Identification of a wheat germ agglutinin-sensitive ATPase in yeast nuclei

We have found that wheat germ agglutinin (WGA), a lectin that specifically binds to N-acetylglucosamine residues inhibits the in vitro transport of plasmid DNA, pJDB219, into yeast nuclei. Histochemical staining of the isolated nuclei with biotinylated WGA and streptavidin-biotinylated peroxidase complex revealed the presence of WGA-binding materials around the nuclear pore under an electron microscope. Using WGA-agarose column chromatography of yeast nuclear extracts, a novel Mg²⁺-dependent ATPase was isolated. Its activity was highly sensitive to WGA and stimulated by Nonidet P-40 or phosphatidylserine. We suggest that the WGA-sensitive ATPase plays a role in yeast nuclear transport of DNA.     

Curing of the 2 mu DNA plasmid from Saccharomyces cerevisiae.

The 2 mu DNA plasmid is often eliminated from yeast cells when they are transformed with the 2 mu DNA-LEU2-pMB9 composite plasmid pJDB219. Since pJDB219 is subsequently lost with high frequency, derivatives lacking all 2 mu DNA can be prepared from any strain.     

Isolation of recipient yeast strains for the stable reproduction of 2-micron DNA vectors

Most Leu- clones of yeast transformants (cir0, pJDB219) can stabilize the replication of 2 micron-vectors with REP3, the stability obtained being comparable to the one for the standard cir0 strain. One of the Leu- clones was used to isolate a plasmid with Rep 1.2 functions ("Rep-helper plasmid"). The plasmid was shown to carry a partially active LEU2 gene by transforming both E. coli and S. cerevisiae to Leu+ phenotype. A restriction analysis performed demonstrated that the Rep-helper plasmid has lost approximately 1.9 kb compared to the parent pJDB219, deletion and rearrangement having taken place at the bacterial and 2 mem components boundary. The Rep-helper plasmid carrying host strains allows to quantify the REP3 function on different 2 microns vectors. Some but not all cir+ stabilized vectors show greater stability in Rep-helper strains compared to the standard cir0 ones. Manipulating the Rep-helper plasmid level, by selecting for Leu+ phenotype, stabilized REP3 +/- plasmid p3030, but mostly destabilizes REP3+ plasmid YEp13HIS3.     

Flavonoids suppresses the enhancing effect of beta-carotene on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A549 cells

This study investigated the individual and combined effects of beta-carotene with a common flavonoid (naringin, quercetin or rutin) on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-related carcinogen in human. A human lung cancer cell line, A549, was pre-incubated with beta-carotene, a flavonoid, or both for 1h followed by incubation with NNK for 4 h. Then, we determined DNA strand breaks and the level of 7-methylguanine (7-mGua), a product of NNK metabolism by cytochrome P450 (CYP). We showed that beta-carotene at 20 microM significantly enhanced NNK-induced DNA strand breaks and 7-mGua levels by 90% (p < 0.05) and 70% (p < 0.05), respectively, and that the effect of beta-carotene was associated with an increased metabolism of NNK by CYP because the concomitant addition of 1-aminobenzotriazole, a CYP inhibitor, with beta-carotene to cells strongly inhibited NNK-induced DNA strand breaks. In contrast to beta-carotene, incubation of cells with naringin, quercetin or rutin added at 23 microM led to significant inhibition of NNK-induced DNA strand breaks, and the effect was in the order of quercetin > naringin > rutin. However, these flavonoids did not significantly affect the level of 7-mGua induced by NNK. Co-incubation of beta-carotene with any of these flavonoids significantly inhibited the enhancing effect of beta-carotene on NNK-induced DNA strand breaks; the effects of flavonoids were dose-dependent and were also in the order of quercetin > naringin > rutin. Co-incubation of beta-carotene with any of these flavonoids also significantly inhibited the loss of beta-carotene incorporated into the cells, and the effects of the flavonoids were also in the order of quercetin > naringin > rutin. The protective effects of these flavonoids may be attributed to their antioxidant activities because they significantly decreased intracellular ROS, and the effects were also in the order of quercetin > naringin > rutin. These in vitro results suggest that a combination of beta-carotene with naringin, rutin, or quercetin may increase the safety of beta-carotene.     

2-micrometers DNA-like plasmids in the osmophilic haploid yeast Saccharomyces rouxii.

DNA plasmids were detected in two independent strains of Saccharomyces rouxii among 100 yeast strains other than Saccharomyces cerevisiae tested. The plasmids, pSR1 and pSR2, had almost the same mass (approximately 4 X 10(6) daltons) as 2-micrometers DNA of S. cerevisiae. pSR1 and pSR2 gave identical restriction maps with restriction endonucleases BamHI, EcoRI, HincII, HindIII, and XhoI, and both lacked restriction sites for PstI, SalI, and SmaI. These maps, however, differed significantly from that of S. cerevisiae 2-micrometers DNA. Restriction analysis also revealed two isomeric forms of each plasmid and suggested the presence of a pair of inverted repeat sequences in the molecules where intramolecular recombination took place. DNA-DNA hybridization between the pSR1 and pSR2 DNAs indicated significant homology between their base sequences, whereas no homology was detected between pSR1 and pJDB219, a chimeric plasmid constructed from a whole molecule of 2-micrometers DNA, plasmid pMB9, and a 1.2-kilobase DNA fragment of S. cerevisiae bearing the LEU2 gene. A chimeric plasmid constructed with pSR1 and YIp1, the larger EcoRI-SalI fragment of pBR322 ligated with a 6.1-kilobase DNA fragment of S. cerevisiae bearing the HIS3 gene, could replicate autonomously in an S. cerevisiae host and produced isomers, presumably by intramolecular recombination at the inverted repeats.     

Intranuclear trafficking of plasmid DNA is mediated by nuclear polymeric proteins lamins and actin

Functions of nuclear polymeric proteins such as lamin A/C and actin in transport of plasmid DNA were studied. The results show that the lamina plays an important role in plasmid DNA's entry into the cell nucleus from the cytoplasm. Selective disruption of lamin A/C led to a halt in plasmid DNA transport through the nuclear envelope. Inside the nucleus, plasmid DNA was frequently localized at sites with impaired genome integrity, such as DNA double-strand breaks (DSBs), occurring spontaneously or induced by ionizing radiation. Polymeric actin obviously participates in nuclear transport of plasmid DNA, since inhibition of actin polymerization by latrunculin B disturbed plasmid transport inside the cell nucleus. In addition, precluding of actin polymerization inhibited plasmid co-localization with newly induced DSBs. These findings indicate the crucial role of polymeric actin in intranuclear plasmid transport.     

Effects of oxidative stress on the nuclear translocation of extracellular superoxide dismutase

The effect of oxidative stress on the cellular uptake and nuclear translocation of extracellular superoxide dismutase (EC-SOD) was investigated. EC-SOD was incorporated from conditioned medium of stable EC-SOD expressing CHO-EK cells into 3T3-L1 cells within 15 min. The uptake was clearly inhibited by the addition of heparin at a concentration of 0.4 microg/ml. Treatment of the 3T3-L1 cells with H(2)O(2) (5 mM for 5 min), followed by incubation with CHO-EK medium downregulated the uptake of EC-SOD. Nuclear translocation of the incorporated EC-SOD was clearly enhanced by H(2)O(2) treatment following incubation with the CHO-EK medium. EC-SOD is the only anti-oxidant enzyme which is known at this time to be actively transported into nuclei. The results obtained here suggest that the upregulation of the nuclear translocation of EC-SOD by oxidative stress might play a role in the mechanism by which the nucleus is protected against oxidative damage of genomic DNA.     

Vectors with restriction-site banks. III. Escherichia coli-Saccharomyces cerevisiae shuttle vectors

The bank of unique restriction sites present in plasmid pJRD158 has been incorporated into new vectors carrying selective markers and replicons derived from commonly used Escherichia coli-Saccharomyces cerevisiae shuttle vectors pJDB207 and YRp7. The new vectors pMH158 and pJO158 have 21 and 23 unique restriction sites, respectively, and their complete DNA sequences are known.     

Liposome-mediated delivery of DNA to carrot protoplasts

The encapsulation of DNA within liposomes and subsequent fusion of the liposomes with carrot (Daucus carota L.) protoplasts were examined to determine optimum conditions for effective liposome-mediated delivery of DNA to protoplasts. Escherichia coli [³H]DNA could be encapsulated with 50% efficiency using encapsulation volumes as low as 0.5 ml. Incorporation of liposome-encapsulated [³H]DNA by carrot protoplasts increased linearly for 2.5 h, and increasing the ratio of protoplasts to liposomes increased the total amount of radioactive label incorporated within the protoplasts. Liposome-mediated incorporation of [³H]DNA by protoplasts increased over a range of polyethylene glycol concentrations up to 20%, but Ca²⁺ did not increase liposome-mediated incorporation when present in the liposome-protoplast incubation mixture. Optimum incorporation was observed when the pH of the liposome-protoplast incubation medium was decreased to 4.8. Encapsulation experiments using DNA of the plasmid pBR322 indicated that an average of 200–1,000 intact copies of pBR322 were sequestered within each nucleus after liposome delivery.     

The metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were 3H-labeled in vitro and injected back into X. laevis oocytes, most 3H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, 3H-TP60 are rapidly degraded with a half-life of 2-3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of 3H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide, a cysteine protease inhibitor, resulting in the accumulation of 3H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.     

Enhanced resistance to nuclease degradation of nucleic acids complexed to asialoglycoprotein-polylysine carriers.

We have previously shown targeting of DNA to hepatocytes using an asialoorosomucoid-polylysine (AsOR-PL) carrier system. The AsOR-PL conjugate condenses DNA and facilitates entry via specific receptor-ligand interactions. In these studies, our objective was to determine if AsOR-PL conjugates protect bound DNA from nuclease attack. Double-stranded plasmid or single-stranded oligonucleotide DNA, alone or bound to conjugate, was incubated under conditions mimicking those encountered during in vitro and in vivo transfections. The results showed that complexed DNA was effectively protected from degradation by serum nucleases. Degradation of single-stranded oligonucleotides was inhibited 3- to 6-fold in serum during 5 hours of incubation. For complexed plasmids, greater than 90% remained full-length during 1.5 and 3 hour incubations in serum or culture medium containing 10% serum, respectively. Uncomplexed plasmid was completely degraded after 15 minutes in serum or 60 minutes in medium. In cell lysates, the conjugate was not effective in inhibiting endonuclease activity; plasmids were readily converted from supercoiled to open circular and linear forms. However, the resultant nicked forms were substantially protected from further degradation during one hour of incubation compared to plasmid alone. Under all conditions complexed DNA did not readily dissociate from the conjugate. Overall, for both single and double-stranded DNA, AsOR-PL conjugates conferred substantial protection from nuclease degradation.     

Vaccinia virus infection of HeLa cells. I. Synthesis of vaccinia DNA in host cell nuclei.

The replication of vaccinia virus is thought to take place exclusively in the cytoplasm of host cells. However, using DNA-DNA hybridization techniques, it can be shown that a significant fraction of the synthesis of vaccinia DNA takes place in the nucleus as well as the cytoplasm. The (3H) thymiding pulse-labeled vaccinia DNA synthesized in the nucleus reaches a maximum at about 3 h after infection, corresponding to the time of maximal DNA synthesis in infected cells. At this time host DNA synthesis drops to about 25% of the rate of the uninfected cells. Even with short labeling times (2 min) the nucleus is found to contain 60% of the incorporated (3H)thymidine, much of which is in vaccinia DNA. Prior inhibition of host nuclear DNA synthesis with mitomycin C, followed by removal of the antibiotic causes a subsequent inhibition of vaccinia DNA synthesis and complete suppression of mature virus. Purified nuclei, isolated from vaccinia-infected cells, also synthesize vaccinia DNA in vitro. Over 90% of the DNA synthesized in vitro by isolated nuclei contain vaccinia-specific sequences.     

Incorporation of exogenous circular DNA into large catenated networks in isolated nuclei. Evidence for involvement of the nuclear scaffold

Circular plasmid DNA was efficiently converted into huge catenated intranuclear networks by incubation with isolated nuclei in the presence of ATP. The network production is abolished by omission of ATP, strongly inhibited by etoposide (VP-16), but only slightly inhibited by antibody to topoisomerase I, indicating that the major enzyme responsible for catenation is DNA topoisomerase II. Under optimal conditions, a single nucleus incorporates about 4.2 x 10(4) DNA rings into its networks. Under the light microscope, networks retrieved from nuclei appear like spheres of various sizes. Sedimentation analysis showed that most of the networks are composed of thousands of catenated rings, which was confirmed by electron microscopy. Data from experiments that caused partial disruption of the networks were submitted to analysis based on probable models of catenane structure. The results suggest that the predominant pattern is a linear alignment of catenated rings. Similar networks are formed when the nuclear scaffold is incubated with circular DNA in the presence of nuclear extract containing topoisomerase II. Titration experiments showed that the scaffold binds a stoichiometric amount of the substrate and that a critical level of DNA is required for network formation. The results are consistent with the idea that DNA-binding sites are fixed on the scaffold and mediate catenation of bound DNA circles by holding them in close proximity to each other. We propose that catenation by the nuclear scaffold also occurs in intact nuclei, suggesting additional roles for the scaffold in vivo.     

Inhibitory effects of dicyclohexylcarbodiimide on spinach cytochrome b6-f complex

The electron transfer activity of purified cytochrome b6-f complex of spinach chloroplast is inhibited by dicyclohexylcarbodiimide (DCCD) in a concentration and incubation time dependent manner. The maximum inhibition of 75% is observed when 300 mole of DCCD per mole of protein (based on cytochrome f) is incubated with cytochrome b6-f complex at room temperature for 40 min. The inhibition of the complex is not due to the formation of cross links between subunits but due to the modification of carboxyls. The amount of DCCD incorporation is directly proportional to the activity loss, suggesting that some carboxyl groups in the complex are directly or indirectly involved in the catalytic function. The incorporated DCCD is located mainly at cytochrome b6 protein. The partially inhibited complex shows the same H+/e-ratio as that of the intact complex when embedded in phospholipid vesicles.     

A stable plasmid carrying the yeast Leu2 gene and containing only yeast deoxyribonucleic acid

The plasmid pSLe1 is a deletion derivative of the yeast-Escherichia coli hybrid plasmid pJDB219, obtained by HindIII digestion, ligation, and transformation directly into Saccharomyces cerevisiae. pSLe1 has only yeast sequences; it contains one of the inverted repeated sequences of plasmid 2muDNA and the LEU2 gene. pSLe1 is stably maintained in yeast cells without selective pressure. pSLe1 is about half as large as 2muDNA, but pSLe1 does not displace the normal 2muDNA.     

Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro.

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.     

Distinct mechanisms of DNA damage in apoptosis induced by quercetin and luteolin

Quercetin has been reported to have carcinogenic effects. However, both quercetin and luteolin have anti-cancer activity. To clarify the mechanism underlying the carcinogenic effects of quercetin, we compared DNA damage occurring during apoptosis induced by quercetin with that occuring during apoptosis induced by luteolin. Both quercetin and luteolin similarly induced DNA cleavage with subsequent DNA ladder formation, characteristics of apoptosis, in HL-60 cells. In HP 100 cells, an H₂O₂-resistant clone of HL-60 cells, the extent of DNA cleavage and DNA ladder formation induced by quercetin was less than that in HL-60 cells, whereas differences between the two cell types were minimal after treatment with luteolin. In addition, quercetin increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in HL-60 cells but not in HP 100 cells. Luteolin did not increase 8-oxodG formation, but inhibited topoisomerase II (topo II) activity of nuclear extract more strongly than quercetin and cleaved DNA by forming a luteolin-topo II-DNA ternary complex. These results suggest that quercetin induces H₂O₂-mediated DNA damage, resulting in apoptosis or mutations, whereas luteolin induces apoptosis via topo II-mediated DNA cleavage. The H₂O₂-mediated DNA damage may be related to the carcinogenic effects of quercetin.     

Molecular and genetic studies of an R factor system consisting of independent transfer and drug resistance plasmids

Certain genetic, structural, and biochemical properties of a class 2 R-factor system consisting of the conjugally proficient transfer plasmid I and the naturally occurring non-conjugative tetracycline (Tc) resistance plasmid 219 are reported. I and 219 exist as separate plasmid deoxyribonucleic acid (DNA) species in both Escherichia coli and Salmonella panama, having molecular weights of 42 x 10(6) and 5.8 x 10(6), respectively. The buoyant densities of I and 219 are 1.702 and 1.710 g/cm(3), respectively, in neutral cesium chloride. Although the Tc resistance plasmid is not transmissible in a normal conjugal mating, it is mobilized in a three-component mating by plasmid I and by certain other conjugative plasmids of the fi(+) or fi(-) phenotype. Mobilization does not appear to involve intermolecular recombination between plasmids, and no covalent linkage of resistance markers and fertility functions is observed. Transformation of CaCl(2)-treated E. coli by plasmid DNA is shown to be a useful procedure for studying the biological properties of different plasmid molecular species that have been fractionated in vitro, and for selectively inserting non-self-transmissible plasmids into specific bacterial strains. The effects of tetracycline on the rate of protein synthesis carried out by plasmid 219 were studied by using isolated E. coli minicells into which this plasmid had segregated. Consistent with the results of earlier investigations showing the inducibility of plasmid-mediated Tc resistance in E. coli, the antibiotic was observed to stimulate protein synthesis in minicells carrying the plasmid 219 and totally inhibit (3)H-leucine incorporation by minicells lacking the Tc resistance marker. Five discrete polypeptide species were synthesized by minicells carrying plasmid 219; exposure of minicells or parent bacteria to Tc resulted in specific and reproducible changes in polypeptide synthesis patterns.     

Triiodothyronine nuclear receptor and the role of non-histone protein factors in in vitro triiodothyronine binding

The rat liver triiodothyronine (T3) nuclear receptor rapidly looses, after a partial purification from the nuclear extract, its ability to bind T3. We previously reported that histones, in the presence of DNA, could protect against inactivation enhancing the T3 binding site concentration and maintaining the high affinity for T3. A nuclear fraction discarded during the receptor purification (fraction A) was also found able to restore T3 binding and was analyzed. As histones + DNA, fraction A stabilized the T3 binding site from irreversible inactivation during incubation with T3, increasing its concentration while keeping the same high affinity for T3. It was active even at relatively high receptor concentration, appeared slightly more active than histones (+ DNA) in the same protein concentration range (up to 50-fold increment of T3 binding at the optimal concentration of 25 micrograms/ml) and was unaffected or slightly inhibited by DNA. Other proteins (ovalbumin, soybean trypsin inhibitor, RNAase) and rat liver cytosol were several times less effective, suggesting a major role of some nuclear constituents. The active factors in fraction A essentially belong to non-histone nuclear proteins. Fraction A was found heterogeneous regarding the molecular size and pHi of the active factors, the existence of subfractions more active on a protein concentration basis being suggested but not yet clearly evidenced. Efficient in vitro T3 binding to the isolated T3 nuclear receptor thus depends on the presence of several different nuclear constituents, histones + DNA or some non-histone proteins. Whether interactions with these constituents could modulate T3 binding within the nucleus remains to be elucidated.