Isolation of UV-sensitive variants of CHO-K1 by nylon cloth replica plating.

Techinques are described which permit the identification and isolation of UV-sensitive variants from mutagenized populations of Chinese hamster ovary (CHO) cells. Identification is based on the observation that within two days after receiving a dose of approximately 240 ergs/mm2 of UV irradiation most of the cells in a colony of CHO detach from the surface of a plastic tissue culture dish. At a lower dose of UV, which does not kill or detach a significant number of parental cells, UV-sensitive colonies are killed and become detached. Thus a clear plaque is produced in a lawn of unirradiated parental cells, marking the site occupied by a sensitive colony. Live cells from such sensitive colonies have been recovered from a nylon cloth replica prepared prior to irradiation and characterized. One UV-sensitive variant (CHO-UV-1) is indistinguishable from parental cells in X-ray resistance, chromosome number, generation time, and duration of the phases of the cell cycle. For UV irradiation the hit number (-n), shoulder width (Dq), and mean lethal dose (Do) for the variant are 2.8, 21 ergs/mm2, and 21 ergs/mm2, respectively, as compared to 2.6, 36 ergs/mm2, and 45 ergs/mm2 for CHO-K1 cells. These values have not changed for a period of eight months in culture.     

Unscheduled incorporation of thymidine in ultraviolet-irradiated human lymphocytes

The nature, degree, and kinetics of unscheduled thymidine incorporation previously shown to occur in 90 % of irradiated lymphocytes was stud-incorporation was sever ely depressed i n t h e presence of 10(-4) M acriflavine and by low temperature, but was unaffected by 10(-3) M hydroxyurea or caffeine. Over a dose range of 25 to 400 ergs/mm2, the uptake of thymidine was increased by a factor of only 1.6, although the survival of lymphocytes, measured 5 days after irradiation, decreased by almost two orders of magnitude. (The survival curve suggests that 90% of the lymphocytes have a D0 of 35 ergs/mm(2) and 10 % have a D0 of 250 ergs/mm(2).) After exposure to 25 ergs/mm(2), over 70 % of the cells survived for 5 days in culture; moreover, cells which had been stimulated by this dose to incorporate thymidine transformed and divided after exposure to phytohema-glutinin. The final uptake of thymidine was significantly greater when a total dose of 75 ergs/mm(2) was fractionated into three doses of 25 ergs/mm(2) given at six hourly intervals than when it was given as a single dose. The degree of thymidine incorporation and the fraction of leukemic cells labeled were not significantly different from those in normal lymphocytes.     

Effect of Ultraviolet Light on Mengovirus: Formation of Uracil Dimers, Instability and Degradation of Capsid, and Covalent Linkage of Protein to Viral RNA

UV irradiation of purified mengovirus resulted in a very rapid inactivation of the infectivity of the virions (D(37) [37% survival dose] = 700 ergs/mm(2)) which correlated in time with the formation of uracil dimers in the viral RNA. During the first 2 min of irradiation, an average of 1.7 uracil dimers were formed per PFU of virus inactivated. Hemagglutination activity of the virions began to decrease only after a lag period of about 5 min and at a much lower rate (D(37) = 84,000 ergs/mm(2)). This decrease coincided in time with the appearance of altered proteins in the capsid and a structural change in the capsid. Although 10- to 20-min irradiated virions appeared intact in the electron microscope and sedimented at 150S in sucrose density gradients, the RNA of the virions became accessible to RNase and extractable by low concentrations of sodium dodecyl sulfate, and the virions broke down upon equilibrium centrifugation in CsCl gradients. During longer periods of irradiation (30 to 60 min), a progressively greater proportion of the virions were converted to 14S protein particles and 80S ribonucleoprotein particles composed of intact viral RNA and about 30% of the capsid proteins, alpha, beta, and gamma. Empty capsids were not detectable at any time during 60 min of irradiation, by which time disruption of the virions was complete. Irradiation of complete virions also resulted in an increased sedimentation rate of the viral RNA and in the covalent linkage to the viral RNA of about 1% of the total capsid protein in the form of heterogeneous low-molecular-weight polypeptides. The two observations seem to be causally related, since irradiation of isolated viral RNA did not result in an increase in sedimentation rate of the RNA, even though uracil dimer formation in viral RNA occurred at about the same rate and to the same extent whether intact virions or viral RNA were irradiated.     

The dose-response relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells.

Exposure of Chinese hamster ovary (CHO) cells clone K1BH4 to ultraviolet (UV) light at doses up to 86 ergs/mm2 did not significantly reduce cell survival, but UV doses of 86-648 ergs/mm2 produced an exponential cell killing. Observed mutation frequency ro 6-thioguannine resistance induced by UV increases approximately in proportion to increasing doses up to 260 ergs/mm2 in a range of 5-648 ergs/mm2 examined. The pooled data of mutation frequency f(X) as a function of dose X from 0-260 ergs/mm2 is adequately described by f(X)=10(-6) (13.6 + 2.04 X). That the UV-induced mutations to 6-thioguanine resistance affects the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus is supported by the observation that all randomly isolated drug-resistant colonies contained highly reduced or undetectable HGPRT activity.     

Relationship Between Radiation Response and the Deoxyribonucleic Acid Replication Cycle in Bacteria: Dependence on the Excision-Repair System

Prestarvation of Escherichia coli for required amino acids results in a marked enhancement in both ultraviolet light (UV) or X-ray resistance for selective strains. Preventing protein synthesis by starvation for required amino acids results in completion of the cycle of chromosomal replication then underway. We have investigated the relationship between starvation-induced resistance enhancement (SIRE) and the excision-repair (Hcr) system in several E. coli strains including E. coli B/r hcr(+) and its isogenic mutant E. coli B/r hcr(-). The following observations were made. (i) The Hcr system is the major component of SIRE in UV-irradiated strain B/r. By using the Hcr(+) strain, SIRE increases the 10% survival dose from approximately 400 ergs to approximately 1,200 ergs/mm(2). With the Hcr cells, the increase is from approximately 45 ergs to 60 ergs/mm(2). (ii) Although prestarvation leads to a moderate enhancement of resistance to X irradiation, this effect is not dependent on the Hcr system. (iii) The double mutant, E. coli B(s-1) (hcr(-)exr(-)) is completely unable to express SIRE whether studied with UV or X irradiation. It is concluded that the Hcr system is the major system responsible for SIRE in UV-treated cells, whereas Exr (resistance to X rays) may be involved to a minor extent. The Exr character appears to be required for SIRE expression in X-ray exposed cells.     

On the use of ultraviolet light to study protein-DNA crosslinking.

Irradiation of Ehrlich ascites chromatin with ultraviolet light (u.v.) leads to protein-DNA crosslinking as determined by CsCl isopycnic ultra-centrifugation and SDS-polyacrylamide gel electrophoresis. At the most 4.5% of the chromatin proteins labelled with (14C)-lysine and (14C)-arginine can be crosslinked to DNA at u.v. doses between 3.6 X 10(4) to 10.8 X 10(4) ergs/mm2. We find however that the crosslinking reaction is hindered by protein breakage induced by u.v. light of wave length of less than 2950 A. Our results point out that caution must be used in the interpretation of studies on protein-nucleic acid interactions using u.v. light.     

Repair of ultraviolet light-induced damage in Micrococcus radiophilus, an extremely resistant microorganism.

Repair of ultraviolet radiation damage was examined in an extremely radioresistant organism, Micrococcus radiophilus. Measurement of the number of thymine-containing dimers formed as a function of ultraviolet dose suggests that the ability of this organism to withstand high doses of ultraviolet radiation (20,000 ergs/mm2) is not related to protective screening by pigments. M. radiophilus carries out a rapid excision of thymine dimers at doses of ultraviolet light up to 10,000 ergs/mm2. Synthesis of deoxyribonucleic acid is reduced after irradiation, but after removal of photodamage the rate approaches that in unirradiated cells. A comparison is drawn with Micrococcus luteus and M. radiodurans. We conclude that the extremely high resistance to ultraviolet irradiation in M. radiophilus is at least partly due to the presence of an efficient excision repair system.     

Dose dependence of the excision of ultraviolet-induced pyrimidine dimers from nuclear deoxyribonucleic acids of haploid and diploid Saccharomyces cerevisiae.

The yield of ultraviolet-induced dimers is similar for a fixed dose in both haploid and diploid Saccharomyces cerevisiae. The excision of these photo-products from the nuclear deoxyribonucleic acids of cells of both ploidies after ultraviolet incident doses of 2 times 10-3 to 4 times 10-3 ergs/mm2 decreased with the corresponding increasing dose. Postirradiation incubation in saline followed by a further incubation in nutrient medium increases the excision as compared to that seen in either nutrient medium or saline alone. Previous data regarding both pyrimidine dimer removal and the survival of haploid and diploid cells after ultraviolet irradiation and either immediate or delayed plating are discussed.     

Incisions in ultraviolet irradiated circular bacteriophage lambda DNA molecules in excision proficient and deficient lysogens of E. coli

Summary The breakage of closed covalent DNA molecules in lysogenic host cells after ultravilet irradiation was investigated. In repair proficient host cells incisions are introduced immediately following irradiation. A steady-state of strand interruptions is observed within 20–50 seconds after irradiation, where the number of broken molecules is dose dependent for doses up to 600 ergs/mm². No ultraviolet promoted strand breaks were observed in uvrA or uvrB lysogens, in accordance with previous results obtained by Shimada, Ogawa & Tomizawa [Molec. gen. Genet. 101, 245 (1968)]. In contrast, uvrC mutants have the ability to form breaks in superinfecting DNA molecules after ultraviolet irradiation. The ultraviolet specific endonucleolytic activity observed in uvrC host cells differs from that observed in uvr ⁺ host cells in that, (i) the first break is introduced at least 15 times slower, (ii) for doses below 300 ergs/mm² the number of strand breaks is higher, (iii) the dose dependence terminates at a lower dose. The possible function of the uvrC gene product in the repair is discussed.     

Photoinhibition of chlamydosporulation of Candida albicans]

Candida albicans produced chlamydospores after 24 h in the dark at 27 degrees C, but the process was inhibited under adequate irradiation of light. The in vivo absorption spectra showed a main peak at 414 nm, and less important peaks at 430, 446, 477, 519, 549 and 560 nm. No bands were detected beyond 600 nm. A total inhibition of chlamydosporulation occurred at 414 nm (monochromatic light) for an initial energy of 2,000 ergs cm-2 s-1. A 4,000 ergs cm-2 s-1 irradiation energy was necessary to observe a marked inhibition at 460, 500 and 530 nm (les chlamydospores and/or immaturity); this energy must be raised to 300,000 ergs cm-2 s-1 to observe a similar effect at 575 and 630 nm. Biological activity spectra were in full concordance with absorption spectra at 414 nm; no interpretation of absorption band at 460 nm is given, but total or partial inhibition could be explained by modulation of protoporphyrin activity.     

Resistance of Spores of Clostridium botulinum 33A to Combinations of Ultraviolet and Gamma Rays

Spores of Clostridium botulinum 33A exhibit a sigmoidal survival curve if subjected to gamma radiation. The present investigation was concerned with two questions: (i) what is the form of an ultraviolet (UV)-survival curve and (ii) what is the combined effect of UV- and gamma radiation? The UV-survival curve was found to be of sigmoidal type with a "shoulder" width of 675 ergs/mm(2) and a D(10) (exp) of 2,950 ergs/mm(2). To test the combination effect, spores were subjected to UV doses of 225, 450, 675, and 900 ergs/mm(2) followed by a series of increasing doses of gamma rays from 200 to 2,000 krad in 200-krad steps. The gamma ray-survival curves showed that increasing UV pretreatment caused a gradual loss of the "Prodiginine" yielding straight line exponential survival curves after preirradiation with UV doses of 675 ergs/mm(2) and above. Simultaneously the D(10) value for gamma-ray irradiation was reduced, e.g. UV preirradiation with 900 ergs/mm(2) reduced the D(10) by 40%. This observation emphasizes the potential practical advantage of combining UV and gamma rays for sterilization of heat-sensitive commodities.     

The chloroplast and cytoplasmic ribosomes of euglena: I. Stability of chloroplast ribosomes prepared by an improved procedure

A new isolation procedure has resulted in an improved yield of stable 68S chloroplast ribosomes from Euglena gracilis var. bacillaris. Chloroplasts are isolated by suspending the cells in buffer I (sorbitol, 250 mm; sucrose, 250 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm) and passing through a French press at less than 1500 pounds per square inch. The crude chloroplasts are purified by three washings with buffer II (sorbitol, 150 mm; sucrose, 150 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm). Stable 68S chloroplast ribosomes are obtained when the isolated chloroplasts are resuspended in ribosome buffer (tris-HCI, pH 7.6, 10 mm; magnesium acetate, 12 mm; KCI, 60 mm) containing spermidine, 0.5 mm; mercaptoethanol, 14 mm; sucrose, 8% (w/w), passed through a French press at 4000 pounds per square inch and extracted with either 0.1% (w/v) sodium deoxycholate or 1.0% (v/v) Triton X-100. At 0 to 4 C in ribosome buffer, the purified 68S chloroplast monosome forms a 53S particle while the 35S particle, an expected product of monosome dissociation, cannot be detected. Spermidine and mercaptoethanol prevent the formation of 53S particles from 68S monosomes. The purified 53S particles derived from 68S monosomes contain 23S RNA as well as a significant amount of 16S RNA, suggesting that this particle may not be a true ribosomal subunit.     

Effects of growth inhibitors and ultraviolet irradiation on F pili

The effects of chloramphenicol, nalidixic acid, mitomycin C, NaCN, and ultraviolet irradiation at 253.7 nm on F pili production by Escherichia coli cells was studied by electron microscopy. The results show that cells contain pools of pili protein, and that assembly does not require synthesis of protein or deoxyribonucleic acid (DNA). NaCN (2 x 10(-2)m) prevents the reappearance of pili and causes existing pili to disappear quickly from the cell surface. This suggests that energy is used in the assembly of pili and to retain pili on the cell. Cells irradiated with high doses (10(4)ergs/mm(2)) of 253. 7 nm light produce fewer pili, and these are shorter than normal. Dose-response curves for number of pili per cell and length of pili resemble single hit kinetics, showing 37% survival at 10(4) ergs/mm(2) and 2 x 10(4) ergs/mm(2), respectively. This suggests that DNA is at the site where pili are produced, and that it may be involved in the assembly of pili.     

The Effect of Hydrogen Peroxide and Ultraviolet Irradiation on Non-sporing Bacteria

A kill of 99.99% was obtained in cell suspensions of Escherichia coli and Streptococcus faecalis by incubation with hydrogen peroxide 1.0% (w/v) for 75 and 180 min respectively. The same kill was produced by 30 s irradiation with ultraviolet (u.v.) light in the presence of hydrogen peroxide 1.0% (w/v). This simultaneous treatment with u.v. and hydrogen peroxide produced a synergistic kill at least 30-fold greater than that produced by irradiation of cell suspensions of Esch. coli with or without subsequent incubation with hydrogen peroxide.     

Increase in UV Mutagenesis by Heat Stress on UV-Irradiated E. coli Cells

When leu- auxotrophs of Escherichia coli, after UV irradiation, were grown at temperatures between 30 and 47°C, the frequency of UV-induced mutation from leu- to leu+ revertant increased as the UV dose and the temperature increased. For cells exposed to a UV dose of 45 J/m2, the mutation frequency at 47°C was 1.9 times that at 30°C; for a dose of 90 J/m2, it was 3.25 times; and for 135 J/m2, it was 4.8 times. Similar enhancement of reversion frequency was observed when the irradiated cells were grown at 30°C in the presence of a heat shock inducer, ethanol (8% v/v). Heat shock-mediated enhancement of UV mutagenesis did not occur in an E. coli mutant sigma 32 (heat shock regulator protein), but sigma 32 overexpression in the mutant strain (transformed with a sigma 32-bearing plasmid) increased the UV-induced mutation frequency. These results suggest that heat stress alone has no mutagenic property, but when applied to UV-damaged cells, it enhances the UV-induced frequency of cell mutation.     

Retardation of Regeneration and Division of Blepharisma by Ultraviolet Radiation and Its Photoreversal

Regeneration of Blepharisma undulans variety japonicus from which the hypostome has been removed is retarded by dosages of 3000 to 4600 ergs/mm.² at wavelength 2654A most strongly when the fragment is exposed soon after cutting. Dosages greater than 4600 ergs/mm.² prevent regeneration. Regeneration is also retarded strongly when the Blepharisma are cut soon after irradiation. Starvation retards regeneration and potentiates the effect of ultraviolet radiations. Division after regeneration of Blepharisma is also retarded by ultraviolet radiations about equally, regardless of when the Blepharisma are cut indicating a more lasting effect of the radiations upon the cells. Blepharisma cut after irradiation usually recover from the effects of the radiations sooner than uncut individuals given the same dosage. Retardation of division by ultraviolet radiation is subject to photoreversal by visible light, especially in a nitrogen atmosphere, provided the ultraviolet dose is not excessive. Visible light alone if prolonged, retards regeneration or may even kill the cut fragments of Blepharisma.     

Properties of Bacteriophage T4 Baseplate Protein Encoded by Gene 8

Gene product 8 (gp8, 344 amino acids per monomer) of bacteriophage T4 is one of the baseplate structural proteins. We constructed an expression vector of gp8 and developed a method for purification of recombinant protein. CD spectroscopy showed that gp8 is an / type structural protein. Its polypeptide chain consists of nearly 40% -structure and 15% -helix. These data agree with results of prediction of secondary structure based on the amino acid sequence of the protein. The sedimentation coefficient under standard conditions (S_20,w) is 4.6S. Analytical ultracentrifugation results demonstrated that gp8 in solution has two types of oligomers—dimer and tetramer. The tetramer of gp8 may be included in the wedge (1/6 of the baseplate), and the dimer may be an intermediate product of association.     

Effect of titanium dioxide concentration on the survival of Pseudomonas stutzeri during irradiation with near ultraviolet light

Cells of Pseudomonas stutzeri in suspensions of TiO₂ ranging in concentration from 0.5 to 4.0 g l^-1 were irradiated with a blacklight blue u.v. source displaying peak emissivity at approximately 370 mm. Irradiation under these conditions is known to result in the generation of lethal free radicals. During irradiation the suspensions were agitated, using a specially modified laboratory shaker, to ensure efficient exposure of the TiO₂. A u.v. radiation dose of 175 kJ m^-2 resulted in cell fractional survival ranging from 5.5 times 10^-5, at the lowest TiO₂ concentration, to 1.0 times 10^-6, at the highest TiO₂ concentration. The advantages of contactors employing TiO₂ suspensions are briefly compared to immobilized TiO₂ systems.