Vol. 124, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Oct. 1975, p. 155-160
Copyright 0 1975 American Society for Microbiology
Labeling the Deoxyribonucleic Acid of Anacystis nidulans L. RESTAINO
AND
E. W. FRAMPTON*
Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115
Received for publication 4 April 1975
Analysis of cell-free extracts of Anacystis nidulans disclosed the absence of both thymidine phosphorylase (EC 2.4.2.4) and thymidine kinase (EC 2.7.1.21) activities. Thymine and thymidine were incorporated inefficiently by intact cells of A. nidulans either in the presence or absence of deoxyguanosine (250 jg/ml). Deoxythymidine monophosphate incorporation was also inefficient. Radioactive deoxyadenosine, at a minimally toxic level (3 jg/ml), was incorporated effectively into the deoxyribonucleic acid (DNA). A cesium chloride-ethidium bromide gradient analysis of the DNA revealed that both the plasmid DNA and the principal DNA of the A. nidulans genome were labeled effectively in cells exposed to [8- l4C Ideoxyadenosine.
The photoautotrophic prokaryotic organisms known traditionally as the blue-green algae (the cyanobacteria) are subjects of increasing scientific interest. Experimentally, however, it has been difficult to label cyanobacterial deoxyribonucleic acid (DNA) even though these organisms can incorporate significant quantities of certain organic compounds into cellular components. Intact cells of Anacystis nidulans incorporate thymine or thymidine inefficiently, although uracil is incorporated into both DNA and ribonucleic acid (RNA) (9, 22). Recently, Ingram and Fisher (12) described the effective incorporation of low levels of deoxyadenosine, an inhibitor of DNA synthesis at higher levels, into the DNA of the marine cyanobacterium, Agmenellum quadruplicatum. In this investigation we have studied (i) whether some of the methods used to enhance thymine and thymidine incorporation and to label Escherichia coli DNA (4-7, 17, 18, 24) could be applied to A. nidulans, (ii) the enzymatic capacities of A. nidularns relative to thymine and thymidine utilization, and (iii) the labeling of A. nidulans DNA using deoxyadenosine. Implications of our finding that thymidine phosphorylase (EC 2.4.2.1) and thymidine kinase (EC 2.7.1.21) activities are absent in A. nidulans cell extracts are discussed in relation to DNA labeling and permeability problems (22) in this organism. MATERIALS AND METHODS Organisms. A. nidulans strain 625 was obtained from the Algae Culture Collection at Indiana University, Bloomington, Ind. E. coli strain B/r was used for controlling the enzyme assays. 155
Media and growth conditions. A. nidulans was grown in BG-11 medium (1, 25) while stock cultures were transferred routinely on Cyanophycean agar (26). A. nidulans was enumerated on agar prepared from double-strength BG-11 medium combined with an equal volume of 3% (wt/vol) agar (Difco) each autoclaved separately and cooled to 55 C before mixing (1). For most experiments, 100-ml volumes of A. nidulans were grown in 250-ml Erlenmeyer flasks stirred with Teflon-covered magnetic stirring bars (2 by X inch [ca. 5.08 by 0.953 cm]). Radioactivity incorporation experiments utilized 20-ml volumes in 50-ml Erlenmeyer flasks stirred by smaller bars (1 in. by % in. [ca. 2.54 by 0.953 cm]). Incubation was accomplished at 39 C under a continuous illumination of 5,000 lux provided by four Cool-White fluorescent lamps (Ken Rad) in a Controlled Environments model E7 environmental chamber. Doubling time under these conditions was about 4.5 h and was monitored by measuring absorbancy increases in a Bausch and Lomb Spectronic-20 colorimeter (3/4-inch [1.91 cm] diameter tube) at 520 nm (16). Bacterial contamination was checked by routinely streaking cultures of A. nidulans on nutrient agar plates which were incubated at 37 C for 48 h. E. coli was grown in C medium (23) at 37 C with forced aeration. Growth experiments with deoxyribonucleosides. Both deoxyadenosine and deoxyguanosine dissolved in BG-11 medium were sterilized by filtration through membrane filters (Schleicher and Schuell, type B-6). For viable cell counts, final dilutions were spread in triplicate on the surface of BG-11 agar plates which were sealed with cellophane tape before incubation for 5 days. Labeling and measurement of radioactivity. Radioactive DNA precursors were added to exponentially growing cultures. The determination of total acid-insoluble radioactivity was similar to the batch procedure described by Ingram and Fisher (12). Triplicate samples (0.1 ml) were removed with an Eppendorf micropipette and deposited on Whatman
156
RESTAINO AND FRAMPTON
no. 3 filter paper disks (2.1-cm diameter). These disks were washed three times for 15 min in cold trichloroacetic acid (5, 10, and 5% wt/vol) and then three times in 95% ethanol. The disks were dried at 60 C for 1 h, added to vials containing a solution of 5 ml of Liquifluor in toluene, and counted in a Beckman LS-230 liquid scintillation counter. Experiments involving [2- 14C Ithymine incorporation employed paper disks, previously washed in 5% trichloroacetic acid for 24 h and dried in order to lower the nonspecific binding of radioactive thymine. The fraction of radioactivity incorp'orated into DNA was determined by removing any RNA present by alkaline hydrolysis. The disks were incubated in 1 N NaOH for a minimum of 4 h (27) before subjecting them to the trichloroacetic acid-ethanol sequence. Resolution of small circular DNA. Resolution of small circular DNA was accomplished by a slight modification of the procedures used by Bazarel and Helinski (3). Briefly, 7.5 ml of radioactive cells (absorbancy= 0.569) were harvested by centrifugation (5,000 x g for 15 min) and washed twice in TES buffer [0.05 M NaCl, 0.005 M ethylenediaminetetraacetate, and 0.05 tris(hydroxymethyl)aminomethane, pH 8.0]. The pellet was suspended in a 1.0-ml solution containing 1.0 mg of lysozyme, 500 Ag of ribonuclease, which had been boiled 10 min to inactivate any contaminating deoxyribonuclease activity, and 100 mg of sucrose in TES buffer, and incubated for 10 min at 37 C. Chilling in an ice bath for 5 min preceded the addition of 0.5 ml of a 2% (wt/vol) solution of sodium dodecyl sarcosinate. After mixing the cells by drawing the solution up and down in a 1-ml pipette, additions were made of 0.5 ml of TES buffer and 0.5 ml of a stock solution of Pronase (1 mg/ml) which had been previously incubated at 37 C for 2 h. After shearing, the lysate (2.0 ml) was added to a vial containing 13.1 g of CsCl, 7.6 ml of glass-distilled water, and 4.0 ml of an ethidium bromide stock solution (700 Mg/ml in 0.1 M sodium phosphate buffer, pH 7.0). After thoroughly mixing the contents of the vial, equal portions were transferred to two polyallomer tubes (% inch diameter by 3 inch [1.6 by 7.6 cm]). The space above the CsCl was filled with paraffin oil before capping. Using a type 65 fixed-angle rotor, centrifugation was accomplished in a Beckman L2-65B ultracentrifuge at 82,663 x g (average) for 24 h at 20 C. Fractions (0.3 ml) were collected from the bottom of the gradient tubes. Duplicate 0.1-ml samples were plated on Whatman no. 3 filter paper disks and passed through the trichloroacetic acid-ethanol sequence before counting. Refractive index measurements were made on every fifth fraction. Preparation of cell-free extracts and enzyme determinations. A. nidulans was grown to an absorbancy of 0.233 (2.6 x 107 cells/ml) while E. coli B/r was grown to an absorbancy of 0.3 measured at 420 nm. Cells of both types were harvested by centrifugation (5,000 x g, 15 min) at 4 C. The cell pellets were washed twice in cold saline (0.85% wt/vol) and the final pellet was suspended in 5 or 10 ml of 0.02 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.2, before being disrupted in a French pressure cell at 10,000 lb/in2. Cell debris was removed
J. BACTERIOL. by centrifugation (3,020 x g, 10 min) and the cell-free extracts were collected by decantation. The protein content of each cell extract was determined by the procedure of Lowry et al. (21). Thymidine kinase (EC 2.7.1.21) activities of both cell extracts were determined by a slight modification of the procedure described by Furlong (8). Dithiothreitol (0.02 Mmol) was substituted for mercaptoethanol. Portions (0.18 ml) of the kinase reaction mixtures were transferred to 12 x 75-mm test tubes. Cell-free extracts (0.04 ml) were added to these tubes and incubated at 37 C. After 0, 10, 20, and 35 min, samples (50 Ml) were removed and mixed with 60 Ml of 0.25 M sodium acetate, pH 4.9. Each tube containing a sample was placed immediately in a 95 C water bath for 3 min before being cooled in an ice bath. Duplicate 50-Ml portions of each sample were deposited on separate diethylaminoethyl-cellulose disks (2.1-cm diameter). One disk was subjected to two 15-min ethanol extractions at room temperature while the other disk remained untreated. The disks were dried and counted as described for the filter paper disks. The percentage of [2-14C ]thymidine converted to deoxythymidine 5'-monophosphate (dTMP) was calculated as described by Furlong (8). Thymidine phosphorylase (EC 2.4.2.4) was determined by the method described by Kammen (17). This method measured the conversion of thymidine to thymine by the change in absorbancy at 300 nm and was read in a Beckman DB-G spectrophotometer. Search for thymineless mutants. Search procedures for thymineless mutants followed closely the methods described by Stacey and Simson (24) and by Little and Hanawalt (20). The mutagen, N-methyl-Nnitrosoguanidine, was utilized at a concentration of 25 Mg/ml (11). Aminopterin and thymine were incorporated into the agar at respective concentrations of 10 and 100 Mg/ml. Chemicals. All radioactive compounds and the Liquifluor were purchased from New England Nuclear Corp., Boston, Mass. Sodium dodecyl sarcosinate (Sarkosyl NL 97) was a product of Geigy Industrial Chemicals, Ardsley, N.Y. N-methyl-Nnitrosoguanidine was a product of Aldrich Chemical Co., Milwaukee, Wis. Aminopterin and thymine were purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Cesium chloride (optical grade) was purchased from Harshaw Chemical Company, Cleveland, Ohio. Deoxythymidine monophosphate, deoxyadenosine, deoxyguanosine, thymidine, ribonuclease, Pronase, lysozyme, trimethoprim, and ethidium bromide were purchased from Calbiochem, San Diego, Calif.
RESULTS Thymidine kinase and thymidine phosphorylase. Crude extracts of A. nidulans were examined for the presence of thymidine phosphorylase and thymidine kinase activities. Neither thymidine phosphorylase nor thymidine kinase activities were detected in crude extracts of A. nidulans, whereas cell-free extracts of E. coli B/r grown in a minimal medium demon-
DNA LABELING IN A. NIDULANS
VoL 124, 1975
157
porated radioactivity, 94% or more of it was localized in the DNA as evidenced by counts resistant to hydrolysis with NaOH. Characterization of the DNA labeled by deoxyadenosine. An analysis of the DNA labeled by the incorporation of radioactive deoxyadenosine is shown in Fig. 1. Two peaks were resolved on CsCl-ethidium bromide gradients. The minor peak (density 1.612 g/cm3) represents the small circular (plasmid) DNA whereas the larger and less dense major peak of radioactivity (density 1.583 g/cm') represents the principal DNA of the A. nidulans genome (2). Effects of deoxyadenosine on growth and viability. There was a pronounced effect of deoxyadenosine on the growth and viability of A. nidulans over a wide concentration range (2 to 200 ug/ml). For purposes of brevity, only the effects of moderately high levels (25 to 75 ,g/ml) (Fig. 2) and the lowest levels (2 to 5 ug/ml) (Fig. 3) are presented here. Although not as dramatic as the levels above 100 gg/ml, the early influences of moderately high levels of deoxyadenoTABLE 1. Thymidine phosphorylase and thymidine sine on cell viability are apparent (Fig. 2B). At all concentrations tested, culture absorbancies kinase activities continued to increase but usually at reduced Enzyme activities rates (Fig. 2A). (nmol/min per mg of protein) At the lowest concentrations (2, 4, and 5 Cell-free extractsa ug/ml) of deoxyadenosine tested, absorbancy Thymidine Thymidine kinase phosphorylase increases were depressed only slightly (Fig. 3A), whereas viable counts declined between 6 and 0.0 0.0 Aracystis nidulans 10.5 h in the presence of either 4 or 5 ug/ml (Fig. A. nidulans (boiled) 0.0 0.0 In the presence of 2 Ag of deoxyadenosine 3B). 54.7 21.3 coli Escherichia absorbancy increases were unaffected ml, per E. coli (boiled) 0.0 0.0 for 23 h although the viable count decreased a E. coli and A. nidulans extracts contained 1.29 mg between 10.5 and 23 h (Fig. 3A and B). and 0.65 mg of protein per ml, respectively. Search for thymine requiring mutants. All
strated readily discernible levels of these enzymes (Table 1). Incorporation of exogenous DNA precursors. A summary of the incorporation of radioactive DNA precursors is presented in Table 2. Deoxyguanosine (250 ,gg/ml) did not influence the incorporation of thymjne or thymidine into either the total nucleic acid fractions (RNA and DNA) or the DNA alone. Similarly, dTMP alone was not incorporated efficiently into either the total nucleic acid fraction or the DNA. More thymine appeared to be incorporated into the total nucleic acid fraction than either thymidine or dTMP. In contrast, radioactive deoxyadenosine was incorporated extensively into the DNA of A. nidulans while deoxyguanosine, a chemically related purine deoxyribonucleoside, was not incorporated effectively. After 12 h, 3.59% of the exogenous [8- "C ]deoxyadenosine present in the medium at the time labeling was initiated was recovered in the total nucleic acid fraction. Of this incor-
TABLE 2. Precursor incorporation Precursora
[2- 1C Jthymine [2- "IC ]thymine + deoxyguanosine [2- 'IC0]thymidine [2-14ITthymidine + deoxyguanosine [2- ITC dTMP [8- 14C deoxyguanosine [8- IC Ideoxyadenosine
,ie (h)
Absorbancyc (520 nm)
RNA + DNA
DNA
22.0 22.0
0.770 0.743
0.116 0.090
0.016 0.013
20.5
0.697
0.029
0.011
20.5
0.688
0.026
0.018
21.0 10.0
0.350
0.028
0.009
0.242 0.484
0.009 3.587
0.005 3.430
Timeb
12.0
change
% Incorporationd
a Speciflc activities: [2-"4C Jthymine (0.307 gCi/6.75 jig per ml); [2- IC ]thymidine (0.2401sCi/12.7 pg per ml); [2-l4C JdTMP (0.244 MCi/14.1 gg per ml); [8-"4CIdeoxyguanosine (2.15 gCi/3.0 Ag per ml); [8-14C ]deoxyadeno-
sine (0.215 ;Ci/3.0 ;g per ml). b Length of exposure to radioactive precursor. c Net change in absorbancy during exposure to the precursor. I Calculated from total radioactivity (counts per minute) present in medium at beginning to acid-insoluble radioactivity in cells at end of exposure period.
158
RESTAINO AND FRAMPTON
J . BACTrERIOL.
dine efficiently into an acid-insoluble fraction (22). We have found that about 90% of the radioactivity incorporated by A. nidulans protoplasts as [2- 4"C ]thymidine is located in the RNA rather than the DNA (unpublished data). This observation supports the data of Glaser et al. (9) who reported that thymine and thymidine are demethylated by intact cells of A. nidulans and are not converted to DNA via a direct pathway. On the basis of this and other observations, Glaser et al. (9) speculated that thymidine phosphorylase and thymidine kinase might be missing in A. nidulans. Thymidine incorporation by A. nidulans protoplasts, therefore, resembles incorporation in certain of the eukary-
z
0 0: LI
FRACTION NUMBER
FIG. 1. Labeling of the small circular DNA by [8- "C]deoxyadenosine. Cultures of A. nidulans were labeled with [8-14C]deoxyadenosine (0.215 jiCi/3.0 ug per ml) by growth for 2.5 to 3 generations in BG-11 medium. Symbols: (0) Density (g/cm') of various fractions; (0) acid-insoluble radioactivity for each fraction.
I
attempts to isolate mutants requiring thymine were unsuccessful. Aminopterin at 25 ug/ml inhibited growth of A. nidulans completely, whereas trimethoprim had no effect on growth at concentrations of 100 ,g/ml. Standard replicating procedures failed to recover any thymine auxotrophs in more than 1,400 clones that were
selected.
TIME (HR)
DISCUSSION The results showing that both thymidine phosphorylase and thymidine kinase activities are absent in A. nidulans (Table 1) are consistent with the observations that the cells are (i) labeled ineffectively by either thymine or thymidine, (ii) that the incorporation of these precursors is not enhanced by addition of a deoxyribonucleoside, and (iii) that thymine auxotrophs could not be isolated whereas auxotrophs of other types (11, 13) have been documented. Complicating the study of nucleic acid precursor incorporation in A. nidulans, however, is the apparent impermeability of this organism to some of these compounds (22). Comparing the incorporation of precursors by A. nidulans protoplasts and by intact cells, Pigott and Carr (22) concluded that the cell wall was partially responsible for the impermeability of this organism to certain nucleic acid precursors. These investigators found that only A. nidulans protoplasts incorporate [2- 4C ]thymi-
JK (A
oi
TIME IHR)
FIG. 2. Effect of deoxyadenosine on the growth and viability of A. nidulans. Symbols: (-) 0 1g/ml; (A) 75 Ag/ml; (0) 50 ug/ml; (A) 25 ,g/ml.
DNA LABELING IN A. NIDULANS
VOL. 124, 1975
A
Is
TIME (MR)
I
Di-i
-i I., 0 w
J
sla 010
0
4
8
12
l6
20
24
TIME (HR)
FIG. 3. Effect of deoxyadenosine on the growth and viability of A. nidulans. Symbols: (a)O tg/ml; (A) 5 ,pg/m I; (O) 4 glgm I; (A) 2 Aglgml.
otic organisms such as Neurospora crassa. This organism lacks thymidine kinase and incorporates about 10 times more radioactivity into its RNA than into the DNA when furnished with [2-14C Ithymidine (10).
Although the growth of certain bacteria, such E. coli strain B, are apparently unaffected by exogenous deoxyadenosine (4), other bacteria are sensitive to its presence. Lark (19) first observed that deoxyadenosine concentrations greater than 10- M blocked DNA synthesis specifically in the bacterium Alcaligenes fecalis. The growth of Neisseria meningitidis is inhibited strongly by deoxyadenosine (500 ug/ml) as
159
(14), but lower concentrations of radioactive deoxyadenosine are incorporated into an acidinsoluble component by this organism (18). Deoxycytidine, deoxyuridine, thymine, thymidine, and dTMP, however, are not incorporated efficiently by N. meningitidis (14, 15, 18). Similar to our findings with A. nidulans, the growth rate of this bacterium is unaffected by deoxyguanosine (500 jg/ml) and it lacks the enzymes thymidine phosphorylase and thymidine kinase (14, 15). In A. quadruplicatum, DNA synthesis is also inhibited specifically by deoxyadenosine addition, and DNA degradation occurs at concentrations exceeding 10 ug/ml (12). Possible biochemical mechanisms involved in these effects in A. quadruplicatum were discussed by Ingram and Fisher (12) and presumably are operating in A. nidulans as well. The viability studies (Fig. 2B and 3B) stress the fact that absorbancy measurements alone are an unreliable indicator of deoxyadenosine toxicity in A. nidulans. The highly specific labeling of A. nidulans DNA by deoxyadenosine (Table 2) can be contrasted with [2- 4C ]uracil incorporation where 92% of the isotope incorporated resides in the RNA (9). This highly specific labeling of DNA by deoxyadenosine can be interpreted as additional evidence that thymidine phosphorylase activity is absent in A. nidulans. The labeling of the plasmid DNA by deoxyadenosine (Fig. 1) produced results similar to those obtained by Asato and Ginoza (2) who used 38phosphorus to label the DNA. Thus, it should be possible to study the plasmids of A. nidulans and other organisms using the incorporation of low levels of isotopically labeled deoxyadenosine. Initial efforts to minimize deoxyadenosine toxicity seem promising. Readily discernible effects on cell viability by low levels (2,ug/ml) of deoxyadenosine were not observed during the first 10.5 h of incorporation (Fig. 3B). In an experiment not included here, when deoxyadenosine (2 ug/ml) was removed after 4 h, the subsequent viability of the culture was unaffected. The possibility of extending this exposure period beyond 4 h is being investigated currently in this laboratory. In contrast to the toxicity resulting from deoxyadenosine addition, we found that relatively high levels of deoxyguanosine (250 sg/ml) were without effect on the growth and viability of A. nidulans. Thus, instead of using deoxyadenosine, as Glaser et al. (9) reported, we used deoxyguanosine to test the possible enhancement of thymine and thymidine incorporation by a deoxyribonucleoside (Table 2). Using an auxanographic assay procedure, A. quadruplicatum was reported to be moderately inhib-
160
RESTAINO AND FRAMPTON
ited by deoxyguanosine (12). The two cyanobacteria may respond differently to this compound, however, we also found that radioactive deoxyguanosine was not incorporated efficiently by A. nidulans (Table 2). Thus, A. nidulans may be either impermeable to deoxyguanosine or lack enzymes required for its conversion into a DNA precursor pool. Guanosine incorporation has been reported to be sensitive to a permeability barrier in intact cells of A. nidulans (22). A continued investigation of the incorporation by protoplasts of such compounds as deoxyguanosine and dTMP should resolve questions relating to the permeability of these and other substances. The isolation of a permeability mutant of A. nidulans would facilitate studies of this kind and possibly provide additional means for labeling the DNA of A. nidulans and other cyanobacteria. ACKNOWLEDGMENTS This investigation was supported by institutional funds from Northern Illinois University. This paper is number 523 from the Department of Biological Sciences. LITERATURE CITD 1. Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:1-4. 2. Asato, Y., and H. S. Ginoza. 1973. Separation of small circular DNA molecules from the blue-green alga Anacystis nidulans. Nature (London) New Biol. 244:133-134. 3. Bazarel, M., and D. R. Helinski. 1968. Circular DNA forms of colicinogenic factors El, E2, and E3 from Escherichia coli. J. Mol. Biol. 36:185-194. 4. Boyce, R. P., and R. B. Setlow. 1962. A simple method of increasing the incorporation of thymidine into the deoxyribonucleic acid of Escherichia coli. Biochim. Biophys? Acta 61:618-620. 5. Breitman, T. R., R. M. Bradford, and W. D. Cannon, Jr. 1967. Use of exogenous deoxythymidylic acid to label the deoxyribonucleic acid of growing wild-type Escherichia coli. J. Bacteriol. 93:1471-1472. 6. Budman, D. R., and A. B. Pardee. 1967. Thymidine and thymine incorporation into deoxyribonucleic acid; inhibition and repression by uridine of thymidine phosphorylase of Escherichia coli. J. Bacteriol. 94:15461550. 7. Friesen, J. D. 1968. Measurement of DNA synthesis in bacterial cells, 625-643. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 12. Academic Press Inc., New York. 8. Furlong, N. B. 1963. A rapid assay for nucleotide kinases using Cl'- or H'-labelled nucleotides. Anal. Biochem. 5:515-522. 9. Glaser, V. M., M. A. Al-Nuri, V. V. Groshev, and S. V. Shestakov. 1973. The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv. Arch. Mikrobiol. 92:217-226.
J. BAScTRioL 10. Grivell, A. R., and J. F. Jackson. 1968. Thymidine kinase: evidence for its absence from Neurospora crassa and some other micro-organisms, and the relevance of this to the specific labelling of deoxyribonucleic acid. J. Gen. Microbiol. 64:307-317. 11. Herdman, M., and N. G. Canf. 1972. The isolation and characterization of mutant strains of the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 70:213-220. 12. Ingram, L. O., and W. D. Fisher. 1972. Selective inhibition of deoxyribonucleic acid synthesis by 2-deoxyadenosine in the blue-green bacterium Agmenellum quadruplicatum. J. Bacteriol. 112:170-175. 13. Ingram, L. O., D. Pierson, J. F. Kane, C. Van Baalen, and R. A. Jensen. 1972. Documentation of auxotrophic mutation in blue-green bacteria: characterization of a tryptophan auxotroph in Agmenellum quadruplicatum. J. Bacteriol. 111:112-118. 14. Jyssum, S. 1971. Utilization of thymine, thymidine and TMP by Neisseria meningitidis. 2. Lack of enzymes for specific incorporation of exogenous thymine, thymidine and TMP into DNA. Acta. Pathol. Microbiol. Scand. Sect. B: 79:778-788. 15. Jyssum, S., and K. Jyssum. 1970. Utilization of thymine, thymidine and TMP by Neisseria meningitidis. 1. Growth response and uptake of labelled material. Acta Pathol. Microbiol. Scand. Sect. B 78:683-691. 16. Kaiser, G., D. L. Lynch, M. J. Starzyk, and M. G. Fenwick. 1968. The effect of temperature and vitamin B1, on the growth of Coelastrum microporum naeg. isolates. Trans. Ill. State Acad. Sci. 61:428-429. 17. Kammen, H. 0. 19.67. Thymine metabolism in Escherichia coli. 1. Factors involved in utilization of exogenous thymine. Biochim. Biophys. Acta 134:301-311. 18. Kingsbury, D. T., and J. F. Duncan. 1967. Use of exogenous adenine to label the nucleic acids of wildtype Neisseria meningitidis. J. Bacteriol. 94:1262-1263. 19. Lark, K. G. 1960. Studies on the mechanism regulating periodic DNA synthesis in synchronized cultures of Alcaligenes fecalis. Biochim. Biophys. Acta 45:121132. 20. Little, J. G., and P. C. Hanawalt. 1973. Thymineless death and ultraviolet sensitivity in Micrococcus radiodurans. J. Bacteriol. 113:233-240. 21. Lowry, 0. H., N. J. Roseborough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 22. Pigott, G. H., and N. G. Caff. 1971. The assimilation of nucleic acid precursors by intact cells and protoplasts of the blue-green alga Anacystis nidulans. Arch. Mikrobiol. 79:1-6. 23. Roberts, R. B., P. H. Abelson, D. B. Bolton, E. F. Cowie, and R. J. Britten. 1955. Studies of biosynthesis in Escherichia coli. Carnegie Inst. Washington Publ. No. 607. 24. Stacey, K. A., and E. Simson. 1965. Improved method for the isolation of thymine-requiring mutants of Escherichia coli. J. Bacteriol. 90:544-555. 25. Stanier, R. Y., R. Kunisawa, M. Mandel, and G. CoherkBazire. 1971. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol. Rev. 35:171-205. 26. Starr, R. C. 1960. The culture collection of algae at Indiana University. Am. J. Botany. 47:67-86. 27. Watson, R., and H. Yamazaki. 1973. Alkali hydrolysis of RNA of Escherichia coli deposited on filter paper disks. Anal. Biochem. 51:312-314.
JOURNAL OF BACTERIOLOGY, Oct. 1975, p. 155-160
Copyright 0 1975 American Society for Microbiology
Labeling the Deoxyribonucleic Acid of Anacystis nidulans L. RESTAINO
AND
E. W. FRAMPTON*
Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115
Received for publication 4 April 1975
Analysis of cell-free extracts of Anacystis nidulans disclosed the absence of both thymidine phosphorylase (EC 2.4.2.4) and thymidine kinase (EC 2.7.1.21) activities. Thymine and thymidine were incorporated inefficiently by intact cells of A. nidulans either in the presence or absence of deoxyguanosine (250 jg/ml). Deoxythymidine monophosphate incorporation was also inefficient. Radioactive deoxyadenosine, at a minimally toxic level (3 jg/ml), was incorporated effectively into the deoxyribonucleic acid (DNA). A cesium chloride-ethidium bromide gradient analysis of the DNA revealed that both the plasmid DNA and the principal DNA of the A. nidulans genome were labeled effectively in cells exposed to [8- l4C Ideoxyadenosine.
The photoautotrophic prokaryotic organisms known traditionally as the blue-green algae (the cyanobacteria) are subjects of increasing scientific interest. Experimentally, however, it has been difficult to label cyanobacterial deoxyribonucleic acid (DNA) even though these organisms can incorporate significant quantities of certain organic compounds into cellular components. Intact cells of Anacystis nidulans incorporate thymine or thymidine inefficiently, although uracil is incorporated into both DNA and ribonucleic acid (RNA) (9, 22). Recently, Ingram and Fisher (12) described the effective incorporation of low levels of deoxyadenosine, an inhibitor of DNA synthesis at higher levels, into the DNA of the marine cyanobacterium, Agmenellum quadruplicatum. In this investigation we have studied (i) whether some of the methods used to enhance thymine and thymidine incorporation and to label Escherichia coli DNA (4-7, 17, 18, 24) could be applied to A. nidulans, (ii) the enzymatic capacities of A. nidularns relative to thymine and thymidine utilization, and (iii) the labeling of A. nidulans DNA using deoxyadenosine. Implications of our finding that thymidine phosphorylase (EC 2.4.2.1) and thymidine kinase (EC 2.7.1.21) activities are absent in A. nidulans cell extracts are discussed in relation to DNA labeling and permeability problems (22) in this organism. MATERIALS AND METHODS Organisms. A. nidulans strain 625 was obtained from the Algae Culture Collection at Indiana University, Bloomington, Ind. E. coli strain B/r was used for controlling the enzyme assays. 155
Media and growth conditions. A. nidulans was grown in BG-11 medium (1, 25) while stock cultures were transferred routinely on Cyanophycean agar (26). A. nidulans was enumerated on agar prepared from double-strength BG-11 medium combined with an equal volume of 3% (wt/vol) agar (Difco) each autoclaved separately and cooled to 55 C before mixing (1). For most experiments, 100-ml volumes of A. nidulans were grown in 250-ml Erlenmeyer flasks stirred with Teflon-covered magnetic stirring bars (2 by X inch [ca. 5.08 by 0.953 cm]). Radioactivity incorporation experiments utilized 20-ml volumes in 50-ml Erlenmeyer flasks stirred by smaller bars (1 in. by % in. [ca. 2.54 by 0.953 cm]). Incubation was accomplished at 39 C under a continuous illumination of 5,000 lux provided by four Cool-White fluorescent lamps (Ken Rad) in a Controlled Environments model E7 environmental chamber. Doubling time under these conditions was about 4.5 h and was monitored by measuring absorbancy increases in a Bausch and Lomb Spectronic-20 colorimeter (3/4-inch [1.91 cm] diameter tube) at 520 nm (16). Bacterial contamination was checked by routinely streaking cultures of A. nidulans on nutrient agar plates which were incubated at 37 C for 48 h. E. coli was grown in C medium (23) at 37 C with forced aeration. Growth experiments with deoxyribonucleosides. Both deoxyadenosine and deoxyguanosine dissolved in BG-11 medium were sterilized by filtration through membrane filters (Schleicher and Schuell, type B-6). For viable cell counts, final dilutions were spread in triplicate on the surface of BG-11 agar plates which were sealed with cellophane tape before incubation for 5 days. Labeling and measurement of radioactivity. Radioactive DNA precursors were added to exponentially growing cultures. The determination of total acid-insoluble radioactivity was similar to the batch procedure described by Ingram and Fisher (12). Triplicate samples (0.1 ml) were removed with an Eppendorf micropipette and deposited on Whatman
156
RESTAINO AND FRAMPTON
no. 3 filter paper disks (2.1-cm diameter). These disks were washed three times for 15 min in cold trichloroacetic acid (5, 10, and 5% wt/vol) and then three times in 95% ethanol. The disks were dried at 60 C for 1 h, added to vials containing a solution of 5 ml of Liquifluor in toluene, and counted in a Beckman LS-230 liquid scintillation counter. Experiments involving [2- 14C Ithymine incorporation employed paper disks, previously washed in 5% trichloroacetic acid for 24 h and dried in order to lower the nonspecific binding of radioactive thymine. The fraction of radioactivity incorp'orated into DNA was determined by removing any RNA present by alkaline hydrolysis. The disks were incubated in 1 N NaOH for a minimum of 4 h (27) before subjecting them to the trichloroacetic acid-ethanol sequence. Resolution of small circular DNA. Resolution of small circular DNA was accomplished by a slight modification of the procedures used by Bazarel and Helinski (3). Briefly, 7.5 ml of radioactive cells (absorbancy= 0.569) were harvested by centrifugation (5,000 x g for 15 min) and washed twice in TES buffer [0.05 M NaCl, 0.005 M ethylenediaminetetraacetate, and 0.05 tris(hydroxymethyl)aminomethane, pH 8.0]. The pellet was suspended in a 1.0-ml solution containing 1.0 mg of lysozyme, 500 Ag of ribonuclease, which had been boiled 10 min to inactivate any contaminating deoxyribonuclease activity, and 100 mg of sucrose in TES buffer, and incubated for 10 min at 37 C. Chilling in an ice bath for 5 min preceded the addition of 0.5 ml of a 2% (wt/vol) solution of sodium dodecyl sarcosinate. After mixing the cells by drawing the solution up and down in a 1-ml pipette, additions were made of 0.5 ml of TES buffer and 0.5 ml of a stock solution of Pronase (1 mg/ml) which had been previously incubated at 37 C for 2 h. After shearing, the lysate (2.0 ml) was added to a vial containing 13.1 g of CsCl, 7.6 ml of glass-distilled water, and 4.0 ml of an ethidium bromide stock solution (700 Mg/ml in 0.1 M sodium phosphate buffer, pH 7.0). After thoroughly mixing the contents of the vial, equal portions were transferred to two polyallomer tubes (% inch diameter by 3 inch [1.6 by 7.6 cm]). The space above the CsCl was filled with paraffin oil before capping. Using a type 65 fixed-angle rotor, centrifugation was accomplished in a Beckman L2-65B ultracentrifuge at 82,663 x g (average) for 24 h at 20 C. Fractions (0.3 ml) were collected from the bottom of the gradient tubes. Duplicate 0.1-ml samples were plated on Whatman no. 3 filter paper disks and passed through the trichloroacetic acid-ethanol sequence before counting. Refractive index measurements were made on every fifth fraction. Preparation of cell-free extracts and enzyme determinations. A. nidulans was grown to an absorbancy of 0.233 (2.6 x 107 cells/ml) while E. coli B/r was grown to an absorbancy of 0.3 measured at 420 nm. Cells of both types were harvested by centrifugation (5,000 x g, 15 min) at 4 C. The cell pellets were washed twice in cold saline (0.85% wt/vol) and the final pellet was suspended in 5 or 10 ml of 0.02 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.2, before being disrupted in a French pressure cell at 10,000 lb/in2. Cell debris was removed
J. BACTERIOL. by centrifugation (3,020 x g, 10 min) and the cell-free extracts were collected by decantation. The protein content of each cell extract was determined by the procedure of Lowry et al. (21). Thymidine kinase (EC 2.7.1.21) activities of both cell extracts were determined by a slight modification of the procedure described by Furlong (8). Dithiothreitol (0.02 Mmol) was substituted for mercaptoethanol. Portions (0.18 ml) of the kinase reaction mixtures were transferred to 12 x 75-mm test tubes. Cell-free extracts (0.04 ml) were added to these tubes and incubated at 37 C. After 0, 10, 20, and 35 min, samples (50 Ml) were removed and mixed with 60 Ml of 0.25 M sodium acetate, pH 4.9. Each tube containing a sample was placed immediately in a 95 C water bath for 3 min before being cooled in an ice bath. Duplicate 50-Ml portions of each sample were deposited on separate diethylaminoethyl-cellulose disks (2.1-cm diameter). One disk was subjected to two 15-min ethanol extractions at room temperature while the other disk remained untreated. The disks were dried and counted as described for the filter paper disks. The percentage of [2-14C ]thymidine converted to deoxythymidine 5'-monophosphate (dTMP) was calculated as described by Furlong (8). Thymidine phosphorylase (EC 2.4.2.4) was determined by the method described by Kammen (17). This method measured the conversion of thymidine to thymine by the change in absorbancy at 300 nm and was read in a Beckman DB-G spectrophotometer. Search for thymineless mutants. Search procedures for thymineless mutants followed closely the methods described by Stacey and Simson (24) and by Little and Hanawalt (20). The mutagen, N-methyl-Nnitrosoguanidine, was utilized at a concentration of 25 Mg/ml (11). Aminopterin and thymine were incorporated into the agar at respective concentrations of 10 and 100 Mg/ml. Chemicals. All radioactive compounds and the Liquifluor were purchased from New England Nuclear Corp., Boston, Mass. Sodium dodecyl sarcosinate (Sarkosyl NL 97) was a product of Geigy Industrial Chemicals, Ardsley, N.Y. N-methyl-Nnitrosoguanidine was a product of Aldrich Chemical Co., Milwaukee, Wis. Aminopterin and thymine were purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Cesium chloride (optical grade) was purchased from Harshaw Chemical Company, Cleveland, Ohio. Deoxythymidine monophosphate, deoxyadenosine, deoxyguanosine, thymidine, ribonuclease, Pronase, lysozyme, trimethoprim, and ethidium bromide were purchased from Calbiochem, San Diego, Calif.
RESULTS Thymidine kinase and thymidine phosphorylase. Crude extracts of A. nidulans were examined for the presence of thymidine phosphorylase and thymidine kinase activities. Neither thymidine phosphorylase nor thymidine kinase activities were detected in crude extracts of A. nidulans, whereas cell-free extracts of E. coli B/r grown in a minimal medium demon-
DNA LABELING IN A. NIDULANS
VoL 124, 1975
157
porated radioactivity, 94% or more of it was localized in the DNA as evidenced by counts resistant to hydrolysis with NaOH. Characterization of the DNA labeled by deoxyadenosine. An analysis of the DNA labeled by the incorporation of radioactive deoxyadenosine is shown in Fig. 1. Two peaks were resolved on CsCl-ethidium bromide gradients. The minor peak (density 1.612 g/cm3) represents the small circular (plasmid) DNA whereas the larger and less dense major peak of radioactivity (density 1.583 g/cm') represents the principal DNA of the A. nidulans genome (2). Effects of deoxyadenosine on growth and viability. There was a pronounced effect of deoxyadenosine on the growth and viability of A. nidulans over a wide concentration range (2 to 200 ug/ml). For purposes of brevity, only the effects of moderately high levels (25 to 75 ,g/ml) (Fig. 2) and the lowest levels (2 to 5 ug/ml) (Fig. 3) are presented here. Although not as dramatic as the levels above 100 gg/ml, the early influences of moderately high levels of deoxyadenoTABLE 1. Thymidine phosphorylase and thymidine sine on cell viability are apparent (Fig. 2B). At all concentrations tested, culture absorbancies kinase activities continued to increase but usually at reduced Enzyme activities rates (Fig. 2A). (nmol/min per mg of protein) At the lowest concentrations (2, 4, and 5 Cell-free extractsa ug/ml) of deoxyadenosine tested, absorbancy Thymidine Thymidine kinase phosphorylase increases were depressed only slightly (Fig. 3A), whereas viable counts declined between 6 and 0.0 0.0 Aracystis nidulans 10.5 h in the presence of either 4 or 5 ug/ml (Fig. A. nidulans (boiled) 0.0 0.0 In the presence of 2 Ag of deoxyadenosine 3B). 54.7 21.3 coli Escherichia absorbancy increases were unaffected ml, per E. coli (boiled) 0.0 0.0 for 23 h although the viable count decreased a E. coli and A. nidulans extracts contained 1.29 mg between 10.5 and 23 h (Fig. 3A and B). and 0.65 mg of protein per ml, respectively. Search for thymine requiring mutants. All
strated readily discernible levels of these enzymes (Table 1). Incorporation of exogenous DNA precursors. A summary of the incorporation of radioactive DNA precursors is presented in Table 2. Deoxyguanosine (250 ,gg/ml) did not influence the incorporation of thymjne or thymidine into either the total nucleic acid fractions (RNA and DNA) or the DNA alone. Similarly, dTMP alone was not incorporated efficiently into either the total nucleic acid fraction or the DNA. More thymine appeared to be incorporated into the total nucleic acid fraction than either thymidine or dTMP. In contrast, radioactive deoxyadenosine was incorporated extensively into the DNA of A. nidulans while deoxyguanosine, a chemically related purine deoxyribonucleoside, was not incorporated effectively. After 12 h, 3.59% of the exogenous [8- "C ]deoxyadenosine present in the medium at the time labeling was initiated was recovered in the total nucleic acid fraction. Of this incor-
TABLE 2. Precursor incorporation Precursora
[2- 1C Jthymine [2- "IC ]thymine + deoxyguanosine [2- 'IC0]thymidine [2-14ITthymidine + deoxyguanosine [2- ITC dTMP [8- 14C deoxyguanosine [8- IC Ideoxyadenosine
,ie (h)
Absorbancyc (520 nm)
RNA + DNA
DNA
22.0 22.0
0.770 0.743
0.116 0.090
0.016 0.013
20.5
0.697
0.029
0.011
20.5
0.688
0.026
0.018
21.0 10.0
0.350
0.028
0.009
0.242 0.484
0.009 3.587
0.005 3.430
Timeb
12.0
change
% Incorporationd
a Speciflc activities: [2-"4C Jthymine (0.307 gCi/6.75 jig per ml); [2- IC ]thymidine (0.2401sCi/12.7 pg per ml); [2-l4C JdTMP (0.244 MCi/14.1 gg per ml); [8-"4CIdeoxyguanosine (2.15 gCi/3.0 Ag per ml); [8-14C ]deoxyadeno-
sine (0.215 ;Ci/3.0 ;g per ml). b Length of exposure to radioactive precursor. c Net change in absorbancy during exposure to the precursor. I Calculated from total radioactivity (counts per minute) present in medium at beginning to acid-insoluble radioactivity in cells at end of exposure period.
158
RESTAINO AND FRAMPTON
J . BACTrERIOL.
dine efficiently into an acid-insoluble fraction (22). We have found that about 90% of the radioactivity incorporated by A. nidulans protoplasts as [2- 4"C ]thymidine is located in the RNA rather than the DNA (unpublished data). This observation supports the data of Glaser et al. (9) who reported that thymine and thymidine are demethylated by intact cells of A. nidulans and are not converted to DNA via a direct pathway. On the basis of this and other observations, Glaser et al. (9) speculated that thymidine phosphorylase and thymidine kinase might be missing in A. nidulans. Thymidine incorporation by A. nidulans protoplasts, therefore, resembles incorporation in certain of the eukary-
z
0 0: LI
FRACTION NUMBER
FIG. 1. Labeling of the small circular DNA by [8- "C]deoxyadenosine. Cultures of A. nidulans were labeled with [8-14C]deoxyadenosine (0.215 jiCi/3.0 ug per ml) by growth for 2.5 to 3 generations in BG-11 medium. Symbols: (0) Density (g/cm') of various fractions; (0) acid-insoluble radioactivity for each fraction.
I
attempts to isolate mutants requiring thymine were unsuccessful. Aminopterin at 25 ug/ml inhibited growth of A. nidulans completely, whereas trimethoprim had no effect on growth at concentrations of 100 ,g/ml. Standard replicating procedures failed to recover any thymine auxotrophs in more than 1,400 clones that were
selected.
TIME (HR)
DISCUSSION The results showing that both thymidine phosphorylase and thymidine kinase activities are absent in A. nidulans (Table 1) are consistent with the observations that the cells are (i) labeled ineffectively by either thymine or thymidine, (ii) that the incorporation of these precursors is not enhanced by addition of a deoxyribonucleoside, and (iii) that thymine auxotrophs could not be isolated whereas auxotrophs of other types (11, 13) have been documented. Complicating the study of nucleic acid precursor incorporation in A. nidulans, however, is the apparent impermeability of this organism to some of these compounds (22). Comparing the incorporation of precursors by A. nidulans protoplasts and by intact cells, Pigott and Carr (22) concluded that the cell wall was partially responsible for the impermeability of this organism to certain nucleic acid precursors. These investigators found that only A. nidulans protoplasts incorporate [2- 4C ]thymi-
JK (A
oi
TIME IHR)
FIG. 2. Effect of deoxyadenosine on the growth and viability of A. nidulans. Symbols: (-) 0 1g/ml; (A) 75 Ag/ml; (0) 50 ug/ml; (A) 25 ,g/ml.
DNA LABELING IN A. NIDULANS
VOL. 124, 1975
A
Is
TIME (MR)
I
Di-i
-i I., 0 w
J
sla 010
0
4
8
12
l6
20
24
TIME (HR)
FIG. 3. Effect of deoxyadenosine on the growth and viability of A. nidulans. Symbols: (a)O tg/ml; (A) 5 ,pg/m I; (O) 4 glgm I; (A) 2 Aglgml.
otic organisms such as Neurospora crassa. This organism lacks thymidine kinase and incorporates about 10 times more radioactivity into its RNA than into the DNA when furnished with [2-14C Ithymidine (10).
Although the growth of certain bacteria, such E. coli strain B, are apparently unaffected by exogenous deoxyadenosine (4), other bacteria are sensitive to its presence. Lark (19) first observed that deoxyadenosine concentrations greater than 10- M blocked DNA synthesis specifically in the bacterium Alcaligenes fecalis. The growth of Neisseria meningitidis is inhibited strongly by deoxyadenosine (500 ug/ml) as
159
(14), but lower concentrations of radioactive deoxyadenosine are incorporated into an acidinsoluble component by this organism (18). Deoxycytidine, deoxyuridine, thymine, thymidine, and dTMP, however, are not incorporated efficiently by N. meningitidis (14, 15, 18). Similar to our findings with A. nidulans, the growth rate of this bacterium is unaffected by deoxyguanosine (500 jg/ml) and it lacks the enzymes thymidine phosphorylase and thymidine kinase (14, 15). In A. quadruplicatum, DNA synthesis is also inhibited specifically by deoxyadenosine addition, and DNA degradation occurs at concentrations exceeding 10 ug/ml (12). Possible biochemical mechanisms involved in these effects in A. quadruplicatum were discussed by Ingram and Fisher (12) and presumably are operating in A. nidulans as well. The viability studies (Fig. 2B and 3B) stress the fact that absorbancy measurements alone are an unreliable indicator of deoxyadenosine toxicity in A. nidulans. The highly specific labeling of A. nidulans DNA by deoxyadenosine (Table 2) can be contrasted with [2- 4C ]uracil incorporation where 92% of the isotope incorporated resides in the RNA (9). This highly specific labeling of DNA by deoxyadenosine can be interpreted as additional evidence that thymidine phosphorylase activity is absent in A. nidulans. The labeling of the plasmid DNA by deoxyadenosine (Fig. 1) produced results similar to those obtained by Asato and Ginoza (2) who used 38phosphorus to label the DNA. Thus, it should be possible to study the plasmids of A. nidulans and other organisms using the incorporation of low levels of isotopically labeled deoxyadenosine. Initial efforts to minimize deoxyadenosine toxicity seem promising. Readily discernible effects on cell viability by low levels (2,ug/ml) of deoxyadenosine were not observed during the first 10.5 h of incorporation (Fig. 3B). In an experiment not included here, when deoxyadenosine (2 ug/ml) was removed after 4 h, the subsequent viability of the culture was unaffected. The possibility of extending this exposure period beyond 4 h is being investigated currently in this laboratory. In contrast to the toxicity resulting from deoxyadenosine addition, we found that relatively high levels of deoxyguanosine (250 sg/ml) were without effect on the growth and viability of A. nidulans. Thus, instead of using deoxyadenosine, as Glaser et al. (9) reported, we used deoxyguanosine to test the possible enhancement of thymine and thymidine incorporation by a deoxyribonucleoside (Table 2). Using an auxanographic assay procedure, A. quadruplicatum was reported to be moderately inhib-
160
RESTAINO AND FRAMPTON
ited by deoxyguanosine (12). The two cyanobacteria may respond differently to this compound, however, we also found that radioactive deoxyguanosine was not incorporated efficiently by A. nidulans (Table 2). Thus, A. nidulans may be either impermeable to deoxyguanosine or lack enzymes required for its conversion into a DNA precursor pool. Guanosine incorporation has been reported to be sensitive to a permeability barrier in intact cells of A. nidulans (22). A continued investigation of the incorporation by protoplasts of such compounds as deoxyguanosine and dTMP should resolve questions relating to the permeability of these and other substances. The isolation of a permeability mutant of A. nidulans would facilitate studies of this kind and possibly provide additional means for labeling the DNA of A. nidulans and other cyanobacteria. ACKNOWLEDGMENTS This investigation was supported by institutional funds from Northern Illinois University. This paper is number 523 from the Department of Biological Sciences. LITERATURE CITD 1. Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:1-4. 2. Asato, Y., and H. S. Ginoza. 1973. Separation of small circular DNA molecules from the blue-green alga Anacystis nidulans. Nature (London) New Biol. 244:133-134. 3. Bazarel, M., and D. R. Helinski. 1968. Circular DNA forms of colicinogenic factors El, E2, and E3 from Escherichia coli. J. Mol. Biol. 36:185-194. 4. Boyce, R. P., and R. B. Setlow. 1962. A simple method of increasing the incorporation of thymidine into the deoxyribonucleic acid of Escherichia coli. Biochim. Biophys? Acta 61:618-620. 5. Breitman, T. R., R. M. Bradford, and W. D. Cannon, Jr. 1967. Use of exogenous deoxythymidylic acid to label the deoxyribonucleic acid of growing wild-type Escherichia coli. J. Bacteriol. 93:1471-1472. 6. Budman, D. R., and A. B. Pardee. 1967. Thymidine and thymine incorporation into deoxyribonucleic acid; inhibition and repression by uridine of thymidine phosphorylase of Escherichia coli. J. Bacteriol. 94:15461550. 7. Friesen, J. D. 1968. Measurement of DNA synthesis in bacterial cells, 625-643. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 12. Academic Press Inc., New York. 8. Furlong, N. B. 1963. A rapid assay for nucleotide kinases using Cl'- or H'-labelled nucleotides. Anal. Biochem. 5:515-522. 9. Glaser, V. M., M. A. Al-Nuri, V. V. Groshev, and S. V. Shestakov. 1973. The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv. Arch. Mikrobiol. 92:217-226.
J. BAScTRioL 10. Grivell, A. R., and J. F. Jackson. 1968. Thymidine kinase: evidence for its absence from Neurospora crassa and some other micro-organisms, and the relevance of this to the specific labelling of deoxyribonucleic acid. J. Gen. Microbiol. 64:307-317. 11. Herdman, M., and N. G. Canf. 1972. The isolation and characterization of mutant strains of the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 70:213-220. 12. Ingram, L. O., and W. D. Fisher. 1972. Selective inhibition of deoxyribonucleic acid synthesis by 2-deoxyadenosine in the blue-green bacterium Agmenellum quadruplicatum. J. Bacteriol. 112:170-175. 13. Ingram, L. O., D. Pierson, J. F. Kane, C. Van Baalen, and R. A. Jensen. 1972. Documentation of auxotrophic mutation in blue-green bacteria: characterization of a tryptophan auxotroph in Agmenellum quadruplicatum. J. Bacteriol. 111:112-118. 14. Jyssum, S. 1971. Utilization of thymine, thymidine and TMP by Neisseria meningitidis. 2. Lack of enzymes for specific incorporation of exogenous thymine, thymidine and TMP into DNA. Acta. Pathol. Microbiol. Scand. Sect. B: 79:778-788. 15. Jyssum, S., and K. Jyssum. 1970. Utilization of thymine, thymidine and TMP by Neisseria meningitidis. 1. Growth response and uptake of labelled material. Acta Pathol. Microbiol. Scand. Sect. B 78:683-691. 16. Kaiser, G., D. L. Lynch, M. J. Starzyk, and M. G. Fenwick. 1968. The effect of temperature and vitamin B1, on the growth of Coelastrum microporum naeg. isolates. Trans. Ill. State Acad. Sci. 61:428-429. 17. Kammen, H. 0. 19.67. Thymine metabolism in Escherichia coli. 1. Factors involved in utilization of exogenous thymine. Biochim. Biophys. Acta 134:301-311. 18. Kingsbury, D. T., and J. F. Duncan. 1967. Use of exogenous adenine to label the nucleic acids of wildtype Neisseria meningitidis. J. Bacteriol. 94:1262-1263. 19. Lark, K. G. 1960. Studies on the mechanism regulating periodic DNA synthesis in synchronized cultures of Alcaligenes fecalis. Biochim. Biophys. Acta 45:121132. 20. Little, J. G., and P. C. Hanawalt. 1973. Thymineless death and ultraviolet sensitivity in Micrococcus radiodurans. J. Bacteriol. 113:233-240. 21. Lowry, 0. H., N. J. Roseborough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 22. Pigott, G. H., and N. G. Caff. 1971. The assimilation of nucleic acid precursors by intact cells and protoplasts of the blue-green alga Anacystis nidulans. Arch. Mikrobiol. 79:1-6. 23. Roberts, R. B., P. H. Abelson, D. B. Bolton, E. F. Cowie, and R. J. Britten. 1955. Studies of biosynthesis in Escherichia coli. Carnegie Inst. Washington Publ. No. 607. 24. Stacey, K. A., and E. Simson. 1965. Improved method for the isolation of thymine-requiring mutants of Escherichia coli. J. Bacteriol. 90:544-555. 25. Stanier, R. Y., R. Kunisawa, M. Mandel, and G. CoherkBazire. 1971. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol. Rev. 35:171-205. 26. Starr, R. C. 1960. The culture collection of algae at Indiana University. Am. J. Botany. 47:67-86. 27. Watson, R., and H. Yamazaki. 1973. Alkali hydrolysis of RNA of Escherichia coli deposited on filter paper disks. Anal. Biochem. 51:312-314.
