Modification of nucleic acids in relation to differentiation

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minidase is an enzyme which cleaves the terminal N-acetyl+glucosamine from cerebral gangliosides and is an example of this kind of enzyme, The acidic form of the glucosaminidase is absent from individuals with Tay-Sachs disease which is associated with increased levels of neuraminidase in the patient [30]. Since the terminal sugar is not removed, the ganglioside is not degraded and brain damage results from the massive accumulation of the glycoprotein. From the few examples outlined here it may be seen that more information on neuraminidase is needed to assess more correctly the significance of this important enzyme in the metabolism and in all aspects of cell behaviour. References 1 Hirst, G. K. (1941) Science 94, 22 2 Hirst, G. K. (1942) J. Exp. Med. 76, 195 3 Burnet, F.M., McCrea, J.F. and Stone, J.D. - (1946) .&it. J. Exp. Parhol. 27,228 4 Gottschalk, A. and Bhargava, A.S. (1971) in The Enzymes (Boyer, P. D., ed.), 3rd edn, Vol. 5, pp. 321-342 5 Klenk, H.D., Compans, R. W. and Choppin, P. W. (1970) Virology 42, 1158-1162 6 Kunim&o, S., Aoyagi, T., Takeuchi, T. and Umezawa, H. (1974) J. Bucteriol. 119, 394-400 7 Ada, G. L. and French, E. L. (1959) Nature, 183, 1740-1741 8 Kraemer, P.M. (1968) Biochirn. Biophys. Acra, 167,205-208 9 Cautrecasas, P. and Illiano, G. (1971) Eiochem. Biophys. Res. Commun. 44.178-184 10 Geisow, M. J. (1975) Biochem. J. 151, 181-183 11 Warren, L. (1959) J. Biol. Chem. 234, 1971-1975 12 Ziegler, P. W. and Hutchinson, H. D. (1972) Appl. Microbial. 23, 1060-1066

13 Bhavananden, V.P., Yeh, 1. K. and Carubelli, R. (1975) Anal. Biochem. 69,385-394 14 Palese, P. and Bodo, P. G. (1970) J. Viral. 6, 556558

15 Bachmayer, H. (1972) FEBS Lett. 23,217-219 16 Hoyle, L. (1969) J. Hyg. 67, 289 17 Kabayo, J.P. and Hutchinson, D. W., to be published 18 Holmquist, L. and Brossmer, R. (1972) 2. Physiol. Chem. 353, 13461350 19 Gottschalk, A. and Drzeniek (1972) in Glycoproteins, Their Composition, Structure and Function

(Gottschalk, A., ed.), pp. 381-402, Elsevier Publishing Co. Amsterdam 20 Holmquist, L. and Gstman, B. (1975) FEBS Lett. 60, 327-330

21 Kilbourne, E. D., Laver, W.G., Schulman, J.L. and Webser, R.G. (1968) J. Viral. 2,281-288 22 Becht, H., Hammerling, U. and Rott, R. (1971) Virology 46,337-343

23 Palese, P., Tobita, K., Ueda, M. and Compans, R. (1974) Virology 61, 397410 24 Tute, M.S., Brammer, K. W., Kaye, B. and Broadbent, R. W. (1970) J. Med. Chem. 13,4448 25 Haksar, A., Maudsley, D.V. and Peron, F. G. (1974) Nature 251, 514-515 26 Cautrecasas, P. (1973) Biochemistry 12, 35583566

27 Lloyd, C. W. (1975) Biol. Rev., 50, 325-350 28 Jancik, J. and Shauer, R. (1974) Z. Physiol. Chem. 355, 395-o

29 Goldstone, A., Konecny, P. and Hoeing, H. (1971) FEBSLert. 13,68-72 30 Okada, S. and O’Brien, J.S. (1969) Science 165, 698-700

Modification of nucleic acids in relation to differentiation Ernest Borek Some of the functions of the modification of nucleic acid are just emerging more than a decade after the discovery of the modifying enzymes. Negative conclusions drawn from ill-designed ‘quickie’ experiments discouraged investigators from entering this fascinating complex field: only one enzyme, RNA polymerase, is needed to synthesize transfer RNA but there are more than 100 enzymes which modtfy it at the macromolecular level.

Nature is a frugal inventor. When She develops a satisfactorily functioning system it is installed in a wide variety of organisms. The awesome achievement of molecular evolution, information storage and retrieval via the complementarity of nucleic acids became the universal, selfperpetuating mechanism of all things living. However, with speciation came problems : since the DNA of all organisms has essentially the same structure, four bases, deoxyribose and phosphate ester bands, how was the individuality of the DNAs to be preserved? How to prevent the integration of the DNA of an invading parasite into that of the host? How to control the orderly, sequential activation of segments of information to achieve differentiation? The solution that emerged was a method of imprinting species-specific modification on nucleic acids. As usual in the history of science this is obvious in hindsight, and, as is also usual, the revelation did not come from a profound insight but rather from the acceptance of the guiding light of serendipity. In 1955 an unusual mutant of Escherichia coli was discovered in which, unlike in all other amino acid requiring organisms, the synthesis of RNA was not turned off in the absence of the required amino acid, methionine. The extraordinary coincidence of this genetic aberration, which was later called RC (relaxed control) with the requirement for methionine, proved to be widely seminal [l]. The presence of methylated bases in DNA was known soon after paper chromatography enabled precise analysis of DNA hydrolysates, but the demonstration of their presence in transfer RNA (tRNA) in which they are more numerous and varied awaited the discovery of tRNA itself. The origin of these modified bases was baffling. The brilliant demonstration by Kornberg of the polymerization of DNA E.B. is Professor of Microbiology and Director of the Basic Oncology Program at the University of Colorado Medical Center, Denver, Colorado, U.S.A.

on a DNA template from monomeric precursors and the similar mechanism subsequently discovered by Samuel Weiss for the synthesis of RNA became the dominant paradigm for the weaving of the structure of nucleic acids. During a seminar on minor components of tRNA by Waldo Cohn, who had discovered pseudouridine in tRNA which is another type of a modified base, it occurred to me that there must be something wrong with the RNA that accumulates in the ‘relaxed’ organism during methionine starvation. The methyl groups on the nitrogens of guanine, adenine and cytosine must come from methionine. A student, L. R. Mandel, demonstrated by painstaking analyses that the RNA that accumulates during methionine starvation indeed does not have the normal complement of methylated bases. Such RNA came to be known as ‘methyl-deficient’. In turn he confirmed by in vivo labelling with [3H]methyl methionine that the methyl groups in tRNA do originate from methionine. His finding of a highly labelled methyl group in ribothymidine which is a ubiquitous component of E. coli tRNA, came as a complete surprise. It had been shown by Kornberg that the methyl group in the thymine of DNA stems from the one carbon pool through the folate pathway. The new pathway for the synthesis of thymine for RNA by the direct transfer of an intact methyl group led us to explore whether these methylated bases were synthesized at the macromolecular level. Another student, Irwin Fleissner, was able to show first with in vivo labelling with [32P]- and [14C]methionine that in the methionine-starved, relaxed organism methyl groups go into tRNA at a much faster rate than 32P, which is the index of polymerization of the nucleosides. Fig. 1 drawn from Fleissner’s findings is the first unequivocal demonstration in vivo of modification of a nucleic acid at the macromolecular level. The next step, the demonstration by Fleissner of the existence of enzymes, the

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anticodan

Generalfeatures of a tRNA molecule (alanine tRNA shown) in the cloverleqfconfiguration.

tRNA methyltransferases, which can methylate RNA at the polymer level in vitro was relatively easy: he had a substrate, methyl-deficient RNA, for assaying activity [2]. Species specificity of nucleic acid modifications The species specificity of these modifying enzymes emerged soon: for studies of the distribution of the tRNA methylating enzymes a control with E. cd’ B tRNA, which is fully methylated by the indigenous enzymes, was always included. While such control was essentially negative when enzymes from E. coli B were used, we found very high incorporation of methyl groups from heterologous enzymes which apparently could seek out sites for methylation which the E. coli B enzymes left vacant. The species specificity of the tRNA methyltransferases promptly pointed the way for searching for the DNA methyltransferases because while at the time there was no natural methyl-deficient DNA, the existence of the enzymes could be demon-

cl4

strated by heterologous interactions. (Methyl-deficient DNA became available relatively recently when we found that the radiation resistant organism, Micrococcus radio&runs, lacks the DNA methyltransferase and consequently its DNA is devoid of methyl groups.) The species-specific modification of tRNA and DNA pointed to us very early that these must have some role which is species specific. On of the roles for the species specificity of DNA became obvious very early. We wrote in 1964, ‘The introduction of methyl groups into DNA undoubtedly alters the conformation of DNA conferring upon it species individuality. Such structural individuality might render difficult the integration of foreign DNA (from some infecting parasite) into the DNA of the host and thus species specific methylation would - serve as a guardian of DNA.’ [3]. After 10 years of brilliant effort on the part mostly of Werner Arber, such a role became well documented by the discovery of modification-restriction enzymes [4]. Since only a small fraction of methyl groups in DNA are involved in modification-restriction we must presume that the others have roles which are yet unknown. Whether they are involved in differentiation is conjectural. There are a variety of claims for a number of functions for methyl groups in DNA but nothing is firm. However, that we must continue to search for other roles of methyl groups is a conviction based on that Francois Jacob calls ‘La logique du vivant’. These enzymes, which perform their function at a high cost in energy, could not have survived the selection pressure of evolution unless they had some useful functions. This assumption buoyed us through the difficult early years when some colleagues concluded on the basis of ill-designed, artefact-laden, in vitro experiments that modification of P32

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nucleic acids has no function at all, neither in protein synthesis nor in modificationrestriction. We needed such buoying for, to borrow from the playwright Lilian Hellman, those were ‘Scoundrel times’ : manuscripts rejected, our major research support discontinued, this in spite of the fact that we had already demonstrated the first qualitatively altered biochemical component of every cancer cell. Modification of tRNA and differentiation Evidence for the simultaneous turning on of the synthesis of several enzymes, and, after all, that is differentiation, came from work with microorganisms. Ames had suggested a regulatory role for tRNAs for the synthesis of enzymes to manufacture the cognate amino acid when the latter falls in short supply. He confirmed this in Salmonella: when the load on the histidine-specific tRNA falls off, transcription of the messenger RNAs (mRNAs) with information for the nine enzymes needed for the biosynthesis of histidine is turned on. As the histidyl tRNA become loaded, the transcription of the mRNAs ceases. The mechanism of this sensitive control is unknown. We do know that the conformation of tRNA is changed reversibly by the loading and unloading of amino acids. However, Ames found a mutant in which the enzymes of histidine biosynthesis are not shut off, regardless of the loading on the tRNA. He and his co-workers examined the histidine-specific tRNA in this mutant in which the enzymes of the histidine pathway are constitutive and found that the tRNA is different in chromatographic properties from the tRNA in the wild type in which the enzymes are inducible. Analysis of the sequence of the two tRNAs revealed but one difference, two uridines in the mutant are not modified to pseudo-uridines. This is a small change in structure to confer such extraordinary attributes on the tRNA but the change is small only in planar representation. From X-ray crystallography we known that tRNA is highly structured. The effect of the non-modification of two uridines upon the tertiary structure of tRNA may be profound. At the present time we can only speculate on the effect of such small modifications upon the interaction of tRNA with some proteins or with DNA itself. It is obvious that a great deal more has to be learned about the interaction of macromolecules before their functional dynamics will emerge [5]. The reader may object that I am stretching the term differentiation with the example I have cited, but if the nine proteins whose synthesis is under the control of

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tRNA were not catalytic but structural, for example, the new proteins of a spore, then it would be readily accepted that this is differentiation controlled by a tRNA modification. Hormonal control of tRNA methyltransferases

In the search for functions of the species specificity of the tRNA methyltransferases we decided to study these enzymes in biological mechanisms which are highly species specific : systems undergoing changes in regulatory processes. In every system examined, and these were numerous, we found profound changes in the tRNA methyltransferases. Subsequent examination of the tRNAs themselves has revealed that in the systems in which the tRNA methyltransferases are changing there are also a few tRNAs which differ from those in stationary systems. The two systems which have been studied most intensively are hormone-stimulated target organs and tumor tissues. We had found that the tRNA methyltransferases are under hormonal control. In the uterus of ovariectomized animals the enzyme activity is one half that in the normal organ. The administration of physiological doses of estrogen restores enzyme activity to normal. We also found a different serine-specific tRNA in the uterus of the ovariectomized animal. Upon administration of estrogen this tRNA disappears, indicating that the structure of tRNA can be under hormonal control. We believe this is the first qualitative effect of a hormone upon the target organ; not more or less tRNA but a different one [6].

Fig. 2. Enhancedefficiency of translation of ovolbuminspectj?cmRNA with tRNAs extroctedfrom the oviduct of estrogen-stimulated immature chicks (upper curve) over tronslotion with tRNAs isolotedfrom the oviducts ofunstimuloted immature chicks. From ref. 6.

5

A possible functional difference among populations of tRNA from hormone-stimulated and unstimulated organs was explored by us using the system developed by Schimke [7]. Immature chicks are stimulated by estrogen which induces them to synthesize ovalbumin prematurely. We have developed a tRNA-dependent in vitro system for the syntheis of ovalbumin from ovalbumin-specific mRNA to which can be added a variety of tRNAs. The population of tRNA isolated from the oviducts of hormone-stimulated chicks can translate ovalbumin messenger twice as efficiently as the population of tRNAs isolated from the oviducts of unstimulated chicks (Fig. 2) [8]. It should be pointed out that the two populations of tRNAs were equally effective in translating globin mRNA. Therefore, it appears that some specific tRNA present in the hormone-stimulated animal is needed for efficient translation of the hormone-induced mRNA. tRNA methyltransferases in tumor tissues

We were prompted to study the tRNA methyltransferases in tumor tissue by an observation by Magee and Farber on the carcinogenic activity of dimethylnitrosamine. They found that the carcinogen alkylates tRNA about 10 times more than DNA [9]. Up until then, all attention had been focused on the alkylation of DNA by chemical carcinogens. Examination of crude enzyme extracts from several tumors revealed hyperactivity of the tRNA methyltransferases. These studies have been extended by many investigators and such hyperactivity has been observed in every tumor examined ; they encompass the whole spectrum of etiology, various growth rates and transplanted as well as spontaneous tumors. There is no exception, apart from some benign tumors [lo]. We also examined the tRNAs themselves in Novikoff hepatoma, by elution chromatography and found three novel species; for histidine, tyrosine and asparagine. These studies have also been extended in laboratories all over the world and again there is no exception. Every tumor examined contains a few novel tRNAs. These are universal qualitative differences. among the biochemical components of tumor tissue : not more or less tRNAs but different ones [l 11. The tRNAs of tumor tissue, however, are not hypermethylated, but there are differences in modification in the tumorspecific tRNAs which have been analyzed and the corresponding counterpart from normal tissue. For example, there is an extraordinarily complex modified base, the Q base, which is present in several

tRNAs in a wide variety of organisms. The Q base has another from which has been named Q*. Recently it has been shown that the starred form is conjugated with some carbohydrate moieties on the hydroxyls in the cyclopentene ring (Fig. 3). The Q* is higher in tumor tRNAs than in the normal counterpart. The concentration of the Q base among the population of tRNAs of Drosophila fluctuates during metamorphosis [ 121.All of this may be coincidental but I doubt it. One possible explanation for the paradox of hyperactive methylating enzymes and essentially normally methylated tRNAs has emerged recently. We find that some of the tRNAs in tumor tissue turn over at a much greater rate than the tRNAs in normal tissues. Perhaps the hypermethylated tRNAs are selectively eliminated. Indeed, there is a very high excretion of most of the modified nucleosides of tRNA in the urine of cancer patients. In view of the high levels of excretion I urged the National Cancer Institute to start a program to ascertain whether the level of excretion products could be used for diagnosis. Preliminary findings indicate that such levels of ‘markers’ correlate in many patients with their tumor burden and, more importantly, with the effectiveness of their therapy [13]. The many functions of tRNA

Transfer RNA is the most complex of all biomacromolecules in both structure and function. We know of over 50 different

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Structure of Q Fig. 3. The structure of the Q base.

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6

modifications which are achieved by probably more than 100 enzymes. Not only does tRNA participate in protein synthesis but it is also a regulatory molecule at both translation and transcription. Moreover, a totally unexpected activity for a tRNA species has been observed by Jacobson, who found that a tyrosyl tRNA in a vermillion mutant of Drosophila inhibits tryptophan pyrrolase, the first enzyme on the pathway of pigment formation. (The Q base appears to be missing from this tyrosyl tRNA [ 141.)This finding opens up a vast new area for exploration for unknown functions of tRNAs. Given all of these potencies of tRNAs, it is untimely to speculate on the molecular mechanism of their action in the regulation of differentiation or in de-differentiation in tumor tissue. However, the unknown must not be a deterrent for intensive investigation, rather it should be a challenge. (A complete bibliography of tRNA modification is given in ref. 15.)

References I Borek, E., Ryan, A. and Rockenbach, J. (1955) J. Bacreriol. 69, 460 2 Fleissner, E. and Borek, E. (1962) Proc. Nat. Acad. Sci. U.S.A. 48, 1199 Srinivasan, P.R. and Borek, E. (1964) Biochemistry 3. 616 Arber, W. (1965) Annu. Rev. Microbial. 19, 365 Singer, C. E., Cortese, R. and Ames, B. N. (1972) Nature 238, 72 Sharma, 0. K. and Borek, E. (1974) Adv. Enzyme Regul. 12, 85 7 Palmiter, R. D., Christensen, A. K. and Schimke, R. T. (1970) J. Biol. Chem. 246,833 8 Sharma, O.K., Beezley, D.N. and Borek, E. (1976) Nature 262, 62 9 Magee, P.N. and Farber, E. (1962) Biochem. J. 83, 114 10 Sheid, B., Lu, T. and Nelson, J. H. (1974) Cancer Res. 34, 2416 II Borek, E. and Kerr, S. J. (1972) Adv. Cancer Res. 15, 173 12 Kasai, H., Kuchino, Y., Nihei, K. and Nishimura, S. (1975) Nucl. Acid Res. 2, 1931 13 Waalkes,P. and Borek, E. (1975) Excerpta Medica, Internat. Congress Series 375, 15-31 14 Jacobsen, K.B. (1971) Nature 231, 17 15 Borek, E. (1974) Control Processes in Neoplasia 4, 147

Gating currents: molecular transitions associated with the activation of sodium channels in nerve Eduardo Rojas and Claude Bergman Elimination of specljk ionic currents across nerve membranes allows the detection of minute currents generated by the displacement of intramembrane charges, the so-called gating currents.

The development of the concept of the sodium channel started with the realization by Bernstein that cellular excitability was a property of the membrane. The starting point at the experimental level was the observation by Cole and Curtis [l] that, concomittant with a propagated electrical impulse (manifestation of cellular electrical excitability) in the squid giant nerve tibre, a decrease in the electrical resistance took place with no detectable change in the membrane capacitance. This result lent strong support to Bernstein’s concept and clearly indicated that the most plastic components of the axolemma, the proteins, E.R. is at the Departmenr of Biophysics, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K. and C.B. is at the Laboratoire de Neurobiologie, Ecole Normale Superieure, Paris5, France.

underwentstructural transitions leading to a transient increase in ionic fluxes. The problem of molecular organization of electrically and chemically excitable membranes is of fundamental importance in explaining Cole and Curtis’s observation and understanding the various mechanisms possible that could generate conductance changes in cellular membranes [2]. For example, it is known that proteins are important membrane constituents; the ratio by weight of proteins to lipids varies between 1.5: 1 and 4: 1 for different membranes. Since the bulk of the membrane capacitance is associated with the lipid phase, the constancy of the membrane capacitance during the passage of an electrical impulse may be interpreted assuming that only a minute fraction of these proteins (a few molecules scattered

throughout within the nerve membrane) underwent transitions inducing dramatic changes in conductivity. Hodgkin and Huxley [3], using the voltage-clamp technique (control of the membrane electrical potential using a feedback amplifier) developed by Cole and Marmont [l], described the kinetic and steady-state properties of the ionic conductance changes typical of the neuron, the most differentiated of the excitable cells. After their experimental analysis of the ionic conductance changes in the squid giant nerve tibre, it was clear that, associated with a step decrease in membrane potential from its controlled level close to the resting value of about -70 mV (the voltage inside the cell is referred to the voltage outside), there were two kinetic components in the membrane currents; these components represent the sodium and the potassium pathways. It was well-established that the currents through these pathways are characterized by driving forces appropriate to cations moving passively down an electrochemical gradient. The essential feature is that these cationic conductances are activated by changes in the electric field across the membrane (electrical excitability). Thus, ionic current records from the squid giant axon were accounted for by postulating the existence of three voltage-dependent, first-order membrane reactions. The fastest of these, described by the HodgkinHuxley variable m, was assumed to control the activation of the inward sodium current, (INa). The slower reactions, h and n, were assumed to control the inactivation of the sodium current and the activation of the outward potassium current, (I,), respectively. Thus, dk/dt=a,(l-k)-bt

(1)

where k = m, h or n and @,kand Z!$are rate coefficients, which are functions of the electrical potential across the membrane. In the description of the conductance change, Hodgkin and Huxley assumed that the sodium and potassium systems were two independent and non-interacting molecular processes. These postulates stimulated much discussion and research in this area of membrane physiology. The controversy of whether Z,, and Ix pass through the membranes in separate structures or through a single set of pathway which conduct first ZNaand then Ix was resolved by showing that the total membrane current could be made to exceed the maximum value of ZNaor Zk, as a single pathway could not simultaneously conduct ZN~and Ix [4]. The sodium and potassium systems were proven to be non-interacting, molecular processes by the experimental observation