Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases

A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl-ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 10(3) and 10(5)-fold decreases in k(cat) observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30-fold decrease in activity and a 16-fold inflation of the K(m) for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphom-evalonate kinase (GHMP kinase) family.     

“Chromosome Memory” of Parental Genomes in Embryonic Hybrid Cells

In the hybrid cells obtained by fusion of embryonic stem cells with adult differentiated cells, homologous chromosomes are in two ontogenetic configurations: pluripotent and differentiated. In order to assess the role of cis- and trans-regulation in the maintenance of these states, we studied a set of clones of hybrid cells of the type embryonic stem cells–splenocytes and used two approaches: segregation of parental chromosomes and comparison of pluripotency of the past hybrid cells and embryonic stem cells. The segregation test showed that the hybrid cells lost only the homologs of the somatic partner and this process was sharply accelerated when the cells were cultivated in nonselective conditions, thus suggesting the full or partial preservation of the initial differences in the organization of parental homologs. The descendants of the former hybrid cells, which had the karyotype similar to that of embryonic stem cells, demonstrated the level of pluripotency, comparable with that of embryonic stem cells despite the long-term effect of trans-acting factors from the somatic partner in the genome of hybrid cells. The data obtained are interpreted in the framework of the concept of chromosome memory, in the maintenance of which the key role is played bycis-regulatory factors.     

Kindling fluorescent proteins for precise in vivo photolabeling

Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.     

Calcium microdomains in aspiny dendrites

Dendritic spines receive excitatory synapses and serve as calcium compartments, which appear to be necessary for input-specific synaptic plasticity. Dendrites of GABAergic interneurons have few or no spines and thus do not possess a clear morphological basis for synapse-specific compartmentalization. We demonstrate using two-photon calcium imaging that activation of single synapses on aspiny dendrites of neocortical fast spiking (FS) interneurons creates highly localized calcium microdomains, often restricted to less than 1 microm of dendritic space. We confirm using ultrastructural reconstruction of imaged dendrites the absence of any morphological basis for this compartmentalization and show that it is dependent on the fast kinetics of calcium-permeable (CP) AMPA receptors and fast local extrusion via the Na+/Ca2+ exchanger. Because aspiny dendrites throughout the CNS express CP-AMPA receptors, we propose that CP-AMPA receptors mediate a spine-free mechanism of input-specific calcium compartmentalization.     

Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.     

Genetic analysis of housekeeping genes of members of the genus Acholeplasma: phylogeny and complementary molecular markers to the 16S rRNA gene

The partial nucleotide sequences of the rpoB and gyrB genes as well as the complete sequence of the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known Acholeplasma species. The same genes of Mesoplasma and Entomoplasma species were also sequenced and used to infer phylogenetic relationships among the species within the orders Entomoplasmatales and Acholeplasmatales. The comparison of the ITS, rpoB, and gyrB phylogenetic trees with the 16S rRNA phylogenetic tree revealed a similar branch topology suggesting that the ITS, rpoB, and gyrB could be useful complementary phylogenetic markers for investigation of evolutionary relationships among Acholeplasma species. Thus, the multilocus phylogenetic analysis of Acholeplasma multilocale sequence data (ATCC 49900 (T) = PN525 (NCTC 11723)) strongly indicated that this organism is most closely related to the genera Mesoplasma and Entomoplasma (family Entomoplasmataceae) and form the branch with Mesoplasma seiffertii, Mesoplasma syrphidae, and Mesoplasma photuris. The closest genetic relatedness of this species to the order Entomoplasmatales was additionally supported by the finding that A. multilocale uses UGA as the tryptophan codon in its gyrB and gyrA sequences. Use of the UGA codon for encoding tryptophan was previously reported as a unique genetic feature of Entomoplasmatales and Mycoplasmatales but not of Acholeplasmatales. These data, as well as previously published data on metabolic features of A. multilocale, leads to the proposal to reclassify A. multilocale as a member of the family Entomoplasmataceae.