Action of T4 endonuclease V on DNA containing various photoproducts

The action of T4 endonuclease V on DNA containing various photoproducts was investigated. (1) The enzyme introduced strand breaks in DNA from ultraviolet-irradiated vegetative cells of Bacillus subtilis but not in DNA from irradiated spores of the same organism. DNA irradiated with long wavelength (360 nm peak) ultraviolet light in the presence of 4,5',8-trimethylpsoralen was not attacked by the enzyme. These results indicate that 5-thyminyl 5,6-dihydrothymine (spore photoproduct) and psoralen mediated cross-links in DNA are not recognized by T4 endonuclease V. (2) DNA of phage PBS1, containing uracil in place of thymine, and DNA of phage SPO1, containing hydroxymethyluracil in place of thymine, were fragmented by the enzyme when the DNA's had been irradiated with ultraviolet light. T4 endonuclease V seems to act on DNA with pyrimidine dimers whether the dimers contain thymine residues or not.     

Analysis of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogen during the cell cycle. II. Ultraviolet light

UV-induction of thymine dimers in cellular DNA and their excision during different phases of the cell cycle of HeLa S3 cells were studied. Induction of thymine dimers was higher in the mitotic phase and the middle of the S phase than in the G₁ phase and from the late S phase to the early G₂ phase which are rather insensitive to UV. However, there is no significant difference in excision rate of UV-induced thymine dimers from the irradiated cells through the cell cycle. These findings indicate that the cyclic variation of UV-survivals during the cell cycle may be due to differences in the amount of thymine dimers in cellular DNA induced by UV-irradiation.     

Photoreactivation of pyrimidine dimers in the DNA of normal and xeroderma pigmentosum cells.

Photoproducts formed in the DNA of human cells irradiated with ultraviolet light (uv) were identified as cyclobuytl pyrimidine dimers by their chromatographic mobility, reversibility to monomers upon short wavelength uv irradiation, and comparison of the kinetics of this monomerization with that of authentic cis-syn thymine-thymine dimers prepared by irradiation of thymine in ice. The level of cellular photoreactivation of these dimers reflects the level of photoreactivating enzyme measured in cell extracts. Action spectra for cellular dimer photoreactivation in the xeroderma pigmentosum line XP12BE agree in range (300 nm to at least 577 nm) and maximum (near 400 nm) with that for photoreactivation by purified human photoreactivating enzyme. Normal human cells can also photoreactivate dimers in their DNA. The action spectrum for the cellular monomerization of dimers is similar to that for photoreactivation by the photoreactivating enzyme in extracts of normal human fibroblasts.     

Occurrence of cytochrome aa3 in Anacystis nidulans

The cytochrome content of membrane fragments prepared from the bluegreen alga (cyanobacterium) Anacystis nidulans was examined by difference spectrophotometry. Two b-type cytochromes and a hitherto unknown cytochrome a could be characterized. In the reduced-minus-oxidised difference spectra the a-type cytochrome showed an α-band at 605 nm and a γ-band at 445 nm. These bands shifted to 590 and 430 nm, respectively, in CO difference spectra. NADPH, NADH and ascorbate reduced the cytochrome through added horse heart cytochrome c as electron mediator. In presence of KCN the reduced-minus-oxidised spectrum showed a peak at 600 nm and a trough at 604 nm. Photoaction spectra of O₂ uptake and of horse heart cytochrome c oxidation by CO-inhibited membranes showed peaks at 590 and 430 nm. These findings are consistent with cytochrome aa₃ being the predominant respiratory cytochrome c oxidase in Anacystis nidulans.     

Probes of DNA Structure and Interactions: Effects of Copper II on Ultraviolet-Induced Pyrimidine Dimer Formation

Cu(II) affects the yield of cyclobutyl dimers induced in DNA by 254 nm radiation. The effects are a function of r, the ratio of Cu(II) to DNA phosphate, and of the ultraviolet (UV) fluence; they seem to reflect two types of copper complexes with DNA. The first probably involves “exterior” binding to the bases of native DNA and increases [unk]TT formation (without affecting [unk]UT yield) by raising the energy levels of bases other than thymine. The second seems to occur only at high ratios (rs) and only after the structure has been opened locally by UV radiation; it involves “interior” binding of Cu(II) to the bases. This complex tends to decrease dimer yield by holding the bases apart and/or by lowering the energy levels of bases other than thymine. These results illustrate the potential use of DNA photoproducts and ligands to probe the structure and interactions of DNA in vitro and perhaps also in vivo.     

Mechanisms of targeted frameshift mutations: Insertions arising during error-prone or SOS synthesis of DNA containing cis-syn cyclobutane thymine dimers

It is still unclear how frameshift mutations arise at cyclobutane pyrimidine dimers. The polymerase model is commonly used to explain the mechanisms of various mutations. An alternative polymerase-tautomer model was developed for UV-induced mutagenesis. A mechanism was proposed for targeted insertions caused by cis-syn cyclobutane thymine dimers. Targeted insertions are frameshift mutations due to addition of one or more nucleotides in a DNA sequence opposite to a lesion capable of stopping DNA synthesis. Among other factors, cyclobutane pyrimidine dimers can cause targeted insertions. UV irradiation can change the tautomeric form of DNA bases. Five rare tautomeric forms are possible for thymine, and they are stable when the thymine is a component of a cyclobutane dimer. A structural analysis showed that none of the canonical nucleotides can be added opposite to a specific rare thymine tautomer so that hydrogen bonds form between the two bases. A single nucleotide gap is consequently left in the corresponding site of the nascent strand when a specialized or modified DNA polymerase drives SOS or error-prone DNA synthesis on a template containing cis-syn cyclobutane thymine dimers with a base occurring in the rare tautomeric form. If the DNA composition is homogenous within the region, the end of the growing DNA strand may slip to form a complementary pair with the nucleotide adjacent to the dimer according to the Streisinger model, thus producing a loop. A targeted insertion is thereby generated to make the daughter strand longer. Targeted insertions were for the first time assumed to result from the cis-syn cyclobutane thymine dimers wherein one or both of the bases occur in the specific tautomeric form that does not allow the addition and hydrogen bonding of any canonical nucleotide in the opposite position. A model was developed to explain how targeted insertions of one or more nucleotides are caused by cis-syn cyclobutane thymine dimers. Thus, the polymerase-tautomer model can explain the nature and formation of targeted frameshift mutations in addition to hot and cold spots or targeted or untargeted nucleotide substitutions.     

γ-Irradiation of Thymine Dimers in Aqueous Solution

Thymine dimers were irradiated in aqueous solution with ⁶⁰Co γ-rays in N₂ or O₂. Thymine and unidentified non-UV-absorbing products appeared. The thymine was identified by spectrophotometry, chromatography, and ability to support the growth of Escherichia coli 15 T^-. Residual dimer was determined by a UV-reversibility assay. The G-values for dimer breakage were approximately equal in N₂ and O₂. At low γ-doses, about two thymines were produced per dimer broken in N₂, whereas only about one thymine appeared per dimer broken in O₂. For dimer irradiated in frozen solution, the yield of thymine was at least 100 times less than in liquid.     

Analysis of DNA Repair and Protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after Exposure to UVC Radiation

Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVC-induced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m² of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy.     

Hymenobacter tibetensis sp. nov., a UV-resistant bacterium isolated from Qinghai–Tibet plateau

A brick-red-pigmented strain (XTM003^T) isolated from the Qinghai–Tibet plateau was investigated using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequence indicated that the organism belonged to the genus Hymenobacter. The predominant menaquinone was MK7. The major fatty acids included iso-15:0, 16:1w5c and summed feature 3 (C_{16: 1}ω7c and/or C_{16: 1}ω6c). The G+C content of the DNA was 55.8%. In addition, DNA–DNA hybridization studies demonstrated that strain XTM003^T had a relatedness value of 50.7% with the phylogenetically most closely related species Hymenobacter norwichensis DSM 15439^T. Based on the results of phenotypic characteristics and DNA–DNA hybridization studies, strain XTM003^T is considered to represent a novel species, for which the name Hymenobacter tibetensis sp. nov. is proposed. The type strain is XTM003^T (=CCTCC AB 207089^T=NRRL B-51271^T).     

Mechanism of Caffeine Enhancement of Mutations Induced by Sublethal Ultraviolet Dosages

Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 μg of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm² of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr⁻. At least 90% of the mutations induced to streptomycin resistance by UV and 85% of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 μg of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r T⁻ was investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers.     

Radiomimetic property of furazolidone and the caffeine enhancement of its lethal action on the vibrios

Sensitivities of the strains belonging to four vibrio biotypes to the action of furazolidone were investigated. Vibrio cholerae (classical) was most and Vibrio parahaemolyticus least sensitive to this drug. Statistical analyses revealed significant differences between any two of the four types of vibrio in respect of their sensitivity to furazolidone. The drug was radiomimetic in action, the doses of UV light (D_UV) and furazolidone (D_f) required for 10% survival of the vibrios being correlated by the equation, D_f = 0.28 exp. (0.008 D_UV). Caffeine exhibited lethal synergism with furazolidone and the synergistic effect depended on the mode of caffeine treatment, the effect being maximum when caffeine was present along with and also after furazolidone treatment. UV spectrophotometric study revealed that caffeine did not bind with native DNA but did so with denatured DNA resulting in a bathochromic shift and a quenching of the caffeine absorption maximum at 209.4 nm. The binding isotherm (Scatchard plot) indicated the presence of a heterogeneity in the binding sites and that the parameters for the strongest mode of binding were n = 0.254 and k = 7.5 × 10⁵ M^?1.     

Streptomyces muensis sp. nov.

A strain of Streptomyces, MBRL 179^T, isolated from a sample from a Limestone quarry located at Hundung, Manipur, India, was characterized by polyphasic taxonomy. The strain formed a monophyletic clade with Streptomyces spinoverrucosus NBRC 14228^T (16S rRNA gene sequence similarity of 99.3 %) in the Neighbour-joining tree. DNA–DNA hybridization experiment gave a DNA–DNA relatedness value of 34.7 % between MBRL 179^T and S. spinoverrucosus NBRC 14228^T. Strain MBRL 179^T contained LL-diaminopimelic acid, xylose, glucose, and mannose in the whole cell-wall hydrolysates along with small amount of ribose. The major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside, with other unknown phospholipids and aminophospholipid. MK-9(H₆), MK-9(H₈) and MK-9(H₄) were the predominant menaquinones detected. The major fatty acids were anteiso-C_16:0 (28.1 %), iso-C_16:0 (20.3 %), C_16:0 (9.4 %) and anteiso-C_17:0 (8.3 %). The G+C content of the genomic DNA was 71.1 %. Based on the polyphasic experiment results, the strain MBRL 179^T merits recognition as a representative of a novel species of the genus Streptomyces for which the name Streptomyces muensis sp. nov. is proposed; the type strain is MBRL 179^T (=JCM 17576^T = KCTC 29124^T).     

Inhibition of inorganic carbon transport by oxygen in a high CO2-requiring mutant (E1) of Anacystis nidulans R2

The effect of O₂ on inorganic carbon (C_i) transport was studied with a high CO₂-requiring mutant (E₁) of Anacystis nidulans R₂. Oxygen (above 2%) inhibited C_i transport by 15–35|X% at CO₂ concentrations above 200 μl/l, but had no apparent effect at low, limiting CO₂ concentration. The action spectra for C_i transport measured in the presence or absence of 20% O₂ showed two peaks around 684 and 625 nm, corresponding to chlorophyll a and phycocyanin absorption, respectively. The difference between these two spectra (anaerobic minus aerobic) showed one peak around 625 nm, which indicates that a linear electron transport from water to O₂ is involved in the O₂ inhibition of C_i transport. Dithiothreitol could overcome the inhibition by O₂. The results suggested that the O₂ inhibition is a result of inactivation of the C_i-transporting system.     

Biased distribution of adenine and thymine in gene nucleotide sequences

We analyzed occurrences of bases in 20,352 introns, exons of 25,574 protein-coding genes, and among the three codon positions in the protein-coding sequences. The nucleotide sequences originated from the whole spectrum of organisms from bacteria to primates. The analysis revealed the following: (1) In most exons, adenine dominates over thymine. In other words, adenine and thymine are distributed in an asymmetric way between the exon and the complementary strand, and the coding sequence is mostly located in the adenine-rich strand. (2) Thymine dominates over adenine not only in the strand complementary to the exon but also in introns. (3) A general bias is further revealed in the distribution of adenine and thymine among the three codon positions in the exons, where adenine dominates over thymine in the second and mainly the first codon position while the reverse holds in the third codon position. The product (A₁/T₁) × (A₂/T₂) × (T₃/A₃) is smaller than one in only a few analyzed genes. Correspondence to: J. Kypr     

Vibrio zhuhaiensis sp. nov., isolated from a Japanese prawn (Marsupenaeus japonicus)

A Gram-negative, oxidase-positive, facultatively anaerobic bacterium, designated strain E20121, was isolated from the digestive tract of a Japanese prawn (Marsupenaeus japonicus) collected from the coastal sea water area of Zhuhai, Guangdong province, China. The new isolate was determined to be closely related to Vibrio ponticus DSM 16217^T, having 97.6 % 16S rRNA gene sequence similarity. Phylogenetic analysis based on recA, pyrH and rpoA also showed low levels of sequence similarities (72.6–96.6 %) with all species of the genus Vibrio. A multigene phylogenetic tree using concatenated sequences of the four genes (16S rRNA, rpoA, recA and pyrH) clearly showed that the new isolate is different from the currently known Vibrio species. DNA–DNA hybridization experiments revealed similarity values below 70 % with the closest related species V. ponticus DSM 16217^T. Several phenotypic traits enabled the differentiation of strain E20121 from the closest phylogenetic neighbours. The DNA G+C content of strain E20121 was determined to be 47.6 mol % and the major fatty acid components identified were C_16:1ω7c and/or C_16:1ω6c (39.8 %), C_18:1ω7c (13.6 %) and C_16:0 (9.6 %). Based on genotypic, phenotypic, chemotaxonomic, phylogenetic and DNA–DNA hybridization analyses, strain E20121 is proposed to represent a novel species of the genus Vibrio for which the name Vibrio zhuhaiensis sp. nov. is proposed. The type strain is E20121^T(=DSM 25602^T = CCTCC AB 2011174^T).     

Deviations from Hardy-Weinberg Proportions: Sampling Variances and Use in Estimation of Inbreeding Coefficients

An analysis is made of the distribution of deviations from Hardy-Weinberg proportions with k alleles and of estimates of inbreeding coefficients (f) obtained from these deviations.—If f is small, the best estimate of f in large samples is shown to be 2Σ _i(T_ii/N_i)/(k - 1), where T_ii is an unbiased measure of the excess of the ith homozygote and N_i the number of the ith allele in the sample [frequency = N_i/(2N)]. No extra information is obtained from the T_ij, where these are departures of numbers of heterozygotes from expectation. Alternatively, the best estimator can be computed from the T_ij, ignoring the T_ii. Also (1) the variance of the estimate of f equals 1/(N(k - 1)) when all individuals in the sample are unrelated, and the test for f = 0 with 1 d.f. is given by the ratio of the estimate to its standard error; (2) the variance is reduced if some alleles are rare; and (3) if the sample consists of full-sib families of size n, the variance is increased by a proportion (n - 1)/4 but is not increased by a half-sib relationship.—If f is not small, the structure of the population is of critical importance. (1) If the inbreeding is due to a proportion of inbred matings in an otherwise random-breeding population, f as determined from homozygote excess is the same for all genes and expressions are given for its sampling variance. (2) If the homozygote excess is due to population admixture, f is not the same for all genes. The above estimator is probably close to the best for all f values.     

Studies on poly(L-lysine50, L-tyrosine50)-DNA interaction

Interaction between poly(Lys⁵⁰, Tyr⁵⁰) and DNA has been studied by absorption, circular dichroism (CD), and fluorescence spectroscopy and thermal denaturation in 0.001M Tris, pH 6.8. The binding of this copolypeptide to DNA results in an absorbance enhancement and fluorescence quenching on tyrosine. There is also an increase in the tyrosine CD at 230 nm. The CD of DNA above 250 nm is slightly shifted to the longer wavelength which is qualitatively similar to, but quantitatively much smaller than, that induced by polylysine binding. At physiological pH the poly(Lys⁵⁰, Tyr⁵⁰)–DNA complex is soluble until there is one lysine and one tyrosine per nucleotide in the complex. The same ratio of amino acid residues to nucleotide has also been observed in copolypeptide-bound regions of the complex. The addition of more poly(Lys⁵⁰, Tyr⁵⁰) to DNA yields a constant melting temperature, T_m′, for bound base pairs at 90°C which is close to that of polylysine-bound DNA under the same condition. The melting temperature, T_m, of free base pairs at about 60°C on the other hand, is increased by 10°C as more copolypeptide is bound to DNA. As the temperature is raised, both absorption and CD spectra of the complexes with high coverage are changed, suggesting structural alteration, perhaps deprotonation, on bound tyrosine. The results in this report also suggest that intercalation of tyrosine in DNA is unlikely to be the mode of binding.     

UV-B-Inducible and Temperature-Sensitive Photoreactivation of Cyclobutane Pyrimidine Dimers in Arabidopsis thaliana

Removal of cyclobutane pyrimidine dimers (CBPDs) in vivo from the DNA of UV-irradiated eight-leaf seedlings of Arabidopsis thaliana was rapid in the presence of visible light (half-life about 1 hour); removal of CBPDs in the dark, presumably via excision repair, was an order of magnitude slower. Extracts of plants contained significant photolyase in vitro, as assayed by restoration of transforming activity to UV-irradiated Escherichia coli plasmids; activity was maximal from four-leaf to 12-leaf stages. UV-B treatment of seedlings for 6 hours increased photolyase specific activity in extracts twofold. Arabidopsis photolyase was markedly temperature-sensitive, both in vitro (half-life at 30°C about 12 minutes) and in vivo (half-life at 30°C, 30 to 45 minutes). The wavelength dependency of the photoreactivation cross-section showed a broad peak at 375 to 400 nm, and is thus similar to that for maize pollen; it overlaps bacterial and yeast photolyase action spectra.     

Unique Properties of Nucleic Acid from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Phage SP-15

BACTERIOPHAGE SP-15 is a large, generalized transducing phage of Bacillus subtilis and B. licheniformis. The DNA extracted from the purified phage has unusual physical properties: its melting temperature (T_m) in 0.15 M NaCl, 0.015 M sodium citrate (SSC) is very low, 61.5° C and its buoyant density in neutral CsCl is very high, 1.761 g/ml.¹. We describe here additional unique features of SP-15 DNA: the presence of (1) a new modified pyrimidine which partially replaces the thymine; (2) a compound which reacts with orcinol as a pentose; (3) alkali-sensitive phosphodiester bonds; and (4) glucose.     

Solubilization and purification of cytochrome a1 from Nitrosomonas

Cytochrome a₁ was solubilized with Triton X-100 from a membrane-envelope preparation of Nitrosomonas and partially purified by repeated fractionation with (NH₄)₂SO₄. The purified fraction of cytochrome a₁ was enriched over the crude extract by a factor of 16 and 300 with respect to protein and c-type cytochrome, respectively. The cytochrome was characterized as cytochrome a₁ on the basis of (a) reduced absorption maxima at 444 nm and 595 nm, (b) acid acetone extractibility and ether solubility of the heme and (c) absorption maximum of 587 nm of the ferro-hemochrome in alkaline pyridine. The α absorption band shifted from 600 nm to 595 nm upon solubilization of the cytochrome with Triton X-100. Spectral shifts were observed in the presence of cyanide and azide and the cytochrome changed with aging to a form with a reduced absorption band at 422 nm. Cytochrome a₁ was reduced anaerobically in the presence of reduced mammalian cytochrome c and was rapidly reoxidized in the presence of O₂. CO caused a shift in the soret peak of the reduced form but did not prevent reoxidation of cytochrome a₁ in the presence of CO-O₂ (95:5, v/v).