An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis

The silencing phenotype in Arabidopsis thaliana lines with an inverted repeat transgene under the control of a phloem-specific promoter was manifested in regions around veins due to a mobile signal of silencing. Genetic analysis implicates RNA-DEPENDENT RNA POLYMERASE2 (RDR2) and an RNA polymerase IVa subunit gene (NRPD1a) in the signaling mechanism. We also identified an SNF2 domain-containing protein (CLASSY1) that acts together with RDR2 and NRPD1a in the spread of transgene silencing and in the production of endogenous 24-nucleotide short interfering RNAs (siRNAs). Cytochemical analysis indicates that CLASSY1 may act in the nucleus with NRPD1a and RDR2 in the upstream part of RNA silencing pathways that generate a double-stranded RNA substrate for Dicer-like (DCL) nucleases. DCL3 and ARGONAUTE4 act in a downstream part of the pathway, leading to endogenous 24-nucleotide siRNA production, but are not required for intercellular signaling. From genetic analysis, we conclude that another downstream part of the pathway associated with intercellular signaling requires DCL4 and at least one other protein required for 21-nucleotide trans-acting siRNAs. We interpret the effect of polymerase IVa and trans-acting siRNA pathway mutations in terms of a modular property of RNA silencing pathways.     

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity.

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.     

v-SNAREs control exocytosis of vesicles from priming to fusion

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.     

Signaling from the membrane via membrane estrogen receptor-alpha: estrogens, xenoestrogens, and phytoestrogens

Estrogen mimetics in the environment and in foods can have important consequences for endocrine functions. When previously examined for action via genomic steroid signaling mechanisms, most of these compounds were found to be very weak agonists. We have instead tested their actions via several membrane-initiated signaling mechanisms in GH3/B6 pituitary tumor cells extensively selected for high (responsive) or low (nonresponsive) expression of the membrane version of estrogen receptor-alpha (mERalpha). We found many estrogen mimetic compounds to be potently active in our quantitative extracellular-regulated kinase (ERK) activation assays, to increase cellular Ca++ levels, and to cause rapid prolactin release. However, these compounds may activate one or both mechanisms with different potencies. For instance, some compounds activate ERKs in both pM and nM concentration ranges, while others are only active at nM and higher concentrations. Compounds also show great differences in their temporal activation patterns. While estradiol causes a bimodal time-dependent ERK activation (peaking at both 3 and 30 min), most estrogen mimetics cause either an early phase activation, a late phase activation, or an early sustained activation. One xenoestrogen known to be a relatively potent activator of estrogen response element-mediated actions (bisphenol A) is inactive as an ERK activator, and only a modest inducer of Ca++ levels and prolactin release. Many different signaling machineries culminate in ERK activation, and xenoestrogens differentially affect various pathways. Clearly individual xenoestrogens must be individually investigated for their differing abilities to activate distinct membrane-initiated signal cascades that lead to a variety of cellular functions.     

Erythrobacter vulgaris sp. nov., a novel organism isolated from the marine invertebrates

Four yellow-pigmented, gram-negative, chemoorganotrophic aerobic bacteria were isolated from starfish Stellaster equestris (strains 022-2-10T, 022-2-9, and 022-2-12) and soft coral (unidentified species) (strain 022-4-7) collected in the South China Sea. 16S rRNA gene sequence-based analyses of the new organisms revealed that Erythrobacter spp. were the closest relatives and shared the highest similarity of 98.7% to E. citreus, 98.5% to E. flavus, 97.9% to E. litoralis and 97.6% to E. longus. The novel organisms were tolerant to 3-6% NaCl, grew between 10 degrees C and 40 degrees C, and were not able to degrade gelatin, casein, and agar, while degraded Tween 80. Two strains (022-2-9 and 022-2-12) could weakly degrade starch. All strains produced a large pool of carotenoids and did not have Bacteriochlorophyll a. Phosphatidylethanolamine (30-36%), phosphatidylglycerol (39-46%), and phosphatidylcholine (21-27%) were the predominant phospholipids. Sphingoglycolipid was not detected. The major fatty acids were 16:0 (6-11%), 16:1omega7 (12-15%), and 18:1omega7 (46-49%). The two-hydroxy fatty acids, 13:0-2OH, 14:0-2OH, 15:0-2OH, 16:0-2OH were also present. The G + C content of the DNAs ranged from 61 to 62 mol%. The level of DNA similarity among four strains was conspecific and ranged from 94% to 98%. Even though new strains and other species of the genus had rather high level of 16S rRNA gene sequence similarities, DNA-DNA hybridization experiments showed only 33-39% of binding with the DNA of the type strains. On the basis of these results and the significant differences demonstrated in the phenotypic and chemotaxonomic characteristics, it is suggested that the new organisms be classified as a novel species; the name Erythrobacter vulgaris sp. nov. is proposed. The type strain is 022-2-10T (= KMM 3465T = CIP 107841T).     

Open reading frame ssr2016 is required for antimycin A-sensitive photosystem I-driven cyclic electron flow in the cyanobacterium Synechocystis sp. PCC 6803

Open reading frame ssr2016 encodes a protein with substantial sequence similarities to PGR5 identified as a component of the antimycin A-sensitive ferredoxin:plastoquinone reductase (FQR) in PSI cyclic photophosphorylation in Arabidopsis thaliana. We studied cyclic electron flow in Synechocystis sp. PCC 6803 in vivo in ssr2016 deletion mutants generated either in a wild-type background or in a ndhB deletion mutant. Our results indicate that ssr2016 is required for FQR and that it operates in a parallel pathway to the NDH1 complex. The ssr2016 deletion mutants are high light sensitive, suggesting that FQR might be important in controlling redox poise under adverse conditions.