TUNEL analyses of bovine blastocysts after culture with EGF and IGF-I

Experiments were carried out to investigate the beneficial effects of IGF-I or EGF on bovine embryo development in chemically defined embryo culture media and resultant incidences of nuclear DNA fragmentation as an indication of embryo quality. Presumptive IVF zygotes were randomly cultured in either control (with no added growth factor) or treatment groups, i.e., with 50 ng/ml IGF-I (experiment 1) or 5 ng/ml EGF (experiment 2). IGF-I supplemented to culture media significantly improved proportions of blastocysts from oocytes inseminated compared to untreated controls (38.0% vs. 28.5%). Only embryos reaching the blastocyst stage on day 8 showed significant effects of IGF-I treatment by resulting in higher blastocyst cell numbers (162 vs. 141) and lower percentages of TUNEL positive nuclei (2.1% vs. 3.3%) when compared to controls. Blastocyst development from oocytes was also improved by EGF supplementation compared to untreated controls (38.5% vs. 30.7%). Cell numbers of either day 7 or day 8 blastocysts were not affected by EGF treatment, nor were percentages of TUNEL positive nuclei when compared with controls. Similar proportions of parthenogenetically activated oocytes developed to blastocysts as for inseminated oocytes (28.8%). Parthenogenetic blastocysts contained fewer cells (93) and an increased percentage of TUNEL positive nuclei (5.7%) than were found for IVF embryos.     

Enhanced Deoxyribonucleic Acid Polymerase Activity in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12

Deoxyribonucleic acid (DNA) polymerase activity was induced at approximately 18 to 20 hr after infection of secondary cultures of human embryonic kidney cells with adenovirus type 2 or type 12, and, at 30 to 50 hr after infection, the activity of this enzyme increased two- to threefold. The activity of thymidine kinase was also induced, but the activity of deoxycytidylic deaminase was not. The DNA content per cell at 71 hr after infection was 1.6-fold greater in adenovirus 2-infected cultures, and approximately 2.4-fold greater in adenovirus 12-infected cultures, than in the noninfected cultures. Several properties of DNA polymerase were studied. The enzymes in normal and adenovirus 2- or 12-infected cell extracts were saturated by approximately the same concentration of heat-denatured salmon sperm DNA primer (160 mug/ml); the enzyme activities had a similar broad pH optimum between 7.5 and 9. Extracts prepared from cells infected by either adenovirus did not activate DNA polymerase from noninfected cells, nor did the noninfected cell extracts inhibit enzyme activity of infected cell extracts. DNA polymerase in both normal and adenovirus 2- or 12-infected cells was located predominantly in the nucleus. In each case, the cytoplasm had only 30% of the enzyme activity of the nucleus. At 40 hr after infection with adenovirus 2 or 12, the activities of the enzyme in the nuclear and cytoplasmic fractions increased two- to threefold. Puromycin, an inhibitor of protein synthesis, prevented DNA polymerase induction when added to cultures during the 18- to 30-hr postinfection period, and it arrested the additional increase in enzyme activity when added after enzyme induction began. However, the increases in both DNA polymerase and thymidine kinase activities took place after treatment of infected cultures with 1-beta-d-arabinofuranosylcytosine, an inhibitor of DNA synthesis and adenovirus growth.     

Comparison among DNA polymerases 1, 2 and 3 from maize embryo axes. A DNA primase activity copurifies with DNA polymerase 2

Three DNA polymerase activities, named 1, 2 and 3 were purified from maize embryo axes and were compared in terms of ion requirements, optimal pH, temperature and KCl for activity, response to specific inhibitors and use of templates. All three enzymes require a divalent cation for activity, but main differences were observed in sensitivity to inhibitors and template usage: while DNA polymerases 1 and 2 were inhibited by N-ethyl maleimide and aphidicolin, inhibitors of replicative-type enzymes, DNA polymerase 3 was only marginally or not affected at all. In contrast, DNA polymerase 3 was highly inhibited by very low concentrations of ddTTP, an inhibitor of repair-type enzymes, and a 100-fold higher concentration of the drug was needed to inhibit DNA polymerases 1 and 2. Additionally, DNA polymerases 1 and 2 used equally or more efficiently the synthetic template polydA-oligodT, as compared to activated DNA, while polymerase 3 used it very poorly. Whereas DNA polymerases 1 and 2 shared properties of replicative-type enzymes, DNA polymerase 3 could be a repair-type enzyme. Moreover, a DNA primase activity copurified with the 8000-fold purified DNA polymerase 2, strenghtening the suggestion that polymerase 2 is a replicative enzyme, of the -type. This DNA primase activity was also partially characterized. The results are discussed in terms of relevant data about other plant DNA polymerases and primases reported in the literature.     

Enhancement of Human DNA Polymerase η Activity and Fidelity Is Dependent Upon a Bipartite Interaction with the Werner Syndrome Protein

We have investigated the interaction between human DNA polymerase η (hpol η) and the Werner syndrome protein (WRN). Functional assays revealed that the WRN exonuclease and RecQ C-terminal (RQC) domains are necessary for full stimulation of hpol η-catalyzed formation of correct base pairs. We find that WRN does not stimulate hpol η-catalyzed formation of mispairs. Moreover, the exonuclease activity of WRN prevents stable mispair formation by hpol η. These results are consistent with a proofreading activity for WRN during single-nucleotide additions. ATP hydrolysis by WRN appears to attenuate stimulation of hpol η. Pre-steady-state kinetic results show that k_pol is increased 4-fold by WRN. Finally, pulldown assays reveal a bipartite physical interaction between hpol η and WRN that is mediated by the exonuclease and RQC domains. Taken together, these results are consistent with alteration of the rate-limiting step in polymerase catalysis by direct protein-protein interactions between WRN and hpol η. In summary, WRN improves the efficiency and fidelity of hpol η to promote more effective replication of DNA.     

A257T linker region mutant of T7 helicase-primase protein is defective in DNA loading and rescued by T7 DNA polymerase

The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ～75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.     

Embryo quality,and not chromosome nondiploidy,affects mitochondrial DNA content in mouse blastocysts

It has been shown recently that there is premature mitochondria biosynthesis in blastocysts from older women whose egg or embryo quality is poor and that aneuploid blastocysts also have a high number of mitochondrial DNA (mtDNA) copies. Whether nondiploidy/aneuploidy or reduced egg or embryo quality causes premature mitochondrial biosynthesis is not known. This study constructed haploid, diploid, triploid, and tetraploid blastocysts by parthenogenetic activation, intracytoplasmic sperm injection with one or two sperm heads, blastomere electrofusion, respectively, and generated reduced cytoplasm quality embryos from diabetic mouse and in vitro fertilization of aged oocytes, and examined whether nondiploidy or reduced cytoplasm quality causes premature mitochondrial biosynthesis. MtDNA numbers of each blastocyst from different models were tested by absolute quantitative real-time polymerase chain reaction. It was found that mtDNA content in preimplantation embryos was not associated with their chromosome ploidy, while mtDNA copy numbers in embryos with suboptimal quality were increased. Therefore, it might be the reduced cytoplasmic quality, and not chromosome nondiploidy, that causes premature mitochondria biosynthesis in blastocysts.     

Dna Synthetic Activity in Tumor-Bearing Mice

The rate of DNA synthesis in normal tissues exhibits circadian rhythmicity. However, there have been conflicting reports of the effects of tumor burden on the circadian rhythm of DNA synthesis in non-cancer tissues. We have developed a mouse colon cancer (MC-26) that exhibits different growth under different photoperiods. The purpose of this study was to analyze DNA synthetic activity in tissues removed from tumor-bearing and tumor-free mice maintained under two different photoperiods. Two groups each of approximately 80 male Balb/c mice were acclimated to one of two light-dark cycles, 12L:12D or 6L:18D. Half of each group were injected with 5.0 × 104 MC-26 cells. Twenty-two days later, all mice were killed in subgroups at 4-6 hr intervals over one 24-hr period. Colons and tumors were removed for measurement of DNA synthesis. Results were analyzed by means of one-way analysis of variance (ANO VA) in order to determine whether DNA synthesis varied significantly within groups over the 24-hr period. The DNA synthetic activity, as measured by uptake of tritiated thymidine, exhibited significant temporal variation in the colons of control (tumor-free) mice under both the 12L:12D and 6L:18D photoperiods. The colons of tumor-bearing mice failed to exhibit a fluctuation under a 12L:12D photoperiod but did show a significant 24-hr rhythm under the 6L:18D photoperiod. The subcutaneously growing cancers did not exhibit a circadian variation in DNA synthetic activity under either photoperiod. Both photoperiod and the presence of cancer appear to affect the DNA synthetic activity observed in mice bearing the MC-26 colon cancer.     

Sphingosine,a Modulator of Human Translesion DNA Polymerase Activity

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼3000 small molecules, including one comprising ∼600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.     

Chimaeric and constitutive DNA fingerprints in the common marmoset (Callithrix jacchus)

Marmosets normally produce dizygotic twins sharing placental blood vessels and exchanging bone marrow cells. Each individual is therefore likely to be a blood chimaera. To date, marmosets had only been DNA fingerprinted using blood samples and probes 33.6 and 33.15, resulting in highly similar fingerprints among litter mates and little variation between other individuals, thereby limiting this method's use for individual identification and parentage testing. In this study, novel probes were applied to detect greater polymorphism and to produce individual-specific DNA fingerprints. As expected, blood DNA profiles of twins and triplets were virtually identical, confirming chimaerism in this tissue and identifying litter mates. Furthermore, these profiles were sufficiently variable to distinguish between sibs from different litters and between all other individuals. To produce individual-specific DNA fingerprints, the use of DNA extracted from tissues poor in leukocytes was essential. The findings demonstrate that, despite extensive blood chimaerism, marmoset colonies can be effectively DNA fingerprinted for indicidual identification, zygosity testing, and relationship studies.     

Influence of DNA aptamer structure on the specificity of binding to Taq DNA polymerase

The secondary structure of DNA aptamer to Taq DNA polymerase was established as a hairpin. Both stem and loop structures of DNA ligand were shown to be involved in the interaction with Taq DNA polymerase. Moreover, the structure and sequence of DNA aptamer that was the most effective inhibitor of DNA polymerase activity were established. This crucial structure was evaluated as a GC-rich stem longer than 17 bp, and a loop consisting of 12 bases with strictly determined nucleotide sequence. It was demonstrated that nucleotide in position 23 counting from the 5"-end of DNA ligand was involved in direct contact with Taq DNA polymerase. The ability of optimized DNA aptamer TQ21-11 to form a complex with the enzyme was increased 5-fold in comparison to the initial aptamer.     

Analysis of DNA fragmentation in mouse embryos exposed to an extremely low-frequency electromagnetic field

Effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on DNA damage in biological systems are still a matter of dispute. The aim of the present study was to investigate the possible effect of electromagnetic field exposure on DNA fragmentation in cells (blastomers) of mouse blastocysts.

Eighty female NMRI mice were randomly divided into 2 groups of 40 animals each. The control group was left unexposed whereas the animals in the EMF-group were exposed to a 50-Hz EMF at 0.5 mT 4 h per day, 6 days a week for a duration of 2 weeks. After the 8^th day of exposure, the female mice in both groups were superovulated (with injections of pregnant mare serum gonadotropin and human chorionic gonadotropin) and then mated overnight. At approximately 4 days after mating (102 h after the human chorionic gonadotropin treatment), blastocysts were obtained by flushing the uterus horns. The mean numbers of pregnant mice, blastocysts after flushing, blastomers within the blastocysts, and the DNA fragmentation index following staining in both groups were compared using statistical methods (SPSS, the Chi-square test, the Student's t-test and the Mann-Whitney U-test, P < 0.05). The results showed that the mean number of blastocysts after flushing was significantly decreased in the EMF-group compared to that of the control group (P < 0.03). The DNA fragmentation index was significantly increased in the EMF-group compared to control (10.53% vs. 7.14%; P < 0.001). However, there was no significant difference in the mean numbers of blastomers and numbers of pregnant mice between the EMF-exposed and control group. Our findings indicate that the EMF exposure in preimplantation stage could have detrimental effects on female mouse fertility and embryo development by decreasing the number of blastocysts and increasing the blastocysts DNA fragmentation.     

Bypass of a nick by the replisome of bacteriophage T7

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.     

Expression of c-fos and c-myc genes in uterus and nonreproductive organs during the implantation period in the rat.

The c-fos and c-myc mRNA levels were investigated in the uterus and nonreproductive organs of rats during the implantation period (from day 5 to 7 of pregnancy). They were determined by densitometric analysis of slot blots and the mean values (n = 4) at a defined age of pregnancy were compared with those observed in nonpregnant control rats (NP group). No significant variations of c-fos level were observed in the liver and brain of pregnant rats. In the uterus, the c-fos level decreased, reached a minimum on day 6 (4-fold decrease compared with the NP group), and then increased. The c-fos level significantly increased in the kidney (2- and 2.5-fold on days 6 and 7), and in the lung (2.5-fold on day 7). The c-myc level did not significantly vary in the uterus, liver, and lung. It significantly increased in the kidney (3-fold on day 7) and in the brain (4.1-, 3.8-, and 6-fold, on days 5, 6, and 7). The changes in c-fos and (or) c-myc gene expression in the uterus and nonreproductive organs could be due to sexual steroids and (or) systemic factors from uterine cells or blastocysts.     

Amplification of bacterial DNA bound on clay minerals by the random amplified polymorphic DNA (RAPD) technique

Abstract: Chromosomal DNA from Bacillus subtilis , bound on the clay minerals, montmorillonite (Wyoming (W) and Apache County (Ap)) and kaolinite (K), was subjected to the random amplified polymorphic DNA (RAPD) technique. DNA bound on the clays was not amplified with 0.625, 1.875, 6.25, and 12.5 U of Taq DNA polymerase, but amplification occurred when the clay-DNA complexes were diluted 10- and 20-fold or when 21 U of Taq DNA polymerase was added. DNA desorbed from the Ap-DNA and K-DNA equilibrium complexes was amplified with 0.625 U of Taq DNA polymerase, whereas amplification of DNA desorbed from the W-DNA complex occurred only after a 10-fold dilution or when 1.875 U of Taq DNA polymerase was used. These observations indicate that clay minerals differentially affect the amplification process, probably by inhibiting the activity of Taq DNA polymerase.     

DNA primase activity from wheat embryos

DNA primase is a recently discovered enzyme capable of synthesizing short primers involved in the initiation of DNA replication.Partially purified preparations from 4 h germinated wheat embryos or commercial wheat germ are able to catalyze the ribonucleoside triphosphate dependent synthesis of DNA with poly dT and M₁₃ single stranded DNA as templates. DNA synthesis is completely dependent on the presence of template and primase. Primase activity from wheat embryos has a molecular weight of about 110000 and a sedimentation coefficient of 5S. The enzyme activity is not inhibited by -amanitin (1 mg/ml) or aphidicolin when the latter is assayed with endogeneous plant DNA polymerase activity. Alkaline hydrolysis of the product synthesized in the presence of [-³²P]dATP and poly dT generates [³²P]-labeled 3(2)AMP showing that a ribo-deoxynucleotide linkage is formed. The size of the oligoribonucleotide primer varies from 2 to 15 residues. Most of the wheat DNA polymerase activity can be eliminated by phosphocellulose chromatography, since the bulk of plant DNA primase is not retained by this resin. Nevertheless, a small but significant amoung of DNA polymerase activity is found associated with DNA primase. By using different inhibitors of DNA polymerase different templates, we have found good indications that DNA polymerase A (-like) is associated with the DNA primase. Moreover, when the previously purified DNA polymerases from wheat embryos (2) were assayed in the presence of primase activity, only DNA polymerase A was able to stimulate DNA synthesis.     

An automated procedure to measure DNA synthesis in preimplantation mouse embryos

This paper describes a sensitive, reproducible, and automated procedure to measure DNA synthesis in preimplantation mouse embryos. Conditions for the DNA synthesis assay have been optimized as follows: (1) 4 μCi/ml³H-thymidine (sp. act. 20 Ci/immole); (2) a labeling period from 2 to 7 hours; (3) a 3-hour preincubation period for blastocysts and from 0 to 7 hours preincubation for 8-cell embryos; and (4) from 1 to 64 embryos per assay. The amount of DNA synthesis per embryo was found to be directly proportional to the number of cells (nuclei) per embryo. The described assay should be useful for future studies on the effect of synthetic and natural compounds on the development of preimplantation mouse embryos, as measured by perturbations in embryonic DNA synthetic activity.     

A stimulatory effect of the fluid from preimplantation rabbit blastocysts upon luteinization of monkey granulosa cell cultures.

Blastocyst fluid was aspirated from Day 6 1/2--7 rabbit blastocysts and was added to cultures of granulosa cells obtained from preovulatory follicles of untreated rhesus monkeys or from follicles of monkeys or from follicles of monkeys treated with PMSG. The stimulation of progesterone secretion was measured and equated with that produced by hCG. The hCG-like activity was also measured in a radioreceptor assay using 125I-labelled hCG and porcine granulosa cells. In 8 out of 10 experiments with cultured cells from untreated monkeys, addition of 20% blastocyst fluid from Days 6--9 of culture stimulated progesterone secretion by 2- to 6-fold. Similar findings were obtained in 5 experiments with cultures from PMSG-treated monkeys except that the blastocyst fluid was added from Days 0 to 6 of culture. The granulosa cells in such cultures underwent morphological luteinization. Compared to a standard of purified hCG the blastocyst fluid contained about 0.76--2.5 ng hCG-like activity/ml which was non-dialysable. The radioreceptor assay indicated the presence of 0.5--2.5 ng hCG-like material/ml.     

A DNA polymerase from maize axes: its purification and possible role

Three different DNA polymerase activities can be resolved by passing a protein extract from 24 h imbibed maize axes through DEAE-cellulose. These activities have been numbered 1, 2 and 3, according to their elution order. One of them, DNA polymerase 2, elutes at 100–120 mM phosphates. This enzyme was further purified by passing it through Heparin-Sepharose, Sephacryl S-300 and DNA cellulose. Purification was nearly 5000-fold. The enzyme needs Mg²⁺, is stimulated by K⁺, has an optimum pH of 7.0 and its optimum temperature is 30–37 °C. Specific inhibitors for different types of polymerases, such as aphidicolin, dideoxythymidine triphosphate and N-ethyl maleimide, gave intermediate values of inhibition, making impossible the definition of the type of enzyme purified by its inhibitory pattern. SDS-PAGE indicated the presence of several bands of molecular masses of 28–40, 56 and 15 kDa. Most of these bands could be visualized when proteins from crude extracts were analyzed by western blot, using an antibody against calf thymus DNA polymerase . A high molecular mass (around 500 kDa) was calculated by western blot of native gels using the same antibody. Finally, specific activity of this enzyme increased 100-fold during maize germination whereas polymerase 3 virtually did not increase. Furthermore, immunoprecipitation experiments with the antipolymerase -antibody showed a decrease in DNA polymerase activity by 70%. The possibility that polymerase 2 is a replicative enzyme is discussed.