A sensitive fluorometric assay for the determination of DNA

A sensitive fluorometric assay for DNA determination using m-diaminobenzoic acid dihydrochloride for the reaction with deoxyribose liberated by perchloric acid is described. Fluorescence is proportional to the amount of DNA over the range 0.05–5 μg of DNA when the reaction is conducted in a volume of 400 μl and over the range 0.01–0.5 μg when the reaction is conducted in a volume of 40 μl. The assay is suitable for estimating DNA both in perchloric acid extracts and in complex mixtures with protein and RNA. The determination of DNA in cells grown as a monolayer by the described method is simple and rapid.     

The oxidative damage of plasmid DNA by ascorbic acid derivatives in vitro: the first research on the relationship between the structure of ascorbic acid and the oxidative damage of plasmid DNA

To study the structure-function relationship of the oxidative-damage effect of ascorbic acid, we have focused on the interaction between plasmid DNA pUC19 and a series of ascorbic acid derivatives modified on different OH groups in the presence of transition metal ions. Some ascorbic acid derivatives can selectively cleave plasmid DNA from Form I to Form II in the presence of low concentration of Cu2+ just like ascorbic acid itself, while other derivatives oxidatively damage plasmid DNA slightly. We found that those derivatives with unattached 2-OH and 3-OH groups retain the ability to cleave the plasmid DNA. The derivatives that have been methylated on 2-OH or 3-OH can only cleave plasmid DNA softly, and those derivatives that have been protected on both 2-OH and 3-OH can hardly exert an oxidative damage on plasmid DNA under the same condition. Form these results, we can draw the conclusion that 2-OH and 3-OH groups of the ascorbic acid molecule contribute most to this biological activity.     

Effects of ascorbic acid on the nonenzymatic binding to DNA and the mutagenicity of N-hydroxylated metabolite of a tryptophan-pyrolysis product

Ascorbic acid enhanced the nonenzymatic binding of the mutagen 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) to DNA with a concomitant increase in the mutagenicity of N-OH-Trp-P-2. The covalent binding of N-OH-Trp-P-2 to DNA was higher at pH 7.4 than pH 6.2 or 5.0. Ascorbic acid increased the binding of N-OH-Trp-P-2 at all pH levels examined. These results indicate that ascorbic acid enhances the DNA damage caused by N-OH-Trp-P-2.     

Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex

Hepatitis C virus (HCV) helicase, non-structural protein 3 (NS3), is proposed to aid in HCV genome replication and is considered a target for inhibition of HCV. In order to investigate the substrate requirements for nucleic acid unwinding by NS3, substrates were prepared by annealing a 30mer oligonucleotide to a 15mer. The resulting 15 bp duplex contained a single-stranded DNA overhang of 15 nt referred to as the bound strand. Other substrates were prepared in which the 15mer DNA was replaced by a strand of peptide nucleic acid (PNA). The PNA–DNA substrate was unwound by NS3, but the observed rate of strand separation was at least 25-fold slower than for the equivalent DNA–DNA substrate. Binding of NS3 to the PNA–DNA substrate was similar to the DNA–DNA substrate, due to the fact that NS3 initially binds to the single-stranded overhang, which was identical in each substrate. A PNA–RNA substrate was not unwound by NS3 under similar conditions. In contrast, morpholino–DNA and phosphorothioate–DNA substrates were utilized as efficiently by NS3 as DNA–DNA substrates. These results indicate that the PNA–DNA and PNA–RNA heteroduplexes adopt structures that are unfavorable for unwinding by NS3, suggesting that the unwinding activity of NS3 is sensitive to the structure of the duplex.     

Site-specific mutagenesis of PRD1 DNA polymerase: mutations in highly conserved regions of the family B DNA polymerase

The PRD1 DNA polymerase is a small multifunctional enzyme containing three major conserved amino acid sequences shared by family B DNA polymerases. Thus, the PRD1 DNA polymerase provides an useful model system with which to study structure-function relationships of DNA polymerase molecules. In order to investigate the functional and structural roles of the highly conserved amino acid sequences, we have introduced mutations into each of the 3 conserved regions of the PRD1 DNA polymerase. Genetic complementation study as well as DNA polymerase assay indicated that each mutation inactivated DNA polymerase catalytic activity, but not the 3' to 5' exonuclease activity.     

Integration of the Deoxyribonucleic Acid of Adenovirus Type 12 into the Deoxyribonucleic Acid of Baby Hamster Kidney Cells

In a previous report, evidence was presented that the deoxyribonucleic acid (DNA) of adenovirus type 12 (Ad12) is integrated by covalent linkage into the DNA of baby hamster kidney cells (BHK-21 cells). These studies have been extended. The DNA of Ad12 and that of BHK-21 cells grown in medium containing 5-bromodeoxyuridine could be separated by equilibrium centrifugation in alkaline CsCl density gradients. BHK-21 cells were infected with (3)H-labeled Ad12, and the total intracellular DNA was analyzed at various times after infection in alkaline CsCl density gradients. The (3)H label in the position of cellular DNA hybridized predominantly with viral DNA and to a lesser extent also with cellular DNA. Replication of viral DNA could not be detected in BHK-21 cells. The appearance of viral (3)H label in the density stratum of cellular DNA was not significantly affected when DNA synthesis in Ad12-infected BHK-21 cells was inhibited >96% by cytosine arabinoside. These findings provided additional evidence for integration of Ad12 DNA into the DNA of BHK-21 cells. It could be calculated that 5 to 55 Ad12 DNA equivalents per cell are integrated. Replication of viral or cellular DNA was not required for integration. Inhibition of protein or ribonucleic acid synthesis interfered with integration only slightly.     

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.     

Isolation and characterization of the structural gene for secreted acid phosphatase from Schizosaccharomyces pombe

The Schizosaccharomyces pombe acid phosphatase structural gene (PHO 1) was isolated by complementation of an S. pombe acid phosphatase mutant with a wild type S. pombe DNA recombinant plasmid library. Northern analysis indicates that acid phosphatase is encoded by a 1.4-kilobase mRNA of which approximately 100 bases are 3'-poly(A). The gene contains no introns and the 3' and 5' untranslated regions are short. According to DNA and amino acid sequence data, the S. pombe acid phosphatase has a molecular weight of 50,600. An 18-amino acid sequence at the N terminus was found that is similar to previously identified signal peptides in other eukaryotic secretory proteins. This signal peptide is apparently removed during secretion, since it is absent in the mature secreted acid phosphatase. The gene can be induced 2--3-fold by starvation for phosphate. The signals required for this induction are contained on the isolated DNA clone. Although the gene can be expressed in Saccharomyces cerevisiae, secretion is abnormal.     

Stimulating effect of histones on DNA synthesis in the regenerating rat liver]

Effect of exogenous histones, nuclear globulins and acid proteins on DNA synthesis is studied in regenerating liver of rats in which the synthesis of their own proteins and thus DNA replication are inhibited by cycloheximide. In these conditions histones from regenerating rat liver are found to stimulate 3H-thymidine incorporation into DNA of hepatectomyzed rat liver. Nuclear globulins and acid proteins from regenerating liver, and histones from intact liver produced no stimulating effect on DNA sythesis.     

Location of the serine residue involved in the linkage between the terminal protein and the DNA of phage phi 29.

B. subtilis phage phi 29 has a terminal protein, p3, covalently linked to the 5' ends of the DNA through a phosphodiester bond between a serine residue and 5'-dAMP. This protein acts as a primer in DNA replication by forming an initiation complex with the 5'-terminal nucleotide dAMP. The amino acid sequence of the terminal protein, deduced from the nucleotide sequence of gene 3, showed the presence of 18 serine residues in a total of 266 amino acids. In this paper we have identified the serine involved in the linkage with the DNA as the residue 232, located close to the C-terminus of the molecule. This result was obtained by amino acid analysis of the peptide that remains linked to the DNA after proteinase K digestion of the terminal protein-phi 29 DNA complex and automated Edman degradation of the corresponding [125I]-labeled tryptic peptide. Prediction of the secondary structure of the terminal protein suggested that the serine residue involved in the linkage with the DNA is placed in a beta-turn, probably located on the external part of the molecule, as indicated by hydropathic values.     

Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a

The Dnmt3a gene, which encodes de novo-type DNA methyltransferase, encodes two isoforms, full-length Dnmt3a and Dnmt3a2, which lacks the N-terminal 219 amino acid residues. We found that Dnmt3a showed higher DNA-binding and DNA-methylation activities than Dnmt3a2. The N-terminal sequence from residues 1 to 211 was able to bind to DNA, but could not distinguish methylated and unmethylated CpG. Its binding to DNA was inhibited by a major groove binder. Four basic amino acid residues, Lys51, Lys53, Arg177 and Arg179, in the N-terminal region were crucial for the DNA-binding activity. The ectopically expressed N-terminal sequence (residues 1-211) was localized in nuclei, whereas that harbouring mutations at the four basic amino acid residues was also detected in the cytoplasm. The DNA-methylation activity of Dnmt3a with the mutations was suppressed under physiological salt conditions, which is similar that of Dnmt3a2. In addition, ectopically expressed Dnmt3a with mutations, as well as Dnmt3a2, could not be retained efficiently in nuclei on salt extraction. We conclude that the DNA-binding activity of the N-terminal domain contributes to the DNA-methyltransferase activity via anchoring of the whole molecule to DNA under physiological salt conditions.     

Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes.     

DNA degradation in the kidney of folic acid-treated guinea pigs

Previous investigators agree on the increased DNA synthesis and destruction of tissues caused by folic acid (FA) administered parenterally. This study aims to clarify whether DNA degradation due to the destruction of cells and nuclei precedes DNA synthesis following FA administration. Forty guinea pigs were divided into four groups: group 1, contained 10 controls; in group 2, ten animals received intraperitoneally 300 mg/kg of body wt FA; in group 3, ten animals received FA and 12 h later frusemide intramuscularly in a dose of 7 mg/kg body wt; and finally in group 4, ten animals received frusemide as in group 3. FA produced necrosis of the epithelial cells of the convoluted tubules as the detection of the beta-aminoisobutyric acid end product of DNA and thymine catabolism indicated. Frusemide administered in group 3 had a favourable effect on the acute renal failure induced by FA.     

A mutation in the Mod subunit of EcoP15I restriction enzyme converts the DNA methyltransferase to a site-specific endonuclease

A closer inspection of the amino acid sequence of EcoP15I DNA methyltransferase revealed a region of similarity to the PDXn(D/E)XK catalytic site of type II restriction endonucleases, except for methionine in EcoP15I DNA methyltransferase instead of proline. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. EcoP15I-M357P DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically EcoP151-M357P.DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically, 5'-CAGCAG(N)(10)-3', as indicated by the arrows, in presence of magnesium ions.     

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.     

Relationship between DNA synthesis and the increase in the level of tubulin during dedifferentiation of isolatedZinnia mesophyll cells

Summary The level of tubulin in cultured matureZinnia mesophyll cells increases to between 4 and 5 times its initial value when DNA synthesis occurs. The rapid increase in the level of tubulin requires the presence of auxin and cytokinin, as does the induction of DNA synthesis. Inhibitors of DNA synthesis suppress the rapid increase in the level of tubulin. These results imply that the rapid increase in the level of tubulin is dependent on the occurrence of DNA synthesis,i.e., on the reinitiation of the cell cycle. The presence of microtubules is not important for either the increase in the level of tubulin or for the induction of DNA synthesis, because both the increase in the level of tubulin and DNA synthesis occur even when microtubules are depolymerized by colchicine.Abbreviations APM amiprophos-methyl - araC arabinosylcytosine - BA 6-benzyladenine - BSA bovine serum albumin - DMSO dimethylsulfoxide - EGTA ethylene glycol bis(2-aminoethylether)tetraacetic acid - FITC fluorescein isothiocyanate - FUdR fluorodeoxyuridine - GA gibberellin A₃ - IAA indole-3-acetic acid - MES 2-(N-morpholino)ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PBS phosphate buffered saline - PCA perchloric acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - SDS sodium dodecyl sulfate     

Alteration of the omega-3 fatty acid desaturase gene is associated with reduced linolenic acid in the A5 soybean genotype

Reducing the linolenic acid (18?:?3^{ω? 3,6,9}) concentration of soybean [Glycine max (L.) Merr.] oil may lessen the need for chemical hydrogenation and enhance flavor stability. Soybean genotypes A5 and A23 have reduced linolenic acid concentration compared with current cultivars. Seed linolenic acid is synthesized primarily by the ω-3 fatty acid desaturase located in the microsomes. The objective of this research was to study whether this enzyme has a role in reducing the fatty acid levels in the soybean genotypes A5 and A23. DNA from A5 and A23 was analyzed by gel-blot hybridization with a cDNA encoding the ω-3 fatty acid desaturase. A5 and lines selected from it have a DNA fragment missing compared to A23 and lines with normal linolenic acid concentration. Seventy F_4:5 lines from a population segregating for linolenic acid concentration were scored for presence or absence of the fragment. The absence of the fragment was significantly (P?0.0001) associated with a reduced linolenic acid level and accounted for 67% of the variation for linolenic acid in the population. These results suggest that the reduced linolenic acid concentration in A5 was at least partially the result of a full or partial deletion of a microsomal ω-3 desaturase gene. No DNA polymorphisms were found for the desaturase gene in A23, so no mutations could be studied in this line.