Gibberellic acid stabilises microtubules in maize suspension cells to cold and stimulates acetylation of alpha-tubulin.
Gibberellic acid is known to stabilise microtubules in plant organs against depolymerisation. We have now devised a simplified cell system for studying this. Pretreatment of a maize cell suspension with gibberellic acid for just 3 h stabilised protoplast microtubules against depolymerisation on ice. In other eukaryotes, acetylation of alpha-tubulin is known to correlate with microtubule stabilisation but this is not established in plants. By isolating the polymeric tubulin fraction from maize cytoskeletons and immunoblotting with the antibody 6-11B-1, we have demonstrated that gibberellic acid stimulates the acetylation of alpha-tubulin. This is the first demonstrated link between microtubule stabilisation and tubulin acetylation in higher plants. (+info
Enhancer-like properties of an RNA element that modulates Tombusvirus RNA accumulation.
Prototypical defective interfering (DI) RNAs of the plus-strand RNA virus tomato bushy stunt virus contain four noncontiguous segments (regions I-IV) derived from the viral genome. Region I corresponds to 5'-noncoding sequence, regions II and III are derived from internal positions, and region IV represents a 3'-terminal segment. We analyzed the internally located region III in a prototypical DI RNA to understand better its role in DI RNA accumulation. Our results indicate that (1) region III is not essential for DI RNA accumulation, but molecules that lack it accumulate at significantly reduced levels ( approximately 10-fold lower), (2) region III is able to function at different positions and in opposite orientations, (3) a single copy of region III is favored over multiple copies, (4) the stimulatory effect observed on DI RNA accumulation is not due to region III-mediated RNA stabilization, (5) DI RNAs lacking region III permit the efficient accumulation of head-to-tail dimers and are less effective at suppressing helper RNA accumulation, and (6) negative-strand accumulation is also significantly depressed for DI RNAs lacking region III. Collectively, these results support a role for region III as an enhancer-like element that facilitates DI RNA replication. A scanning-type mutagenesis strategy was used to define portions of region III important for its stimulatory effect on DI RNA accumulation. Interestingly, the results revealed several differences in the requirements for activity when region III was in the forward versus the reverse orientation. In the context of the viral genome, region III was found to be essential for biological activity. This latter finding defines a critical role for this element in the reproductive cycle of the virus. (+info
A function for the plasmalemma grooves of a fission yeast.
Ultrastructural studies on regenerating protoplasts of Schizosaccharomyces pombe show that the spatial differentiation of the plasmalemma into grooves and flat areas is reflected in a functional differentiation in cell-wall synthesis. The grooves are the initial site of production of wall fibrils. (+info
Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity.
Mechanosensitive channels are ubiquitous amongst bacterial cells and have been proposed to have major roles in the adaptation to osmotic stress, in particular in the management of transitions from high to low osmolarity environments. Electrophysiological measurements have identified multiple channels in Escherichia coli cells. One gene, mscL, encoding a large conductance channel has previously been described, but null mutants were without well-defined phenotypes. Here, we report the characterization of a new gene family required for MscS function, YggB and KefA, which has enabled a rigorous test of the role of the channels. The channel determined by KefA does not appear to have a major role in managing the transition from high to low osmolarity. In contrast, analysis of mutants of E.coli lacking YggB and MscL shows that mechanosensitive channels are designed to open at a pressure change just below that which would cause cell disruption leading to death. (+info
The pro1(+) gene from Sordaria macrospora encodes a C6 zinc finger transcription factor required for fruiting body development.
During sexual morphogenesis, the filamentous ascomycete Sordaria macrospora differentiates into multicellular fruiting bodies called perithecia. Previously it has been shown that this developmental process is under polygenic control. To further understand the molecular mechanisms involved in fruiting body formation, we generated the protoperithecia forming mutant pro1, in which the normal development of protoperithecia into perithecia has been disrupted. We succeeded in isolating a cosmid clone from an indexed cosmid library, which was able to complement the pro1(-) mutation. Deletion analysis, followed by DNA sequencing, subsequently demonstrated that fertility was restored to the pro1 mutant by an open reading frame encoding a 689-amino-acid polypeptide, which we named PRO1. A region from this polypeptide shares significant homology with the DNA-binding domains found in fungal C6 zinc finger transcription factors, such as the GAL4 protein from yeast. However, other typical regions of C6 zinc finger proteins, such as dimerization elements, are absent in PRO1. The involvement of the pro1(+) gene in fruiting body development was further confirmed by trying to complement the mutant phenotype with in vitro mutagenized and truncated versions of the pro1 open reading frame. Southern hybridization experiments also indicated that pro1(+) homologues are present in other sexually propagating filamentous ascomycetes. (+info
RNA polymerase I transcription in a Brassica interspecific hybrid and its progenitors: Tests of transcription factor involvement in nucleolar dominance.
In interspecific hybrids or allopolyploids, often one parental set of ribosomal RNA genes is transcribed and the other is silent, an epigenetic phenomenon known as nucleolar dominance. Silencing is enforced by cytosine methylation and histone deacetylation, but the initial discrimination mechanism is unknown. One hypothesis is that a species-specific transcription factor is inactivated, thereby silencing one set of rRNA genes. Another is that dominant rRNA genes have higher binding affinities for limiting transcription factors. A third suggests that selective methylation of underdominant rRNA genes blocks transcription factor binding. We tested these hypotheses using Brassica napus (canola), an allotetraploid derived from B. rapa and B. oleracea in which only B. rapa rRNA genes are transcribed. B. oleracea and B. rapa rRNA genes were active when transfected into protoplasts of the other species, which argues against the species-specific transcription factor model. B. oleracea and B. rapa rRNA genes also competed equally for the pol I transcription machinery in vitro and in vivo. Cytosine methylation had no effect on rRNA gene transcription in vitro, which suggests that transcription factor binding was unimpaired. These data are inconsistent with the prevailing models and point to discrimination mechanisms that are likely to act at a chromosomal level. (+info
Long-distance RNA-RNA interactions and conserved sequence elements affect potato virus X plus-strand RNA accumulation.
Conserved octanucleotide sequences located upstream of two major potato virus X (PVX) subgenomic RNAs (sgRNAs), as well as elements in the 5' end of the genome, affect accumulation of sgRNA. To determine if complementarity between these sequences is important for PVX RNA accumulation, we analyzed the effects of mutations within these elements and compensatory mutations in a tobacco protoplast system and in plants. Mutations in the 5' nontranslated region (NTR mutants) that reduced complementarity resulted in lower genomic RNA (gRNA) and sgRNA levels, whereas mutations to the octanucleotide elements affected only the corresponding sgRNA levels. However, for both the NTR and octanucleotide mutants, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Mutants containing changes in the NTR and compensatory changes in one of the octanucleotide elements restored levels of gRNA and the other sgRNA species with an unaltered octanucleotide element to those of wild-type. Although compensatory changes significantly increased levels of the sgRNA species with the modified octanucleotide element, levels were not restored to those of wild-type. Our data indicate that long distance RNA-RNA interactions and the sequences of the interacting elements are required for PVX plus-strand RNA accumulation. (+info
Development of a self-cloning system for Actinomadura verrucosospora and identification of polyketide synthase genes essential for production of the angucyclic antibiotic pradimicin.
A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 10(5) CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis. (+info