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In the accompanying article, using molecular dynamics calculations, we found that the 66-77 and 122-138 domains in ras-p21 and the 821-827, 832-845, 917-924, 943-953, and 1003-1020 domains in GAP have different conformations in complexes of GAP with wild-type and oncogenic ras-p21. We have now synthesized peptides corresponding to each of these domains and coinjected them into oocytes with oncogenic p21, which induces oocyte maturation, or injected them into oocytes incubated with insulin that induces maturation by activating wild-type cellular ras-p21. We find that all of these peptides inhibit both agents but do not inhibit progesterone-induced maturation that occurs by a ras-independent pathway. The p21 66-77 and 122-138 peptides cause greater inhibition of oncogenic p21. On the other hand, the GAP 832-845 and 1003-1021 peptides inhibit insulin-induced maturation to a significantly greater extent. Since we have found that activated wild-type and oncogenic p21 activate downstream targets like raf differently, these GAP peptides may be useful probes for identifying elements unique to the wild-type ras-p21 pathway.

Oncogenic ras (Val 12-containing)–p21 protein induces oocyte maturation by a pathway that is blocked by peptides from effector domains of ras –p21, i.e., residues 35–47 (that block Val 12–p21-activated raf ) and 96–110 and 115–126, which do not affect the ability of insulin-activated cellular p21 to induce maturation. Oncogenic p21 binds directly to jun –N-terminal kinase (JNK), which is blocked by the p21 96–110 and 115–126 peptides. This finding predicts that oncogenic p21, but not insulin, induces maturation by early and sustained activation of JNK. We now directly confirm this prediction by showing that oncogenic p21 induces activating phosphorylation of JNK (JNK-P) and of ERK (MAP kinase) (MAPK-P), whose levels correlate with oocyte maturation. p21 peptides 35–47 and 96–110 block formation of JNK-P and MAPK-P, further confirming this correlation and suggesting, unexpectedly, that raf –MEK–MAPK and JNK– jun pathways strongly interact on the oncogenic p21 pathway. In contrast, insulin activates only low levels of JNK-P, and, surprisingly, we find that insulin induces only low levels of MAPK-P, indicating that insulin and activated normal p21 utilize MAP kinase-independent signal transduction pathways.

Purpose: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. Methods: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MAP kinase-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. Results: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. Conclusion: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MAP kinase inhibitors. The results imply that blockade of both MAP kinase and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras- induced tumors.

We have previously found that a peptide corresponding to residues 35-47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras-p21 peptide corresponding to residues 96-110 that blocks ras-jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation. We now find that the p21 35-47, but not the 96-110, peptide completely blocks c-raf-induced maturation. This finding suggests that the 35-47 peptide blocks oncogenic ras at the level of raf; that activated normal and oncogenic ras-p21 have differing requirements for raf-dependent signaling; and that the two oncogenic-ras-selective inhibitory peptides, 35-47 and 96-110, act at two different critical downstream sites, the former at raf the latter at JNK/jun, both of which are required for oncogenic ras-p21 signaling.

Minor leucine tRNA species, tRNA and tRNA , from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman, E., Holmes, W. M., and Hatfield, G. W. (1979) J. Mol. Biol. 129, 567−585]. In the present study, these two tRNALeu species were purified from E. coli A19, and the nucleotide sequences were determined by a post-labeling method. tRNA was found to correspond to the tRNA gene reported as su°6 tRNA [Yoshimura, M., Inokuchi, H., and Ozeki, H. (1984) J. Mol. Biol. 177, 627−644]. The first letter of the anticodon was identified to be 2‘-O-methylcytidine (Cm). tRNA was identified as the minor leucine tRNA that has been sequenced previously (tRNA ) [Yamaizumi, Z., Kuchino, Y., Harada, F., Nishimura, S., and McCloskey, J. A. (1980) J. Biol. Chem. 255, 2220−2225]. There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA . Nucleoside N* was isolated to homogeneity (1 A260 unit). By 1H NMR spectroscopy, nucleoside N* was found to be a 2‘-O-meth...

In the preceding paper we found from molecular dynamics calculations that the structure of the ras-binding domain (RBD) of raf changes predominantly in three regions depending upon whether it binds to ras-p21 protein or to its inhibitor protein, rap-1A. These three regions of the RBD involve residues from the protein–protein interaction interface, e.g., between residues 60 and 72, residues 97–110, and 111–121. Since the rap-1A–RBD complex is inactive, these three regions are implicated in ras-p21-induced activation of raf. We have therefore co-microinjected peptides corresponding to these three regions, 62–76, 97–110, and 111–121, into oocytes with oncogenic p21 and microinjected them into oocytes incubated in in insulin, which activates normal p2l. All three peptides, but not a control peptide, strongly inhibit both oncogenic p21- and insulin-induced oocyte maturation. These findings corroborate our conclusions from the theoretical results that these three regions constitute raf effector domains. Since the 97–110 peptide is the strongest inhibitor of oncogenic p21, while the 111–121 peptide is the strongest inhibitor of insulin-induced oocyte maturation, the possibility exists that oncogenic and activated normal p21 proteins interact differently with the RBD of raf.

The small-angle X-ray scattering technique was used to characterize the structure in solution of wild type ras p21 as well as the oncogenic proteins mutated at residue 12, 59, or 61. In the presence of GDP, the radius of gyration, Rg, determined for wild type ras p21 was 16.89 +/- 0.01 A, while the wild type ras p21 bound to the GTP analogue GDPNHP (5'-guanyl imido diphosphate beta-gamma-imidoguanosine 5'-triphosphate) showed an Rg value of 17.46 +/- 0.01 A, which is 3.3% larger. The result shows that ras p21 expands upon GTP binding. The Rgs of mutated proteins were 17.04 +/- 0.01, 16.98 +/- 0.01, and 17.03 +/- 0.01 A for the Gly-12 to Val, Ala-59 to Thr, and Gln-61 to Leu mutants, respectively. The scattering profiles were analyzed by simulation of hydrated ras p21, based on the crystal atomic coordinates, and it was concluded that the ras p21 molecule incorporates 20% more bulk water upon GTP binding. The increase of bulk water is especially conspicuous around the interface between switch I (residues 32-40) and switch II (residues 60-66) regions. This suggests that hydration plays an important role in the interaction with GAP.

By monitoring the mutagenicity to a new Salmonella tester strain, YG1024, which has a much higher level of O-acetyltransferase activity than S.typhimurium TA98, we found two new mutagenic compounds in bacteriological-grade beef extract. One of them (compound I), which had a similar UV spectrum to that of 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), was isolated and shown to account for approximately 2% of the total mutagenicity of the materials adsorbed to blue cotton, and its concentration was estimated to be 6.0 ng/g beef extract. This amount of compound in beef extract was insufficient to allow measurements of various spectra, but its level was increased approximately 9-fold by heating beef extract with creatine and threonine at 200 degrees C for 5 h. From UV and mass spectra of the compound obtained from beef extract heated with creatine plus threonine, it was deduced to be a hydroxymethyl derivative of aminodimethylimidazo-quinoxaline. Compound I was isolated from the urine of rats given 4,8-DiMeIQx and identified as 2-amino-4-hydroxymethyl-3,8-dimethylimidazo[4,5-f]quinoxaline (4-CH2OH-8-MeIQx) by 1H-NMR analysis. 4-CH2OH-8-MeIQx induced 326,000 revertants of YG1024 and 99,000 revertants of TA98 per micrograms in the presence of S9 mix.

We previously found two new mutagens, compounds I and II, in bacteriological-grade beef extract by monitoring the mutagenicity to a new Salmonella strain, YG1024; compound I was identified as 2-amino-4-hydroxymethyl-3,8-dimethylimidazo[4,5-f]quinoxaline (4-CH2OH-8-MeIQx). In the present study, we isolated compound II from the beef extract, which accounted for 2% of the total mutagenicity of materials adsorbed on blue cotton. Further, we found that a large quantity of compound II was produced by heating a mixture of creatine, threonine and glucose (1:1:0.5) at 200 degrees C for 5 h, the level being 860-fold of that in the beef extract. The structure of this compound was determined to be 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx) by X-ray crystallography. The amount of 7,9-DiMeIgQx in bacteriological-grade beef extract was estimated to be 53 ng/g. This compound induced 13 800 and 670 revertants of S. typhimurium YG1024 and TA98 respectively, per micrograms in the presence of S9 mix.

The argU (dnaY) gene product, a minor tRNAArg, from Escherichia coii has the anticodon N*CU with an unidentified modified nucleoside N* in position 34 [Kiesewetter, S., Fisher, W. & Sprinzl, M. (1987) Nucleic Acids Res. 15, 3184]. In the present study, argU tRNA was purified from E. coli A 19 strain and nucleoside N* was characterized by the TLC and HPLC analyses. Nucleoside N* was found to be different from any naturally occurring modified nucleosides. From unfractionated E. coli tRNA species, nucleoside N* was prepared in an amount sufficient for 1H-NMR experiments. By the analyses of one-dimensional and two-dimensional NMR spectra, nucleoside N* was suggested to be 5-methylaminomethyluridine (mnm5U), which was confirmed by comparison with a chemically synthesized preparation of mnm5U. Thus, the occurrence of mnm5U in mature tRNA was found for the first time. Further, the modification of U(34) to mnm5U in this tRNA was found to contribute to the strict recognition of two degenerate codons terminating in A and G.

Normal RAS proteins play a key role of molecular switch in the transduction of the growth signal from extracellular to intracellular space. The state of the switch is "on" when GTP is bound and "off" when GDP is bound to the protein. The crystal structure of a complex between a nonhydrolyzable GTP analog and the catalytic domain of a RAS protein has been determined by a rotation-translation search method. The orientations and positions of four independent molecules have been determined using a single molecule as a probe in the search. The crystal structure reveals that the gamma phosphate of the GTP analog induces extensive conformational changes on two loop regions of the protein.

Purpose: We have previously found that a synthetic peptide corresponding to ras-p21 residues 96–110 (PNC2) selectively blocks oncogenic (Val 12-containing) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferation, we synthesized an inducible plasmid that expressed this peptide sequence. Our purpose was to test this expression system in oocytes to determine if it was capable of causing selective inhibition of oncogenic ras-p21. Methods: We injected this plasmid and a plasmid expressing a control peptide into oocytes either together with oncogenic p21 or in the presence of insulin (that induces maturation that is dependent on normal cellular ras-p21) in the presence and absence of the inducer isopropylthioglucose (IPTG). Results: Microinjection of this plasmid into oocytes together with Val 12-p21 resulted in complete inhibition of maturation in the presence of inducer. Another plasmid encoding the sequence for the unrelated control peptide, X13, was unable to inhibit Val 12-p21-induced maturation. In contrast, PNC2 plasmid had no effect on the ability of insulin-activated normal cellular or wild-type ras-p21 to induce oocyte maturation, suggesting that it is selective for blocking the mitogenic effects of oncogenic (Val 12) ras p21. Conclusion: We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p21 and may therefore be highly effective in blocking proliferation of ras-induced cancer cells. Also, from the patterns of inhibition, by PNC2 and other ras- and raf-related peptides, of raf- and constitutively activated MEK-induced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.

We have obtained evidence that oncogenic and activated normal ras-p21 proteins utilize overlapping but distinct signal transduction pathways. Recently, we found that ras-p21 binds to both jun and its kinase, jun kinase (JNK). We now present evidence that suggests that oncogenic but not normal activated p21 depends strongly on early activation of JNK/jun. This early activation most likely involves direct interaction between oncogenic p21 and JNK/jun because p21 peptides that blocked the binding of p21 to JNK and jun strongly inhibited oncogenic p21-induced oocyte maturation while they did not inhibit insulin-activated normal cellular p21-induced maturation. Very similar results were also obtained for a newly characterized specific inhibitor of JNK which blocked oncogenic but not normal activated p21-induced oocyte maturation. We also found that both jun and JNK strongly enhanced oncogenic p21-induced oocyte maturation while they inhibited insulin-activated normal p21-induced oocyte maturation. These results suggest that the peptides and JNK inhibitor may be useful agents in selectively blocking the effects of oncogenic but not normal p21 in cells.

Codon recognition by Escherichia coli tRNALeu4 and tRNALeu5 was investigated by analysis of the competition between two aminoacyl-tRNA species in an in vitro protein synthesis. Both tRNA species strictly obey the wobble rule when they are in competition with other tRNA species. This is probably due to the post-transcriptional modifications at the first position of the anticodon of these tRNALeu species, supporting the proposal that the conformational rigidity of post-transcriptionally modified pyrimidine nucleotides guarantees the correct codon recognition.

We have recently shown that a peptide (residues 35-47) from a functional region of the ras p21 protein, thought to be involved in the binding of p21 to GTPase activating protein, the antibiotic azatyrosine, known to induce the ras-recision gene, and the selective protein kinase C inhibitor, CGP 41,251, all inhibit oncogenic p21 protein-induced maturation of oocytes in a dose-dependent manner. We now show that these three agents only partially inhibit insulin-induced oocyte maturation, known to be dependent on activation of cellular p21 protein. On the other hand, the anti-p21 protein antibody Y13-259 completely inhibits both insulin- and oncogenic p21 protein-induced maturation as does a tetrapeptide, CVIM, known to block the enzyme farnesyl transferase which covalently attaches the farnesyl moiety to the p21 protein allowing it to attach to the cell membrane. Our results suggest that while the oncogenic and insulin-activated normal p21 proteins share certain elements of their signal transduction pathways in common, these pathways diverge and allow for selective inhibition of the oncogenic pathway.

The ras -oncogene-encoded p21 protein is known to produce malignant transmation of NIH 3T3 cells as well as maturation of Xenopus oocytes when microinjected into these cells. p21 protein is known to bind a GTPase activating protein (GAP) intracellularly; residues 32–45 have been implicated in interacting with GAP. We demonstrate here that a peptide corresponding to residues 35–47 of p21 as well as the antibiotic azatyrosine inhibit the ras -induced maturation of Xenopus oocytes in a dose-related manner upon microinjection. We have previously shown that this p21 peptide and azatyrosine could inhibit the effects of p21 protein on cell transformation and pinocytosis in NIH 3T3 cells. In the present study, in which we have extended these results to the oocyte system, we also demonstrate that both partially inhibit insulin-induced oocyte maturation, a process which is thought to involve activation of endogenous p21 protein; on the other hand, both agents fail to inhibit oocyte maturation induced by progesterone, which is known not to act through p21 protein activation. Control studies with other peptides and tyrosine analogues support the selective nature of these events. These results suggest that both the p21-related peptide and azatyrosine have potent anti- ras effects intracellularly.

Ras proteins participate as a molecular switch in the early steps of the signal transduction pathway that is associated with cell growth and differentiation. When the protein is in its GTP complexed form it is active in signal transduction, whereas it is inactive in its GDP complexed form. A comparison of eight three-dimensional structures of ras proteins in four different crystal lattices, five with a nonhydrolyzable GTP analog and three with GDP, reveals that the "on" and "off" states of the switch are distinguished by conformational differences that span a length of more than 40 A, and are induced by the gamma-phosphate. The most significant differences are localized in two regions: residues 30 to 38 (the switch I region) in the second loop and residues 60 to 76 (the switch II region) consisting of the fourth loop and the short alpha-helix that follows the loop. Both regions are highly exposed and form a continuous strip on the molecular surface most likely to be the recognition sites for the effector and receptor molecule(or molecules). The conformational differences also provide a structural basis for understanding the biological and biochemical changes of the proteins due to oncogenic mutations, autophosphorylation, and GTP hydrolysis, and for understanding the interactions with other proteins.

The potent mutagens Trp-P-1 (3-amino-1, 4-dimethyl-5H-pyrido-[4,3-b]-indole) and Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) are known to be produced by pyrolysis of tryptophan [8]. To determine whether such mutagens are produced by cooking foods, the fractions obtained from broiled sardines cooked in the ordinary way were analysed by gas chromatography/mass spectrometry. The results showed that 13.3 ng of Trp-P-1 and 13.1 ng of Trp-P-2 were, in fact, present per gram of broiled sardines.

The N3 imino units of dihydrouridine were identified in samples of 15N-labeled Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR. The peaks for dihydrouridine had high field 1H (9.7-9.8 ppm) and 15N (147.8-149.5 ppm) chemical shifts. Assignments were made by 1H-15N chemical shift correlation based on values obtained in model studies with tri-O-benzoyl- and tri-O-acetyldihydrouridine. The rates of exchange of the imino protons with water suggest that the D-loop in tRNAfMet is less stable than the D-loops in tRNALys or tRNAPhe. Closely spaced peaks were observed for the two dihydrouridines in tRNAPhe in a high resolution spectrum.

We have identified phosphoethanolamine as the pituitary-derived growth-promoting material that specifically stimulates the rat mammary carcinoma cell line 64-24. We have been studying the growth characteristics of the 64-24 cell line, which was isolated from a highly hormone-dependent tumor and which retains in culture many characteristics of the original tumor. Previously, crude bovine pituitary extract was shown to contain a significant amount of growth-stimulating activity for these cells, and a growth factor from this extract was purified to homogeneity. This report describes the identification of the growth factor as phosphoethanolamine. Further, the biological activity of phosphoethanolamine was found to be virtually identical to that of the purified growth factor. A possible role of phosphoethanolamine in the growth of mammary tumor cells as well as of normal mammary epithelial cells and other tissues is discussed.

Three species of methionine tRNAs and phenylalanine, tyrosine, and isoleucine tRNAs were purified from an extreme thermophile, Thermus thermophilus HB8. Formylation studies of the three methionine tRNAs and their codon-specific binding activities to ribosomes showed that two of them (named tRNAf1Met and tRNAf2Met) were initiator tRNAs and the other (named tRNAmMet) was a non-initiator. The tRNAs from T. thermophilus all had melting temperatures of up to ten degrees higher than the corresponding species from E. coli. Most of the species also had slightly higher G+C contents than the corresponding species of E. coli, and each of them contained one mol each of the modified nucleosides, O2'-methylguanosine (Gm), 2-thioribothymidine (s2T), and 1-methyladenosine (m1A). Their high melting temperatures could be explained by their high G+C contents and the presence of the modified nucleosides, espically s2T. Comparison of the melting temperatures of T. thermophilus tRNAf2Met with those of E. coli tRNAfMet and tRNAmMet at different magnesium concentrations showed that magnesium was also a factor in the thermostability of the thermophile tRNA.

The N1 imino units in Escherichia coli tRNAfMet, tRNAGlu, tRNAPhe, and tRNATyr were studied by 1H-15N NMR using three different techniques to suppress signals of protons not attached to 15N. Two of the procedures, Fourier internuclear difference spectroscopy and two-dimensional forbidden echo spectroscopy permitted 1H and 15N chemical shifts to be measured simultaneously at 1H sensitivity. The tRNAs were labeled by fermentation of the uracil auxotroph S phi 187 on a minimal medium containing [1-15N]uracil. 1H and 15N resonances were detected for all of the N1 psi imino units except psi 13 at the end of the dihydrouridine stem in tRNAGlu. Chemical shifts for imino units in the tRNAs were compared with "intrinsic" values in model systems. The comparisons show that the A X psi pairs at the base of the anticodon stem in E. coli tRNAPhe and tRNATyr have psi in an anti conformation. The N1 protons of psi in other locations, including psi 32 in the anticodon loop of tRNAPhe, form internal hydrogen bonds to bridging water molecules or 2'-hydroxyl groups in nearby ribose units. These interactions permit psi to stabilize the tertiary structure of a tRNA beyond what is provided by the U it replaces.

The primary structure of Escherichia coli tRNA UUR Le which recognizes the UU series of codons has been determined. The sequence is pG-C-C-C-G-G-A-s4U-G-G-U-G-G-A-A-D-C-Gm-C-D-A-G-A-C-A-C-A-A-G-G-G-A-psi-U-N-A-A-ms2i6A-A-psi-C-C-C-C-U-C-G-G-C-G-G-C-G-U-U-C-G-C-G-C-U-G-U-G-C-G-G-G-T-psi-C-A-A-G-U-C-C-C-G-C-U-C-C--G-G-G-U-A-C-C-A. The chain length of tRNA UUR Leu is 87 residues, the same as other E. coli tRNA Leu s and T4 phage-coded tRNA Leus. Its sequence is especially similar to that of E. coli tRNA2 Leu in the Darm and T psi C arm regions. E. coli tRNA UUR Leu contains an unknown modified nucleoside in the first position of the anticodon and was shown by mass spectrometry and chemical degradation to be an adenosine derivativee. Addition of tRNA UUR Leu to a cell-free protein-synthesizing system with high Mg2+ concentration resulted in the formation of polyleucine miscoded by poly(U), indicating that the unknown modified nucleoside exhibits a tendency to recognize U under certain conditions.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a potent mutagen and carcinogen present in heated foodstuffs. The covalent binding of MeIQx to calf thymus DNA and calf liver RNA with microsomal activation was demonstrated. A major metabolite which exerts a direct mutagenic effect on S. typhimurium TA98 was found by HPLC analysis after incubation of MeIQx with rat liver microsomal fraction. The metabolite was identified as 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx). Synthetic N-OH-MeIQx was found to bind non-enzymatically to DNA and RNA at neutral pH even at 0 degrees C. Addition of acetic anhydride increased the binding of N-OH-MeIQx to DNA 10 times. These results suggest that MeIQx is metabolized to N-OH-MeIQx by microsomal cytochrome P-450 and further activated to an acetylated form that binds efficiently to nucleic acids in rat liver. Preferential modification of polyguanylic acid suggests that guanine residues of DNA are mainly modified with MeIQx. Synthetic N-OH-MeIQx exerted direct mutagenic activity on S. typhimurium TA98 inducing 150,000 rev/micrograms. Pentachlorophenol (PCP) caused a dose-dependent inhibition of this mutagenic effect, but 2,6-dichloro-4-nitrophenol (DCNP) did not. Thus the acetyltransferase of S. typhimurium seems to be important for the high mutagenicity of MeIQx after its microsomal activation.

A Y-base derivative was isolated from rat liver tRNAPhe and its structure was assigned to be alpha-(carboxyamino)-beta-hydroxy-4,9-dihydro-4,6-dimethyl-9-oxo-1H-imidazol[1,2-a]purine-7-butyric acid dimethyl ester (hydroxy-Y), based on the results of mass spectrometry and chemical degradation. This modified base seems to be the major fluorescent component of rat liver tRNAPhe; the peroxy-Y base previously isolated from rat liver tRNAPhe and characterized by Nakanishi and his coworkers (1,2) was not present in our preparation.

Two compounds forming oxygen radicals were isolated from heated starch by monitoring the potency to form 8-hydroxydeoxyguanosine from deoxyguanosine during purification procedures. These compounds were identified as hydroxymethylreductic acid and methylreductic acid. The former compound was mutagenic to Salmonella strains TA100, TA102 and TA104 and the latter compound induced sister-chromatid exchanges in human NL3 cells. Hydroxymethylreductic acid was found to be a novel compound. Considerable amounts of these compounds were detected in various heat-processed foods.

Directly combined high performance liquid chromatography-mass spectrometry (LC/MS) has been studied as a method of analysis of heterocyclic aromatic mutagens in cooked foods, in the parts per billion concentration range. Identification and semiquantitative estimation of mutagens is based on accurate measurement of chromatographic retention (k') and molecular weight-selective detection of mutagens, which are protonated during passage of the chromatographic eluant into a thermospray interface of a quadrupole mass spectrometer. Standard chromatographic retention (k') values in two reversed-phase systems and data from thermospray mass spectra from nine mutagens are reported. An isolation scheme employing CH3OH extraction, acid-base partition, cellulose-trisulfo-Cu-phthalocyanine adsorption, and normal-phase HPLC was used prior to LC/MS analysis. Initial applications have been demonstrated in the analysis of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in broiled salmon flesh. Levels measured were estimated to be in the range 0.2 to 0.4 microgram/kg IQ and 0.4 to 0.9 microgram/kg MeIQ. The method is judged to be generally applicable with minimal sample prefractionation to detection of mutagens at the parts per billion level in cooked foods.

Poly([14C]adenosine diphosphate ribose) was synthesized from [14C]NAD+ with calf thymus nuclei. The fraction containing poly(adenosine diphosphate ribose) eluted with 0.22--0.40 M phosphate buffer (pH 6.8) from a hydroxylapatite column, was completely hydrolyzed with venom phosphodiesterase, and was separated by DEAE-Sephadex A-25 column chromatography in 7 M urea. A new compound, which constituted 2% of the products from poly(adenosine diphosphate ribose), was found in addition to the expected products--i.e., 5'-AMP, 2'-(1''-ribosyl)adenosine-5',5''-bis(phosphate), and its derivatives. This compound was identified as 2'-[1''-ribosyl 2''-(or 3''-)(1'''-ribosyl)]adenosine-5',5'',5'''-tris(phosphate). The existence of this compound is evidence of a branching structure of poly(adenosine diphosphate ribose), which was previously thought to be a linear molecule. The content of this compound suggests that the frequency of branching is about 1 per 20--30 adenosine diphosphate ribose residues of high molecular weight poly(adenosine diphosphate ribose).

The effect of various guanine ribonucleotides on the antitumor activity of 5-fluorouracil (FUra) was investigated by its action on adenocarcinoma 755, 5′-GDP and 5′-GMP were both equally effective in potentiating the antitumor activity of FUra without increasing toxicity. 5′-GTP and 5′-IMP also potentiated the activity but not as much as 5′-GMP. 2′-GMP and 3′-GMP did not enhance the antitumor activity. In contrast, cGMP antagonized the effects of FUra. The incorporation of 3H -labeled FUra into RNA or DNA showed there was no obvious association between the incorporation and antitumor activity after any treatment with guanine ribonucleotides. The combination of FUra and 5′-GMP produced the greatest inhibition of RNA synthesis. The combination of FUra and 2′-GMP had no effect on RNA synthesis. The inhibition of RNA synthesis may be the result of decreased pyrimidine pool size and increased incorporation of FUra into RNA. Potentiation of the antitumor activity of FUra by 5′-GMP was reversed by the injection of cytidine. Moreover, the combination of 5-fluorocytidine (FCyd) and 5′-GMP showed greater antitumor activity than FCyd alone. These results indicate that a decreased CTP pool potentiates the antitumor activity of FUra. Thus, 5′-GMP or 5′-GDP strongly enhanced the antitumor activity of FUra, and the potentiation resulted from the inhibition of RNA synthesis caused by reduction of the CTP and UTP pool sizes and increased incorporation of FUra into RNA.

In order to study the possible detoxification mechanisms of the carcinogenic arylamine, 2-amino-6-methyldipyrido[1,2- a : 3′,2′- d ]imidazole (GluP-1), the in vitro non-enzymatic reaction of 2-nitroso-6-methyldipyrido[1,2- a : 3′,2′- d ]imidazole (NOGluP-1) with reduced glutathione (GSH) was examined at pH 7.4 under both aerobic and anaerobic conditions. Two GSH-arylamine adducts were isolated and found to contain the GluP-1 and GSH moieties in a 1:1 molar ratio via an N-S linkage. Their structures were assigned as sulfinamide (-NH-SO-) and N -hydroxy-sulfonamide (-N(OH)-SO 2 -) by their behaviour under acidic and basic conditions and by UV-VIS, 1 H-NMR, infrared and mass spectrometries. Also, a N -hydroxy-sulfonamide adduct was produced when NOGluP-1 and cysteine were reacted at pH 7.4. The N -hydroxy-sulfonamide structure is a new binding form between arylnitroso compounds and thiols. The formation of these adducts may also take place in vivo as a detoxification of toxic arylamines since GSH is abundant in organs such as liver or kidney.

The nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8, was determined by a combination of classical methods using unlabeled samples to determine the sequences of the oligonucleotides of RNase T1 and RNase A digests and a rapid sequencing gel technique using 5'-32P labeled samples to determine overlapping sequences. Formylmethionine tRNA from T. thermophilus is composed of two species, tRNAf1Met and tRNAf2Met. Their nucleotide sequences are almost identical, and are also almost identical with that of E. coli tRNAfMet, except for slight modifications and replacements. Both species have modifications at three points which do not exist in E. coli tRNAfMet: 2'-O-methylation at G19, N-1-methylation at A59 and 2-thiolation at T55. Moreover U51 in E. coli tRNAfMet is replaced by C51 in both species, so that a G-C pair is formed between this C51 and G65. tRNAf2Met has a reversed G-C pair at positions 52 and 64 compared with those in tRNAf1Met and E. coli tRNAfMet. Other regions are mostly the same as those in all prokaryotic initiator tRNAs so far reported. The thermostability of these thermophile initiator tRNAs is discussed in relation to their unique modifications.

Two indole compounds isolated from fresh Chinese cabbage were shown to be mutagen precursors that yielded direct-acting mutagens on treatment with nitrite. They were identified as 4-methoxyindole-3-acetonitrile and 4-methoxy-indole-3-aldehyde. When these compounds were treated with 50 mM nitrite at pH 3.0 for 1 h at 37 degrees C, 4-methoxyindole-3-acetonitrile induced 31,800 and 10,000 revertants of Salmonella typhimurium TA100 and TA98, respectively, per mg of mutagen precursor in the absence of S9 mix, and 4-methoxyindole-3-aldehyde induced 156,900 revertants of TA100 and 26,800 revertants of TA98 per mg in the absence of S9 mix.

A method of general applicability for the detection and quantification of mutagens in cooked foods at the ppb level is presented. A minimal sample prefractionation is employed and [Me-2H3]-labeled analogs of the compounds of interest are added for identification and quantification of mutagens by accurate measurement of chromatographic retention (K') in reverse-phase high-performance liquid chromatography (HPLC), and by measurement of the ratio of response of the protonated molecular ions of analyte and internal standard by directly coupled liquid chromatography-mass spectrometry (LC/MS). Initial application is demonstrated in the analysis of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in broiled salmon. Measured levels of IQ and MeIQ in broiled salmon flesh were 0.3-1.8 ppb and 0.6-2.8 ppb, respectively, and for the skin of broiled salmon 1.1-1.7 ppb and 1.5-3.1 ppb, respectively. Results on cooked beef and sardine are also reported.

Structure of a mutagenic compound (Me-IQ) isolated from broiled fish was determined to be 2-amino-3,4-dimethylimidazo[4,5-f]quinoline based on the mass-, UV- and 1H-NMR-spectra and chemical synthesis. Me-IQ showed strong mutagenic activity towards Salmonella typhimurium TA98 in the presence of S-9 mix.

After treatment with nitrite, Chinese cabbage showed direct-acting mutagenicity on Salmonella typhimurium TA100 inducing 3100 revertants per g. One of the mutagen precursors that became mutagenic after nitrite treatment was isolated, and identified as indole-3-acetonitrile. After treatment with nitrite, 1 mg of indole-3-acetonitrile induced 17 400 revertants of TA100 and 21 000 revertants of TA98 without S9 mix.

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

For use in screening for environmental mutagens and carcinogens, a highly fluorescent derivative of guanosine, 2'-deoxy-2'-(2",3"-dihydro-2",4"-diphenyl-2"-hydroxy-3"-oxo-1"-pyrrol yl) guanosine (FG), was synthesized. When incubated with FG in aqueous solution, mutagens form adducts that can be analyzed with an HPLC-fluorescence detector-system. By this method, mutagens such as glyoxal, methylglyoxal, 2-(2-furyl)-3-(5-nitrofuryl) acrylamide and 4-nitroquinoline-N-oxide, used as model compounds, were detected rapidly with high sensitivity. Reaction with isopropylideneguanosine (IPG), followed by isolation and characterization of the mutagen-IPG-adduct was found to be a useful method for identifying unknown mutagens in crude samples. This method was successfully applied in identification of the mutagens in heated glucose (200 degrees C, 20 min); glyoxal-IPG and 8-hydroxy-IPG were identified in the reaction mixture.

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.

Escherichia coli 5S RNA labeled with 15N at N3 of the uridines was isolated from the S phi-187 uracil auxotroph grown on a minimal medium supplemented with [3-15N]uracil. 1H-15N multiple quantum filtered and 2D chemical shift correlated spectra gave resonances for the uridine imino 1H-15N units whose protons were exchanging slowly with solvent. Peaks with 1H/15N shifts at 11.6/154.8, 11.7/155.0, 11.8/155.5, 12.1/155.0, and 12.2/155.0 ppm were assigned to GU interactions. Two labile high-field AU resonances at 12.6/156.8 and 12.8/157.3 ppm typical of AU pairs in a shielded environment at the end of a helix were seen. Intense AU signals were also found at 13.4/158.5 and 13.6/159.2 ppm where 1H-15N units in normal Watson-Crick pairs resonate. 1H resonances at 10.6 and 13.8 ppm were too weak, presumably because of exchange with water, to give peaks in chemical shift correlated spectra. 1H chemical shifts suggest that the resonance at 13.8 ppm represents a labile AU pair, while the resonance at 10.6 ppm is typical of a tertiary interaction between U and a tightly bound water or a phosphate residue. The NMR data are consistent with proposed secondary structures for 5S RNA.

The molecular conformations of naturally occurring 2-thiopyrimidine nucleosides (5-methylaminomethyl-2-thiouridine, 5-methoxycarbonylmethyl-2-thiouridine and 2-thiocytidine) and 5'-mononucleotides (5-methylaminomethyl-2-thiouridine 5'-monophosphate and 2-thiocytidine 5'-monophosphate) in 2H2O solution were elucidated by analyses of the proton NMR spin-coupling constant, nuclear Overhauser effect, and lanthanide-induced shifts and relaxation enhancements. As monomers, these nucleotides are almost exclusively in the 3E-gg-anti form, even in the absence of ordinary stabilizing factors of this form; i. e., base-stacking and base-pairing interactions with other nucleotide units. This inherent conformational rigidity of the 2-thiopyrimidine units probably contributes to stability of the conformation of tRNA.

The conformational properties of Q-nucleoside-5′-monophosphate (pQ) in aqueous solution was investigated by (1) H-NMR. By the analyses of spin-coupling constants, NOE and spin-lattice relaxation rates, (2) E-form for ribose ring and anti-form for N-glycoside bond are found to be the main conformations. Further, the conformation of -CH (2) -N (+) H (2) - linkage is discussed.

One of the E. coli mutants selected for deficiency of modified nucleoside Q was found to contain an analogue of Q and normal guanosine in place of Q. The analogue of Q, designated as preQo, was isolated on a large scale from purified tRNATyr containing preQo. The structure of preQo was determined to be 7-(cyano)-7-deazaguanosine by comparison of its ultraviolet absorption spectra, thin-layer chromatographic mobility and mass spectrum with those of synthetic material.

Results form /sup 1/H NMR studies of /sup 15/N-labeled Escherichia coli tRNA/sub f//sup Met/ indicated an unambiguous assignment for the G-U pair and detection of a uridine resonance at 11.4 ppm. Results indicated that signals for only 24-25 imino protons are clearly seen for E. coli tRNA/sub f//sup Met/ at 35/sup 0/C. Since 21 imino hydrogens participate in presumably more stable secondary interactions, only 3 or perhaps 4 peaks for tertiary hydrogen bonds are visible. Our results with /sup 15/N-labeled material indicate that uridine imino hydrogens in s/sup 4/U8-A14, rT54-A58, and G18-psi55-P58 (or U60) account for three of these tertiary signals.

2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) was found in bacteriological-grade beef extract by liquid chromatographies with electrochemical detection and a photodiode array detector. 4,8-DiMeIQx was estimated to be present at a level of 10.0 ng per g of beef extract, and to account for 9% of the total mutagenicity of the extract.

The hyper-modified nucleoside Q (queuosine) is exclusively located in the wobbling position of anticodons of tRNATyr, tRNAHis, tRNAAsn and tRNAAsp that recognise codons NAcU (ref. 1). Queuosine and its hexose-containing derivatives2,3 are widely distributed in microorganisms, animals and plants4,5. We confirm here the chemical structure of queuosine as 7-(3, 4-trans-4, 5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine2,6 (Fig. 1). The unique structural features of Q are the unusual cyclopentenediol side chain and the 7-deazaguanine ring. Queuosine is one of the most complicated modified nucleosides found thus far7.