Schizosaccharomyces pombe Ddb1 is functionally linked to the replication checkpoint pathway

Schizosaccharomyces pombe Ddb1 is homologous to the mammalian DDB1 protein, which has been implicated in damaged-DNA recognition and global genomic repair. However, a recent study suggested that the S. pombe Ddb1 is involved in cell division and chromosomal segregation. Here, we provide evidence that the S. pombe Ddb1 is functionally linked to the replication checkpoint control gene cds1. We show that the S. pombe strain lacking ddb1 has slow growth due to delayed replication progression. Flow cytometric analysis shows an extensive heterogeneity in DNA content. Furthermore, the Deltaddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the Deltaddb1 strain exhibits a high level of the Cds1 kinase activity during passage through S phase. Moreover, mutation of the cds1 gene relieves the defects observed in Deltaddb1 strain. The results suggest that many of the defects observed in Deltaddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1.     

Paternal influence on timing of pronuclear DNA synthesis in naturally ovulated and fertilized mouse eggs

Fertilized oocytes of the inbred genotypes AKR (AK), C57BL/6 (B6), DBA/2 (D2), and CBA (CB) and the hybrid genotypes F1 (female AK X male B6) and F1 (female B6 X male AK) were collected by flushing the oviducts of female mice every 2 h from 2 until 26 h post coitum. Developmental stages of the embryos and DNA content of the pronuclei were estimated by morphological criteria and cytofluorometric measurement of the pronuclei (ethidium bromide-stained DNA), respectively. In all genotypes, S-phase started about 4 h post conception (h.p.c.). The duration of S-phase amounted to 5.9 h (F1 [female B6 X male AK]), 6.4 h (AK), 8.5 h (B6), 9.4 h (F1 [female AK X male B6]), 9.8 h (D2), and 11.4 h (CB). In each of the reciprocal F1 hybrids, the length of S-phase differed from the maternal genotype (p less than 0.01) and resembled closely the paternal genotype (p greater than 0.25). Cleavage from one-cell stage to two-cell stage occurred between 16 and 21 h.p.c.     

Paternal age is positively linked to telomere length of children

Telomere length is linked to age-associated diseases, with shorter telomeres in blood associated with an increased probability of mortality from infection or heart disease. Little is known about how human telomere length is regulated despite convincing data from twins that telomere length is largely heritable, uniform in various tissues during development until birth and variable between individuals. As sperm cells show increasing telomere length with age, we investigated whether age of fathers at conception correlated with telomere length of their offspring. Telomere length in blood from 125 random subjects was shown to be positively associated with paternal age (+22 bp yr -1, 95% confidence interval 5.2-38.3, P = 0.010), and paternal age was calculated to affect telomere length by up to 20% of average telomere length per generation. Males lose telomeric sequence faster than females (31 bp yr -1, 17.6-43.8, P < 0.0001 vs. 14 bp yr -1, 3.5-24.8, P < 0.01) and the rate of telomere loss slows throughout the human lifespan. These data indicate that paternal age plays a role in the vertical transmission of telomere length and may contribute significantly to the variability of telomere length seen in the human population, particularly if effects are cumulative through generations.     

Translation termination is involved in histone mRNA degradation when DNA replication is inhibited

The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3' untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.     

Sit4 phosphatase is functionally linked to the ubiquitin-proteasome system

Using a synthetic lethality screen we found that the Sit4 phosphatase is functionally linked to the ubiquitin-proteasome system. Yeast cells harboring sit4 mutations and an impaired proteasome (due to pre1-1 pre4-1 mutations) exhibited defective growth on minimal medium. Nearly identical synthetic effects were found when sit4 mutations were combined with defects of the Rad6/Ubc2- and Cdc34/Ubc3-dependent ubiquitination pathways. Under synthetic lethal conditions, sit4 pre or sit4 ubc mutants formed strongly enlarged unbudded cells with a DNA content of 1N, indicating a defect in the maintenance of cell integrity during starvation-induced G(1) arrest. Sit4-related synthetic effects could be cured by high osmotic pressure or by the addition of certain amino acids to the growth medium. These results suggest a concerted function of the Sit4 phosphatase and the ubiquitin-proteasome system in osmoregulation and in the sensing of nutrients. Further analysis showed that Sit4 is not a target of proteasome-dependent protein degradation. We could also show that Sit4 does not contribute to regulation of proteasome activity. These data suggest that both Sit4 phosphatase and the proteasome act on a common target protein.     

Methylations of histone H3 lysine 9 and lysine 36 are functionally linked to DNA replication checkpoint control in fission yeast

Recently, histone H4 lysine 20 and H3 lysine 79 methylations were functionally linked to DNA damage checkpoint. The crosstalk between histone methylation and the S-M checkpoint, however, has remained unclear. Here, we show that H3 lysine 9 (K9) and lysine 36 (K36) methylations catalyzed by two histone methyltransferases Clr4 and Set2 are involved in hydroxyurea (HU)-induced replication checkpoint. The clr4-set2 double mutants besides histone H3-K9 and K36 double mutants exhibited HU-sensitivity, a defective HU-induced S-M checkpoint, and a significant reduction of HU-induced phosphorylation of Cdc2. Intriguingly, the clr4-set2 double mutations impaired the HU-induced accumulation of a mitotic inhibitor Mik1. Double mutants in Alp13 and Swi6, which can specifically bind to H3-K36 and K9 methylations, exhibited phenotypes similar to those of the clr4-set2 mutants. Together, these findings suggest that methylations of histone H3-K9 and K36 by Clr4 and Set2 are functionally linked to DNA replication checkpoint via accumulation of Mik1.     

Serial pronuclear transfer increases the developmental potential of in vitro-matured oocytes in mouse cloning

In vitro-matured germinal vesicle oocytes are an interesting source of cytoplast recipients in both animal and human nuclear transfer (NT) experiments. We investigated two technical aspects that might improve the developmental potential of nuclear transfer mouse embryos constructed from in vitro-matured germinal vesicle oocytes. In a first step, the effect of two maturation media on the embryonic development of NT embryos originating from in vitro-matured oocytes was compared. Supplementation of the oocyte maturation medium with serum and gonadotrophins improved the developmental rate of NT embryos constructed from in vitro-matured oocytes, but it was still inferior to that obtained with in vivo-matured metaphase II (MII) oocytes. Second, we investigated the effect of serial pronuclear transfer from NT zygotes originating from both in vitro- and in vivo-matured oocytes to in vivo-fertilized zygotic cytoplasts. Blastocyst quality was evaluated by counting nuclei from trophectoderm and inner cell mass cells using a differential staining. Sequential pronuclear transfer significantly improved the blastocyst formation rate of NT embryos originating from in vitro-matured oocytes up to the rate obtained with in vivo-matured MII oocytes. We conclude that the developmental potential of NT embryos constructed from in vitro-matured oocytes can be optimized by serial pronuclear transfer to in vivo-produced zygotic cytoplasts.     

Double-strand breaks induce short-scale DNA replication and damage amplification in the fully grown mouse oocytes

Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2’-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.     

UV-induced DNA repair is not detectable in pre-dictyate oocytes of the mouse

Oocytes from fetal and neonatal mice were UV-irradiated, cultured in medium containing tritiated thymidine and analysed following autoradiography. As previously reported irradiated dictyate cells clearly displayed an increased grain count. However we detected no UV-induced response in oocytes at the leptotene, zygotene or pachytene stages of meiosis. This contrasts with the situation in spermatogenesis where high levels of repair can be induced in equivalent early prophase stages.     

The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis

Testicular mitochondria were previously shown to contain an abundance of peripheral-type benzodiazepine recognition site(s)/receptor(s) (PBR). We have previously purified, cloned, and expressed an Mr 18,000 PBR protein (Antkiewicz-Michaluk, Mukhin, A. G., Guidotti, A., and Krueger, K. E. (1988) J. Biol. Chem. 263, 17317-17321; (Sprengel, R., Werner, P., Seeburg, P. H., Mukhin, A. G., Santi, M. R., Grayson, D. R., Guidotti, A., and Krueger, K. E. (1989) J. Biol. Chem. 264, 20415-20421); and in this report, we present evidence that PBR are functionally linked to Leydig cell steroid biosynthesis. A spectrum of nine different ligands covering a range of over 4 orders of magnitude in their affinities for PBR were tested for their potencies to modulate steroidogenesis in the MA-10 mouse Leydig tumor cell line. The Ki for inhibition of [3H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide binding and the EC50 for steroid biosynthesis for this series of compounds showed a correlation coefficient of r = 0.95. The most potent ligands stimulated steroid production by approximately 4-fold in these cells. This stimulation was not inhibited by cycloheximide, unlike human chorionic gonadotropin- or cyclic AMP-activated steroidogenesis. The action of PBR ligands was not additive to stimulation by human chorionic gonadotropin or cyclic AMP, but was additive to that of epidermal growth factor, another regulator of MA-10 Leydig cell steroidogenesis. Moreover, PBR ligands stimulated, in a dose-dependent manner, pregnenolone biosynthesis by isolated mitochondria when supplied with exogenous cholesterol. This effect was not observed with mitoplasts (mitochondria devoid of the outer membrane). Cytochrome P-450 side chain cleavage activity, as measured by metabolism of (22R)-hydroxycholesterol, was not affected by PBR ligands in intact cells. Similar results were also obtained with purified rat Leydig cells. In conclusion, PBR are implicated in the acute stimulation of Leydig cell steroidogenesis possibly by mediating the entry, distribution, and/or availability of cholesterol within mitochondria.     

A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane.

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.     

Degradation of insulin in isolated liver endosomes is functionally linked to ATP-dependent endosomal acidification

The degradation of insulin in isolated liver endosomes and the relationships of this process with ATP-dependent endosomal acidification have been studied. Incubation of endosomal fractions containing 125I-insulin in isotonic KCl at 30 degrees C resulted in a rapid loss of insulin integrity as judged from trichloroacetic acid precipitability, Sephadex G-50 chromatography, immunoreactivity and receptor binding ability, with a maximum at pH 5-6 (t1/2: 10, 10, 6 and 6 min, respectively). On a log/log plot, the amount of acid-soluble products generated was linearly related to the amount of insulin associated with endosomes (slope, 0.80). Upon incubation, virtually all acid-soluble products diffused out of endosomes as judged from their solubility in aqueous poly(ethyleneglycol). In permeabilized endosomes, intact insulin was also released in part extraluminally, but only when degradation was inhibited did this release increase with lowering pH. ATP shifted the pH for maximal insulin degradation to about 7.5-8.5 and caused endosomal acidification as judged from the uptake of acridine orange and the fluorescence of internalized fluorescein-labeled dextran and galactosylated bovine serum albumin (delta pH about 0.8-0.9). GTP, ITP and UTP exerted comparable effects but with lower potencies. The ability of ATP to alter the pH dependence of insulin degradation was maximal in the presence of Cl-, other anions being less effective (Br- greater than gluconate = SO4(2-) greater than NO3- = sucrose = mannitol) and/or inhibitory (NO3-). Na+, K+ and Li+ supported more effectively ATP-dependent insulin degradation than did choline. Divalent cations were required for the ATP effect (Mg2+ = Mn2+ greater than Co2+ greater than Ni2+ = Zn2 greater than Ca2+). Little or no effects of ATP occurred in the presence of proton ionophores such as monensin and carbonyl cyanide chlorophenylhydrazone, and inhibitors of the proton ATPase such as N-ethylmaleimide. The abilities of nucleotides, ions and inhibitors to support or inhibit ATP-dependent insulin degradation were well correlated with their abilities to affect ATP-dependent acidification. The acidotropic agents chloroquine and quinacrine caused a leftward shift in the pH dependence of insulin degradation and a decrease in maximal degradation; in the presence of ATP, chloroquine almost completely inhibited degradation at pH 5-9. It is concluded that ATP-dependent acidification, in part by enhancing the dissociation of the insulin-receptor complex, is required for optimum degradation of insulin within liver endosomes.     

Male pronuclear formation and sperm mitochondria in porcine oocytes following intracytoplasmic injection of pig or mouse sperm

In the present study we determined the chromatin organization and fate of introduced mitochondria in porcine embryos following intracytoplasmic injection of pig or mouse sperm cells. At 3, 6, 9 and 12 h following injection of pig or mouse spermatozoa or isolated sperm heads, the oocytes were fixed and stained with propidium iodide. Between 3 and 6 h following injection, both porcine and murine sperm chromatin developed into pronuclei. The male and female pronuclei were apposed within 12 h in porcine oocytes following sperm injection from either source. We also introduced foreign mitochondria from either mouse or pig sperm midpiece into porcine oocytes following sperm injection. While porcine sperm mitochondria rapidly disappeared from the actively developing porcine oocytes, mouse sperm mitochondria remained in the embryos until the 8-cell stage. These results suggest that pronuclear formation and movement occur between 6 and 12 h following sperm incorporation into the cytoplasm, and that foreign mitochondria are selectively removed in a species-specific manner.     

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3′ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.     

DNA supercoiling by gyrase is linked to nucleoid compaction

The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a nucleoid to fit inside the 1-2 m cell in a functional format. To examine the role of DNA supercoiling as nucleoid compaction force we modulated the activity of DNA gyrase by electronic, genetic, and chemical means. A model based on physical properties of DNA and other cell components predicts that relaxation of supercoiling expands the nucleoid. Nucleoid size did not increase after reduction of DNA gyrase activity by genetic or chemical means, but nucleoids did expand upon chemical inhibition of gyrase in chloramphenicol-treated cells, indicating that supercoiling may help to compress the genome.     

Differential inspiratory timing is genetically linked to mouse chromosome 3

Genetic control ofdifferential inspiratory timing(TI) at baseline has beenpreviously demonstrated among inbred mouse strains. The inheritancepattern for TI between C3H/HeJ(C3; 188 ± 3 ms) and C57BL/6J (B6; 111 ± 2 ms) progenitors wasconsistent with a two-gene model. By using the strain distributionpattern for recombinant inbred strains derived from C3 and B6progenitors, 100% concordance was established betweenTI phenotypes and DNA markers onmouse chromosome 3. This genotype-phenotype hypothesis was tested bytyping 52 B6C3F₂(F₂) progeny by using simplesequence repeat DNA markers (n = 21)polymorphic between C3 and B6 strains on mouse chromosome 3. Linkageanalysis compared marker genotypes to baseline ventilatory phenotypesby computing log-likelihood values. A putative quantitative trait locuslocated in proximity to D3Mit119 wassignificantly associated with baselineTI phenotypes. At the peak(log-likelihood = 3.3), the putative quantitative trait locusdetermined 25% of the phenotypic variance inTI among F₂ progeny. In conclusion, thisgenetic model of ventilatory characteristics demonstrated an importantlinkage between differential baseline TI and a candidate genomicregion on mouse chromosome 3.

     

Sequence-dependent DNA replication in preimplantation mouse embryos.

Circular, double-stranded DNA molecules were injected into nuclei of mouse oocytes and one- or two-cell embryos to determine whether specific sequences were required to replicate DNA during mouse development. Although all of the injected DNAs were stable, replication of plasmid pML-1 DNA was not detected unless it contained either polyomavirus (PyV) or simian virus 40 (SV40) DNA sequences. Replication occurred in embryos, but not in oocytes. PyV DNA, either alone or recombined with pML-1, underwent multiple rounds of replication to produce superhelical and relaxed circular monomers after injection into one- or two-cell embryos. SV40 DNA also replicated, but only 3% as well as PyV DNA. Coinjection of PyV DNA with either pML-1 or SV40 had no effect on the replicating properties of the three DNAs. These results are consistent with a requirement for specific cis-acting sequences to replicate DNA in mammalian embryos, in contrast to sequence-independent replication of DNA injected into Xenopus eggs. Furthermore, PyV DNA replication in mouse embryos required PyV large T-antigen and either the alpha-beta-core or beta-core configuration of the PyV origin of replication. Although the alpha-core configuration replicated in differentiated mouse cells, it failed to replicate in mouse embryos, demonstrating cell-specific activation of an origin of replication. Replication or expression of PyV DNA interfered with normal embryonic development. These results reveal that mouse embryos are permissive for PyV DNA replication, in contrast to the absence of PyV DNA replication and gene expression in mouse embryonal carcinoma cells.