The Escherichia coli threonyl-tRNA synthetase gene contains a split ribosomal binding site interrupted by a hairpin structure that is essential for autoregulation

The expression of the gene encoding Escherichia coli threonyl-tRNA synthetase (ThrRS) is negatively autoregulated at the translational level. ThrRS binds to its own mRNA leader, which consists of four structural and functional domains: the Shine–Dalgarno (SD) sequence and the initiation codon region (domain 1); two upstream hairpins (domains 2 and 4) connected by a single-stranded region (domain 3). Using a combination of in vivo and in vitro approaches, we show here that the ribosome binds to thrS mRNA at two non-contiguous sites: region −12 to +16 comprising the SD sequence and the AUG codon and, unexpectedly, an upstream single-stranded sequence in domain 3. These two regions are brought into close proximity by a 38-nucleotide-long hairpin structure (domain 2). This domain, although adjacent to the 5' edge of the SD sequence, does not inhibit ribosome binding as long as the single-stranded region of domain 3 is present. A stretch of unpaired nucleotides in domain 3, but not a specific sequence, is required for efficient translation. As the repressor and the ribosome bind to interspersed domains, the competition between ThrRS and ribosome for thrS mRNA binding can be explained by steric hindrance.     

Water stress and photoinhibition in acclimatization ofRosa hybrida plantlets

Summary MicropropagatedRosa hybrida plantlets were simultaneously rooted and acclimatized under 100 and 200 μmol m⁻² s⁻¹ light for 2 wk. At the end of the first week of acclimatization, the plantlets were transferred onto a low water potential medium (from −0.06 MPa to −0.3 MPa). Dry weight was decreased by increased hight and low water potential. Photoinhibition of photosynthesis, expressed as a decrease in Fv/Fm ratio and ΦPSII and an increase in 1 −qp, occurred in plants grown under 200 μmol m⁻² s⁻¹. When high light (200 μmol m⁻² s⁻¹) and water stress were applied simultaneously, their effects on chlorophyll fluorescence parameters depended on stress duration; after 1 d of water stress, photoinhibition was more pronounced; after 7 d of stress, Fv/Fm ratio and ΦPSII were higher than after 1 d of stress; photoinhibition was reduced. This suggests that after a 1-d stress, the effect of water stress alone included a superimposed effect of photoinhibition to which the water-stressed plants were sensitized; after 7 d, plantlets had adapted to water stress. The photoprotective effects under high light might result in energy dissipative mechanisms linked to photochemical and nonphotochemical quenching other than CO₂ fixation.     

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.     

The Cytoplasmic Zinc Finger Protein ZPR1 Accumulates in the Nucleolus of Proliferating Cells

The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells.     

Intrageneric Phylogeny ofAcomys(Rodentia, Muridae) Using Mitochondrial Gene Cytochromeb

This paper investigates interspecies relationships within the genusAcomys(spiny mice) by analyzing entire mitochondrial cytochromebgene (1141 bp). This gene provides strong phylogenetic signal, as shown by high support of the topology obtained (bootstrap value and RNA support number). The phylogeny is congruent with inferences from allozymes for the species considered. Controversial taxonomy ofAcomys cahirinus, dimidiatus, airensis,andignitusis clarified, with their specific ranks confirmed on the basis of tree topology and nucleotide distances. Phylogenetic relationship between the undescribed speciesAcomyssp. from west Africa andA. airensisargue in favor of two distinct colonization events in this zone.     

Cloning, Expression, and Pharmacology of Four Human 5-Hydroxytryptamine4 Receptor Isoforms Produced by Alternative Splicing in the Carboxyl Terminus

Abstract: We report here the molecular cloning of three new splice variants of the human serotonin 5-hydroxytryptamine₄ (h5-HT₄) receptor, which we named h5-HT_4(b), h5-HT_4(c), and h5-HT_4(d). The sequence following the splicing site at Leu₃₅₈ in the C-terminal tail of h5-HT_4(b) displays a 74% protein identity with the same region in the long form of the rat 5-HT₄ receptor (r5-HT_4L) but is shorter by 18 amino acids compared to its rat counterpart. The splice variants h5-HT_4(c) and h5-HT_4(d) are the first of their kind to be described in any animal species. The C terminus of h5-HT_4(c) displays a high number of putative phosphorylation sites. The h5-HT_4(d) isoform corresponds to an ultrashort form of the receptor, with a truncation two amino acids after the splicing site. Tissue distribution studies revealed some degree of specificity in the pattern of expression of the different isoforms within the human body. The four splice variants transiently expressed in COS-7 cells displayed an identical 5-HT₄ pharmacological profile and showed a similar ability to stimulate adenylyl cyclase activity in the presence of 5-HT. The stimulatory pattern of cyclic AMP formation in response to the 5-HT₄ agonist renzapride was found to be significantly different between h5-HT_4(a) and the other h5-HT₄ isoforms, indicating that the splice variants may differ in the way they trigger the signal transduction cascade following receptor activation.     

Development of a Quantitative Polyacrylamide Gel Electrophoresis Analysis Using a Multichannel Radioactivity Counter for the Evaluation of Oligonucleotide-Bound Drug Carrier

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of³³P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.     

Establishment and Characterization of Cell Line LSV5 That Retains the Altered Adhesive Properties of Human Junctional Epidermolysis Bullosa Keratinocytes

Herlitz junctional epidermolysis bullosa (H-JEB) is characterized by hampered expression of the adhesion ligand laminin-5. Thus far, analysis of the processes underlying the epithelial–mesenchymal dysadhesion marking this disease has been limited by the reduced growth and adhesive capabilities of the epithelial cells derived from H-JEB patients. To overcome these difficulties, we used SV40 virus to immortalize H-JEB keratinocytes with a homozygous nonsense mutation in the gene that encodes the γ2 chain of laminin-5. Cell lines (LSV) derived from infected keratinocytes maintain a stable karyotype, grow independent of 3T3 feeder layers and are not tumorigenic. Further analysis of clone LSV5 showed an increased secretion of laminin-6 and fibronectin compared to normal keratinocytes. Similar to parental H-JEB keratinocytes, these cells regenerate stratified epidermisin vitroand, inin vivomodels, they synthesize a basement membrane lacking laminin-5. LSV cells show hypermotility and reduced adhesive properties resulting from an incomplete association with the underlying culture substrate. These results demonstrate that LSV5 cells retain the pathologic phenotype of H-JEB keratinocytes and can serve as a model system to study the adhesion processes mediated by laminin-5.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.