.

The study on the rate of initiation of model gene cat transcription under the control of E. coli (Plac UV5, Ptrp, Pcat, Ptac), phage lambda (PL, PR), phi X174 (PD) promotors was carried out by means of hybridization of pulse labelled in vivo mRNA with the DNA coding parts. The presence of gene bla(Apr) with its own constitutive promoter in all the recombinants permitted to use the level of appropriate mRNA in the cell as an internal standard. This method allowed to evaluate the true efficiency of the promoters in question. The strength of the promoters studied is shown to be equal within the limit of one order value.

[Analysis of the nucleotide sequence of a small colicinogenic plasmid Cold gene coding for lysis]. / Analiz nukleotidnoi posledovatel'nosti gena lizisa maloi kolitsinogennoi plazmidy Cold.

The nucleotide sequence of a DNA fragment of a small colicigonenic plasmid Cold-CA23 containing the lysis gene cdl has been defined. An open reading frame has been found within the fragment for 48 amino acid polypeptide with the molecular mass 4920 Da. The polypeptide is homologous to the lysis proteins encoded by the small colicinogenic plasmids ColE1 and CloDF13. The homology was also found for the structural and functional arrangement of the cdl gene and the plasmid CloDF13 lysis gene. The analysis of the possible secondary structures of the lysis protein encoded by cdl gene has revealed the amino acid sequences able to form the alternative structures. The described feature is supposed to be required for lysis protein translocation from cytoplasmatic to outer membrane of Escherichia coli cells.

[Expression of the chloramphenicol acetyltransferase gene is under control of various promoters of E. coli and phage lambda]. / Ekspressiia gena khloramfenikolatsetiltransferazy pod kontrolem nekotorykh promotorov E. coli i bakteriofaga lambda.

Plasmids have been constructed with the structural region of the cat gene being under the control of the lactose (lacUV5), tryptophane (trpOP), operons of Escherichia coli, the hybrid trp-lac (tac) promoter and early bacteriophage lambda promoters (PL, PR and PLIT). The expression of chloramphenicolacetyltransferase gene in Escherichia coli cells harbouring such recombinant plasmids and pBR325 as well has been examined by determining the chloramphenicol resistance and studying the enzyme activity of Cm-acetylase. A high level of enzyme synthesis is connected with transcription from PL, PR and tac promoters.

[Plasmid vectors of "insertion inactivation" of the trimethoprim resistance marker]. / Plazmidnye vektory "insertsionnoi inaktivatsii" markera rezistentnosti k trimetoprimu.

We demonstrate the possibility of using the T4 phage frd gene as an insertion inactivation marker within pBR322, in plasmids with changing copy number and expression of foreign genes under control. The structural part of the frd genes contains unique recognition sites for EcoRI and SalI endonucleases. Transformants with recombinant plasmids carrying the frd gene grow on media with up to 500 mkg/ml trimethoprim, whatever the gene dosage.

Making a friend from a foe: expressing a GroEL gene from the whitefly Bemisia tabaci in the phloem of tomato plants confers resistance to tomato yellow leaf curl virus.

Some (perhaps all) plant viruses transmitted in a circulative manner by their insect vectors avoid destruction in the haemolymph by interacting with GroEL homologues, ensuring transmission. We have previously shown that the phloem-limited begomovirus tomato yellow leaf curl virus (TYLCV) interacts in vivo and in vitro with GroEL produced by the whitefly vector Bemisia tabaci. In this study, we have exploited this phenomenon to generate transgenic tomato plants expressing the whitefly GroEL in their phloem. We postulated that following inoculation, TYLCV particles will be trapped by GroEL in the plant phloem, thereby inhibiting virus replication and movement, thereby rendering the plants resistant. A whitefly GroEL gene was cloned in an Agrobacterium vector under the control of an Arabidopsis phloem-specific promoter, which was used to transform two tomato genotypes. During three consecutive generations, plants expressing GroEL exhibited mild or no disease symptoms upon whitefly-mediated inoculation of TYLCV. In vitro assays indicated that the sap of resistant plants contained GroEL-TYLCV complexes. Infected resistant plants served as virus source for whitefly-mediated transmission as effectively as infected non-transgenic tomato. Non-transgenic susceptible tomato plants grafted on resistant GroEL-transgenic scions remained susceptible, although GroEL translocated into the grafted plant and GroEL-TYLCV complexes were detected in the grafted tissues.

Cellular distribution of COT1 kinase in Neurospora crassa.

The Neurospora crassa cot-1 gene encodes a Ser/Thr protein kinase, which is involved in hyphal elongation. Many vacuoles, abnormally shaped mitochondria, and nuclei, along with differences in the structure of the cell wall and hyphal septa, were observed in hyphae of the cot-1 mutant shortly after a shift to the restrictive temperature. Immunolocalization experiments indicated that COT1 was associated with the cytoplasmic membrane; COT1 was also detected in the cytoplasm. The membrane-associated COT1 was absent from the cot-1 mutant when shifted to the restrictive temperature, as was a lower molecular weight isoform of COT1. We propose that COT1 may be involved in several cellular processes, and the spatial and temporal regulation of COT1 activity involves trafficking of the kinase within the fungal cell and its possible interaction with additional proteins.

Glutamine synthetase protects against neuronal degeneration in injured retinal tissue.

The neurotransmitter glutamate is neurotoxic when it is accumulated in a massive amount in the extracellular fluid. Excessive release of glutamate has been shown to be a major cause of neuronal degeneration after central nervous system injury. Under normal conditions, accumulation of synaptically released glutamate is prevented, at least in part, by a glial uptake system in which the glia-specific enzyme glutamine synthetase (GS) plays a key role. We postulated that glial cells cannot cope with glutamate neurotoxicity because the level of GS is not high enough to catalyze the excessive amounts of glutamate released by damaged neurons. We examined whether elevation of GS expression in glial cells protects against neuronal degeneration in injured retinal tissue. Analysis of lactate dehydrogenase efflux, DNA fragmentation, and histological sections revealed that hormonal induction of the endogenous GS gene in retinal glial cells correlates with a decline in neuronal degeneration, whereas inhibition of GS activity by methionine sulfoximine leads to increased cell death. A supply of purified GS enzyme to the culture medium of retinal explants or directly to the embryo in ovo causes a dose-dependent decline in the extent of cell death. These results show that GS is a potent neuroprotectant and that elevation of GS expression in glial cells activates an endogenous mechanism whereby neurons are protected from the deleterious effects of excess glutamate in extracellular fluid after trauma or ischemia. Our results suggest new approaches to the clinical handling of neuronal degeneration.

Developmental changes in the expression and compartmentalization of the glucocorticoid receptor in embryonic retina.

Inducibility by glucocorticoids of the glutamine synthetase gene in chicken embryo retina and the transcriptional activity of the glucocorticoid receptor (GR) greatly increase between embryonic days 6 and 10 (E6, E10), although the level of GR does not markedly change during that time. This apparent discrepancy was investigated by examining the pattern of GR expression in undifferentiated E6 retina and in E10 retina, which consists mostly of differentiated cells. Two GR isoforms, 90 and 95 kDa, were found to be expressed at both of these ages at a similar total level but in different proportions: in E6 retina the level of the 90-kDa isoform was higher, whereas in E10 retina the 95-kDa receptor was higher. However, following treatment of the retinas with cortisol, the 95-kDa isoform became the predominant receptor at both ages. Immunohistochemical analysis revealed that the cellular localization of GR markedly changed in the course of development: in the undifferentiated E6 retina GR was expressed in virtually all cells, whereas in the more differentiated E10 and E12 retina, GR was detected only in Müller glia cells. The latter represent approximately 20% of the cells in this tissue and are the only cells in which glucocorticoid hormone induces the glutamine synthetase gene. We suggest that the compartmentalization of GR in Müller glia is a major aspect of the mechanism that modulates receptor activity during retina development and results in the temporal increase in the inducibility of glutamine synthetase and its specific localization in Müller glia cells.

A mutation within the catalytic domain of COT1 kinase confers changes in the presence of two COT1 isoforms and in Ser/Thr protein kinase and phosphatase activities in Neurospora crassa.

Neurospora crassa grows by forming spreading colonies. cot-1 belongs to a class of N. crassa colonial temperature-sensitive (cot) mutants and encodes a Ser/Thr protein kinase. We have mapped the cot-1 mutation to a single base change resulting in a His to Arg substitution at amino acid 351, which resides within the catalytic domain. Antibodies raised against COT1 detected and immunoprecipitated a predominant 73-kDa polypeptide in N. crassa extracts, whose abundance was constant under all growth conditions tested. An additional, lower MW COT1 isoform (67-kDa) present in the wild-type was not detected in cot-1 grown at the restrictive temperature. Similarly, this isoform was not detected in cot-3 or cot-5 strains, when grown at restrictive temperatures. Reduced levels of Ser/Thr kinase activity and an increase in type 1 and type 2B phosphatase (calcineurin) activities were measured in a cot-1 background. Apparent changes in the phosphorylation state of the p150(Glued) subunit of the dynactin cytoskeletal motor component (encoded by ro-3, a suppressor of cot-1) and evidence of in vitro physical interactions between COT1 and calcineurin indicate a functional linkage among COT1 kinase, type 2B phosphatase, and dynactin.

The Neurospora crassa colonial temperature-sensitive 3 (cot-3) gene encodes protein elongation factor 2.

At elevated temperatures, the Neurospora crassa mutant colonial, temperature-sensitive 3 (cot-3) forms compact, highly branched colonies. Growth of the cot-3 strain under these conditions also results in the loss of the lower molecular weight (LMW) isoform of the Ser/Thr protein kinase encoded by the unlinked cot-1 gene, whose function is also involved in hyphal elongation. The unique cot-3 gene has been cloned by complementation and shown to encode translation elongation factor 2 (EF-2). As expected for a gene with a general role in protein synthesis, cot-3 mRNA is abundantly expressed throughout all asexual phases of the N. crassa life cycle. The molecular basis of the cot-3 mutation was determined to be an ATT to AAT transversion, which causes an Ile to Asn substitution at residue 278. Treatment with fusidic acid (a specific inhibitor of EF-2) inhibits hyphal elongation and induces hyperbranching in a manner which mimics the cot-3 phenotype, and also leads to a decrease in the abundance of the LMW isoform of COT1. This supports our conclusion that the mutation in cot-3 which results in abnormal hyphal elongation/branching impairs EF-2 function and confirms that the abundance of a LMW isoform of COT1 kinase is dependent on the function of this general translation factor.