Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

Different activities of the adenovirus types 5 and 12 E1A regions in transformation with the EJ Ha-ras oncogene.

We have compared the capacities of the E1A regions of nononcogenic adenovirus type 5 (Ad5) and highly oncogenic Ad12 to cooperate with the EJ bladder carcinoma Ha-ras-1 oncogene in the transformation of primary baby rat kidney cells. Both E1A regions, when cotransfected with the Ha-ras oncogene, transformed the primary cells with a low frequency. Ad5 E1A plus Ha-ras-transformed cells differed in phenotype from cells transformed by Ad12 E1A plus Ha-ras. The cells expressing Ad5 E1A appeared highly transformed and practically failed to adhere to plastic. This phenotype may be due to the virtually complete absence of fibronectin gene expression in these cells. In contrast, the cells expressing Ad12 E1A were flatter and adhered to plastic, whereas fibronectin gene expression was reduced but not absent. The oncogenic potential of the two types of E1A plus ras-transformed cells was tested by their injection into both athymic nude mice and weanling syngeneic rats. The Ad5 E1A plus ras-transformed cells were found to be highly oncogenic in both animal species, whereas the Ad12 E1A plus ras-transformed cells were only weakly oncogenic in both syngeneic rats and nude mice. The difference in oncogenic potential of the Ad5 E1A plus ras- and the Ad12 E1A plus ras-transformed cells is discussed in terms of the different capacities of the Ad5 and Ad12 E1A-encoded proteins to modulate cellular gene expression.     

Oncogenicity by adenovirus is not determined by the transforming region only.

We have constructed a nondefective recombinant virus between the nononcogenic adenovirus 5 (Ad5) and the highly oncogenic Ad12. The recombinant genome consists essentially of Ad5 sequences, with the exception of the transforming early region 1 (E1) which is derived from Ad12. HeLa cells infected with the recombinant virus were shown to contain the Ad12-specific E1 proteins of 41 kilodaltons (E1a) and 19 and 54 kilodaltons (both encoded by E1b). The recombinant virus replicated efficiently in human embryonic kidney cells and HeLa cells, showing that the transforming regions of Ad5 and Ad12 had similar functions in productive infection. After the recombinant virus was injected into newborn hamsters, no tumors were produced during an observation period of 200 days. Thus, despite the fact that all products required for oncogenic transformation in vitro were derived from the highly oncogenic Ad12, the recombinant virus did not produce tumors in vivo. These data show that tumor induction by adenovirus virions is not determined only by the gene products of the transforming region.     

Qualitative and quantitative immunofluorescence studies of chloroplast ferredoxin

Antibodies were raised in rabbits against 2Fe–2S ferredoxin from N. tabacum L. The antibodies showed partial cross-reactivity in the double diffusion test with ferredoxins from Spinacia oleracea L., Petunia inflata Fries., P. axillaris Lam., Phaseolus vulgaris L., Chlamydomonas remhardii Dang. A complete cross-reaction was observed with ferredoxins from five other Nicotiana species, thus with this test it was impossible to discriminate between these ferredoxins. Therefore the following test was performed. Heterologous ferredoxin (i.e., ferredoxin other than from N. tabacum) was coupled covalently to Sepharose beads. Rabbit anti-N. tabacum-serum was then pre-incubated with this ferredoxin which resulted in complete abolition of cross-reactivity with free heterologous ferredoxin. However, the serum retained antibody activity against specific antigenic determinants of N. tabacum ferredoxin. When this serum was tested against ferredoxin purified from the hybrid: N. tabacum ()xN. glutinosa () it gave a positive reaction. The relative content of maternal N. tabacum ferredoxin in the hybrid was estimated by using a fluorescent derivative of this specific antibody and estimating the cross-reactivity compared with that of artificial mixtures of pure N. tabacum and N. glutinosa ferredoxins. The hybrid contained 50% of maternal ferredoxin. This technique was also applied to ferredoxins of other species of Nicotiana and to the ferredoxin from the hybrid N. clevelandii ()xN. glutinosa (). We conclude that it provides a good test system for the study of the expression of chloroplast ferredoxin in Nicotiana hybrids in general.Abbreviations PBS phosphate buffered saline - FITC fluorescein isothiocyanate - S.E.M. standard error of means     

The cis-expression of the coat protein of turnip mosaic virus is essential for viral intercellular movement in plants

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant-infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double-fluorescent (green and mCherry) protein-tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N-terminus (amino acids 1–97), the core (amino acids 98–245), and the C-terminus (amino acids 246–288). We found that deletion of CP or its segments amino acids 51–199, amino acids 200–283, or amino acids 265–274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6–50 in the N-terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell-to-cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans-expression of the wild-type CP either by TuMV or through genetic transformation-based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis-expressed CP is functional.     

A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55γ as a Stress-Sensitive Inhibitor of c-SRC

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55γ and PR55δ as inhibitors of c-Jun NH₂-terminal kinase (JNK) activation by UV irradiation. We show that PR55γ binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55γ and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55γ.     

The poly(phenolic) domain of potato suberin: a non-lignin cell wall bio-polymer.

Suberized plant cell walls have three distinguishing features: (1) tissue specificity, (2) a poly(aliphatic) domain and (3) a unique, "lignin-like" poly(phenolic) domain. With respect to the latter, comparisons have often been made to lignin, but the unique phenolic composition of suberized cells yields a unique polymer better designated as a poly(phenolic) domain. Potato tubers that have been induced to suberize through wounding make an excellent model system with which the chemistry, biochemistry and macromolecular assembly of the suberin poly(phenolic) domain can be monitored. For example, wound healing potato tubers have been used to determine the unique hydroxycinnamic acid nature of its poly(phenolic) domain using specific carbon-13 labeling studies and specific chemical degradation techniques (e.g. thioacidolysis). Furthermore, a suberization-associated anionic peroxidase has been purified from suberizing potato tubers and subsequently shown to oxidize hydroxycinnamic acids (and their derivatives) in preference to monolignols, as well as yield an unique polymer in vitro. We have since extended these studies to begin analyzing the macromolecular assembly process leading to the deposition of this suberized tissue specific domain. To this end we have begun to describe an H(2)O(2)-generating system with NAD(P)H-dependent oxidase-like properties that is temporally associated with the formation of potato suberin poly(phenolics) during suberization. Herein we describe our progress to date.     

Reactive oxygen species production in association with suberization: evidence for an NADPH-dependent oxidase

In response to wounding, potato tubers generate reactive oxygen species (ROS) in association with suberization. Immediately following wounding, an initial burst of ROS occurs, reaching a maximum within 30 to 60 min. In addition to this initial oxidative burst, at least three other massive bursts occur at 42, 63 and 100 h post-wounding. These latter bursts are associated with wound healing and are probably involved in the oxidative cross-linking of suberin poly(phenolics). The source of ROS is likely to be a plasma membrane NADPH-dependent oxidase immunorelated to the human phagocyte plasma membrane oxidase. The initial oxidative burst does not appear to be dependent on new protein synthesis, but the subsequent bursts are associated with an increase in oxidase protein components. Oxidase activity is enhanced in vitro by hydroxycinnamic acids and conjugates associated with the wound healing response in potato.