Synthesis of Deoxyribonucleic Acid After Ultraviolet Irradiation of Sensitive and Resistant Haemophilus influenzae

Synthesis of deoxyribonucleic acid (DNA) has been measured as a function of ultraviolet (UV) radiation dose in wild-type and seven UV-sensitive strains of Haemophilus influenzae. At the UV doses used, all strains were able to resume DNA synthesis, even those which are unable to excise pyrimidine dimers from their DNA. These excisionless strains showed longer UV-induced delays in DNA synthesis than all but one of the other strains. The longest delay was shown by DB117, a strain which can excise dimers but which is recombination deficient and unable to rejoin X ray-induced single-strand breaks. All strains showed a progressive decrease in sensitivity as they approached the stationary phase.     

Repair of Single-Strand Deoxyribonucleic Acid Breaks in Ultraviolet Light-Irradiated Haemophilus influenzae

The wild-type strain and mutants of Haemophilus influenzae, sensitive or resistant to ultraviolet light (UV) as defined by colony-forming ability, were examined for their ability to perform the incision and rejoining steps of the deoxyribonucleic acid (DNA) dark repair process. Although UV-induced pyrimidine dimers are excised by the wild-type Rd and a resistant mutant BC200, the expected single-strand DNA breaks could not be detected on alkaline sucrose gradients. Repair of the gap resulting from excision must be rapid when experimental conditions described by us are employed. Single-strand DNA breaks were not detected in a UV-irradiated sensitive mutant (BC100) incapable of excising pyrimidine dimers, indicating that this mutant may be defective in a dimer-recognizing endonuclease. No single-strand DNA breaks were detected in a lysogen BC100(HP1c1) irradiated with a UV dose large enough to induce phage development in 80% of the cells.     

Specificity in Deoxyribonucleic Acid Uptake by Transformable Haemophilus influenzae

Cells of Haemophilus influenzae strain Rd competent for genetic transformation irreversibly bound approximately five molecular fragments of H. influenzae deoxyribonucleic acid (DNA) per cell; under identical conditions, DNA derived from Escherichia coli B was not taken up (<1 molecule per 50 cells). Similarly, DNA from Xenopus laevis was not taken up by competent H. influenzae. Of the heterologous DNAs tested, only DNA from H. parainfluenzae interfered with the uptake of H. influenzae DNA, as judged by competition experiments employing either DNA binding or genetic transformation as the test system. The extracellular heterologous DNA did not suffer either single- or double-strand breakage upon exposure to competent H. influenzae.     

Integration and Repair of Ultraviolet-Irradiated Transforming Deoxyribonucleic Acid in Haemophilus influenzae

The extent of association between donor transforming deoxyribonucleic acid (DNA) and recipient DNA in Haemophilus influenzae as a function of ultraviolet (UV) dose to the transforming DNA has been measured by isopycnic analysis of lysates of (3)H-labeled recipient cells exposed to DNA labeled with (32)P and heavy isotopes. Except for doses above 15,000 ergs/mm(2), the results of these measurements are in good agreement with previous estimates made by another technique. Experiments with a mutant temperature sensitive for DNA synthesis and another mutant defective in excision of pyrimidine dimers suggest that the discrepancy between the methods of high doses results from DNA synthesis, in which portions of the associated donor DNA containing pyrimidine dimers are excised and broken down, and the components are reutilized for synthesis.Repair of UV-irradiated, transforming DNA during incubation of recipient cells is observed as an increase in transforming ability when fractions from CsCl gradients of cell lysates are assayed on excision-deficient cells. When transforming DNA containing markers of different UV sensitivities is used, repair of the UV-resistant nov marker by excision proficient cells takes place exclusively in the donor DNA that is associated with recipient DNA, and this repair is observed even in the absence of DNA synthesis. However, no repair is observed in the case of the more UV-sensitive str marker, possibly because excision events may remove a large fraction of the integrated str markers in addition to repairing a small fraction of the integrated DNA containing this marker.     

Fate of Recipient Deoxyribonucleic Acid During Transformation in Haemophilus influenzae

During genetic transformation of Haemophilus influenzae, segments of the host deoxyribonucleic acid (DNA) corresponding to the integrating donor DNA were degraded and liberated into the medium. This degradation was detected by the release of the radioactive label from host DNA during a time period matching the time of development of maximal linkage between donor and host markers. The host label released above that released from nontransformed, control cultures was equivalent to about 2% of the host genome or 16 x 10(6) daltons of DNA. The released, labeled material was acid-soluble and dialyzable. The label release from control cultures was unaffected at 30 C; at this temperature, the recombination-specific release from transformed cells was suppressed. High molecular weight fragments of host DNA corresponding in size to the donor fragments could not be found free within the cell, weakly bound to other host DNA, or bound to non-integrated donor DNA by a reciprocal cross mechanism.     

Photodynamic Action on Native and Denatured Transforming Deoxyribonucleic Acid from Haemophilus influenzae

The photodynamic inactivation of native or denatured transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae is described. The inactivation at the same pH was higher for denatured than native DNA. At acidic pH, the inactivation both for native and denatured DNA was faster than at alkaline pH. The guanine content of photoinactivated native DNA at neutral pH was less than untreated DNA. The inactivation of biological activity was more extensive than the alteration of guanine. The absorption spectrum of photoinactivated native or denatured DNA was only slightly different than the control DNA at the different experimental conditions.     

Effect of Ethylene on Cell Division and Deoxyribonucleic Acid Synthesis in Pisum sativum

Ethylene and supraoptimal levels of 2,4-dichlorophenoxyacetic acid inhibit the growth of the apical hook region of etiolated Pisum sativum (var. Alaska) seedlings by stopping almost all cell divisions. Cells are prevented from entering prophase. The hormones also retard cell division in intact root tips and completely stop the process in lateral buds. The latter inhibition is reversed partially by benzyl adenine. In root tips and the stem plumular and subhook regions, ethylene inhibits DNA synthesis. The magnitude of this inhibition is correlated with the degree of repression of cell division in meristematic tissue, suggesting that the effect on cell division results from a lack of DNA synthesis. Ethylene inhibits cell division within a few hours with a dose-response curve similar to that for most other actions of the gas. Experiments with seedlings grown under hypobaric conditions suggest that the gas naturally controls plumular expansion and cell division in the apical region.     

Genetic Integration in the Heterospecific Transformation of Haemophilus influenzae Cells by Haemophilus parainfluenzae Deoxyribonucleic Acid

The in vivo chemical linkage of Haemophilus parainfluenzae deoxyribonucleic acid (DNA) with the H. influenzae genome has been found to occur at a much higher level than is suggested by the low efficiency of the heterospecific transformation of an antibiotic resistance marker. This linkage, about 60% of the level with homospecific DNA, was found to involve alkali-stable bonding. The amount of host DNA label released (about 60%) was about the same as that released during homospecific transformation. Also, over 60% of the H. influenzae cells adsorbing H. parainfluenzae DNA could not form colonies upon plating. This lethality of the heterospecific transformation was not immediate but followed considerable metabolic activity of the host cells. These data are presented to show that the "limited-pairing" hypothesis may be only a partial explanation for the low efficiency of heterospecific transformation. Another hypothesis is presented which takes into account the lethal effect of this kind of transformation.     

Thymine and Thymidine Uptake by Haemophilus influenzae and the Labeling of Deoxyribonucleic Acid

The influence of ribo- and deoxyribonucleosides and ribo- and deoxyribonucleotides on the uptake of radiolabeled thymidine and thymine by Haemophilus influenzae during growth was investigated. A nucleoside-degrading enzyme similar to that reported in Escherichia coli was found to break down thymidine unless other nucleosides were present to divert its action. The presence of other nucleosides permitted a nearly quantitative uptake of even low levels of thymidine. This quantitative uptake of thymidine in the presence of an excess of other nucleosides suggests that the uptake mechanism for thymidine is specific in this organism. Under optimal conditions, as much as 50% of the deoxyribonucleic acid (DNA) thymine was derived from exogenous thymidine. Thymine was not taken up by H. influenzae but, in the presence of purine deoxynucleosides, labeled thymine entered the cells, presumably as thymidine. Ribonucleosides or ribonucleotides inhibited thymine conversion to thymidine, but, as noted above, they were degraded by a cellular enzyme. Thus, unless the ribonucleoside level was excessively high, a cell level of growth was reached at which the inhibiting ribonucleoside was broken down and labeled thymine appeared in the cells at an increasing rate. Twenty-five per cent of the DNA thymine of this organism was labeled with exogenous thymine. The information noted above serves as the basis for isotopically labeling the DNA.     

Mechanism of Inactivation of Haemophilus influenzae Transforming Deoxyribonucleic Acid by Sonic Radiation

Transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae was exposed to sonic radiation of various durations. Reductions in transforming ability of the DNA, cellular DNA uptake, and integration into the genome, and single- and double-stranded molecular weights of the transforming DNA were measured and compared. We conclude that (i) sonic radiation causes DNA strand breaks (almost always double-strand breaks with relatively few alkaline-labile bonds), the number increasing with exposure until the double-stranded molecular weight is reduced to less than 10(6) daltons; and (ii) since transformation is reduced about as much as integration and much more than uptake, inactivation of transforming DNA by sonic radiation appears to be caused mostly by failure of Haemophilus cells to integrate the transforming DNA that is taken into the cells. These results are similar to those for inactivation by X radiation but differ from those for ultraviolet radiation. A strand break caused by sonic radiation, however, does not necessarily inactivate the transforming DNA, whereas in the case of ionizing radiation it may. The results may be fit by the model proposed by Cato and Guild. From our data and the equation of Lacks, the minimum active site of DNA necessary for transformation and the frequency of exchanges between donor and recipient strands upon integration of transforming DNA were estimated as 0.35 x 10(6) to 0.7 x 10(6) daltons and 0.15 to 0.4 switches per 10(6) daltons, respectively.     

Fate of Donor Deoxyribonucleic Acid in a Highly Transformation-Deficient Strain of Haemophilus influenzae

A transformation-deficient strain of Haemophilus influenzae (efficiency of transformation 10⁴-fold less than that of the wild type), designated TD24, was isolated by selection for sensitivity to mitomycin C. In its properties the mutant was equivalent to recA type mutants of Escherichia coli. The TD24 mutation was linked with the str-r marker (about 30%) and only weakly linked with the nov-r2.5 marker. The uptake of donor deoxyribonucleic acid (DNA) was normal in the TD24 strain, but no molecules with recombinant-type activity (molecules carrying both the donor and the resident marker) were formed. In the mutant the intracellular presynaptic fate of the donor DNA was the same as that in the transformation-proficient (wild-type) strain, and the radioactive label of the donor DNA associated covalently with the recipient chromosome in about the same quantity as in the wild type. However, many fewer donor atoms were associated with segments of the mutant's recipient chromosome as compared with segments of the wild-type chromosome. In the mutant the association was accompanied by complete loss of donor marker activity. The lack of donor marker activity of the donor-recipient complex of DNA isolated from the mutant was not due to lack of uptake of the complex by the second recipient and its inability to associate with the second recipient's chromosome. Because the number of donor-atom-carrying resident molecules was higher than could be accounted for by the lengths of presynaptic donor molecules, we favor the idea that the association of donor DNA atoms with the mutant chromosome results from local DNA synthesis rather than from dispersive integration of donor DNA by recombination.     

Homology Between the Deoxyribonucleic Acids of Haemophilus influenzae and Haemophilus parainfluenzae

To determine the degree of homology between deoxyribonucleic acid (DNA) from Haemophilus influenzae and that from Haemophilus parainfluenzae, the two DNAs were hybridized by the membrane-filter technique. It was found that 44% of the DNA from each species was sufficiently homologous to allow hybrid formation.     

Ultraviolet-Induced Decrease in Integration of Haemophilus influenzae Transforming Deoxyribonucleic Acid in Sensitive and Resistant Cells

The decrease in integration of transforming deoxyribonucleic acid (DNA) caused by ultraviolet irradiation of the DNA was found to be independent of the presence or absence of excision repair in the recipient cell. Much of the ultraviolet-induced inhibition of integration resulted from the presence in the transforming DNA of pyrimidine dimers, as judged by the photoreactivability of the inhibition with yeast photoreactivating enzyme. The inhibition of integration made only a small contribution to the inactivation of transforming ability of the DNA by ultraviolet radiation.     

Killing of Haemophilus influenzae Cells by Integrated Ultraviolet-Induced Lesions from Transforming Deoxyribonucleic Acid

Highly competent cultures of Haemophilus influenzae are inactivated by exposure to transforming deoxyribonucleic acid (DNA) irradiated with ultraviolet light (UV). As a function of UV dose to the DNA, the killing goes to a maximum and then decreases. The killing of H. influenzae by unirradiated H. parainfluenzae DNA, reported by other workers, is enhanced by low doses of UV, but drops off at high doses. Since there are no such lethal effects in a strain of H. influenzae that takes up DNA normally but does not integrate it, it is concluded that the killing is associated with integrated UV lesions. All the killing of wild-type cells due to irradiated DNA is eliminated by photoreactivation of the DNA. The killing of an excisionless strain of H. influenzae, however, is not eliminated by maximal photoreactivation of the irradiated transforming DNA. The nonphotoreactivable fraction of killing in the excisionless strain increases with increasing dose. The kinetics of the killing-dose curves may be explained only partially in terms of UV-induced loss of integration. It is postulated that the number of pyrimidine dimers relative to other DNA components integrated decreases at higher UV doses.     

Acid-Soluble Breakdown of Homologous Deoxyribonucleic Acid Adsorbed by Haemophilus influenzae: Its Biological Significance

Competent bacteria of Haemophilus influenzae strain Rd were exposed to various kinds of radioactive deoxyribonucleic acid (DNA) for short periods of time and at relatively low temperature. The fate of phage HP1 DNA was studied most extensively. Adsorbed DNA was partially acid solubilized by lysogens and by nonlysogens with very similar kinetics. The biological activity of the DNA decreased extensively in both lysogenic and nonlysogenic recipients. 2,4-Dinitrophenol had no effect on the acid solubilization but largely abolished the biological inactivation. Inactivation kinetics for three different markers and for the triple combination were roughly the same. The presence of 2,4-dinitrophenol in the medium, or the HP1 prophage in the chromosome, did not alter this observation. This suggests that acid solubilization involves the destruction of whole DNA molecules. In view of the absence of DNA homology between phage and host, it is concluded that acid-soluble breakdown of adsorbed transforming DNA is not an integral part of the donor DNA integration process. Behavior of mutant bacteria indicates that neither exonuclease III nor exonuclease V is involved.     

Postreplication Repair of Ultraviolet Damage in Haemophilus influenzae

The deoxyribonucleic acid (DNA) synthesized following ultraviolet (UV) irradiation of wild-type (Rd) and recombination-defective strains of Haemophilus influenzae has been analyzed by alkaline sucrose gradient sedimentation. Strain Rd and a UV-resistant, recombination-defective strain Rd(DB117) (rec-) are able to carry out postreplication repair, i.e., close the single-strand gaps in the newly synthesized DNA; in the UV-sensitive, recombination-defective strain DB117, the gaps remain open. The lack of postreplication repair in this strain may be the result of degradation of the newly synthesized DNA.     

Deoxyribonucleic Acid Repair Replication after Ultraviolet Light or X-Ray Exposure of Bacteria

A comparison of repair synthesis after ultraviolet light (UV) or X-ray exposure was made in Escherichia coli strains 15T(-) (555-7) and B/r by use of a D, (15)N, (13)C density labeling system. During the initial 15 min of incubation after UV irradiation, both a "repair" synthesis and a reduced semiconservative deoxyribonucleic acid (DNA) synthesis occurred. In the so-called "physiological" dose range used, the latter was greater than the former. X-irradiation of cells, at doses producing similar levels of cell death as in the UV-exposed cultures, did not lead to a similar repair replication process. However, a density heterogeneity of the DNA synthesized in the initial 10 min after exposure was observed. This is interpreted in terms of X ray-induced DNA degradation. Normal cells showed only a semiconservative type of replication and, therefore, within the limits of resolution of the system used (the incorporation of 1,000 to 5,000 nucleotides per replicating chromosome could be measured), DNA in normal cells did not appear to undergo a repair synthesis involving thymine exchange. These results indicate that not all repair mechanisms mimic that found after UV exposure.     

Unlinking of Cell Division from Deoxyribonucleic Acid Replication in a Temperature-sensitive Deoxyribonucleic Acid Synthesis Mutant of Escherichia coli

A new type of temperature-sensitive deoxyribonucleic acid (DNA) synthesis mutant, which can divide without a completion of DNA replication, was isolated from a thymidine-requiring Escherichia coli strain by means of photo-bromouracil selection after nitrosoguanidine mutagenesis. In this mutant, in spite of the fact that DNA synthesis stopped immediately after the temperature shift from 30 to 41 C, cells could continue to divide, though at a reduced rate. This cell division without DNA synthesis at 41 C is further supported by the following results. (i) Cell division took place at high temperature without addition of thymidine but not at all at 30 C. The parent strain of the mutant did not divide at 41 C without thymidine. (ii) Smaller cells isolated from the culture grown at 41 C did not contain DNA. This was shown by chemical analysis of the smaller cells and on electron micrographs. Ability of cells to divide was examined according to sizes of cells. By using the culture at 30 C, cells of various sizes were separated by means of sucrose-density gradient centrifugation. It was found that all cell fractions, including the smallest one, could divide at high temperature. These results suggest that in this mutant the completion of DNA replication is not required for triggering cell division at high temperature. Heat sensitivity of a factor which links cell division with DNA replication appears to be responsible. Some possible mechanisms of the coordination between cell division and DNA replication are discussed.     

Cell Division During Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli

When cultures of Escherichia coli B/r growing at various rates were exposed to ultraviolet light, mitomycin C, or nalidixic acid, deoxyribonucleic acid (DNA) synthesis stopped but cell division continued for at least 20 min. The chromosome configurations in the cells which divided were estimated by determining the rate of DNA synthesis during the division cycle. The cultures were pulse-labeled with (14)C-thymidine, and the amount of label incorporated into cells of different ages was found by measuring the radioactivity in cells born subsequent to the labeling period. The cells which divided in the absence of DNA synthesis were those which had completed a round of chromosome replication prior to the treatments. It was concluded that completion of a round of replication is a necessary and sufficient condition of DNA synthesis for cell division.