Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

Rapid and transient thrombin stimulation of phosphatidylinositol 4,5-bisphosphate synthesis but not of phosphatidylinositol 3,4-bisphosphate independent of phospholipase C activation in platelets

When platelets are stimulated by thrombin they immediately undergo inositol lipid hydrolysis via phospholipase C activation. However, subsequently an increased production of phosphatidylinositol 4,5-bisphosphate is observed. Phospholipases C were inhibited by lowering the cytoplasmic free calcium concentration by preincubation with Quin-2-tetra(acetoxymethyl) ester. Aggregation and secretion were also totally suppressed. Under these conditions we observed an increased labeling of phosphatidylinositol 4,5-bisphosphate, indicating a stimulation of inositol lipid kinases, independent of lipid hydrolysis by phospholipase C. Conversely the production of phosphatidylinositol 3,4-bisphosphate was totally abolished. These results suggest a different regulation of the kinases/phosphatases responsible for the production of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate.     

Transglutaminase-like activity in Chrysanthemum leaf explants cultivated in vitro in relation to cell growth and hormone treatment

In Chrysanthemum leaf explants cultivated in vitro the capacity to covalently link polyamines to protein substances exists. This plant enzyme activity shows some similarities with mammalian transglutaminases. In foliar explants cultured on a medium promoting bud or root formation increasing levels of transglutaminase-like activity occurred during the first days of culture when cell multiplication was rapid then the levels declined as the rate of cell division decreased and differentiation occurred. Undifferentiated callus exhibited low transglutaminase-like activity. Transglutaminase-like activity also increased in rapidly proliferating and growing organs (roots and buds initiated from the foliar explants) and decreased during maturity. The relationship among transglutaminases-like activity, cell division, bud and root formation is discussed.     

Molecular Cloning of a Gene on Chromosome 19q12 Coding for a Novel Intracellular Protein: Analysis of Expression in Human and Mouse Tissues and in Human Tumor Cells, Particularly Reed–Sternberg Cells in Hodgkin Disease

A novel protein, named NNX3, was molecularly characterized by cloning its cDNA, and its gene was mapped to chromosome 19q12. The equivalent mouse cDNA and gene were also cloned to allow us to analyze expression in murine in addition to human cells and tissues. Human and mouse NNX3 genes are composed of nine exons coding for proteins that are unrelated to any known protein. Signal peptides and hydrophobic domains are absent, corroborating their localization in the cytoplasm in transfected Cos cells. In Western blotting and immunoprecipitation, human NNX3 appeared as a doublet ofM_r64K–66K, while mouse NNX3 was a 70-kDa protein, both apparently much larger than the predicted 50 kDa, due in part to a stretch of 16–18 acidic residues hinging two nearly equally sized domains. In addition, phosphorylation of serine residues was demonstrated. Putative nuclear targeting signals were predicted, but NNX3 protein and two truncated versions remained localized in the cytoplasm of transfected Cos cells. NNX3 was expressed in embryonic and adult mouse tissues, particularly in brain, muscle, and lung. The expression of human NNX3 was most notable in human skeletal muscle and in ganglion cells and was also evident in human tumors and derived cell lines. This was confirmed by entries appearing in the GenBank EST database during the later phase of this study, representing partial NNX3 cDNA isolated from diverse neoplastic and developing tissues. Surprisingly, NNX3 was immunochemically detected in Reed–Sternberg cells of Hodgkin disease, in parallel with restin, a cytoplasmic protein we previously characterized (J. Delabieet al.,1993,Leuk. Lymphoma 12,21–26). The cloning and comprehensive molecular analysis of NNX3 as presented will form the basis for elucidating its function and, conversely, will constitute a marker for Reed–Sternberg cells in Hodgkin disease.     

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.