(CANCER RESEARCH 26 Part 1, 2026-2040, September 1966]

Control Mechanisms in Ribonucleic Acid Synthesis1

VINCENT G. ALLFREY

The Rockefeller University, New York, Xew York

Summary

The study of RNA synthesis in isolated calf thymus lymphocyte nuclei indicates that most of the DNA is nonfunctional as atemplate in RNA synthesis. This view is supported by highresolution autoradiography and by fractionation procedures,which permit a separation of the active and inactive states ofthe chromatin. Because the addition of histones to nuclei inhibits many of their biosynthetic activities, including RNAsynthesis, while the removal of nuclear histones leads to increasedrates of RNA synthesis, studies were made of histone metabolism in cells in which RNA synthesis can be altered experimentally. Striking changes were observed in histone acetylation, areaction in which the terminal amino groups in the histonesaccept acetyl groups from acetyl-coenzyme A. Histone acetylation correlates very well with gene activation in lymphocytesstimulated to enlarge and divide by the addition of phytohemag-glutinin. An early acetylation of the histones appears to signala later change in the capacity of the chromatin to synthesizeRNA. Hormonal stimulation of RNA synthesis in the liver ofadrenalectomized rats also has a parallel in increased histoneacetylation. Cells in which RNA synthesis is inhibited (by theaddition of phytohemagglutinin) show a corresponding decreasein histone acetylation. Autoradiography shows that acetylationoccurs on the chromosomes in Chironomus salivary gland cells.It is also more pronounced in active chromatin fractions than infractions which are relatively inactive in RNA synthesis. Suchchromatin fractions in thymus nuclei also differ with respect totheir content of phosphoproteins. A study of phosphoproteinmetabolism in isolated calf thymus nuclei indicates that thephosphorylation of serine and threonine in nuclear proteins is anenergy-dependent reaction, that it is reversible, and that theturnover of phosphate in proteins is coupled to other energy-dependent reactions in the nucleus. A possible role of phosphoproteins in the structure and function of the chromatin is discussed.

From the viewpoint of biochemical genetics, the major bio-synthetic events that characterize cell type and cell functionare directly or indirectly under the control of the DNA of thechromosomes. The major function of the chromosomal materialin an interphase cell is regarded as essentially synthetic, and anactive locus is a site of RNA synthesis. The immediate geneproducts, messenger, ribosomal, and amino acid transfer RNA's,

then direct the synthesis of the proteins, structural and enzymic,

1This work was supported in part by a research grant (GM-04919) from the USPHS.

which are characteristic of the cell type, the individual, and thespecies.

This view, emphasizing as it does the central role of the cellnucleus, is given considerable weight in the following discussion,but it should be stressed at the outset that cellular controlprocesses are varied and that they include mechanisms, such asalterations in enzyme structure and activity, which are not considered here. The origins and functions of extranuclear DNA incytoplasmic organelles, certain to be important in the analysisof cellular control processes, are also overlooked in favor of themore weighty aspects of nuclear DNA function and its regulation.

Regulatory mechanisms have often been regarded as 1-wayavenues of communication out of the nucleus, with the rest ofthe cell playing an essentially passive role. Such a unidirectionalconcept of control is certainly not in accord with the resultsof classical embryologie experiments, especially those involvinga translocation of nuclei to different areas of cytoplasm, sincesuch experiments indicate clearly that the nucleus is responsiveto the environment in which it operates; nor does it accord withmore recent studies on the induction of chromosomal RNAsynthesis by steroid hormones. This type of feedback to thechromosomes, a major variable in the regulation of cell function,will not be emphasized in the experiments to be described here,although a few instances are given. It should be noted, however,that there is good evidence that the interphase nucleus is responsive to changes in nutrition, to stress, to hormonal stimuli,to ionic fluxes, to inductive effects from neighboring tissues, tocircadian rhythms, etc., and there is no longer any reason toregard the chromosomes or chromosomal activity as completelysheltered, immune to the environment, or beyond hope of correction.

In considering problems of regulation at the chromosomallevel, much can be learned from a study of the differentiated cellsin higher organisms. What types of control processes may beinvoked to explain the high degree of specialization and thedifferences between cells in different somatic tissues? In recentyears it has become increasingly clear that this control involves aselection process in which various portions of the genome arecalled into activity at different times during the life of the cell.The great differences in morphology, chemistry, and behaviorof differentiated cells can, in this view, be traced ultimately tothe selection mechanisms that decide which genetic loci will beactive and which will not.

We begin with a realization that, by and large, the varioussomatic cells of an organism, despite their differences, carryequivalent genetic endowments. This is evident in the equivalence of chromosome numbers in diploid cells of various specialized tissues. It is emphasized by the classical genetic observations

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Control Mechanisms in RATA Synthesis

that eye color, wing form, bristle number, and other externaltraits in the fruit fly, Drosophila, can be correlated with mappositions along the giant polytene chromosomes of the salivarygland. It is established at the chemical level by the findings ofMirsky and Ris (53) and of Boivin and the Vendrelys (76)that the nuclei of different somatic cells of an organism containequal or nearly equal amounts of the genetic material DNA,while sperm cells contain half that amount. Tumor cells, especially those perpetuated in long-term tissue cultures, may deviatefrom the norm in both chromosome number and DNA content,but there are good reasons to believe that this is a result of tumorprogression rather than tumor initiation (30). The DNA constancy rule may be complicated in virus-initiated tumors by thepresence of viral DNA in the host genome (11), but even in thecase of polyoma virus-induced tumors, it has been shown thatthe DNA extracted from the tumor has the same nucleotidesequences (as judged by "hybridization" experiments) as do theDNA's prepared from other somatic tissues of the same organ

ism (11).It follows that, for all its specialization, each cell is endowed

with a totality of genetic information characteristic of theorganism; yet this potential is only partially expressed. Lymphocytes, for example, do not synthesize hemoglobin, anderythrocytes do not synthesize myosin, nerve-sheath proteins,or digestive enzymes. Similarly, tumor cells usually lose thespecialized functions typical of their cells of origin and takeon other properties instead (65).

What are the mechanisms by which cells choose which geneticloci will be active and which loci will remain nonfunctional?This problem, apart from its fundamental interest in the molecular biology of the nucleus, is particularly germane to the studyof development and may be presumed to have some directrelevance to the breakdown of control mechanisms in cancercells.

One approach to this central problem begins with a study offunction in isolated cell nuclei. Using a combination of biochemical and cytologie procedures, correlations can be madebetween the activity of the genetic material in RNA synthesisand its morphology; and fine structure of the chromatin can,in turn, be related to its composition and metabolic activity.Some experiments of this sort will now be discussed. The workto be described was done in collaboration with A. E. Mirsky,V. C. Littau, B. G. T. Fogo, A. 0. Pogo, J. H. Frenster, and L.Kleinsmith. We are particularly indebted to T. A. Langan,whose findings with F. Lipmann on nuclear phosphoproteinswere made available to us before their publication (Ref. 47;T. A. Langan and F. Lipmann, manuscript in preparation).

RNA Synthesis in Isolated Nuclei

About 10 years ago it was observed that nuclei isolated fromcalf thymus lymphocytes still retained a number of biosyntheticactivities, including a capacity to synthesize RNA's from low

molecular weight precursors (9, 10). The activity of the nucleuscan be readily followed by tracer technics in which suspensionsof nuclei in buffered sucrose media are incubated with 14C-or3H-labeled purines, purine and pyrimidine nucleosides, or ortho-phosphate-32?, followed by isolation and characterization of the

radioactive RNA formed.

Fortunately, the isolated thymus lymphocyte nucleus retainsthe ability to synthesize ATP2 aerobic-ally as well as by gly-

colysis (9, 60); this is necessary for the further biosyntheticreactions in which nucleosides and free purines are converted tothe corresponding nucleoside triphosphates—the immediateprecursors of the RNA poly nucleotide chain.

As judged by the extent of incorporation of isotopie precursors, the isolated nucleus synthesizes appreciable amounts ofRNA, but different types of RNA are made at widely differentrates. This becomes evident when nuclear RNA's are fractionated

after short-term labeling experiments. The distribution ofisotope in the RNA fractions separated by solubility differences(6), by Chromatographie technics, or by modifications of thephenol procedure (72) indicates that the lymphocyte nucleus isnot synthesizing large amounts of ribosomal RNA at this time,but that it is rapidly assembling RNA's which resemble thymus

DNA in their average base composition. Indeed, nuclear RNAfractions with base compositions approximating those of calfthymus DNA have been isolated in bulk and analyzed chemically (7, 72), and such DNA-like RNA fractions have beenobserved to stimulate amino acid incorporation by ribosomesprepared from nuclei (8). Although it is now clear that suchmethods for the fractionation of nuclear RNA yield imperfectand degraded products, because of RNase activity during theisolation, they do indicate the great metabolic heterogeneity ofRNA's in the nucleus.

RNA Involvement in Nuclear Protein Synthesis

The need for RNA in nuclear protein synthesis became apparent when it was shown that amino acid incorporation in thenucleus, as in the cytoplasm, involves amino acid transfer RNA's

(25) and nuclear ribosomes (24, 66). Evidence for what nowwould be called "messenger RNA" came early. In 1957, it was

observed that nuclear RNA synthesis must continue if proteinsynthesis is to proceed at maximal rates (10). Agents whichblocked RNA synthesis, such as DRB (10, 75) or actinomycinD (8, 70), also induced a progressive inhibition of amino aciduptake into nuclear proteins. This is the expected outcome ifthe messenger RNA fraction of the nucleus includes componentswith high rates of replacement and decay. Of course, in thecells of higher organisms, messenger RNA's may be expected to

differ in their stability and turnover rates.

The Role of DNA in Nuckar RNA Synthesis

The synthesis of RNA's in the nucleus is DNA dependent,

as expected, but it is important to stress that not all of the DNAis actually functioning as a template for RNA synthesis. Thisfact has been demonstrated directly by experiments in whichthe DNA was progressively removed from nuclei exposed toincreasing concentrations of DNase. Such nuclei were thentested for the effects of DNA depletion on nuclear function(7, 10). It was found that 70-80% and possibly more of the totalDNA could be removed from nuclei with no apparent effect on

2The following abbreviations are used: ATP, adenosine tri-phosphate; DRB, 5,6-dichloro-ii-D-ribofuranosylbenzimidazole.lys/arg ratio, lysine-to-arginine ratio; PHA, phytohemagghitinin;DEAE, diethylaminoethyl ; UTP, uridine triphosphate.

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the amount of RNA synthesized. These experiments led toanother interesting finding concerning the effects of histoneson nuclear function. The synthetic activity of nuclei was impaired when histones were released at the time DNA was degraded. Protection against the adverse effects of histones wasprovided by the addition of polyanions, such as polyethylenesulfonate or polyacrylic acid, which could form complexes withthe basic proteins. In the presence of such protecting agents,RNA synthesis was apparently normal in nuclei that had lostmost of their DNA. It was concluded that the DNA removedmust have been nonfunctional as far as RNA synthesis wasconcerned; i.e., it was not available as a template for the RNApolymerase reaction (39, 74, 76). However, if all of the remaining DNA was removed, RNA synthesis came to a halt. If only aportion of the remaining DNA was removed, the rate of RNAsynthesis decreased proportionately. This led to a linear correspondence between the amount of DNA remaining in thenucleus and its capacity for RNA synthesis (7).

It follows that the DNA of the interphase lymphocyte nucleusmust be sequestered in ways that permit a small portion of thetotal genome to be functional as a template for RNA synthesis,while most of the genetic information is maintained in an inactive condition. As will be seen later, activation of previouslyinactive loci is possible and can be followed by changes in thechemistry of the chromatin.

Active Chromatiii and Inactive Chromatin

The lymphocyte nucleus offers an interesting structural corollary to support the previous conclusions about active and inactive DNA. Electron microscopy of such nuclei in tissue sections,or following their isolation, reveals that the chromatin is distributed into dense clumps of compacted fibrils and diffuseregions of extended filaments of about 100-150 A diameter

(48, 49). It has been shown that the electron density of thesenuclear areas after uranyl acetate staining corresponds closelyto their DNA content as visualized by Feulgen staining ofmatching thick sections (48). From such comparisons it can beestablished that most of the DNA of the nucleus resides in theelectron-dense areas, while the diffuse chromatin contains onlya fraction of the total DNA.

The biosynthetic activity of different regions of the nucleuscan be visualized directly by the technics of high-resolutionautoradiography (18, 48). Fig. 1 shows the distribution of radioactive RNA hi thymus nuclei following a 30-min incorporationof uridine-3H. Two features of such autoradiographs are striking:

the vast majority of the grains occur over or near the areas ofdiffuse chromatin, while few grains are seen over the large massesof condensed chromatin. Since most of the DNA of the thymusnucleus is present in the compact masses of ehromatin, it followsthat most of the DNA is inactive in promoting RNA synthesis.These conclusions support the inferences drawn from the DNaseexperiments summarized in the preceding section. They arealso in agreement with other cytologie investigations, e.g., thefinding by Hsu that chromocenters in mouse cells (strain H4c)are relatively inert in the incorporation of uridine into RNA(36), and a recent demonstration by Granboulan and Gran-boulan that RNA synthesis occurs preferentially in the diffusechromatin of kidney cell nuclei (28). In this connection, reference

should be made also to the superb autoradiographic study ofDNA synthesis in interphase nuclei of proliferating cells of thesalamander made by Hay and Revel (31). They found thatthymidine-3H incorporation occurs rapidly only in the DNA ofthe diffuse chromatin. This result, however, raises some interesting questions, because all of the DNA of the nucleus must bereplicated. It follows that all of the chromatin of a dividing cellmust spend some time in the diffuse state. This, in turn, suggests that the physical state of the chromatin is subject totransformation, presumably by active mechanisms. A possiblerole of the histones in chromatin structural changes is discussedbelow in connection with histone acetylation and histone interaction with phosphoproteins in the nucleus.

Chromatin Fractionation

A procedure has been developed which permits the isolationof chromatin subfractions which resemble those of the interphaselymphocyte nucleus in their morphology and biosynthetic activity (26). In this method, isolated nuclei are washed withdilute buffers at neutral pH and then allowed to swell in cation-free sucrose solution. A brief sonication of the swollen nucleidisrupts chromatin structure and releases the clumps from mostof the adhering diffuse fibrillar material. These fractions can beseparated by differential centrifugation. As expected, most ofthe DNA is recovered in the easily sedimented masses of condensed chromatin. The diffuse chromatin fraction is collected byhigh-speed centrifugation; it contains less of the total DNA,but its RNA content is appreciably higher than that of theclumps (26).

When such a fractionation is carried out after incubatingnuclei in the presence of radioactive RNA precursors, it is foundthat the specific activity of the RNA in the diffuse chromatinfraction may exceed that of the RNA in the condensed chroma-tin by a factor of 3 (26). Although this is less of a difference inactivity than one would predict on the basis of the autoradiographs presented in Fig. 1, the utility of the bulk fractionationprocedure for further biochemical studies is obvious. Its defectsare due largely to the imperfect nature of the sonication step:a short sonication leaves mam- of the fibrils still adhering to

the condensed chromatin clumps, while on the other hand, thehigh shearing forces developed in the sonic field may be expectedto fragment and disperse some of the clumps. The method iscurrently being modified in an attempt to sharpen the selectivityof the sonication procedure, and bulk preparations of active andinactive chromatin are currently available. They offer an opportunity to investigate differences in the chemistry of functionaland nonfunctional nuclear regions. Comparisons have alreadyrevealed differences in histone acetylation and in the phosphoryla-tion of proteins in the different chromatin fractions; these differences will be discussed below.

Histones and Nuclear Function

In 1961 it was observed that the addition of thymus histonesto isolated thymus nuclei led to an inhibition of protein and RNAsynthesis (1, 2, 5). This effect has been studied in some detailin the hope that it might lead to a clearer understanding of thechemical factors controlling chromosomal activity. The importance of the histones in nuclear economy is indicated by their

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Control Mechanisms in RNA Synthesis

high concentration in nuclei and by their presence in chromosomes. The average thymus nucleus, as isolated, contains abouti of its mass as histones. These basic proteins can be extractedin dilute acids, or in strong salt solutions (16, 54); they can alsobe displaced from the DNA by the addition of more basic proteins such as the protamines of fish sperm (54). The histones ofthe thymus comprise a mixture of proteins, and they have beenfractionated in many ways, the details of which need not bepresented here, except to note that much of the progress inbulk separation procedures is due to the work of J. A. V. Butlerand his co-workers; Daly and Mirsky; Crampton, Moore, andStein; K. Murray; L. Hnilica; H. Cruft; and H. Busch; the readeris referred to recent reviews (16, 58, 63) for more detailed accounts of this field. Chromatography of the thymus histones onion exchange columns usually yields 3 or 4 major fractions whichdiffer in amino acid composition, particularly with respect totheir lysine-to-arginine ratios. Although histones differ in otherrespects as well, the lys/arg ratio has become a convenientstandard upon which to base their classification. This has provenuseful even though practically all of the histone fractions so farisolated are themselves mixtures, as judged by electrophoreticanalysis on starch gels (16, 33, 59) or polyacrylamide gels (16,20, 51). In the interests of simplifying the study of their metabolism and function, one can conveniently group the histones into3 classes: lysine-rich, arginine-rich, and intermediate. Indeed,for some purposes, we have found it convenient to separate thehistones into only 2 major groups, using electrophoresis at pH 9on cellulose polyacetate strips; this procedure yields a fast-moving band of lysine-rich histones (lys/arg ratio: 14/1),followed by a broad, diffuse band containing the arginine-richfraction. Histone fractions separated in this way differ strikinglyin rates of synthesis and acetylation (see below), and onlythe slower band contains methyl groups attached to the lysineresidues in the polypeptide chains (57).

In considering the role of the histones in nuclear function, itshould be recalled that the addition of histones to nuclei inhibitsmany of their biosynthetic activities. For example, histones havebeen shown to inhibit nuclear ATP synthesis (52), to block aminoacid transport (2), and to reduce amino acid uptake by nucleiand isolated ribosomes (25). Histones have a widespread toxicityfor other enzyme systems as well—cytochrome oxidase, forexample (62)—and they effectively block mitochondrial respiration and phosphorylation (52). In view of this wide spectrumof inhibitory effects, considerable caution must be placed on theinterpretation of the observation that histones also inhibitnuclear RNA synthesis.

In another approach to the study of histone function, examining the UNA polymerase activity of chromatin fractions prepared from pea-seedling nuclei, Huang and Bonner observedthat the chromatin (containing the histone proteins) was lesseffective as a primer or template for RNA synthesis than wasfree DNA (37). [In the DNA-dependent RNA polymerasereaction, the ribonucleoside triphosphates are utilized as precursors and assembled into polynucleotide chains, the nucleo-tide sequence and specificity of which are directed by the natureof the DNA template (39, 74, 78).] Similar observations indicating an inhibition of RNA polymerase activity by added histones have been made by Karr and Butler (13), Hindley (32),Hurwitz et al. (40), and in this laboratory (4, 5, 16). This work

will not be reported in detail except to note that, in general,histones inhibit the uptake of isotopie RNA precursors, thoughthere is no general agreement on which histone fractions aremost inhibitory. Here, too, caution is needed in the interpretation of the results. Many of these effects can be mimicked byother basic proteins and by synthetic polycations, such aspolylysine. The enzymology of the RNA-synthesizing systemsis complex, and the assay is complicated by the tendency ofnucleohistone aggregates to precipitate from the medium. Thesecomplexities have led to conflicting results and to wide differencesof opinion on the significance of the findings. For example, recentwork by Sonnenberg and Zubay indicates that a sonically dispersed DNA-histone complex is an effective template for RNAsynthesis, and they have questioned whether the low primeractivity usually observed in chromatin fractions, or in DNA-histone complexes, is due primarily to the inaccessible physicalstate of the nucleohistone, rather than to a specific represserfunction of the histones themselves (73). Huang and Bonner, onthe other hand, have taken considerable pains to assure thataggregation and precipitation of the nucleohistones in their assaymedium are minimal and have concluded that histones do act asrepressers of RNA synthesis.

For these reasons we have been inclined to follow a differentapproach. Beginning with tests for RNA synthesis in nucleifrom which the histones have been removed, we have progressedto the study of histone structural transformations in cells duringperiods of gene activation or repression. Some of these experiments will now be described.

Effects of Histone Removal on Nuclear RNA Synthesis

Histones can be preferentially removed from isolated calfthymus nuclei by selective tryptic digestion. Thus, treatment ofnuclear suspensions with beef pancreatic trypsin under theproper conditions leads to a removal of about 70% of the totalhistone, without destruction of RNA polymerase activity, andwithout hydrolyzing more than 5% of the nonhistone protein (9).After such treatment, the rate of RNA synthesis by the nucleiincreases and may be 300-400 % higher than that observed inuntrypsinized controls. Chart 1 shows how the uptakes of guano-sine-8-14Cand adenine-8-14C are accelerated after histone removal.

The lower curves show the extent of RNA labeling in the controlnuclei; the upper curves show the corresponding activity inhistone-depleted nuclei. In some experiments the action of theproteolytic enzyme was moderated by the addition of the soyabean trypsin inhibitor (Curve 2o) ; the increase in RNA synthesiswas less marked, but still evident. Moreover, the addition offresh histone to such nuclei leads to an immediate inhibition offurther RNA synthesis (Curve 26).

Fractionation of the nuclear RNA's after such experiments

shows that removal of the histones is followed by an increasedsynthesis of the DNA-like or messenger RNA fraction; isotopeincorporations are increased by about 300% (9). The questionarises whether removal of the histones has induced the synthesisof new types of RNA, i.e., RNA species that are not normallymade in the lymphocyte nucleus because the corresponding DNAtemplates are not available. Attempts have been made to answerthis question by comparing the incorporations of different RNAprecursors in normal and trypsm-treated nuclei. Although differ-

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Guanosine-8- C Adenine-8-l4C

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CHART 1. The effects of removing histones on RNA synthesis by isolated calf thymus nuclei. The incorporation of guanosine-8-14Cand adenine-8-14C are plotted as a function of the time of incubation at 37°C.The lower curve (Curve 3) shows the time course of 14C

uptake into the RNA of control nuclei. The upper curve (Curve 1) shows the corresponding uptake in nuclei which were treated withtrypsin under conditions which removed 70% of the total histones. Curve Za, is for nuclei treated with trypsin + soy inhibitor tomoderate the enzyme activity. Curve 2b shows that the addition of histone to trypsin-treated nuclei again suppresses nuclear RNA

synthesis.

enees in uptake ratios have been observed, it cannot yet be concluded that new RNA's have been formed because of the likeli

hood that trypsin treatment of the nuclei alters the pool sizesof different nucleotides to different extents. A more promisingapproach is to test for new RNA's by measuring the extent ofhybridization of the newly synthesized RNA's with denatured

calf thymus DNA. Such experiments are now in progress. In thisconnection the autoradiographic studies of Robert and Kroeger(70) deserve special mention. They observed that trypsin treatment of the polytene chromosomes in insect cells leads to anincrease in RNA synthesis at the chromosomal loci or "puffs"

that were already engaged in RNA synthesis. This is an especially interesting result because it strongly suggests that the activeloci of the chromosomes still contain histone proteins. Thiswould argue against the simple view that DNA function requireshistone removal. In addition, the increased susceptibility of thehistones at active loci to tryptic attack indicates that they areless protected than are histones at inactive regions of the chromosome—a conclusion which raises the possibility that histone-

DNA linkages are not fixed, but can be modified in the transitionfrom "inactive" to "active" chromatin. More direct chemical

evidence in favor of both of these ideas is presented below.

Reactions ¿fodifying Histone Structure in the Nucleus

Thymus histones contain acetyl and methyl groups in additionto their amino acid constituents. The presence of JV-terminal

acetyl groups was first detected by Phillips (64), and the occurrence of e-iV-methyl lysine in hydrolyzates of the histones was

first described by Murray (56).We and others have been interested in the mechanisms by

which these substituents become incorporated into the histonemolecule. The results to date can be summarized briefly. It isnow clear that both acetylation and methylation take place aftercompletion of the polypeptide chain. Such group addition reactions can be readily distinguished from histone synthesis by theuse of puromycin; this antibiotic blocks amino acid incorporationinto the proteins of the nucleus, including the histones, but ithas no effect on either acetylation or methylation of the histones(4). Both acetylation and methylation are enzymic processes,which can be studied in isolated nuclei, in nuclear extracts, and inchromatin fractions (3). In acetylation, the donor group isacetyl coenzyme A (3); in methylation the original CH3-groupdonor is methionine (4, 56), and the immediate donor is adenosyl-methionine (61).

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Control Mechanisms in RNA Synthesis

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CHART2. Separation of I4C-acetyl-labeled histories on carboxymethylcellulose (CMC) columns after incubating isolated thymusnuclei in the presence of Na acetate-2-14C. The total histone fraction was extracted in dilute acid, precipitated in acetone, redissolvedand adsorbed to CMC. The elution was carried out in a salt gradient at pH 5.0, followed by 0.1 N HC1. The specific activity of thedifferent histone fractions is indicated by the height of the corresponding cross-hatched areas. The continuous line indicates the opticaldensity of the fractions in the elution profile.

Correlations between Histone Acetylation and Chromosomal Activity

In the hope that these remarkable structural modifications ofthe histones may offer clues to the regulation of chromatinfunction, studies have been made comparing the acetylation ofdifferent types of histones in different fractions of chromatin, inresting nuclei, and in nuclei triggered into new biosyntheticactivities. Some of these results will now be described.

When nuclei are incubated in the presence of acetate-2-14C(or acetyl-coenzyme A-14C), and the histones are subsequently

fractionated by column chromatography on carboxymethylcellulose (Chart 2), it is observed that the highest extent oflabeling occurs in the arginine-rich histone fraction (4). Similarresults are obtained if the histones are separated by zonal electro-phoresis on cellulose polyacetate strips.

We had observed previously (5, 16) that the arginine-richhistone fraction (prepared by carboxymethylcellulose chromatography) was a potent inhibitor of the RNA polymerase reaction, but the degree of inhibition was quite variable. The dis-cover}' of the relatively high rate of acetate incorporation intothe arginine-rich histones raised the possibility that acetylation

of the histones might affect their interaction with the DNA

template. This prompted an experiment in which the histonewas acetylated chemically and then tested for its effects on theRNA polymerase reaction (4). The histone was acetylated usingvery small amounts of acetic anhydride under conditions recommended for the gentle acetylation of the growth hormone; according to Reid, this method gives preferential substitution ofthe a-amino groups with only limited acetylation of the «-aminogroups of the lysine residues in the protein.

Three different RNA polymerase systems were tested, derivedfrom calf thymusnuclei,A'se/ierw-/iíaroZ¿,and Azotobacter vindandii,

respectively. All systems were inhibited by the arginine-richhistone fraction as isolated, but in all cases the acetylated histones failed to inhibit RNA synthesis as effectively as did theparent histone fraction; yet these derivatives were still highlybasic proteins with a strong affinity for DNA; moreover, theywere taken up rapidly by nuclei from which the native histoneshad previously been removed (4). Thus, it was clear that theresults obtained were not due simply to a loss of basicity of thehistone proteins. It was further concluded that comparativelysubtle changes in histone chemical structure could influenceDNA-histone interactions in vitro and modify the template activity of the DNA in the RNA polymerase assay system.

Now, considerable caution must be used in interpreting the

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results of such experiments with chemically modified histonefractions, but they have raised an interesting question: Does theenzymatic acetylation of histones—a process which proceeds inthe nucleus independently of histone synthesis—alter chromâtin

structure and function?To test this hypothesis, we have investigated the intranuclear

sites of histone acetylation and have extended our observationsto include a number of correlations between histone acetylationand RXA synthesis in vivo.

Histone AcetylatioTìir Chromatin Fractions

Several double-labeling experiments have been carried outwith isolated thymus nuclei, using uridine-2-14C or orotic acid-6-14Cas RNA precursors and sodium acetate-2-14C as a precursor

of the histone acetyl groups. Following incubation, the nucleiwere washed, broken by sonication, and fractionated to yield theactive and inactive chromatin subfractions (3, 26). These werethen extracted with dilute acid to yield the histones. The specificactivity of the total histone was measured by technics describedelsewhere (4). The specific activity of the RNA in each fractionwas also determined. The results of 4 experiments are summarizedin Table 1. It is clear that histone acetylation, like RNA synthesis, is much more pronounced in the "active" chromatin fraction.

Although this need not indicate any direct relationship betweenhistone structural modifications and RXA synthesis, a numberof other correlations support this view.

Histone acetylation is a chromosomal event. This is seen inautoradiographic studies of acetate-3H and uridine-3H uptake inpolytene chromosomes in the isolated salivan- glands of Chirono-

mus tenions. Uptake of both isotopes was clearly chromosomal(V. C. Littau, V. G. Allfrey, and A. E. Mirsky, manuscript inpreparation), but it remains to be seen whether histone acetylation, like RNA synthesis, can be induced at specific "puffing"

loci by steroid hormones (19) or by altering intranuclear ionconcentrations (46).

TABLE 1

COMPARISONOF HISTONE ACETYLATIONANDRXA SYNTHESISINSUBNUCLEAR FRACTIONS"

Experimenti234Precursorconcentrationuic/ml)61.03.05.01.0Chromatinfraction0IIIIIIIIIIIIIIIISpecific

activityof histones(cpm/mg)"432108568331007395109279824Specific

activity ofRNA

(cpm/mg)'20204030101)003330011402980

" Nuclei incubated for 30 min at 37°Cin the presence of Xaacetate-2-14C, or orotic acid-(i-l4C.

6 Concentration of sodium acetate-2-l*C.' Fraction I is the condensed, inactive chromatin fraction;

Fraction III is the diffuse chromatin active in RXA synthesis.d Determined by steam distillation of acetic acid-14C from 6 N

HjPOi hydrolyzates of histones.«Determined us described in Allfrey and Mirsky ((>).

TABLE 2A COMPARISONOFACETATE-UCANDLYSINE-UC INCORPORATIONS

INTO THE HISTONES OF ISOLATEDCALF THYMUS XUCLEIAND INTACT DUCK ERYTHROCYTES"

Kquul volumes of thymus nuclear suspension and duck erytliro-cyte suspensions were incubated for 30 min at 37°Cin the presenceof 5 nc of coenzyme A-acetate-l-14C and DL-lysine-l-14C-2 HC1.

Following incubation, thymus histones were extracted with 0.2 NHC1 as described elsewhere (10). The erythrocytes were hemolyzedwith Tergitol TP-9 and their nuclei collected by differentialcentrifugation. Histones were extracted in 0.2 N HC1.

SOURCEOFHISTONESThymus

nucleiDuck erythrocytesSPECIFIC

ACTIVITY OF HISTONES LABELEDWITHCoA-acetate-l-»C

(cpm/mg)"1340

139Lysine-l-'<C

(cpm/mg)19.3

13.8

" Determined by steam distillation of acetic acid-l-14C from the

acid hydrolyzates of the dialyzed histone fraction (10).

Another type of correlation between RNA synthesis andhistone acetylation became evident in tracer studies of nuclearmetabolism in cells in which the genetic apparatus appears toexist in a highly repressed state. The mature, nucleated red cellsof the duck, for example, incorporate only negligible amounts ofisotopie precursors into RNA. A comparison was made, therefore,of histone acetylation and RNA synthesis in these inactive cells,and the results were contrasted with the relatively high RNAsynthetic rates and histone acetylation activity in lymphocytenuclei (Table 2). The findings support the notion that cells whichare not actively engaged in RNA synthesis are not activelyacetylating their histones either.

An interesting extension of these experiments is now possible.Recent work by Harris (29) has shown that, in multinucleatehybrid cells formed by the hybridization of HeLa cells with avianerythrocytes, the erythrocyte nuclei resume the synthesis ofRNA. It should be possible, using autoradiographic procedures,to determine whether these nuclei resume histone acetylation atthe same time.

Histone Acetylation in Human Lymphocytes during Gene Activation

In a search for more direct evidence relating the acetylation ofhistones to the activity of chromatin, our attention has latelybeen directed toward the response of human peripheral lymphocytes to phytohemagglutinin (55). This system has yielded thebest evidence to date for the functional significance of histoneacetylation. Some experiments done with Beatriz G. T. Fogowill now be descrilx>d.

It is well known that human lymphocytes maintained in tissueculture will rarely go on to divide. However, when 1'HA (aprotein fraction derived from the common liean Phaseolus vid-garis) is added to the cell suspension, the results are immediateand dramatic. Nearly all of the cells are transformed, increasingin size and in over-all metabolic activity; they soon begin tosynthesize new proteins, and to increase their rates of RNAsynthesis. At a relatively late stage in their response to phytohemagglutinin, they synthesize DNA and histones, and after

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Control Mechanisms in RNA Synthesis

48-72 hr, 70-90% of the cells go on to divide. The reader is referred to recent reviews for more detailed accounts of this phenomenon (69, 71).

Our interest has focused on the early changes in nuclear structure and metabolism induced by the addition of the proteinstimulus. Lymphocytes maintained in tissue culture for 20-24 hr

respond immediately to added PHA (67). The response involvesa synthesis of new proteins and new types of RNA. Changes inamino acid incorporation into proteins become evident in 15min, but the increase in amino acid uptake can be blocked byboth puromycin and actinomycin D (67). The latter result makesit likely that the synthesis of new types of RNA is a prerequisitefor the increased synthesis of protein that is observed.

What happens to the metabolism of the hist ones when lymphocyte genetic activity is triggered in this way?

Repeated experiments have shown that histone acetylation,like RNA synthesis, is augmented soon after the addition ofphytohemagglutinin to human lymphocytes (67). As in isolatedthymocyte nuclei, the arginine-rich histone fraction shows ahigher rate of acetate-14C uptake than does the lysine-rich frac

tion. In these experiments the histones were prepared fromhighly purified lymphocyte nuclei, by extraction in dilute acids,followed by electrophoresis at pH 9 on cellulose polyacetatestrips to remove nonbasic protein contaminants (67). Typicalspecific activity figures showed 0.400 /i/umole of acetate incorporated per 100 /ujumolesof histone in the slow-moving arginine-rich band, while the corresponding figure for the fast-movinglysine-rich band was only 0.225 /¿jumólesincorporated in thesame time period (67).

Tracer studies also show that acetylation of the histones is notdependent on histone synthesis, which does not become appreciable for another 24 hr in culture, nor is acetylation blocked whenhistone synthesis is inhibited by the addition of puromycin (67).The difference between acetate-14C uptake and synthesis isillustrated by a comparison of the incorporations of acetate-2-14Cand alanine-l-14C into the arginine-rich histone fraction of PHA-

stimulated cells. After 1 hr of incubation, 0.01 jujumoleof alaninehad been taken up into the histones, a small effect compared with0.385 /u/umoleof acetate incorporated in the same time interval.

The PHA-stimulated cell acetylates its histones much morerapidly than does the control lymphocyte. In 4 experimentsmeasuring acetate-14C incorporation over a 1-hr period, acetylation of the arginine-rich fraction in PHA-treated cells exceededthat of the control cells by 400%.

Even more striking was a comparison of the ways in whichacetate uptake into histones and uridine uptake into RNA changewith time after addition of PHA. This was followed in pulse-labeling experiments; a uniform cell culture was divided equally—one-half received a PHA supplement, the other half served as acontrol. Aliquots of the 2 cultures were withdrawn at 15-minintervals and incubated for a fixed time period (15 min) in thepresence of uridine-2-14C or Na acetate-2-14C (or tritiated ace

tate). The radioactivity of the electrophoretically purified histonefractions was then determined and plotted as a function of thetime elapsed since adding the PHA to the culture. A similar plotwas prepared for the pulse-labeled RNA in the 2 cultures. Theresults (Chart 3) showed that the control lymphocytes incorporated acetate into histones, or uridine into RNA, at essentiallya uniform rate over the time-period studied. However, in PHA-

stimulated cells, the rate of acetylation increased rapidly withtime; indeed, it was evidently higher right from the outset.Moreover, the increase in the rate of histone acetylation appearsto precede the increased uptake of RNA precursors (Chart 3).This is additional support for the view that a change in the structure of the chromatin—brought about by, or coincident with,histone acetylation—is a necessary prerequisite for the synthesisof new RNA's at previously repressed gene loci.

This aspect of control has, as yet, little direct evidence tosupport it, but we have tested to see whether other examples ofgene activation also involve increased acetylation of the histoneproteins. For example, the administration of cortisol to adrenalec-tomized rats is known to cause an increase in RNA synthesisin the liver (as the Feigelsons and F. Kenney and co-workershave shown). We have recently observed that histone acetylationis also augmented in hormone-treated animals, but not in theadrenalectomized controls (A. 0. Pogo, V. G. Allfrey, and A. E.Mirsky, manuscript in preparation).

Equally striking are some correlations between the inhibitionof nuclear RNA synthesis and a cessation of histone acetylation.Human polymorphonuclear leukocytes, for example, are inhibited when phytohemagglutinin is added to the culture medium. The synthesis of RNA falls abruptly, and the acetylationof the arginine-rich histone fraction is inhibited to about thesame extent (67).

Similarly, when viral infection of mammalian cells in cultureleads to a depression of host cell RNA synthesis (34), thereappears to be a decrease in histone acetylation (V. Holoubek,personal communication).

Histone acetylation is only 1 aspect of the complex and changing chemistry of chromatin. Other features which have recentlyattracted our attention are histone methylation, histone phos-phorylation, and the dynamic metabolism of chromâtin-associated phosphoproteins. The last-named subject offers someinteresting new insights into the chemistry of the cell nucleus.

Nuclear Phosphoprotein Metabolism

It has been known for some time that cells exposed to ortho-phosphate-32P in tracer studies of RNA and DNA synthesis alsoincorporate 32P into phosphoprotein linkages (22, 41). The oc

currence and formation of phosphoserine in protein fractionsderived from cell nuclei have recently come under intensiveinvestigation by T. Langan and F. Lipmann.3 In a fine series of

experiments, they have amassed convincing evidence for thenuclear localization of a highly phosphorylated protein fraction,which appears to contain tracts of adjacent serine-phosphateresidues and phosphothreonine as well. Such proteins have beenfound in the nuclei of calf thymus lymphocytes and rat livernuclei, where they occur in high concentrations. A strikingillustration of their nuclear localization is the fact that they arereadily detected in the nucleated RBC of birds but not in thenonnucleated erythrocytes of mammals.3

Following T. A. Langan's lead, we have recently begun to

examine the phosphorylation of proteins in isolated calf thymusnuclei, paying particular attention to the nature of the phosphorylation reaction, the linkage between phosphate and pro-

8T. A. Langan and F. Lipmann, manuscript in preparation.

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Vincent G. Auf rey

I60C

O 30

Time of Incubation in Minutes afterPHA60

CHART3. Comparison of historie acetylation and RNA synthesis in "resting" lymphocytes and in cells stimulated by phytohemag-glutinin. Aliquots were taken from control and PHA-stimulated cell cultures at the indicated times and then incubated for 15 min inthe presence of uridine-2-14C or Na acetate-(methyl-3H). Following incubation, the histones were extracted with acid, precipitated withacetone, and then purified by electrophoresis on cellulose polyacetate at pH 9. The specific activities of the arginine-rich and lysine-rich bands are plotted separately as a function of the time after addition of PHA. The specific activity of the RNA was measuredby methods described elsewhere (67) and is also plotted against the time elapsed following addition of PHA. Note that the increasein rate of histone acetylation appears to precede the increase in the rate of RNA synthesis.

tein, and the metabolic stability of the phosphate previouslyincorporated.

It is a matter of some interest that chromatin fractions which,differ in their rates of RNA synthesis also differ in their contentof phosphoproteins. An analysis of the chromatin subfractionsprepared from thymocyte nuclei by the method of Frenster et al.(26) has been carried out by T. A. Langan. The fractions werefreed of RNA, DNA, and lipids, and the remaining protein wasanalyzed for its content of alkali-labile phosphate groups. Theresults were expressed in terms of the number of jumólesofalkali-labile phosphate groups per 100 mg of DNA in eachchromatin fraction. The active chromatin contained 13.5 /¿molesof P as phosphoprotein, while the inactive chromatin clumpscontained only 3.8 /umoles of P in an equivalent stretch of DNA(47).

In this connection, it is worth noting that the chromatin fractions, as isolated (26), do not differ appreciably in their histone-to-DNA ratios, although the active fraction may have slightlyless acid-extractable protein per unit weight of DNA (23). Thepresence of high phosphoprotein concentrations in the activechromatin is an interesting clue because it raises the possibilitythat phosphoproteins, by complexing with histones, may change

the affinity between histones and DNA and thus modify thetemplate activity of the chromatin in RNA synthesis. Indeed,complexes between isolated phosphoproteins and histones canbe formed in vitro (47), and their formation does diminish theinhibitory effects of added histones on the RNA polymerasereaction (47).

L. J. Kleinsmith in our laboratory has performed some experiments which cast some new light on the phosphorylation ofproteins in the cell nucleus. One surprising observation (whichwill warrant further study) is that histone fractions purified bycolumn chromatography on carboxymethylcellulose, or by electrophoresis at pH 9 on cellulose polyacetate, can be labeled withorthophosphate-32P and shown to contain radioactive phos-

phoserine (44). Yet, fractionation of the nuclear proteins aftersuch labeling experiments indicates that most of the radioactivityand the greater part of the alkali-labile phosphate remain in theresidual protein after extracting the nuclei with neutral buffersand dilute acids; only a small portion of the total radioactivity isfound in the readily soluble nuclear proteins or in the acid-extractable histones.

Other studies have established that the phosphorylation ofnuclear proteins is an energy-dependent reaction; agents such as

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Control Mechanisms in RNA Synthesis

iodoacetate, which blocks nuclear glycolysis, or 2,4-dinitro-phenol, which blocks aerobic ATP synthesis in thymus nuclei(52), also prevent the incorporation of 32P-labeled orthophosphateinto protein ester linkages.

The phosphorylation of nuclear proteins does not appear to betightly coupled to protein synthesis. The independent nature ofphosphorylation can be shown by the use of puromycin to inhibitprotein synthesis. The effectiveness of the antibiotic in blockingthe incorporation of serine-14C is indicated by the experimentssummarized in Chart 4. Note that phosphorylation, as judged

250 serine-'4C

10

100r

80 -

60 -

40

20

0

orthophosphate-32P

Control

20 3020 30 0 10Time of incubation (minutes)

CHART4. A comparison of the effects of puromycin on theincorporation of serine-14C and orthophosphate-32P into the proteins of isolated calf thymus nuclei. The specific activity of theprotein is plotted against the time of incubation. Note that puromycin effectively inhibits serine incorporation without affectingphosphate uptake into the phosphoprotein fraction.

by the incorporation of phosphate-32?, proceeds as usual. It

follows that phosphorylation probably involves the esterificationof serine and threonine residues in a previously existing poly-peptide chain. This conclusion is in accord with the findings ofLangan and Lipmann that rat liver nuclei contain an ATP-dependent protein phosphokinase.3

A clear distinction between protein phosphorylation andsynthesis became evident in "cold chase" experiments, in whichthe retention of previously incorporated phosphate-32? wascompared with that of serine-3-14C.Nuclei were incubated in thepresence of either serine-14Cor phosphate-32? for 15 min. Theywere then washed to remove the radioactive precursors and rein-cubated in media containing a great excess of phosphate-31? andserine-12C. The retention of 32P and 14Cin the nuclear protein

was then measured as a function of the time of incubation in the"cold" medium. The results are shown in Chart 5. It is clear thatserine-14C, once incorporated, remains stable for the duration ofthe experiment, but protein-bound phosphate groups turn overat a high rate. We have found that this "turnover" is itself energy

dependent and have proposed that the phosphoester linkages innuclear phosphoproteins are not broken by a simple hydrolyticreaction but that they are coupled to other energy-dependentreactions in the nucleus (44).

What is the significance of a rapid "turnover" of phosphate

groups linked to protein in the chromatin? The notion thatregions of high negative charge density in phosphoproteins mightinfluence DNA-histone interactions and so modify the structureand function of the chromatin has already been mentioned.Since the phosphoprotein concentrations of active chromatinfractions greatly exceed those of inactive chromatin fractions,and since the phosphate groups of these proteins turn overrapidly, a mechanism is suggested for influencing DNA-histoneinteractions in a dynamic way, and perhaps shifting the con

30 45 60 75 90

Time of incubation (minutes)

105 120

CHART5. Evidence for the turnover of protein-bound phosphate in isolated calf thymus nuclei. The nuclei were incubated for 15min in the presence of serine-14Cand phosphate-32?. They were washed and resuspended in radioisotope-free medium for the indicatedtime periods, and the retention of isotope previously incorporated was measured as a function of time during the "cold chase." Notethat serine-14C, once incorporated, is retained in the nuclear proteins, while phosphate-32? is rapidly lost from the phosphoproteinfraction.

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Vincent G. Allfrcy

densed, inactive state of the chromatin to a more open, functionalstate. Dephosphorylation of the diffuse chromatin, on the otherhand, could again lead to tighter coiling of the DNA-histone-protein complex. Such a spooling mechanism could begin toaccount for the observed autoradiographic evidence (31) that inDNA synthesis all of the chromatin must spend part of its time inthe diffuse state.

Since RNA synthesis in lymphocyte nuclei appears to requirethat the template DNA exist in the diffuse state, one would predict an increase in nuclear phosphorylation during periods ofgene activation. This has been tested in human lymphocytesstimulated by phytohemagglutinin (44). In one test, after 60min of incubation with PHA, followed by 15 min of labeling withphosphate-32?, the specific activity of the phosphoproteins inPHA-treated cells was 209% greater than that observed in thecorresponding controls. Moreover, the kinetics of 32P uptake

suggest that protein phosphorylation, like histone acetylation,is an early event in the course of gene activation in this system.Nuclear protein phosphorylation may, of course, have verydifferent meanings from those suggested above, but preliminarytests have failed to correlate this phenomenon with membrane-bound "transport" reactions (44); nor does it seem likely that

the phosphorylation of nuclear proteins is coupled to RNAsynthesis in a direct way, because agents which block nuclearRNA synthesis (such as DRB) (75) do not prevent phosphorylation of the proteins (44). Other possible roles of protein-boundserine phosphates include DNA binding (14), use as a stablephosphate pool, or use as intermediary agents in nuclear phos-phoryl transfer reactions. At present, the correlation with geneactivation seems the most promising lead, and kinetic studiesof protein phosphorylation and turnover under varying physiologic conditions in different nuclear types are being activelypursued.

On the Specificity of Control Mechanisms in RNA Synthesis

The problem of specifically inducing or repressing RNAsynthesis at relatively few loci in the chromosomes is not readilyanswerable in terms of histone interaction with DNA, assumingthat histones do repress RNA synthesis. Although it is clear thatdifferent histones can influence the structure of the chromatinin different ways (49) and that histone structures become modified in the course of gene activation (67), the question of histoneplacement on different cistrons along the DNA double helix isnot yet resolved.

It is clear that a control mechanism which permitted generecognition by complementary base pairing between DNA nu-cleotides and a complementary nucleotide sequence could resolvethis problem. In this view, genetic control would involve thedelivery and binding of regulatory proteins at sites dictated bythe recognition sequences of attached polynucleotides—an attractive alternative to the view that proteins alone must recognize and influence the function of thousands of different geneticloci in the chromosomes.

There are many reports in the literature of unusual or lowmolecular weight RNA's in the cells of higher organisms (e.g.,27, 38, 45, 77). Apart from the 5 S RNA's, which appear to be a

component of the ribosomes (27), some of these ribonucleic acids

appear to be associated with the proteins of the chromatin (38).For the past year we have been testing newer methods for the

isolation and purification of RNA's from isolated lymphocyte

nuclei, from Xenopus oocytes, and from Ilyanassa embryos atdifferent stages in development (E. H. Davidson, F. Kramer,M. Grippa, V. G. Allfrey, and A. E. Mirsky, manuscript inpreparation). Following extraction in phenol-cresol in the presence of sodium naphthalene-disult'onate, bentonite, and detergents, the RNA's are separated by chromatography on Ecteolaor DEAE-Sephadex columns.

In the expectation that lymphocyte nuclei (which cannotcarry on many of the functions of the liver) might contain certain RNA's which could repress chromosomal function in the

liver, we added small amounts of the thymus nuclear RNAfractions to suspensions of purified liver nuclei and then testedthe RNA polymerase activity of the latter. In one such test, inwhich the control nuclei incorporated 68 ¿t/iinolesof uridylic acid-14C(derived from TJTP-14C),the liver nuclei receiving the thymus

nuclear RNA (Fraction I from an Ecteola column precipitatedwith ethanol and redissolved) incorporated only 19.9 /u/nmolesof the labeled precursor (A. O. Pogo, V. G. Allfrey, and A. E.Mirsky, manuscript in preparation).

Such effects must, of course, be interpreted with extremecaution, especially in the light of the incisive investigations byS. Weiss and co-workers on RNA inhibition of RNA polymeraseactivity. It remains to be seen whether this relatively directapproach will be meaningful in the analysis of nuclear controlmechanisms.

Some Implications Relevant to the Cancer Problem

Up to this point very little has been said about the tumor cellor about the relevance of the control of RNA synthesis to thecontrol of cancer. There is good reason for this reluctance toextrapolate these early and inadequate findings about thebiochemistry of chromatin to yet another theory of cancer induction or cancer control ; at least 4 new aspects of chromosomestructure and metabolism have come to light in the past year,and intensive research in this area has hardly begun. Nevertheless, there are experimental data which suggest strongly that thetumor cell has altered its pattern of RNA synthesis and is "reading" parts of the genome which would ordinarily have been

suppressed or inhibited as far as their template activity is concerned. One of the best examples of this is provided by thesuperb experiments of Kidson and Kirby (42). They have shown,by counter-current distribution analysis of the rapidly labeledRNA's of normal and hepatoma tissue, that the tumor tissue has

deviated from the normal in its spectrum of messenger RNAproduction. This is illustrated in Chart 6, taken from their 1964paper (42). Even more striking are the results of later experiments (43), which showed that the shift away from the normalpattern of RNA synthesis is gradual and becomes more extremeduring prolonged feeding of the carcinogenic azo dyes, such as4'-fluoro-4-dimethylaminoazobenzene and aminoazobenzene. It is

important to stress that these changes in liver RNA metabolismare reversible, if azo dye feeding is stopped in time. This impliesthat feedback to the chromosomes can be effective as a cancercontrol mechanism. Moreover, this reversibility need not beregarded as peculiar to the precarcinogenic state induced by azo

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Control Mechanisms in RNA Synthesh

1500

«IOC

500UI—

C/l

250

OO

200

150

tx

«.0

LIVER

WEEKS AFTER TUMORTRANSPLANTATION

HEPATOMA

10 20 30 ¿0 50 60

TRANSFER No.TO 80 90

CHART6. Comparison by countercurrent distribution of therapidly labeled RNA fractions of normal liver and hepatomas.From Kidson and Kirby (42).

dyes, since reversion of hamster cells transformed by Roussarcoma virus has also been reported (50).

There has been a frequently recurring notion that tumor cellsare abnormal or deficient in histone content (e.g., 21). Thisinteresting possibility cannot be ruled out at present, but reportsto this effect have usually been criticized on technical grounds,and the present consensus is that normal and tumor cells neednot display major differences in their histone compositions. Onthe other hand, there is good evidence for change in other proteinfractions of the nucleus. For example, Bakay and Sorof, in adetailed electrophoretic study of the soluble nuclear proteins ofnormal liver and azo dye-induced hepatomas, reported that thehepatomas appeared to have less of the basic and near basiccomponents and more of the acidic proteins (12). This interesting result indicates that changes have taken place in the environment of the chromatin, and that nuclear charge distributions arealtered in the cancer cell. Since charge is 1 of the major variablesaffecting DNA-histone interactions, the shift toward negativitycould have a great influence on the fine structure of the chroma-tin and the template activity of the DNA. However, it remainsto be seen whether such changes in nuclear protein compositionare themselves responsible for a loss of genetic control or whetherthey are simply indicative of an abnormal protein metabolismfollowing deranged chromatin function.

A Final Note

In the preceding paragraphs emphasis has been placed on thesupposition that the carcinogenic process is not immune tooutside influences and that it can be reversed. The great experiments of Braun on plant tumors show that reversion tonormal function can be achieved (17). This is a goal which seemsfeasible for animal tumors as well, at least in the early stages ofhyperplasia.

Since it is likely that the cancer cell is defective in its controlover RNA synthesis, reversion to normal pat terns of growth andfunction will require understanding and manipulation of thecontrol mechanisms in chromosomes. Even in cases where reversion to the normal state seems most unlikely (as may be thecase in highly aneuploid tumor cell lines) control of cell divisionshould still be possible by blocking the synthesis or the initiationof synthesis of critical RNA's necessary for cell replication.

Prospects of achieving this goal may seem remote, but there isgood reason to believe that the field of control mechanisms inhigher organisms will soon have access to new and direct technicsfor selectively influencing nuclear function.

References

1. Allfrey, V. G. Observations on the Mechanism and Control ofProtein Synthesis in the Cell Nucleus. In: (). Lindberg (ed.),Functional Biochemistry of Cell Structures VI. è:127—47.Oxford: Pergamon Press, 1961.

2. — —. Factors Controlling Protein Synthesis in NuclearRibosomes in Vitro and in Vivo. In: The Molecular Basis ofNeoplasia, pp. 581-604. Austin: University of Texas Press,1962.

3. . Structural Modifications of Histones and Their Possible Role in the Regulation of Ribonucleic Acid iSynthesis.In: Canadian Cancer Conferences, Vol. 6, pp. 313-35.New York :Pergamon Press, 1964.

4. Allfrey, V. G., Faulkner, R., and Mirsky, A. E. Acetylationand Methylation of Histones and Their Possible Role in theRegulation of RNA Synthesis. Proc. Nati. Acad. Sei. U. S., 61:786-94, 1964.

5. Allfrey, V. G., Littau, V. C., and Mirsky, A. E. On the Role ofHistones in Regulating RNA Synthesis in the Cell Nucleus.Ibid., 49: 414-21, 19G3.

6. Allfrey, V. G., and Mirsky, A. E. Some Aspects of RibonucleicAcid Synthesis in Isolated Cell Nuclei. Ibid., 43: 821-26, 1957.

7. . Evidence for the Complete DNA-Dependence of RNASynthesis in Isolated Thymus Nuclei. Ibid., 48: 1590-96, 1962.

8. — —.Mechanisms of Synthesis and Control of Protein andRibonucleic Acid Synthesis in the Cell Nucleus. Cold SpringHarbor Symp. Quant. Biol., 28: 247-62, 1963.

9. Allfrey, V. G., Mirsky, A. E., and Osawa, A. Protein Synthesisin Isolated Cell Nuclei. Nature, 176: 1042^9, 1955.

10. . Protein Synthesis in Isolated Cell Nuclei. J. Gen.Physiol., 40: 451-90, 1957.

11. Axelrod, IX, Habel, K., and Bolton, E. T. Polyoma VirusGenetic Material in a Virus-free Polyoma Induced Tumor.Science, 146: 1466-68, 1964.

12. Bakay, B., and Sorof, S. Soluble Nuclear Proteins of Liver andTumor in Azo-dye Carcinogenesis. Cancer Res., 34: 1814-26,1964.

13. Barr, G. C., and Butler, J. A. V. Histones and Gene Function.Nature, 199: 1170-77,1963.

14. Bendich, A., Borenfreund, E., Korngold, G. C., Krim, M.,and Balis, M. E. Amino Acids or Small Peptides as Punctua-

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tion in the Genetic Code of DNA. In: Acidi nucleici e lorofunzioni biologica. Convegno Antonio Baselli, IstitutoLombardo, Accademia di Scienze e Lettere, pp. 214-37. Pavía,Italy: Tipografia Successori Fusi, 1964.

15. Bonner, J., and Huang, R.-C. Properties of ChromosomalNucleohistone. J. Mol. Biol., 6: 169-174, 1963.

16. Bonner, J., and Ts'o, P. O. P. (eds.). The Nucleohistones.San Francisco: Holden-Day, Inc., 1964.

17. Braun, A. C. A Demonstration of the Recovery of the Crown-gall Tumor Cell with the Use of Complex Tumors of SingleCell Origin. Proc. Nati. Acad. Sei. U. S., 45: 932-38, 1959.

18. Caro, L. G., and vanTubergen, R. P. High-Resolution Auto-radiography. J. Cell Biol., 15: 173-88, 1962.

19. Clever, U. Actinomycin and Puromycin: Effects on SequentialGene Activation by Ecdysone. Science, 146: 794-95, 1964.

20. Cruft, H. J. The Electrophoresis of Histones in Polyacryl-amide Gels. Biochem. J., 84: 47P-48P, 1962.

21. Cruft, H. J., Mauritzen, C. M., and Stedman, E. AbnormalProperties of Histones from Malignant Cells. Nature, 174:580-85, 1954.

22. Davidson, J. N., and Smellie, M. The Incorporation of Radioactive Phosphorus into the Ribonucleotide Fraction of LiverTissue. Biochem. J., 52: 599-606, 1952.

23. Frenster, J. H. Nuclear Polyanions as De-repressors of Synthesis of Ribonucleic Acid. Nature, 206: 680-83, 1965.

24. Frenster, J. H., Allfrey, V. G., and Mirsky, A. E. Metabolismand Morphology of Ribonucleoprotein Particles from theCell Nucleus of Lymphocytes. Proc. Nati. Acad. Sei. U. S.,46: 432-44, 1960.

25. . In vitro Incorporation of Amino Acids into the Proteins of Isolated Nuclear Ribosomes. Biochim. Biophys.Acta, 47: 130-37, 1961.

26. — —. Repressed and Active Chromatin Isolated from Inter-phase Lymphocytes. Proc. Nati. Acad. Sei. U. S., SO: 1026-32,

1963.27. Galibert, F., Larsen, C. J., Lelong, J. C., and Boiron, M.

RNA of Low Molecular Weight in Ribosomes of MammalianCells. Nature, 207: 1039-41, 1965.

28. Granboulan, N., and Granboulan, P. Cytochimie Ultrastruc-turale du Nucléole. II. Étudedes Sites de Synthèse du RNAdans le Nucléoleet le Noyau. Exptl. Cell Res., 38: 604-19,1965.

29. Harris, H. Behaviour of Differentiated Nuclei in Hetero-karyons of Animal Cells from Different Species. Nature, 206:583-88, 1965.

30. Hauschka, T. S. The Chromosomes in Ontogeny and Oncog-eny. Cancer Res., el: 957-74, 1961.

31. Hay, E. D., and Revel, J. P. The Fine Structure of the DNPComponent of the Nucleus. An Electron Microscope StudyUtilizing Autoradiography to Localize DNA Synthesis. J.Cell Biol., 16: 29-51, 1963.

32. Hindley, J. The Relative Ability of Reconstituted Nucleohistones to Allow DNA-Dependent RNA Synthesis. Biochem.Biophys. Res. Commun., 12: 175-79, 1963.

33. Hnilica, L., Johns, E. W., and Butler, J. A. V. Observationson the Species and Tissue Specificity of Histones. Biochem.J., 82: 123-29, 1962.

34. Holoubek, V., and Reuckert, R. D. Studies of Nuclear ProteinMetabolism After Infection of Ehrlich Ascites Cells withMaus-Elberfeld (ME) Virus. Biochem. Biophys. Res. Commun., IS: 166-71, 1964.

35. Hopkins, J. W., III. Amino Acid Activation and Transfer toRibonucleic Acids in the Cell Nucleus. Proc. Nati. Acad. Sci.U. S., 46: 1461-70, 1959.

36. Hsu, T.C. Differential Rate in RNA Synthesis between Euchro-

matin and Heterochromatin. Exptl. Cell Res., 87: 332-34,1962.

37. Huang, R.-C., and Bonner, J. Histone, a Suppressor of Chromosomal RNA Synthesis. Proc. Nati. Acad. Sei. U. S., 48:1216-22, 1962.

38. - —. Histone-bound RNA, a Component of Native Nucleohistone. Ibid., 54: 960-67, 1965.

39. Hurwitz, J., Bresler, A., and Diringer, R. The EnzymaticIncorporation of Ribonucleotides into Polyribonucleotidesand the Effect of DNA. Biochem. Biophys. Res. Commun.,S: 15-19, 1900.

40. Hurwitz, J., Evans, A., Babinet, C., and Skalka, A. On theCopying of DNA in the RNA Polymerase Reaction. ColdSpring Harbor Symp. Quant. Biol., 28: 59-65, 1963.

41. Johnson, R. M., and Albert, J. Incorporation of P*2 into the"Phosphoprotein" Fraction of Mammalian Tissue. J. Biol.Chem., 200: 335-14, 1953.

42. Kidson, C., and Kirby, K. S. Recognition of Altered Patternsof Messenger-RNA Synthesis in a Mouse Hepatoma. CancerRes. ,24: 1604-11, 1964.

43. . Selective Alteration of Rapidly-labeled RNA Synthesisin Rat Liver during Azo-dye Carcinogenesis. Ibid., 25: 472-77,1965.

44. Kleinsmith, L. J., Allfrey, V. G., and Mirsky, A. E. Phosphoprotein Metabolism in Isolated Lymphocyte Nuclei. Proc.Nati. Acad. Sei. U. S., 55: 1182-89, 1966.

45. Koteies, G. J., Antoni, F., and Radnot, M. The Nucleic AcidContent of the Lens and Some Properties of Its "Soluble-RNA." Acta Med. Acad. Sci. Hung., 19: 271-83, 1963.

46. Kroeger, H. Chemical Nature of the System Controlling GeneActivities in Insect Cells. Nature, 200: 1234-35, 1963.

47. Langan, T. A., and Smith, L. Interaction of Histones with aPhosphoprotein Preparation from Rat Liver and Its Effectson Histone Inhibition of RNA Synthesis. NIH InformationExchange Group No. 7, Scientific Memo No. 113, 1965.

48. Littau, V. C., Allfrey, V. G., Frenster, J. H., and Mirsky,A. E. Active and Inactive Regions of Nuclear Chromatin asRevealed by Electron Microscope Autoradiography. Proc.Nati. Acad. Sci. U. S., 52: 93-100, 1964.

49. Littau, V. C., Burdick, C. J., Allfrey, V. G., and Mirsky, A. E.The Role of Histones in the Maintenance of Chromatin Structure. Ibid., 54: 1204-12, 1965.

50. MacPherson, I. Reversion in Hamster Cells Transformed byRous Sarcoma Virus. Science, 148: 1731-33, 1965.

51. McAllister, H. C., Wan, Y. C., and Irvin, J. L. Electrophoresisof Histones and Histone Fractions on Polyacrylamide Gels.Anal. Biochem., 5: 321-29, 1963.

52. McEwen, B. S., Allfrey, V. G., and Mirsky, A. E. Studies ofEnergy-Yielding Reactions in Thymus Nuclei. I. Comparisonof Nuclear and Mitochondrial Phosphorylation. J. Biol.Chem., 238: 758-66, 1963.

53. Mirsky, A. E., and Ris, H. Variable and Constant Componentsof Chromosomes. Nature, 163: 666-67, 1949.

54. — —.Composition and Structure of Isolated Chromosomes.J. Gen. Physiol., 34: 475-92, 1951.

55. Moorhead, P. J., Nowell, P. C., Mellman, W. J., Battips,D. M., and Hungerford, D. A. Chromosome Preparations ofLeukocytes Cultured from Human Peripheral Blood. Exptl.Cell Res., 20: 613-16, 1960.

56. Murray, K. The Occurrence of Epsilon-N-methyl Lysine inHistones. Biochemistry, S: 10-15, 1964.

57. . The Heterogeneity of Histones. In J. Bonner andP. O. P. Ts'o (eds.), The Nucleohistones, pp. 21-35. San

Francisco: Holden-Day, 1964.

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-. The Basic Proteins of Cell Nuclei. Ann. Rev. Biochem.,58. -34: 209-16, 1965.

59. Neelin, J. N., and Connell, G. E. Zone Electrophoresis ofChicken Erythrocyte Histone in Starch Gel. Biochim. Bio-phys. Acta, SI: 539-41, 1959.

60. Osawa, S., Allfrey, V. G., and Mirsky, A. E. Mononucleotidesof the Cell Nucleus. J. Gen. Physiol., 40: 491-513, 1957.

61. Paik, W. K., and Kim, S. Study of the Origin of Epsilon-N-methyl Lysine in Proteins. J. Biol. Chem., 240: 4629, 1965.

62. Person, P., and Fine, A. Reversible Inhibition of Beef HeartCytochrome c Oxidase by Polyionic Molecules. Science, 1S2:43-44, 1960.

63. Phillips, D. M. P. The Histones. Progr. Biophys. and Biophys.Chem., le: 211-80, 1962.

64. — —. The Presence of Acetyl Groups in Histones. Biochem.J., 87: 258-63, 1963.

65. Pitot, H. C. Some Biochemical Essentials of Malignancy.Cancer Res., 23: 1474-82, 1963.

66. Pogo, A. O., Pogo, B. G. T., Littau, V. C., Allfrey, V. G.,Mirsky, A. E., and Hamilton, M. G. The Purification andProperties of Ribosomes from the Thymus Nucleus. Biochim.Biophys. Acta, SB: 849-64, 1962.

67. Pogo, B. G. T., Allfrey, V. G., and Mirsky, A. E. HistoneAcetylation and RNA Synthesis in Lymphocytes Stimulatedby Phytohemagglutinin. Proc. Nati. Acad. Sei. U. S., 55:805-12, 1966.

68. Reich, E., Franklin, R. M., Shatkin, A. J., and Tatum, E^L.Effect of Actinomycin D on Cellular Nucleic Acid Synthesisin Virus Production. Science, 1S4: 556-57, 1961.

Control Mechanisms in RNA Synthesis

69. Robbins, J. H. Tissue Culture Studies of the Human Lymphocyte. Ibid., 146: 1648-54, 1965.

70. Robert, M., and Kroeger, H. Lokalisation zusätzlicher RNS-Synthese in Trypsin-behandelten Riesenchromosomen vonChironomus Thummi. Experientia, 2/: 326-28, 1965.

71. Rubin, A. D., and Cooper, H. L. Evolving Patterns of RNAMetabolism during Transition from Resting State to ActiveGrowth in Lymphocytes Stimulated by Phytohemagglutinin.Proc. Nati. Acad. Sei. U. S., 64: 469-76, 1965.

72. Sibatani, A., de Kloet, S. R., Allfrey, V. G., and Mirsky, A. E.Isolation of a Nuclear RNA Fraction Resembling DNA inIts Base Composition. Ibid., 48: 471-77, 1962.

73. Sonnenberg, B. P., and Zubay, G. Nucleohistone as a Primerfor RNA Synthesis. Ibid., 54: 415-20, 1965.

74. Stevens, A. Incorporation of Adenine Ribonucleotide intoRNA by Cell Fractions from E. coli B. Biochem. Biophys.Res. Commun., S: 92-96, 1960.

75. Tamm, I., Folkers, K., and Shunk, C. H. High InhibitoryActivity of Certain Halogenated Ribofuranosylbenzimida-zoles on Influenza B Virus Multiplication. J. Bacteriol., 7%:54-58, 1956.

76. Vendrely, R., and Vendrely, C. La teneur du noyau en acidedesoxyribonucleique a travers les organes, les individus, etles espècesanimales. Experientia, 4: 434-36, 1948.

77. Virmaux, N., Mandel, P., and Urban, P. F. Evidence of TwoSoluble Types of RNA in Eye Lens. Biochem. Biophys. Res.Commun., 16: 308-13, 1964.

78. Weiss, S. B. Enzymatic Incorporation of RibonucleosideTriphosphates into the Interpolynucleotide Linkages ofRNA. Proc. Nati. Acad. Sei. U. S., Iß:1020-30, 1960.

SEPTEMBER 1966 2039

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Vincent G. Allfrcy

FIG. 1. Electron microscope autoradiographs of sections of isolated calf thymus nuclei following incubation in the presence of uri-dino-3H for 30 min. Note the prevalent localization of the silver grains over the diffuse areas of the chromatin, indicating that theseare the regions of RNA synthesis. The line in the lower left corner is 1 />•X 21,000.

2040 CANCER RESEARCH VOL. 26

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1966;26:2026-2040. Cancer Res   Vincent G. Allfrey  Control Mechanisms in Ribonucleic Acid Synthesis

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