previously1 were used with minor modifications in the studies described

INHIBITION BY CERTAIN PTERIDINES OF RIBOSOMAL RNA AND DNA SYNTHESIS IN DEVELOPING ONCOPELTUS EGGS* BY S. E. HARRIS AND H. S. FORREST GENETICS FOUNDATION, THE UNIVERSITY OF TEXAS, AUSTIN Communicated by Wilson S. Stone, May 15, 1967 In a previous paper, we have shown that during normal development in the egg of the milkweed bug, Oncopeltus fasciatus (Dallas), RNA and DNA synthesis are discontinuous. Most strikingly, with regard to RNA, there is a burst of ribosomal RNA (rrna) synthesis between the 4th and 96th hour of development (at 21), before and after which this synthesis can barely be detected.1 The concentration of the pteridine, xanthopterin (which actually exists in the egg as 7,8-dihydroxanthopterin2), falls throughout development, the major changes taking place at about the same time as rrna synthesis is at its maximal rate. (Xanthopterin has been shown to be converted into the red erythropterin, this change being responsible for the characteristic color change of Oncopeltus eggs from yellow to orange-red.2) In order to test if these two events-ribosomal RNA synthesis and disappearance of xanthopterin-were in any way related, the rate of rrna synthesis in partially dechorionated eggs was measured, using the technique previously described,2 in the presence and in the absence of exogenous xanthopterin. Indeed, in partially dechorionated eggs, xanthopterin, at a concentration of 1-3 l in Ringer's solution, was found to bring about 25 per cent inhibition of the rate of incorporation of radioactive uridine into rrna over the period of maximum rate of synthesis under our conditions. Surprisingly, however, in an experiment originally designed as a control, isoxanthopterin (at the same concentration) was found to be an even better inhibitor; yet, at least by the assay method we have used, isoxanthopterin concentration does not change during development. In a similar manner, both isoxanthopterin and xanthopterin also inhibited the rate of DNA synthesis, but other closely related compounds, e.g., 2-amino-4-hydroxypteridine, 2-amino-4-hydroxy-6-hydroxymethylpteridine, or leucopterin, had essentially no effect on the rate of either DNA or rrna synthesis. Materials and Methods.-M\ethods for assaying RNA and DNA synthesis described previously1 were used with minor modifications in the studies described here. The main difference was that labeling experiments with radioactive precursors had to be carried out at ph 1.5 in order to achieve, and maintain, a reasonable concentration of the pteridines (all of which were of synthetic origin) in solution. Thus eggs (.3 gm) were dechorionated and incubated for 2 hours in 2 ml of insect Ringer's solution at ph 7.; they were collected as described before and placed in a standard incubation medium (1 ml) containing insect Ringer's solution, Tris buffer, ph 9., 32 Amoles of sodium hydroxide (bringing the ph of the reaction mixture to 1.5), 1 jlmoles of the pteridine under investigation, and either 5 4c H3-5T-uridine (23. c/mmole) or 5 tic H3-6T-thymidine (14.5 c/mmole). A control without pteridine was always run on eggs at the same developmental stage. All subsequent extractions and analyses were as described previously. At ph 1.5 there is a 3 per cent decrease, as compared with ph 7.5, in the total tissue uptake of isotope, and a 4 per cent decrease in the rate of synthesis of 89

9 ZOOLOGY: HARRIS AND FORREST PRoc. N. A. S. 2 125 Total RNA 2 125- cof Oj1 C,U C X U3) C X I D A H L FIG. 1.-Effects of various pteridines on RNA synthesis in 68-hr-old Oncopeltus eggs. The values are expressed as per cent of control. C, control; X, xanthopterin; I, isoxanthopterin; D, 7,8-dihydroxanthopterin; A, 2-amino-4-hydroxypteridine; H, 2- amino-4-hydroxy-6-hydroxymethylpteridine; L, leucopterin. rrna and DNA. However, exactly the same pattern of DNA and RNA synthesis during development was found with eggs assayed at ph 1.5 as with eggs assayed at ph 7.5. Results.-Effect of pteridines on rrna synthesis: Ribosomal RNA synthesis is proceeding at its maximum rate in the 68-hour embryo. The effects of the following pteridines were examined at this time: 7,8-dihydroxanthopterin, xanthopterin, isoxanthopterin (all of which occur in Oncopeltus eggs), 2-amino4-hydroxypteridine, 2-amino-4-hydroxy-6-hydroxymethylpteridine, and leucopterin (all of which are naturally occurring compounds which have not, however, been demonstrated to occur in Oncopeltus eggs). The results are recorded in Figure 1. 2-Amino-4- hydroxypteridine, 2-amino-4-hydroxy-6-hydroxymethylpteridine, and leucopterin had essentially no effect on rrna synthesis. Significantly, 7,8-dihydroxanthopterin also had no effect, but xanthopterin caused a 15-3 per cent inhibition, and isoxanthopterin a 5 per cent inhibition. In this figure are also recorded the results of the effects of the compounds tested on "small-molecular-weight" RNA. Although the bulk of this material is probably soluble RNA, the figures are not very reliable because no further attempt was made to fractionate the materials which might be present in this area of the gradients. It is of interest, however, that the trends in incorporation of radioactive precursor are the same as those observed for ribosomal RNA synthesis. Because of these observations, the effects of the two active compounds were examined at other stages in the development of the eggs, particularly during the period (68-116 hr) when dynamic alterations in rrna synthesis are occurring (Fig. 2). Thus, at around 96 hours, the extent of inhibition (2%) achieved using xanthopterin was the sarme as at 68 hours, although the rate of rrna synthesis

VOL. 58, 1967 ZOOLOGY: HARRIS AND FORREST 91 ::; z C-) co,!-- Cl vn 68 92 96 98 HOURS OF DEVELOPMENT 116 FIG. 2.-Effects of xanthopterin. and isoxanthopterin on rrna synthesis at various developmental stages in Oncopeltus eggs. a Control; isoxanthopterin; E xanthopterin. was 4 per cent less than the rate at 68 hours. Shortly thereafter (i.e., at 98 hr), xanthopterin stimulated slightly the incorporation of isotope into rrna. Similar but more dramatic effects were obtained with isoxanthopterin. Up to 96 hours, taking into account the changing rate of rrna synthesis, about the same level of inhibition was observed. After this time, the inhibition caused by isoxanthopterin changed to a steadily increasing stimulation so that, by 116 hours, the level of Io O 11 C a- o Fraction Number Fraction Number Fraction Number FIG. 3.-Sucrose density gradients of RNA extracted from Oncopeltus eggs after incubation with H3-uridine in the presence or absence of isoxanthopterin or xanthopterin, Conditions for extraction of RNA and for running the gradients are given in ref, 1, Nu

z a 8 92 ZOOLOGY: HARRIS AND FORREST PROC. N. A. S. 6 CL G) C X I D H A FIG. 4.-Effects of various pteridines on DNA synthesis in 68-hour-old Oncopeltus eggs. Symbols for compounds used are as in Fig. 1. incorporation of isotope into rrna was 35 per cent above the control. Sucrose gradients for the two critical time periods are shown in Figure 3. Effects of pteridines on DNA synthesis: DNA synthesis in developing eggs was followed using tritiated thymidine, and the effects of the following compounds on its rate of synthesis were-studied: 2-amino-4-hydroxypteridine, 2-amino-4-hydroxy- 6-hydroxymethylpteridine, 7,8-dihydroxanthopterin, xanthopterin, and isoxanthopterin. The results are shown in Figure 4. The first two compounds had little or no effect on the incorporation of radioactivity into DNA, while dihydroxanthopterin had a slight effect. (In this case, the question of its having any activity is complicated by its ease of oxidation, during incubation, to xanthopterin which may then exert its particular effect.) Xanthopterin brought about a 45 per cent inhibition of the rate of DNA synthesis, a relatively greater effect than its effect on rrna synthesis, and isoxanthopterin inhibited DNA synthesis to about the same extent (55%O of control) as it did rrna synthesis. In the 116-hour-old embryo, the rate of DNA synthesis is about 2 per cent of the level observed in the 68 hour embryo. At this stage xanthopterin and isoxanthopterin had no significant effect on the rate of DNA synthesis (Fig. 5). Discussion.- Although the phenomena described above are undoubtedly real, their underlying basis is obscure. It could be argued, for example, that xanthopterin and isoxanthopterin affect entry of the precursor into the partially dechorionated egg. There are two arguments against a mechanism of this sort. Firstly, a

VOL. 58, 1967 ZOOLOGY: HARRIS AND FORREST 93 Fia. 5. Effects of xanthopterin and iso- MM xamithopterin on ])NA synthesis at two de- X velopmenital stages in Oncopeltus eggs.. E IS Control; isoxarit-hopterin; BI xanthopterin. 68 hrs. -116 hrs. differential response to both compounds is observed at different developmental stages. Secondly, the ratio of the amount of synthesis to total precursor uptake (per cent ethanol-insoluble radioactivity, Table 1) is lower when either isoxanthopterin or xanthopterin is present in the test system for measurement of rrna synthesis. Assuming that the amount of precursor (2.2 jsmoles of uridine in 1 ml incubating medium) is well below the amount needed to saturate the precursor pool within the eggs, then if either of these pteridines were inhibiting a transport system in the cell membrane, the ratio of synthesis to total uptake would be expected to be equal to or greater than the control. The reverse is the case. Another possible interpretation of the data is that these pteridines affect key enzymes involved in the synthesis of precursors of RNA and DNA, or of these macromolecules themselves. Any one of a number of enzymes could be involved (e.g., dihydrofolic reductase, thymidylic synthetase), but again the compelling argument against enzyme inhibition, at least as a primary effect, is the differential response of the dechorionated eggs to the two compounds. Thus, for example, DNA synthesis in 116-hour-old embryos is not affected at all by isoxanthopterin or xanthopterin, although synthesis is still proceeding at a substantial rate (2% of the rate in 68-hour-old embryos). The most attractive hypothesis to explain the results is that isoxanthopterin and/or xanthopterin interact directly with the template for the synthesis of rrna or DNA, i.e., with DNA itself or a DNA-histone complex. (Of course, this, in turn, could affect the activities of both DNA and RNA polymerases.) Thus, in the 68-hour-old embryo, when synthesis of rrna is proceeding at its most rapid rate, it is possible that products of this rapid synthesis could bring about an activation of endogenous isoxanthopterin or xanthopterin which would eventually build up in concentration to a sufficient extent to inhibit polymerase activity by affecting the efficiency of the template. Whether this would be a specific effect on that part of the genome concerned with rrna transcription is not known (although it can be pointed out that the synthesis of low-molecular-weight RNA is also affected). The effect of these compounds might be much more general, Eand the specificity for

94 ZOOLOCY: HIARRIS AND FORREST PROC. N. A. S. TABLE 1 EFFECT OF ISOXANTHOPTERIN ON THE INCORPORATION OF H3-URIDINE INTO RNA AT VARIOUS STAGES OF DEVELOPMENT IN Oncopeltus -68 hr-..-92 hr-- -96 hr--.-98 hr*, -116 hr--e C I C I C I C I C I Total tissue radioactivity (cpm X 1-6) 2.2 1.33 2.6 1.32 1.53 1.34 1.47 1.5 1.5 1.53 Ethanolinsoluble radioactivity (cpm X 1-6).35.19.22.14.33.22.12.14.11.13 Per cent ethanolinsoluble 17.3 14.3 1.5 9.4 21.6 16.4 7.8 8.9 7.3 8.5 Specific activity (cpm/pg RNA) rrna 37. 19.4 19.3 11.9 22.5 15.5 12.5 15.9 7.3 9.8 srna 25.5 83.6 13.3 7.8 175.7 117.6 54. 72.5 38.5 58.3 * Average of duplicate experiments. rrna synthesis observed in these experiments might only stand out because this type of RNA appears to be the major product of polymerase activity in the developing egg. The fact that the level of 7,8-dihydroxanthopterin (which does not inhibit the synthesis of rrna or DNA) begins to fall at about the same time as rrna and DNA synthesis begin to increase, suggests the possibility that increasing metabolic activity might convert it into xanthopterin which, after having exercised its influence on template efficiency of the DNA, is, in turn, converted into erythropterin. The situation with isoxanthopterin must be more complex, since its concentration (as measured by paper chromatography of developing eggs) remains unchanged during development. We have some evidence that at least a portion of the isoxanthopterin in the egg is associated with a protein from which it could be released at the appropriate time to exert its effect on template efficiency. We have some preliminary evidence also that isoxanthopterin does, in fact, bind to double-stranded DNA, but the nature of this binding, and its biological significance, remain to be explored. Summary.-In developing Oncopeltus eggs, in which ribosomal RNA is the major type of RNA synthesized, the rate of this synthesis, when it is at its maximum, can be affected by incubating partially dechorionated eggs with either of two naturally occurring pteridines, isoxanthopterin or xanthopterin. Isoxanthopterin is more effective, bringing about a 5 per cent inhibition of incorporation of radioactive isotope into rrna. DNA synthesis is also affected by the same two compounds. At later stages of development these inhibitory effects disappear, and indeed, with respect to rrna synthesis, the pteridines stimulate incorporation of precursor. * Supported by USPHS grants GM 12323, 2T1GM 337-6, and GM 337, and by the Robert A. Welch Foundation, Houston, Texas. 1 Harris, S. E., and H. S. Forrest, Science, 156 (3781), 1613 (1967). 2 Forrest, H. S., S. E. Harris, and L. J. Morton, J. Insect Physiol., 13, 359 (1967).