DNA Polymerase δ Is Required for Early Mammalian Embryogenesis

Background

In eukaryotic cells, DNA polymerase δ (Polδ), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Polδ in mammalian development has not been thoroughly investigated.

Methodology/Principal Findings

To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Polδ-null (Pold1 ^−/−) mice and D400A exchanged Polδ (Pold1 ^exo/exo) mice. The D400A exchange caused deficient 3′–5′ exonuclease activity in the Polδ protein. In Polδ-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1 ^−/− mice suffered from peri-implantation lethality. Although Pold1 ^−/− blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Polδ participates in DNA replication during mouse embryogenesis. In Pold1 ^exo/exo mice, although heterozygous Pold1 ^exo/+ mice were normal and healthy, Pold1 ^exo/exo and Pold1 ^exo/− mice suffered from tumorigenesis.

Conclusions

These results clearly demonstrate that DNA polymerase δ is essential for mammalian early embryogenesis and that the 3′–5′ exonuclease activity of DNA polymerase δ is dispensable for normal development but necessary to suppress tumorigenesis.     

Inhibition of ACh-stimulated exocytosis by NSAIDs in guinea pig antral mucous cells: autocrine regulation of mucin secretion by PGE2

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 microM) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 microM; an inhibitor of the EP1-EP4 prostaglandin receptors) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89, 20 microM; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE(2) (1 microM) prevented the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-1) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). ACh and ionomycin increased PGE(2) release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in [Ca(2+)](i) activates Ca(2+)-regulated exocytotic events and PGE(2) release mediated by COX-1. The released PGE(2) induces the accumulation of cAMP, which enhances the Ca(2+)-regulated exocytosis. The autocrine mechanism mediated by PGE(2) maintains the high-level mucin release from antral mucous cells during ACh stimulation.     

Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale ( Balaenoptera bonaerensis )

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against alpha-tubulin 4-6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.     

Enzymatic and nonenzymatic formation of reactive oxygen species from 6-anilino-5,8-quinolinequinone

The nonenzymatic and enzymatic formation of reactive oxygen species (ROS) from LY83583 (6-anilino-5,8-quinolinequinone) was investigated by electron paramagnetic resonance (EPR) spectroscopy. In the presence of thiol compounds such as glutathione and L-cysteine, LY83583 underwent a one-electron reduction due to low redox potential (-0.3+/-0.01 V vs. SCE), followed by formation of LY83583 semiquinone anion radical. This species was characterized by EPR spectroscopy under an argon atmosphere at neutral pH. Under an aerobic condition, this species interacts with molecular oxygen to form a superoxide anion radical. GSH-conjugated LY83583 was also identified by NMR and FAB-MS. When LY83583 was applied to PC12 cells, ROS formation was completely inhibited by both the flavoenzyme inhibitor DPI and the DT-diaphorase inhibitor dicumarol. On the other hand, ROS generation occurred independent of intracellular GSH level. These results indicate that LY83583 can generate ROS both enzymatically and nonenzymatically, although the enzymatic formation is dominant over the nonenzymatic system in PC12 cells.     

Molecular cloning and genetic mapping of perennial ryegrass casein protein kinase 2 α-subunit genes

The α-subunit of the casein protein kinase CK2 has been implicated in both light-regulated and circadian rhythm-controlled plant gene expression, including control of the flowering time. Two putative CK2α genes of perennial ryegrass (Lolium perenne L.) have been obtained from a cDNA library constructed with mRNA isolated from cold-acclimated crown tissue. The genomic organisation of the two genes was determined by Southern hybridisation analysis. Primer designs to the Lpck2a-1 and Lpck2a-2 cDNA sequences permitted the amplification of genomic products containing large intron sequences. Amplicon sequence analysis detected single nucleotide polymorphisms (SNPs) within the p150/112 reference mapping population. Validated SNPs, within diagnostic restriction enzyme sites, were used to design cleaved amplified polymorphic sequence (CAPS) assays. The Lpck2a-1 CAPS marker was assigned to perennial ryegrass linkage group (LG) 4 and the Lpck2a-2 CAPS marker was assigned to LG2. The location of the Lpck2a-1 gene locus supports the previous conclusion of conserved synteny between perennial ryegrass LG4, the Triticeae homoeologous group 5L chromosomes and the corresponding segment of rice chromosome 3. Allelic variation at the Lpck2a-1 and Lpck2a-2 gene loci was correlated with phenotypic variation for heading date and winter survival, respectively. SNP polymorphism may be used for the further study of the role of CK2α genes in the initiation of reproductive development and winter hardiness in grasses.     

Involvement of Ca2+-dependent proteasome in the degradation of both cyclin B1 and Mos during spontaneous activation of matured rat oocytes

In matured rat oocytes, spontaneous activation from the metaphase-II (MII) stage occurred after collection from the oviducts. It is well known that the mitogen-activated protein kinase (MAPK) pathway and p34(cdc2) kinase play an important role in the arrest at MII in other species. However, there is no information about the difference in these factors among strains of rats. In the present study, in spontaneously activated oocytes from the Wistar rat, the Mos protein level and the activity of MAPK kinase (MEK)/MAPK were decreased at 120 min (13.8, 25.7, and 19.3, respectively, P<0.05), whereas Sprague-Dawley (SD) oocytes, which were not spontaneously activated, had a high level of Mos protein and MEK/MAPK activity (75.9, 76.2, and 87.9, respectively, P<0.05). Phosphorylation of MAPK in the SD oocytes was significantly suppressed by MEK inhibitor, U0126 at 60 min; this treatment decreased p34(cdc2) kinase activity via cyclin B1 degradation in a time-dependent manner. The treatment with proteasome inhibitor, MG132 or Ca2+-chelator, BAPTA-AM, overcame the spontaneous degradation of both Mos and cyclin B1 in a dose-dependent manner in Wistar oocytes. More than 90% of Wistar oocytes treated with BAPTA-AM were arrested at MII until 120 min. In conclusion, SD oocytes carrying Mos/MEK/MAPK, maintained a high activity of p34(cdc2) kinase by stabilizing cyclin B1, thus involved in their meiotic arrest. In contrast, Wistar oocytes had a relatively low cytostatic factor activity; rapid decrease of Mos/MEK/MAPK failed to stabilize both cyclin B1 and Mos, and these oocytes were likely to spontaneously activate.     

Production of transgenic rats by ooplasmic injection of spermatogenic cells exposed to exogenous DNA: a preliminary study

The aim of the present study was to investigate the efficiencies of producing transgenic rats by the ooplasmic injection of sperm heads (intracytoplasmic sperm injection: ICSI) and elongating spermatids (elongating spermatid injection: ELSI) exposed to the EGFP DNA solution. A slightly lower proportion of ICSI oocytes using sperm heads exposed to a concentration of 0.5 microg/ml DNA solution for 1 min developed into offspring (13.3%, 48/361) when compared to that of oocytes injected with nontreated sperm heads (19.4%, 32/165). Eight ICSI offspring were found to be EGFP-carrying transgenic rats (16.7% per offspring; 2.2% per embryo). After a 1-min exposure of the elongating spermatids to 5 microg/ml of DNA solution, 8.8% (45/511) of the ELSI oocytes developed into offspring while 12.7% (22/173) of the ELSI oocytes using nontreated spermatids developed. Six ELSI offspring carried the EGFP DNA (13.3% per offspring; 1.2% per embryo). The conventional pronuclear microinjection of 5 microg/ml of DNA solution resulted in the higher production of offspring (29.7%, 104/350) and the birth of three transgenic rats (2.9% per offspring; 0.9% per embryo). Thus, sperm heads and elongating spermatids were practically useful as the vector of exogenous DNA if the DNA-exposed spermatogenic cells were microinseminated into rat oocytes.     

Real-time RT-PCR profiling of transcription factors including 34 MYBs and signaling components in white lupin reveals their P status dependent and organ-specific expression

Two controlled microcosm experiments aimed at a critical re-assessment of the contributions of divergent arbuscular mycorrhizal (AM) fungi to plant mineral nutrition were established that specifically targeted Plantago lanceolata–Glomus intraradices (B.B/E) and –Gigaspora margarita (BEG 34) symbioses developed in a native, nutrient limited, coastal dune soil. Plant tissue nitrogen (N), phosphorus (P) and potassium (K) status as well as plant growth parameters and levels of mycorrhizal colonization were assessed at harvest. In addition to the general well-established mycorrhizal facilitation of P uptake, the study was able to demonstrate a G. intraradices-specific contribution to improved plant nitrogen and potassium nutrition. In the two respective experiments, G. intraradices-inoculated plants had 27.8% and 40.8% more total N and 55.8% and 23.3% more total K when compared to Gi. margarita inoculated counterparts. Dissimilar overall contribution of the two isolates to plant nutrition was identified in AM-genus specific differences in plant tissue N:P:K ratios. G. intraradices inoculated and non-mycorrhizal plants generally exhibited N:P:K ratios indicative of P limitation whereas for Gi. margarita mycorrhizal plants, corresponding ratios strongly implied either N or K limitation. The study provides further evidence highlighting AM functional biodiversity in respect to plant nutrient limitation experienced by mycorrhizal P. lanceolata in an ecologically relevant soil system.     

The Identification of a Novel Gene,MAPO2, That Is Involved in the Induction of Apoptosis Triggered by O6-Methylguanine

O⁶-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU). This report describes the identification of a novel gene, MAPO2 (O⁶-methylguanine-induced apoptosis 2), which is originally annotated as C1orf201. The MAPO2 gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the MAPO2 gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G₂/M phase, however, the production of the sub-G₁ population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the MAPO2 gene product might positively contribute to the induction of apoptosis triggered by O⁶-methylguanine.