Queuine (q), a cyclopentendiol derivative of 7-aminomethyl-7-deazaguanine, is a nutrient factor for lower and higher eukaryotes, except yeast; it is synthesized in eubacteria partly at the level of tRNA. In eukaryotes q is preferentially inserted into the wobble position of specific tRNAs in differentiated and adult tissues, but occurs mainly free in embryonic and fast proliferating cells. HeLa cells grow to a higher cell densitiy under aerobic than under hypoxic conditions only when supplemented with q. Here we show that in hypoxically grown HeLa cells, sufficiently supplied with q, free q accumulated when serum factors become limiting while the respective tRNAs remained completely q deficient. In these cells the levels of lactate dehydrogenase A (LDH A) mRNA and of LDH A protein were at least twofold higher than in aerobically grown cells, independent of the absence or presence of q. In response to q the LDH A, isoenzyme was further activated by a post-translational mechanism. In q-deficient HeLa cells the activity of the major anoxic stress protein, LDHk, increased as a result of hypoxia; this increase was suppressed by q. In aerobically grown, q-deficient cells significant activities of LDH A, and LDH, were present ; both activities were markedly lowered by q, while the mitochondrial electron flow was improved. The results show that free q is essential for relieving hypoxic stress in HeLa cells that results from oxygen limitation. The modified base queuine [q; 7-5-( [(lS,4S,5R)-4,5-di-anine], is synthesized in eubacteria only. During the biosynthesis GTP is converted to the precursor 7-aminomethyl-7-deazaguanine by so far unknown steps. The tRNA guanine transglycosylase (Tgt) exchanges this precursor specifically for the guanine residue in the anticodons GUN of tRNAs specifying the amino acids Asn, Asp, His and Tyr [l]. The cyclopentendiol moiety is derived from the ribose residue of S-adenosylmethionine and is attached to the modified tRNA precursor by a novel AdoMet-dependent reaction that is catalyzed by the tRNA ribosyltransferase-isomerase (QueA) [2]. During the QueA reaction, epoxy-queuosine is formed that is finally reduced to queuosine (Q) by a vitamin-B ,,-dependent, unidentified enzyme [3]. The genes, tgt and queA, are localized on the Escherichia coli genome in one operon [4].Mammals obtain q from nutrients or the intestinal flora [5-91. Specific eukaryotic Tgt enzymes insert the q-factor in a practically irreversible reaction into the wobble position of cytosolic and of mitochondrial tRNA,,, species [5-71.

Addition of the q-base to q-deficient non-transformed mammalian cells stimulated their proliferation. The q-base also improved proliferation of some cancer-derived cell lines, but inhibited proliferation of others. The proliferation of HeLa-S3 carcinoma cells was stimulated by q under aerobic conditions, but was inhibited when the cells had shifted their energy metabolism towards glycolysis as the result of oxygen limitation. Q-deficient cells could not adapt their proliferation to the respective oxygen tension. The q-base stimulated the proliferation of non-transformed fibroblasts but inhibited proliferation of the same cell line, when aerobic glycolysis was increased after transformation with the ras gene. The results suggest that the q-base permits mammalian cells to adapt their proliferation to their specific metabolic state.

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The eukaryotic tRNA:guanine transglycosylase (TGT) catalyses the base-for-base exchange of guanine for queuine (the q-base) a nutrition factor for eukary-

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