Identification of coiled bodies inBrassica napus nuclei during embryogenesis and early germination

Summary Nucleolus-associated bodies characterize interphase nuclei of many plant species. The recent demonstration that such bodies contain small nuclear ribonucleoproteins as well as coilin clearly indicates that they belong to a larger family of nuclear structures, known as coiled bodies, that have been intensively studied in a variety of animal cell types. In a previous work, we have shown that coiled bodies were present in close association with the nucleolus inZea mays dry seeds as well as during subsequent stages of germination. This study reveals that similar nuclear structures were also present duringBrassica napus embryogenesis starting at the torpedo stage and that they were, likewise, generally located on the nucleolar surface. As in the case ofZ. mays, coiled bodies were observed in cells of dry seeds as well as in those of early germinating tissues. These bodies were labelled with monoclonal antibody K121, an antibody reacting with the unique 5-terminal cap structure containing 2,2,7-trimethylguanosine that characterizes small nuclear RNAs. Owing to their intimate association with the nucleolus in all stages studied, the possibility is considered that, in these plant cells, coiled bodies are assembled on an organizer element located within this organelle.Abbreviations BSA bovine serum albumin - IgM immunoglobulin M - NAB nucleolus-associated body - NAC nucleolus-associated chromatin - PBS phosphate-buffered saline - snRNA small nuclear ribonucleic acid - snRNP small nuclear ribonucleoprotein     

Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA

Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.     

PARK3 influences age at onset in Parkinson disease: a genome scan in the GenePD study

Parkinson disease (PD) is a late-onset neurodegenerative disorder. The mean age at onset is 61 years, but the disease can range from juvenile cases to cases in the 8th or 9th decade of life. The parkin gene on chromosome 6q and loci on chromosome 1p35-36 and 1p36 are responsible for some cases of autosomal recessive early-onset parkinsonism, but they do not appear to influence susceptibility or variability of age at onset for idiopathic PD. We have performed a genomewide linkage analysis using variance-component methodology to identify genes influencing age at onset of PD in a population of affected relatives (mainly affected sibling pairs) participating in the GenePD study. Four chromosomal loci showed suggestive evidence of linkage: chromosome 2p (maximum multipoint LOD [MaxLOD] = 2.08), chromosome 9q (MaxLOD = 2.00), chromosome 20 (MaxLOD = 1.82), and chromosome 21 (MaxLOD = 2.21). The 2p and 9q locations that we report here have previously been reported as loci influencing PD affection status. Association between PD age at onset and allele 174 of marker D2S1394, located on 2p13, was observed in the GenePD sample (P=.02). This 174 allele is common to the PD haplotype observed in two families that show linkage to PARK3 and have autosomal dominant PD, which suggests that this allele may be in linkage disequilibrium with a mutation influencing PD susceptibility or age at onset of PD.     

The viroid and viroid-like RNA database.

The viroid and viroid-like RNA database is a compilation of all natural sequences published in journals or available from the GenBank and EMBL nucleotide sequence libraries. Several information regarding these RNA species such as the position of their self-catalytic domains and the open reading frame of the human hepatitits delta virus are provided. The database also includes a determination of the likely ancestral sequence of most species and a prediction of the most stable secondary structures of these sequences. This online database is available on the World Wide Web (http://www.callisto.si.usherb.ca/[symbol: see text]jpperra ). It should provide an excellent reference point for further phylogenetic and structure-function studies of these RNA species.     

Jack pine of all trades: Deciphering intraspecific variability of a key adaptive trait at the rear edge of a widespread fire-embracing North American conifer

Premise

Understanding mechanisms fostering long-term persistence of marginal populations should provide key insights about species resilience facing climate change. Cone serotiny is a key adaptive trait in Pinus banksiana (jack pine), which shows phenotypic variation according to the fire regime. Compared to range-core populations within the fire-prone boreal forest, low and variable serotiny in rear-edge populations suggest local adaptation to uncommon and unpredictable wildfire regime. We assessed environmental/physiological factors that might modulate intraspecific variation in cone serotiny.

Methods

We experimentally subjected closed cones to incrementing temperatures, then tested seed germination to determine whether and how various ecological factors (cone age, branch height, tree size, tree age) are related to cone dehiscence and seed viability in jack pines from rear-edge and range-core populations in eastern Canada.

Results

Cones from rear-edge populations dehisce at a lower opening temperature, which increases with cone age. Cones from range-core stands open at a more constant, yet higher temperature. Cones from rear-edge stands take between 13 and 27 years to reach the level of serotiny achieved at the range core. At the rear edge, seed viability is steady (51%), whereas it decreases from 70% to 30% in 20 years at the range core.

Conclusions

We inferred the mechanisms of a bet-hedging strategy in rear-edge populations, which ensures steady recruitment during fire-free intervals and successful postfire regeneration. This capacity to cope with infrequent and unpredictable fire regime should increase the resilience of jack pine populations as global changes alter fire dynamics of the boreal forest.     

Far upstream activating promoter regions are responsible for expression of the BnC1 cruciferin gene from Brassica napus

Summary Cruciferin is the major seed storage protein in Brassica napus. As much as 1.9 kbp of the BnC1 cruciferin gene promoter have been sequenced and analyzed. Promoter fragments with 5 deletions from –2500 to –v202 were fused with the ß-glucuronidase reporter gene and used for Nicotiana tabacum transformation. ß-glucuronidase could be specifically expressed in transgenic tobacco seeds under the control of the BnC1 promoter and regulatory elements were found to be dispersed over 1903 bp. An almost 5-fold increase in ß-glucuronidase expression was obtained when the promoter length was increased from –379 to –498, and another 10-fold increase was observed when sequences between –1266 and –1903 were added. Histochemical analysis shows that the region between –844 and –1266 directs the expression of the chimeric gene specifically to the root apical meristem.Abbreviations GUS ß-glucuronidase - MU 4-methyl umbelliferone - MUG 4-methyl-umbelliferyl-ß-D-glucuronide - X-gluc 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide     

A Proline-, Threonine-, and Glycine-Rich Protein Down-Regulated by Drought Is Localized in the Cell Wall of Xylem Elements

A cDNA clone encoding a proline-, threonine-, and glycine-rich protein (PTGRP) was isolated from a wild tomato species (Lycopersicon chilense) (L.X. Yu, H. Chamberland, J.G. Lafontain, Z. Tabaeizadeh [1996] Genome 39: 1185-1193). Northern-blot analysis and in situ hybridization studies revealed that PTGRP is down-regulated by drought stress. The level of the mRNA in leaves and stems of 8-d drought-stressed plants decreased 5- to 10-fold compared with that in regularly watered plants. The mRNA re-accumulated when drought-stressed plants were rewatered. Antibodies raised against a glutathione S-transferase/PTGRP fusion protein were used to elucidate the subcellular localization of the protein by immunogold labeling. In regularly watered L. chilense plants, PTGRP protein was found to be localized in xylem pit membranes and disintegrated primary walls. Examination of sections from drought-stressed plants revealed a significant decrease in the levels of labeling. In these samples, only a few scattered gold particles were detected in the same areas. In the leaf tissues of plants that had been rewatered for 3 d following an 8-d drought stress, the labeling pattern was similar to that of the regularly watered plants. To our knowledge, PTGRP is the first drought-regulated protein that has been precisely localized in the cell wall.     

Nucleotides -1 to -4 of hepatitis delta ribozyme substrate increase the specificity of ribozyme cleavage

In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides. Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding with the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme. Some substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently cleaved substrates. The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be -1HRHY(-4) (H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.