Unusual features of fibrillarin cDNA and gene structure in Euglena gracilis: evolutionary conservation of core proteins and structural predictions for methylation-guide box C/D snoRNPs throughout the domain Eucarya

Box C/D ribonucleoprotein (RNP) particles mediate O^2′-methylation of rRNA and other cellular RNA species. In higher eukaryotic taxa, these RNPs are more complex than their archaeal counterparts, containing four core protein components (Snu13p, Nop56p, Nop58p and fibrillarin) compared with three in Archaea. This increase in complexity raises questions about the evolutionary emergence of the eukaryote-specific proteins and structural conservation in these RNPs throughout the eukaryotic domain. In protists, the primarily unicellular organisms comprising the bulk of eukaryotic diversity, the protein composition of box C/D RNPs has not yet been extensively explored. This study describes the complete gene, cDNA and protein sequences of the fibrillarin homolog from the protozoon Euglena gracilis, the first such information to be obtained for a nucleolus-localized protein in this organism. The E.gracilis fibrillarin gene contains a mixture of intron types exhibiting markedly different sizes. In contrast to most other E.gracilis mRNAs characterized to date, the fibrillarin mRNA lacks a spliced leader (SL) sequence. The predicted fibrillarin protein sequence itself is unusual in that it contains a glycine-lysine (GK)-rich domain at its N-terminus rather than the glycine-arginine-rich (GAR) domain found in most other eukaryotic fibrillarins. In an evolutionarily diverse collection of protists that includes E.gracilis, we have also identified putative homologs of the other core protein components of box C/D RNPs, thereby providing evidence that the protein composition seen in the higher eukaryotic complexes was established very early in eukaryotic cell evolution.     

Localization of the genes for the 100-kDa complement-activating components of Ra-reactive factor (CRARF and Crarf) to human 3q27–q28 and mouse 16B2–B3

Human and mouse genes for the complement-activating component (P100) of Ra-reactive factor, a novel bactericidal factor (CRARF and Crarf), were mapped to R-banded metaphase chromosomes by fluorescence in situ hybridization with human and mouse P100 cDNA 2.7 and 2.0 kb long, respectively. The localization of fluorescent signals showed that CRARF and Crarf mapped to human 3q27–q28 and mouse 16B2–B3, respectively. This evidence is consistent with the previous assumption that the distal portion of the long arm of human chromosome 3 is homologous to the proximal portion of mouse chromosome 16.     

Differences between BALB/c and C57BL/6 mice in mouse hepatitis virus replication in primary hepatocyte culture.

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) resulted in acute hepatic failure accompanying extremely elevated viral growth in the liver in interferon-gamma-deficient BALB/c (BALB-GKO), but not C57BL/6 (B6-GKO) mice. To examine the basis of the strain difference against MHV infection in interferon-gamma-deficient mice, viral replication in primary hepatocyte cultures from BALB/c and B6 mice with or without the IFN-gamma gene was compared in vitro. The MHV replication in BALB/c hepatocytes with or without the IFN-gamma gene was significantly higher than that in B6 hepatocytes with or without the IFN-gamma gene, suggesting that there is a strain difference in MHV replication in hepatocytes. Since a significant difference in MHV replication in hepatocytes was not observed between wild type and IFN-gamma-deficient mice of the same genetic background, the phenomenon is thought to be independent of IFN-gamma. However, pretreatment of hepatocytes with recombinant mouse interferon-gamma inhibited MHV replication in a dose-dependent fashion. The results are discussed with respect to the pathology of MHV infection in mice with or without the IFN-gamma gene.     

Functional reconstitution of a crenarchaeal splicing endonuclease in vitro

Sulfolobus tokodaii strain 7 is one of Crenarchaea whose entire genome has been sequenced. The genome sequence revealed that it possesses two open reading frames (ORFs) that are homologous to EndA, a protein responsible for splicing endonuclease activity in Archaea. Interestingly, one of the two ORFs lacks a putative catalytic amino acid residue for the nuclease activity. To investigate their functions, the two ORF products were individually expressed in Escherichia coli, partially purified, and tested for their nuclease activities in vitro. Using in vitro transcribed tRNA precursor as a substrate, we found that the two ORF products are concurrently required to cleave exon-intron junctions. Our finding implies that the splicing endonuclease for the organism is a multi-subunit complex composed of the two endA gene products.     

A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of T-armless tRNAs in nematode mitochondria

Nematode mitochondria possess extremely truncated tRNAs. Of 22 tRNAs, 20 lack the entire T-arm. The T-arm is necessary for the binding of canonical tRNAs and EF (elongation factor)-Tu (thermo-unstable). The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm-binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D-arm. This is the first example of an EF-Tu recognizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.     

Gain and loss of an intron in a protein-coding gene in Archaea: the case of an archaeal RNA pseudouridine synthase gene

Background  

We previously found the first examples of splicing of archaeal pre-mRNAs for homologs of the eukaryotic CBF5 protein (also known as dyskerin in humans) in Aeropyrum pernix, Sulfolobus solfataricus, S. tokodaii, and S. acidocaldarirus, and also showed that crenarchaeal species in orders Desulfurococcales and Sulfolobales, except for Hyperthermus butylicus, Pyrodictium occultum, Pyrolobus fumarii, and Ignicoccus islandicus, contain the (putative) cbf5 intron. However, the exact timing of the intron insertion was not determined and verification of the putative secondary loss of the intron in some lineages was not performed.     

A unique serine-specific elongation factor Tu found in nematode mitochondria

The translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNAs to ribosomes by recognizing the tRNA acceptor and T stems. However, the unusual truncation observed in some animal mitochondrial tRNAs seems to prevent recognition by a canonical EF-Tu. For instance, nematode mitochondria contain tRNAs lacking a T or D arm. We recently found an atypical EF-Tu (EF-Tu1) specific for nematode mitochondrial tRNAs that lack the T arm. We have now discovered a second factor, EF-Tu2, which binds only to tRNAs that lack a D arm. EF-Tu2 seems unique in its amino acid specificity because it recognizes the aminoacyl moiety of seryl-tRNAs and the tRNA structure itself. Such EF-Tu evolution might explain tRNA structural divergence in animal mitochondria.     

Chimeric analysis of EGFP and DsRed2 transgenic mice demonstrates polyclonal maintenance of pancreatic acini

The pancreatic islet is an assembly of specific endocrine cells. There are many conflicting reports regarding whether the acinus develops from single or multiple progenitor cells. This study investigated the development and maintenance clonality of the pancreatic acinus and duct using a chimeric analysis with EGFP and DsRed2 transgenic mice. Chimeric mice (G-R mice) were obtained by the aggregation method, using 8-cell stage embryos from EGFP and DsRed2 transgenic mice. The islets from the G-R mice were chimeric and mosaic, consisting of either EGFP- or DsRed2-positive populations, as in previous reports. On the other hand, most acini developed from either EGFP or DsRed2 origin, but some were chimeric. Interestingly, these chimeric acini were clearly separated into two-color regions and were not mosaic. Some large intralobular pancreatic ducts consisting of more than 10 cells were found to be chimeric, but no small ducts made up of less than 9 cells were chimeric. Our histological observations suggest that the pancreatic acinus polyclonally and directionally is maintained by multiple progenitor cells. Pancreatic large ducts also seem to develop polyclonally and might result from the assembly of small ducts that develop from a single origin. These findings provide useful information for further understanding pancreatic maintenance.     

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands

Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺]_i in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺]_i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺]_i increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺]_i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺]_i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺]_i, although UTP (a P2Y_2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺]_i, but α,β-meATP (a P2X_1,3 receptor agonist) had no effect. RT-PCR indicated that P2X_2,3,4,5,6,7 and P2Y_1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.     

Ubc9 is essential for viability of higher eukaryotic cells

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.