Interaction of berberine chloride with deoxyribonucleic acids: evidence for base and sequence specificity

The interaction of berberine chloride with natural and synthetic DNAs of differing base composition and sequences was followed by various spectroscopic and viscometric studies. The binding of berberine chloride was characterized by hypochromism and bathochromism in the absorption bands, enhancement of fluorescence intensity, stabilization against thermal denaturation, perturbations in the circular dichroic spectrum, increase in the contour length of sonicated rod-like DNA and induction of unwinding-rewinding process of covalently closed superhelical DNA, depending on the base composition and sequences of base pairs. Binding parameters determined from absorbance and fluorescence titration by Scatchard analysis, according to an excluded-site model, indicated a very high specificity of berberine to AT-rich DNAs and alternate AT polymer. Fluorescence quantum yield was maximum for the complexes with AT-rich DNAs and alternate AT polymer. Taken together, these results suggest that berberine chloride exhibits considerable specificity towards alternating AT polymer and binds to AT-rich DNAs by a mechanism of classical intercalation.     

Mechanism and base specificity of DNA Breakage in intact cells by neocarzinostatin

When electrophoresed on an agarose gel, the DNA isolated from neocarzinostatin- (NCS-) treated HeLa cells migrates in a ladder of discrete bands indicative of preferential breakage in the linker region of the nucleosomes. The 5'-termini of the drug-induced DNA strand breaks were characterized by reduction of the nucleoside 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase and treatment of the DNA with hot alkali and alkaline phosphatase prior to the kinase assay to give the total 5'-termini. In DNA isolated from NCS-treated cells, nucleoside aldehyde accounts for 30-45% of the drug-generated 5' ends; the remainder have PO4 termini. By contrast, 5'-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5' ends. Of the 32P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP much greater than CMP greater than GMP, a distribution in excellent agreement with that obtained for in vitro drug-treated DNA. DNA sequencing experiments, using the 340 base pair alphoid DNA fragment isolated from drug-treated cells, show that the pattern of breakage produced by NCS within a defined sequence of DNA in intact cells is similar to that in the in vitro reaction, with a preferential attack at thymidylate residues, but a much higher concentration of the drug was required to cause comparable breakage in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of DNA base release by bleomycin.

DNA was treated with bleomycin in the presence of Fe2+ and 2-mercaptoethanol under conditions where only a few percent of the bases were released. Release of all four bases was a linear function of bleomycin concentration, but the amount of thymine released was twice that of cytosine, 7 times that of adenine, and twelve times that of guanine. Unidentified minor products of thymine, of cytosine and of a purine were also released. Bromouracil did not sensitize DNA to bleomycin-induced breakage, and was released at the same rate as thymine.     

Cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for bovine preproinsulin

A cDNA library was prepared from poly(A+) RNA isolated from fetal bovine pancreas. Bacterial colonies were screened for sequences homologous to a rat preproinsulin I cDNA probe. Ten positive clones were selected at random and further studied. Northern blot analyses revealed that seven of these clones hybridized to a single RNA species, of approximately 400 nucleotides. Sequence analysis of one of these clones (pbI2885) revealed the entire structural region of bovine preproinsulin mRNA including a 72 nucleotide region encoding a signal peptide enriched in hydrophobic residues. The overall nucleotide homology between bovine and human preproinsulin mRNA was 76% for the preregion, 89% for the A chain, 83% for the B chain, and 68% for the C peptide (including a 15 nucleotide deletion).     

Single-strand and double-strand deoxyribonucleic acid breaks produced by several bleomycin analogues

Production of single-strand breaks (ssb) and double-strand breaks (dsb) of PM2 phage DNA by several structurally related bleomycin (BLM) analogues was studied by gel electrophoresis. BLM A2 and BLM B2 produced a comparable extent of dsb. In various experiments, BLM A2 and BLM B2, at 22-41 ng/mL, degraded 50% of the form I DNA into 33-38% form II and 12-17% form III DNA. BLM B1' produced ssb and dsb at a ratio similar to that of BLM A2, but both at a rate less than half that of BLM A2. Phleomycin (PLM) D1 induced an equivalent amount of ssb but only one-eighth of dsb induced by BLM B2. The relatively lower extent dsb production for PLM D1 was observed either in borate buffer (pH 9.5) or in Tris-HCl buffer (pH 7.5) and in the presence or absence of exogenous Fe(II). Deamido-BLM A2 produced ssb to an extent approximately half that of BLM A2 and dsb to less than one-eighth that of BLM A2. The following conclusions were drawn. (1) BLM analogues produced ssb and dsb to different extents and ratios. (2) The ratio of dsb to ssb varied depending on the analogue, indicating a lack of a direct correlation between ssb and dsb. (3) The extent of ssb and dsb was affected by modifications on both the C- and N-terminal half-molecules of BLM: modification of either the N-terminal amide or the bithiazole greatly reduced dsb, whereas changes in structure or charge in the C-terminal amine affected ssb and dsb to a similar extent.     

Effect of deoxyribonucleic acid on the production of reduced oxygen by bleomycin and iron

The binding of bleomycin to DNA in the presence and absence of ferric iron was measured by fluorescence spectroscopy. In millimolar concentrations of tris(hydroxymethyl)aminomethane, pH 7.5, approximately 80% of the bleomycin binds to DNA. Ferric iron seems to have no significant effect on the binding of DNA to bleomycin. The induction of oxygen uptake by ferrous iron and bleomycin was monitored in the presence and absence of DNA. DNA has no effect on the rate of oxygen uptake. Therefore, the iron binding site and the DNA binding site appear to be independent of each other. Under conditions where 80% of the bleomycin is bound to DNA, the ferrous iron-bleomycin-induced reduction of oxygen follows Michaelis-Menten kinetics. Ferrous iron autoxidation produces ethylene from methional. The addition of bleomycin greatly increases ethylene production. DNA, under conditions where 80% of the bleomycin is bound to DNA, inhibits ethylene production. Since ethylene is a measure of hydroxyl radical production, we conclude that DNA is able to compete with methional for the hydroxyl radical. We postulate a mechanism for DNA double-strand breaks in which the bleomycin selectively binds to DNA and recurrently produces the hydroxyl radical at that site. The localized generation of many hydroxyl radicals as provided by the proposed oxidation-reduction cycle mechanism may cause multiple strand breaks taking place on both strands of the DNA duplex leading to double-strand breaks. Since catalase, but not superoxide dismutase, is able to inhibit ferrous iron-bleomycin-induced products of the hydroxyl radical, hydrogen peroxide, but not the superoxide radical, is the immediate precursor of the hydroxyl radical.     

Occurrence of insertion sequence (IS) regions on plasmid deoxyribonucleic acid as direct and inverted nucleotide sequence duplications.

Insertion sequence (IS) regions have been identified previously as a cause of strongly polar mutations in Escherichia coli and several bacteriophages. The present experiments indicate that genetically characterized IS regions occur on bacterial plasmid deoxyribonucleic acid (DNA) as both direct and inverted DNA sequence duplications. The DNA insertion which has been shown previously (Sharp et al., 1973) to control expression of tetracycline resistance in the R6-5 plasmid, and which occurs as directly and inversely repeated DNA sequences adjacent to the region believed to contain the tetracycline resistance gene, has been identified as IS3. A second genetically characterized insertion sequence (IS1) has been identified as a direct DNA duplication occurring at both junctions of the resistance transfer factor and R-determinant components of R6-5 and related plasmids. A model is presented for the reversible dissociation of resistance transfer factor and R-determinant components of co-integrate R plasmids at the sites of DNA sequence homology provided by the repeated IS regions.     

Stoichiometry of DNA strand scission and aldehyde formation by bleomycin

A colorimetric assay of DNA breakage by bleomycin has been standardized and indicates that strand scission is stoichiometric with the formation of a single equivalent of an aldehyde compound consisting of base plus deoxyribose carbons 1' to 3'. Both strand scission and aldehyde formation require the presence of O2. An alternate DNA lesion inflicted by bleomycin, alkali labilization, is O2-dependent, as is the accompanying release of free bases.     

The sequence specificity of bleomycin damage in three cloned DNA sequences that differ by a small number of base substitutions

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, has been investigated in three clones of human alpha RI-DNA. The three 340-base pair alpha RI-DNA sequences were almost identical in their nucleotide sequence enabling the study of subtle effects of base substitutions on bleomycin cleavage. By utilizing densitometer scanning and statistical analysis of the degree of bleomycin DNA cleavage, we found 17 significant differences between the three DNA sequences. Eleven of these differences could be attributed to base substitutions close to the dinucleotide cleavage site. However, six of the differences were at positions two or more base pairs from the base substitution sites. The significant differences were up to 12 base pairs from base substitutions. It is proposed that these long range effects are due to base substitutions causing microvariation in the DNA structure to which bleomycin cleavage is sensitive.     

A putative tRNATrp gene cloned from Dictyostelium discoideum: its nucleotide sequence and association with repetitive deoxyribonucleic acid

Using a total tRNA population labeled with 32P, we have cloned a number of tRNA genes from Dictyostelium discoideum. A partial sequence of a cloned 1250-base-pair DNA insert, pDT-513, revealed the occurrence of a putative tRNATrp gene. In addition to the cloverleaf secondary structure, the tRNATrp gene contained all of the invariant and semiinvariant residues found in most tRNA sequences and has a 13-base-pair intron which is located one base removed from the 3' residue of the anticodon. The genomic distribution of the tRNA gene and its flanking sequences was examined via Southern annealing experiments. The structural gene is represented on at least six EcoRI fragments in the D. discoideum genome. Sequences flanking the 5' terminus of the cloned gene are repeated many times in the genome while the sequence flanking the 3' terminus of the pDT-513 DNA insert structural tRNA gene is present only once in the genome.     

Base sequence homology and renaturation studies of the deoxyribonucleic acid of extremely halophilic bacteria

The genetic relatedness among various strains of halophilic bacteria has been assessed by deoxyribonucleic acid-deoxyribonucleic acid (DNA-DNA) duplex formation and ribosomal ribonucleic acid (RNA) hybridization. All of the strains of extremely halophilic rods are closely related, and the extent of divergence of base sequence is similar for the major and minor DNA components. Parallel experiments with ribosomal RNA revealed a relationship between the extremely halophilic rods and cocci and a more distant relationship to moderate halophiles and to a photosynthetic extreme halophile. Renaturation studies of halophile DNA exclude the possibility that the satellite DNA represents multiple copies of a small episomal element. The kinetics of DNA renaturation show that the genome size of the extreme and moderate halophiles is similar to that of Escherichia coli.