Structure and function of the fourth subunit (Dpb4p) of DNA polymerase epsilon in Saccharomyces cerevisiae

DNA polymerase (Pol) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS–PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Pol. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (Δdpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the Δdpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying Δdpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Pol in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S.cerevisiae Pol is conserved in eukaryotes.     

Attenuation of expression of gamma-glutamylcysteine synthetase by ribozyme transfection enhance insulin secretion by pancreatic beta cell line, MIN6

Low levels of intracellular antioxidant enzyme activities as well as glutathione (GSH) concentrations have been described in pancreatic beta cells. We examined the effects of intracellular GSH depletion on insulin secretion and the role of intracellular GSH in signal transduction in beta cell line, MIN6 cells. Anti-gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit ribozyme was stably transfected to MIN6 cells to reduce intracellular GSH concentration. In the presence of 10 mM glucose, ribozyme-transfected cells (RTC) increased insulin secretion from 0.58 microg/10(6) cells/h in control cells (CC) to 1.48 microg/10(6) cells/h. This was associated with increased intracellular Ca(2+) concentration in RTC, detected by fluo-3 staining. Our results demonstrated that intracellular GSH concentration might influence insulin secretion by MIN6 cells, and suggest that enhanced insulin secretion by beta cells conditioned by chronic depletion of GSH is mediated by increased intracellular Ca(2+) concentration.     

Modeling the Expansion of an Introduced Tree Disease

Pine wilt disease is caused by the introduced pinewood nematode, Bursaphelenchus xylophilus, for which the vector is the pine sawyer beetle, Monochamus alternatus. Native Japanese pines, black pine (Pinus thunbergii) and red pine (P. densiflora), are extremely sensitive to the nematode's infection, and the parasite has been expanding nationwide in the last few decades, despite intensive control efforts. To understand the parasite's range expansion in Japan, we modeled the dynamics of the pines and the beetle that disperses the nematode, using an integro-difference equation in a one-dimensional space. Based on field data collected in Japan, we investigated the dependence of the parasite's rate of range expansion on the eradication rate of the beetle, the initial pine density, and the beetle dispersal ability. Our model predicts several results. (1) The Allee Effect operates on beetle reproduction, and consequently the parasite cannot invade a pine stand, once the beetle density decreases below a threshold. (2) The distribution of the dispersal distance of the beetles critically affects the expansion rate of the disease. As the fraction of the beetles that travel over long distance increases from zero, the range expansion accelerates sharply. (3) However, too frequent long-range dispersal results in a failure of the parasite invasion due to the Allee Effect, suggesting the importance of correctly assessing the beetle's mobility to predict the speed of range expansion of the parasite. (4) As the eradication rate is increased, the range expansion speed decreases gradually at first and suddenly drops to zero at a specific value of the eradication rate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Investigation of various genotype characteristics for inosine accumulation in Escherichia coli W3110

For the derivation of an inosine-overproducing strain from the wild type microorganism, it is known that the addition of an adenine requirement, removal of purine nucleoside hydrolyzing activity, removal of the feedback inhibition, and repression of key enzymes in the purine nucleotides biosynthetic pathway are essential. Thus, the disruption of purA (adenine requirement), deoD (removal of purine nucleosides phosphorylase activity), purR (derepression of the regulation of purine nucleotides biosynthetic pathway), and the insensitivity of the feedback inhibition of phosphoribosylpyrophosphate (PRPP) amidotransferase by adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) were done in the Escherichia coli strain W3110, and then the inosine productivity was estimated. In the case of using a plasmid harboring the PRPP amidotransferase gene (purF) that encoded a desensitized PRPP amidotransferase, purF disrupted mutants were used as the host strains. It was found that the innovation of the four genotypes brought about a small amount of inosine accumulation. Furthermore, an adenine auxotrophic mutant of E. coli showed inappropriate adenine use because its growth could not respond efficiently to the concentration of adenine added. As the presence of adenosine deaminase is well known in E. coli and it is thought to be involved in adenine use, a mutant disrupted adenosine deaminase gene (add) was constructed and tested. The mutant, which is deficient in purF, purA, deoD, purR, and add genes, and harboring the desensitized purF as a plasmid, accumulated about 1 g of inosine per liter. Although we investigated the effects of purR disruption and purF gene improvement, unexpectedly an increase in the inosine productivity could not be found with this mutant.     

Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190,000

A heat-stable microtubule-associated protein (MAP) with apparent molecular weight of 190,000 is a major non-neural MAP which distributes ubiquitously among bovine tissues (termed here MAP-U). Previously we reported that microtubule-binding chymotryptic fragments of MAP-U and tau contain a common assembly-promoting (AP) sequence of 22 amino acid residues (Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1989) J. Biol. Chem. 264, 5885-5890). We isolated cDNA clones for MAP-U containing the whole coding sequence. Northern blot analysis revealed that a major species of MAP-U mRNA is 5 kilobases in length and is expressed ubiquitously among bovine tissues. Nucleotide sequence analysis revealed the complete amino acid sequence of MAP-U which consists of 1,072 amino acid residues. Analysis of the deduced amino acid sequence of MAP-U indicated that this molecule is clearly divided into two domains in terms of electrostatic charge distribution: an amino-terminal acidic domain (residues 1-640) and a carboxyl-terminal basic domain (residues 641-1072). The amino-terminal domain of MAP-U shows no significant sequence homology with other known protein sequences including neural MAPs, tau, and MAP-2. The amino-terminal domain of MAP-U contains unique 18 1/2 repeats of 14-amino acid motif which have not been observed in other MAPs. The carboxyl-terminal domain of MAP-U is further divided into three regions: a Pro-rich region (residues 641-880), an AP sequence region (residues 881-1003), and a short hydrophobic tail (residues 1004-1072). The Pro-rich region is mainly composed of five species of amino acid residues, Pro, Ala, Lys, Ser, and Thr. The AP sequence region contains four tandem repeats of AP sequences, and thus, this region is considered to play a leading role in the interaction of MAP-U with microtubules.     

Site-specific phosphorylation by protein kinase C inhibits assembly-promoting activity of microtubule-associated protein 4

We have examined the phosphorylation of bovine microtubule-associated protein 4 (MAP4), formerly named MAP-U, by protein kinase C (PKC). When MAP4 was incubated with PKC, about 1 mol of phosphate was incorporated/mol of MAP4. Phosphorylation of MAP4 caused a remarkable decrease in the ability of the MAP to stimulate microtubule assembly. MAP4 consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain is subdivided into a Pro-rich region and an assembly-promoting (AP) sequence region containing four tandem repeats of AP sequence that is conserved in MAP4, MAP2, and tau [Aizawa et al. (1990) J. Biol. Chem. 265, 13849-13855]. In order to identify the site of MAP4 phosphorylated by PKC, a series of expressed MAP4 fragments was prepared and treated with the kinase. A fragment corresponding to the Pro-rich region (P fragment) was phosphorylated, while fragments corresponding to the projection domain and the AP sequence region were not. In addition, chymotryptic digestion of an authentic MAP4 prephosphorylated by PKC revealed that phosphate was incorporated almost exclusively into a 27-kDa fragment containing the carboxyl-terminal half of the Pro-rich region. We investigated the phosphorylation site in MAP4 using the P fragment and found that Ser815 was phosphorylated almost exclusively. We conclude that the phosphorylation of a single Ser residue in the Pro-rich region negatively regulates the assembly-promoting activity of MAP4.     

Molecular cloning and sequencing of cDNA encoding the phosphatidylinositol kinase from rat brain.

A complementary DNA (cDNA) clone that encodes phosphatidylinositol 4-kinase (PI 4-kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence of 697 residues revealed that the protein contains two putative transmembrane sequences and that the N-terminal part of the protein has several sequences representing potential phosphorylation sites for cAMP- and calmodulin-dependent kinase. The C-terminal region is probably a phosphotransferase domain homologous to the kinase region of protein kinase family proteins. Specific antibody against the protein expressed in Escherichia coli successfully immunoprecipitated rat brain PI 4-kinase. The messenger RNA for PI 4-kinase was found predominantly in brain and rat neural cell lines. This PI kinase may play a specific role in neural signal transduction.     

Microtubule destabilization by cdc2/H1 histone kinase: phosphorylation of a "pro-rich region" in the microtubule-binding domain of MAP-4.

Microtubule-associated protein-4 (MAP-4), a major MAP in proliferating cells, consists of a microtubule-binding domain and a projection domain protruding from the microtubule wall. The former contains a Pro-rich region and an assembly-promoting (AP) sequence region which is common to the neuron-specific MAPs, MAP-2 and tau1. In this paper, we describe the phosphorylation of the Pro-rich region of MAP-4 and the suppression of its assembly-promoting activity by cdc2/H1 histone kinase. This inactivation of MAP-4 may cause disassembly of the interphase microtubular network at the end of the G2 phase of the cell cycle.     

Basic steroid saponins from aerial parts of Fritillaria Thunbergii

From the aerial parts of Fritillaria thunbergii, three glycosidal Solanum alkaloids (basic steroid saponins) were isolated together with minor