Chromosomal proteins and the regulation of gene expression in normal and neoplastic cells

Leukemia Research, Vol. 1, No. 4, lap. 351-384. Pergamon Press, 1977. Printed in Great Britain.

REVIEW

CHROMOSOMAL PROTEINS AND THE REGULATION GENE EXPRESSION IN NORMAL AND N E O P L A S T I C CELLS 1

OF

GARY S. STEIN~, JANET L. STEINa and JUDITH A. THOMSON~ J. Hillis Miller Health Center, University of Florida, Gainesville, FL 32610, U.S.A. (Received 4 April 1977. Accepted 27 April 1977) IT is becoming increasingly apparent that chromosomal proteinsmhistones and nonhistone chromosomal proteins--play an important role in dictating structural and functional properties of the eukaryotic genome. The histones, or basic chromosomal proteins, have been shown to function as repressors of DNA-dependent R N A synthesis, as well as to be involved in packaging o f the genome. Components of the nonhistone chromosomal proteins may also be involved in the maintenance o f genome structure, but additionally several lines of evidence suggest that amongst this complex and heterogeneous class of chromosomal proteins are macromolecules which are responsible for rendering defined genetic sequences transcribable. In the present article we will attempt to characterize the genome-associated proteins with respect to their structural as well as functional properties. Understanding the manner in which these macromolecules interact with the information encoded in the nucleotide sequences of the D N A double helix undoubtedly will enhance our comprehension of a broad spectrum of normal biological processes, such as growth, development, differentiation and maintenance of cellular phenotype. Furthermore, it is reasonable to anticipate that elucidation of the mechanisms by which gene readout is controlled will facilitate deciphering the basis for the aberrations in gene expression which accompany disease processes such as neoplasia. The potential involvement of chromosomal proteins in neoplasia will be considered in some detail.

1 Studies from our laboratory described in this review were supported by research grants GM-20535 from the National Institutes of Health, BMS-7518583 from the National Science Foundation and F75UF-4 from the American Cancer Society. = Department of Biochemistry and Molecular Biology. 3 Department of Immunology and Medical Microbiology. 351

GENE REGULATION IN EUKARYOTIC CELLS There are several levels at which gene expression in eukaryotic cells may be controlled. Within the nucleus regulation may reside at the level of the genome. Such transcriptional control may involve the interactions of chromosomal proteins with D N A sequences and/or the specificity of R N A polymerases which are responsible for the transcription of genetic information. Processing of R N A transcripts also occurs within the nucleus and is a potential level of regulation. Within the cytoplasm regulation o f gene expression may involve further processing of R N A transcripts or any of the complex steps required for protein synthesis. Additionally, post-translational modifications of proteins, either in the nucleus or in the cytoplasm, may influence gene expression. In any specific biological situation, control o f gene expression may reside at any one level or at several levels. Furthermore, the level of control of a given genetic sequence may vary depending upon the biological circumstances. I n the present article we will focus attention on transcriptional control, since it appears that regulation of gene expression, at least in part, resides at this level. A viable model for transcriptional control of gene expression in eukaryotic cells must effectively deal with three fundamental phenomena. First is the quantitative as well as qualitative similarity of D N A in all diploid nuclei of an organism. Thus, every somatic cell possesses a complete and identical set of genetic information. Second is the restricted availability of genetic information for transcription. In differentiated eukaryotic cells only 2-20 ~ of the genome is transcribable at any time, and the specific genetic sequences expressed are different in each cell type, reflecting the metabolic requirements of the cell. That is not to say that all cells express a totally distinct set of genes unexpressed in other cell types. Rather, in addition to expression of genes which are shared in common by many cells, e.g. genes which code for "general housekeeping enzymes", restricted expression of certain genes which often define unique

352

GARY S. S'rEIN et aL

cellular phenotype occurs on a cell or tissue specific basis. F o r example, globin genes are expressed only in erythroid cells, and the expression of ovaibumin genes is observed only in the oviduct. Third is the ability of cells to modulate gene expression in response to specific demands. Such modifications in gene readout occur during development and differentiation, during the cell cycle, in response to hormones, and in general provide a cell with the flexibility required to deal with changes in the intracellular and extracellular environment. The question which therefore arises is how specific regions of the genome are rendered transcribable-or how genes are "turned on" and "turned off". In microbial systems significant inroads have been made towards understanding the mechanism by which genes are regulated. Specific represser proteins have been isoleted which interact with defined genetic sequences and render genes non-transcribable [133, 303]. Specific activators have been shown to modify the interactions o f these repressors with D N A and hence permit transcription [4, 185]. The two prokaryotic systems which have been most extensively characterized are the lac operon [29] and the bacteriophage lambda [164]. While our understanding of prokaryotic gene regulation has progressed to a sophisticated level, caution must be exercised in assuming that analogous mechanisms are operative in eukaryotic cells. In eukaryotic cells the problem of gene regulation has been considerably more difficult to approach for several reasons: (1) In comparison with the prokaryotic genome the eukaryotic genome is considerably more complex. A human cell contains 10a times the amount of D N A present in the bacterium Escherichia coli. However, it is not clear if all the D N A sequences in eukaryotic cells function as genetic information. In addition to the vast increase in the amount of D N A present in eukaryotic cells, genetic sequences are represented as single copies or in certain cases as repeated sequences. Single-copy D N A sequences make up the majority of the genome, and most proteins are coded for by these unique sequences. A small percentage of the D N A of most eukaryotic cells consists of highly repeated sequences which are not transcribed. The function of these highly reiterated sequences is, at present, undetermined. The eukaryotic genome also contains moderately reiterated sequences which include those that code for ribosomal and transfer R N A and for the histories. It has been observed that other short, moderately reiterated sequences are interspersed with unique sequences and these short, moderately reiterated sequences have been proposed to function as regulatory elements. (2) Availability of large

numbers of mutants has facilitated implementation of genetic approaches for studying the regulation of gene expression in prokaryotic cells. However. mutants of eukaryotic cells are difficult to isolate and characterize because of the diploid nature of the genome. Progress has recently been made in genetic analysis of eukaryotic cells by use of the technique of somatic cell hybridization [322], and this approach offers considerable promise. (3) Unlike the prokaryotic genome which consists primarily of D N A , the eukaryotic genome is associated with large quantities of heterogeneous proteins. Although specific regulatory proteins in eukaryotic cells have to date not been identified, evidence is rapidly accumulating which suggests that chromosomal proteins play a key role in dictating the structural properties of the genome and in determining the availability of genetic sequences for transcription. HISTONES Five defined chromosomal proteins have historically been designated as histones. The histones are metabolically stable, positively charged chromosomal proteins enriched in arginine and lysine residues and completely lacking the amino acid tryptophane. Histones as a total class are associated with D N A in a l : l ratio (w/w) and are intimately involved in structural and functional properties of the genome. Because of their basic nature these proteins are readily extractable in dilute mineral acid---one method utilized for their preparation. However, acid treatment may denature the histones. Extraction with salt or displacement of histones with protamine may therefore be the method of choice for histone preparation under certain circumstances. The five principal classes of histones are, with few positive exceptions, present throughout the plant and animal kingdoms. Although all five histones bear a net positive charge, sequence analysis reveals that each histone exhibits a different non-random distribution of amino acids. F o r example, in the lysine-rich histones, polar (charged) amino acids are clustered near the NH~ end of the molecule while the COOH end is enriched in the non-polar residues. This asymmetric arrangement of amino acids in the various histones may provide an important clue to the mode of interaction of specific histones with one another and with other genome components. Involvement of histories in the regulation of gene expression was first postulated by the Stedmans in the early 1940s [369]. However, studies carried out by Huang & Bonner [173] and by Allfrey [10] provided the first biochemical evidence that histones are represser macromolecules. Huang and Bonner

Chromosomal proteins and gene expression showed that when increasing amounts of histones a r e complexed with D N A a progressive decrease in DNA-dependent R N A synthesis is observed and that transcription is completely inhibited at a I:1 histone to D N A r a t i o - - t h e ratio of histone to D N A generally found in the genome of intact cells [172]. Allfrey et aL demonstrated that progressive removal of histones from nuclei results in an activation of R N A transcription [10]. While numerous lines of evidence have substantiated a repressor function for histones, their limited heterogeneity makes it unlikely that, by themselves, histones have the capability to recognize specific gene loci. The similarity of histones in all tissues of an organism is a clear indication of the lack of specificity of these proteins. Although in a few instances an additional histone has been identified (e.g. H5 in avian erythrocytes) and in several lower eukaryotes specific histone fractions appear to be absent, these situations constitute isolated exceptions. The absence of histone fractions cannot be conclusively established until co-electrophoresis with other histones, variations in histone extractibility and proteolytic degradation of histories during isolation have been eliminated. The presence of only two substitutions in the entire amino acid sequence of one arginine-rich histone fraction (H4) which has been compared in calf thymus and in pea bud [89] additionally suggests the evolutionary stability of the histones and the structural and functional constraints which have been imposed on mutational variability of these molecules. Histone modifications

Some heterogeneity exists in individual histone fractions, which is largely attributable to modifications of the proteins in the nucleus or in the cytoplasm of cells following completion of synthesis. Without changes in the amino acid sequences of histones, acetate [summarized in 324], phosphate [17, 224, 248] and methyl groups [8, 43, 61, 62, 285] may be covalently added to and removed from the amino acids of certain histones. Additionally, the sulfur in cysteine can be modified. In the case of phosphorylation, while it has been known for some time that phosphate groups may be post-translationally added to serine and threonine, recently it has been observed that acid-labile amino phosphate is associated with other amino acids [71, 355]. The latter mode of phosphorylation was previously undetected due to the acid extraction procedures commonly used for the preparation of histories. Acetyl CoA is the principal donor of acetate groups [7, 324], and enzymes which are involved with acctylation and de-acetylation of histones have been

353

isolated [221, 264, 426]. S-Adenosyl-methionine is the primary methyl donor and several methylases have been identified [285]. The addition of phosphate to histones is catalyzed by a series of histone kinases [224], and phosphatases which are involved in the removal of phosphate from histones have been observed. One arginine-rich histone (H3) contains cystein residues which chemically and perhaps also enzymatically undergo oxidation of sulfhydryl groups (SH-~SS) and reduction of disulfide linkages (SS--~SH + SH). Post-translational modifications of histone fractions in cells have been correlated with changes in transcriptional, replicative and structural properties of the genome (reviewed by Hnilica [166]). These modifications in the histories are thought to alter h i s t o n e - D N A binding and histone-histone relationships as well as the modes of interaction of histones with other genome-associated proteins. Acetylation and phosphorylation of histones precede or occur concomitantly with the activation of transcription associated with numerous biological processes, e.g. stimulation of cellular proliferation, stimulation by hormones, development, and transformation by oncogenic viruses. Changes in the phosphorylation of specific histories also occur at defined points during the cell cycle. Removal of acetate and phosphate groups from histories which occurs as a function of time may provide a mechanism for repression of activated genetic sequences. Recent studies have provided evidence that the metabolism of phosphate and acetate groups associated with defined amino acid residues of historic fractions is regulated by specific enzymes. Langan & Hohmann [225] have demonstrated that in non-proliferating cells the serine 37 residue of the lysine-rich histone HI is phosphorylated by a cyclic AMP-dependent protein kinase. These investigators have also shown that in proliferating cells phosphorylation of the lysine-rich histone involves sites other than serine 37 and occurs to a large extent on threonine as well as serine residues. The enzyme responsible for the growth-associated phosphorylation, in contrast to the enzyme present in non-growing cells, is unaffected by cyclic nucleotides. Sequence analysis of peptides containing the phosphorylated sites on the lysine-rich histone recently carried out by Langan (personal communication) may provide insight into the mechanism by which histone kinases recognize the appropriate amino acid residues to be phosphorylated. Understanding histone phosphorylation at this level should enhance our knowledge of the biological relevance of the phenomenon. Alifrey and coworkers have identified a deacetylating enzyme which specifically utilizes acetylated lysine residues

354

GARY S. STEINet al.

of arginine-rich histories as substrate [221,426]. The enzyme is ineffective in removing acetate groups from proteins other than histories and exhibits specificity for particular amino acid residues. Methylation and changes in the oxidation state of the sulfhydryl groups of arginine-rich histones have been purported to alter the structural properties of the genome [63, 413]. These latter two post-translational modifications of histories have been correlated with chromosomal condensation during mitosis. However, the evidence for a role in mitosis for disulfide linkages between H3 histone molecules is very weak, particularly when one recognizes that one of the H3 cysteine residues is not conserved over evolution, while mitosis is. In contrast to histone acetylation and phosphorylation, which are reversible processes, histone methylation appears to be irreversible. 1-[istones and genome replication

Since the eukaryotic genome is a p r o t e i n - D N A complex, a comprehensive understanding of genome replication requires consideration of D N A synthesis and the synthesis of chromosomal p r o t e i n s - histones as well as nonhistone chromosomal proteins. Defining the manner in which genome replication occurs is a prerequisite to elucidating the control of cell proliferation--a biological process intimately associated with normal and neoplastic growth. A functional relationship between histone synthesis and D N A replication is suggested by the tight coupling of these events. Histone synthesis and D N A replication occur during a restricted period of the cell cycle (S phase) in cells in culture as well as in vivo, and inhibition of D N A replication results in a rapid shutdown of histone synthesis [44, 359]. Further evidence for the coupling of histone synthesis and D N A replication comes from the presence of histone messenger RNAs on polyribosomes only during the S phase of the cell cycle [42, 44, 57, 123, 124, 182, 393] and the transcription of histone genes only at this time [385, 386, 390]. The coupling of histone gene expression--histone polypeptide synthesis and histone m R N A transcription--with D N A replication has been observed in continuously dividing populations of cells and in nondividing cells following stimulation to proliferate. Although a definitive explanation for the concomitant synthesis of D N A and histones cannot be provided at this time, it is reasonable to speculate that histones are required to complex with newl~¢ replicated DNA. Neither nucleoplasmic nor cytoplasmic pools of histones are present and histones are needed for repression of those D N A sequences which are not to be immediately transcribed and for imposition of the appropriate structure to the genome, i.e. pack-

aging of the newly replicated DNA. While the mode of histone gene expression described above, which appears to involve regulation primarily at the transcriptional level, is the general situation, exceptions have been noted. In sea urchins and in xenopus, initial stages of embryonic development, which involve rounds of successive D N A replication and cell division (cleavage and blastulation), can occur in the presence of inhibitors of R N A synthesis

[150, 351]. Thus, a biologically important situation exists where D N A replication takes place in the absence of histone gene transcription. Essentially this indicates that during early development in these organisms regulation of histone gene expression does not residue at the transcriptional level. In fact, it has been demonstrated that under these conditions maternal messenger RNAs--synthesized and unexpressed in the unfertilized oocyte--serve as the templates for histone synthesis which is activated following fertilization [103, 121, 151]. While the studies to date dealing with histone gene expression during early stages of development have been restricted to invertebrates and amphibians, it is possible that a similar mechanism is operative in higher organisms. Another important biological situation where an apparent uncoupling of histone synthesis and D N A replication has been reported is following radiation or carcinogen induced damage to DNA. Such perturbations of D N A result in excision of the damaged / nucleotide bases and "unscheduled", or "repair" D N A synthesis to replace these nucleotides and restore the biological integrity of the D N A . Stimulation of histone synthesis has not been shown to accompany this D N A repair synthesis [384]. The question which then arises is how, during replicative D N A synthesis, preexisting and newly synthesized histories are distributed between the preexisting and newly replicated DNA. Although a semi-conservative mode of D N A replication has been clearly documented in prokaryotic [255] as well as in eukaryotic cells [405], the fate of pre-existing and newly synthesized histones within this context remains to be established. While Chalkley and his collaborators [180, 18 I] have presented evidence for a random distribution of newly synthesized histones, Tsanev & Russev [419] have concluded that at replication old histones remain associated with the old D N A strands and newly synthesized histone is associated with the new D N A strand. Hopefully, utilization of a broad spectrum of covalent crosslinking agents coupled with high resolution centrifugation techniques will permit further qualifications of this perplexing problem.

Chromosomal proteins and gene expression

Histones and genome structure

Attention has recently been focused on the arrangement of histones along the D N A molecule. Electron microscopic and biochemical evidence from several laboratories has revealed clusters of protein approx. 1130 A, in diameter containing two each of histones H~a, H~b, Ha and H4 and one or possibly two molecules of H~ histone [142, 165, 214, 215, 233, 268, 277, 284, 300, 315, 358, 439]. These clusters, referred to as "nu bodies" or "nucleosomes", are produced from nuclei or chromatin by mild nuclease digestion and are associated with D N A of approx. 200 base pairs in length. It appears that the D N A is located on the external aspect of the particles. Such h i s t o n e - D N A complexes have been observed in all plant and animal cells thus far examined and have also been reported in some D N A tumor viruses [I 31, 147, 239, 300]. Stretches of nucleotides which are nuclease sensitive [80, 81] are presumably located between the clusters. When examined under the electron microscope the observed configuration is that of beads on a string with spaces--presumably D N A - - b e t w e e n the nucleosome particles. However, the packing ratio of D N A and protein crosslinking data suggests that adjacent nucleosomes are contiguous--perhaps joined by H~ histone. This apparent contradiction may be explained if one assumes that the nucleosomes are separated during preparation of nuclei and chromatin for electron microscopy. The localization of the nonhistone chromosomal proteins in relation to the nucleosomes is only beginning to be elucidated. It has been observed that the genetic complexity of the D N A (diversity of genetic sequences) associated with isolated nucleosomes is the same as that of the total D N A [223]. This finding suggests that the histone complexes are located randomly along the D N A and that no specific D N A sequence is required for nucleosome formation. The lack of preferential association of histones with defined genetic sequences is supported by formation of nucleosome-type complexes between histones and viral as well as bacterial DNAs. Furthermore, the frequency of sequences coding for m R N A s is the same in D N A of isolated nu bodies as in the total nuclear D N A , suggesting that template active regions of the D N A are also associated with the historic complexes [223]. Other recent evidence has suggested, however, that there may be some subtle differences in the association of chromosomal proteins with actively transcribed sequences. These studies have shown that globin and ovalbumin genes in tissues which are actively transcribing these m R N A s have a different sensitivity to nuelease digestion than

355

giobin and ovalbumin genes in tissues which are not synthesizing globin- and ovalbumin-specific R N A [16, 127, 438]. The relationship between nucleosomes, transcription and units of replication clearly warrant further study. Probing these questions may produce important information regarding the structural and functional properties of the eukaryotic genome--the two properties often being extremely difficult to separate. From the preceding discussion, it should be evident that histones play an important role in the maintenance of genome structure and in the repression of DNA-dependent R N A synthesis. When h i s t o n e - D N A interactions are altered, modifications in gene readout and in the structural organization of the genome are observed. These findings lead to the conclusion that histones are structural as well as regulatory macromolecules. However, their limited heterogeneity and lack of specificity suggest that histories do not by themselves possess the ability to recognize defined gene loci. NONHISTONE CHROMOSOMAL PROTEINS Recently a considerably amount of attention has been focused on the nonhistone chromosomal protein portion of the genome due to evidence which suggests that amongst these complex and heterogeneous proteins are macromolecules which are involved in the regulation of gene expression. The chemical and biological properties of the nonhistone chromosomal proteins have been extensively reviewed [26, 64, 98, 189, 246, 291,293, 366, 373, 380, 388, 389, 394]. Amino acid analysis of the nonhistone chromosomal proteins as a total class reveals an enrichment in acidic amino acid residues, principally glutamic acid and aspartic acid. However, the individual components of this class consist of acidic, basic, and neutral proteins. In contrast to the histones which are extremely stable proteins, the nonhistone chromosomal proteins exhibit a spectrum of stabilities with half lives ranging from several minutes to several cell generations [45, 88]. Although the messenger R N A s for specific nonhiston¢ chromosomal proteins have not been identified or isolated, metabolism studies indicate that the stabilities of messenger R N A s for the nonhistone chromosomal proteins also cover a broad range [382]. Furthermore, unlike the histones whose synthesis is tightly coupled with D N A replication, various classes of nonhistone chromosomal proteins are synthesized throughout the cell cycle [35, 45, 195, 299, 327, 371, 374] and their synthesis is generally unaffected by inhibition of D N A replication [374, 392].

356

GARYS. S'rEI~et al.

Many of the nonhistone chromosomal proteins have the capacity to undergo post-translational modifications including acetylation [399], methylation [119] and phosphorylation [208, 209]. Acetyl CoA serves as the principal acetate donor, Sadenosyl methioninc as the primary source of methyl groups and phosphorylation is cataiyzed by cyclic nuclentide-dependent and independent protein kinases. Chains of poly (ADP-ribose) are found associated with certain nonhistone chromosomal proteins [48, 68, 357, 397] and glycoproteins [387] as well as glycosaminogiycans [387] have been shown to be components of defined molecular weight classes of nonhiston¢ chromosomal protein. Carbohydrate groups associated with chromosomal proteins may provide a means for interactions of the genome with the nuclear membrane. Alternatively, these carbohydrate-containing vroteins may be involved with genome structure. Additionally, cystein containing chromosomal proteins exhibit oxidation of suifhydryl groups and reduction of disulfide bonds [149]. These post-translational modifications confer the potential for increased specificity on the nonhistone chromosomal proteins. However, direct evidence for a functional relationship between these post-translational modifications of chromosomal proteins and alterations in gene expression remains to be established. Due to the complex and heterogeneous nature of the nonhistone chromosomal proteins, effective fractionation is a prerequisite to examination of their chemical, metabolic and biological properties. Numerous approaches to fractionating the nonhistoric chromosomal proteins have been pursued-a major problem being the insolubility of many of these macromolecules under conditions frequently employed for classical protein fractionation. Such insolubility may in part be attributable to the hydrophobic nature of many nonhistone chromosomal proteins. Fractionation approaches fall into two general categories: (l) analytical methods, solely for identification, quantitation and studies dealing with metabolic properties of the nonhistone chromosomal proteins, and (2) preparative methods for isolation of nonhistone chromosomal protein fractions. Analytical methods include molecular weight fractionation by SDS-polyacrylamide gel ¢iectrophoresis and recently developed high resolution separation by two-dimensional methods employing isoelectric focusing in one dimension and SDS--polyacrylamide gel electrophoresis in the second dimension. While such analytical approaches permit effective separation of nonhistone chromosomal proteins, these methods are limited by the small amounts of protein resolvable and the denaturation of most proteins.

Preparative methods include molecular weight sieving, ion exchange chromatography and a broad spectrum of affinity techniques. These preparative methods may provide chromosomal protein fractions which can be assayed for biological activity. It should be noted, however, that some preparative methods too may result in protein denaturation. Functionally, the nonhistone chromosomal proteins can be defined as structural, enzymatic and regulatory macromolecules. Genome-associated contractile proteins [94, 186, 230, 231] may in part assume structural responsibility, although this role is probably not fulfilled solely by contractile proteins. The contractile proteins which have been identified as genome components include actin, myosin, and tubulin. Modifications in the circular dichroism spectrum of chromatin following extraction of 0.25 MNaCl-soluble nonhistone chromosomal proteins suggest that components of this class of chromosomal proteins are involved in determining structural properties of the genome [77, 235]. The principal enzyme systems associated with the nonhistone chromosomal proteins are summarized in Table l--the variety of complex enzyme systems reflecting the functional diversity of these proteins. Enzymatic components of the nonhistone chromosomal proteins include enzymes involved in RNA synthesis and processing; DNA replication and repair; processing and degradation of protein; modification of nucleic acids; and modification of proteins by addition or removal of acetate, methyl, phosphate and ADP-ribose groups. Chromatin should be viewed as being operationally defined. Hence, the proteins found associated with the isolated genome reflect the methods of chromatin preparation. Techniques utilized for isolation of nuclei from which chromatin is prepared are extremely important. Inclusion of non-ionic detergents at appropriate concentrations during nuclear isolation results in removal of the outer aspects of the nuclear envelope and thus assures the absence of adhering cytoplasmic material. Conditions utilized for lysis of nuclei and washing of chromatin--particularly divalent cations and ionic strengths, as well as the degree of shearing--influence the protein to DNA ratio in chromatin. Transcriptional properties of chromatin prepared by different methods may also vary. During chromatin isolation and purification one must consider loss of genomeassociated proteins as well as adherence of nucleoplasmic proteins. However, the presence of common elements to chromatin and nucleoplasmic fractions need not necessarily be interpreted as an artifact of the preparation. Rather, this similarity may reflect a nucleoplasmic pool of macromolecules which exists

Chromosomal proteins and gene expression

357

TABLE1. ENZYMECOMPONENTSOFTHENONH1STONECHROMOSOMALPROTEINS Nucleic acids as substrates DNA polymerases RNA polymerases Nucleases Nucleotide ligase

Reference

Function

[70, 170, 171,238, 289, 421,422] [86, 126, 229, 260, 403, 440] [273, 424] [129]

Polymerization of deoxyribonucleotides into DNA Polymerization of ribonucleotides into RNA Processing or degradation of RNA and/or DNA Joining of DNA segments during DNA replication and repair Addition of nucleotides to the ends of nucleic acids Methylation of DNA or RNA

Nucleotide exotransferases

[328, 367, 432]

Methylases Chromosomal proteins as substrates Proteases Methylases Kinases Poly (adenosine diphosphate ribose polymerase) Poly (adenosine diphosphate ribose glycohydrolase) Acetylases Deacetylases

[25, 66, 79, 128] [63, 84] [71, 72, 205, 355, 401] [397, 445] [259] [125, 305, 306] [221,426]

in a dynamic equilibrium with the genome and may be functionally important [391]. Under certain conditions used for prepartion of chromatin rearrangement of proteins may occur; this is an especially important consideration when the physical relationship of chromosomal proteins is being studied, as with covalent crosslinking agents. Quantitative and qualitative differences in nonhistone chromosomal proteins have been correlated with alterations in the biological states of cells and tissues which reflect modifications in gene expression. Such differences in the nonhistone chromosomal proteins are consistent with a regulatory role for these macromolecules and have been extensively discussed in several reviews [26, 98, 246, 293, 366, 373,380, 388, 389]. Increased amounts of nonhistone chromosomal proteins have been observed in metabolically and transcriptionally active compared with inactive tissues [93]. An increased nonhistone chromosomal protein to DNA ratio is also found in transcriptionally active (euchromatin) compared with inactive chromatin (heterochromatin) [36, 118, 141, 244, 250, 311]. Qualitative variations in nonhis[one chromosomal proteins reflect their species and tissue specificity [97, 245, 314, 344, 409, 434] as well as differences in active compared with inactive chromatin [141]. Additionally, changes in the corn-

Processing or degradation of proteins Methylation of histones and nonhistone chromosomal proteins Phosphorylation of his[ones and nonhistone chromosomal proteins Addition of ADP-ribose moieties to chromosomal proteins Removal of ADP-ribose moieties from chromosomal proteins Acetylation of his[ones and nonhistone chromosomal proteins Removal of acetate groups from histone and nonhistone chromosomal proteins

position and metabolism of various molecular weight classes of nonhistone chromosomal proteins accompany modifications in gene expression associated with development [187, 344, 442], differentiation both in rive [298, 427] and in vitro [395, 456], stimulation of cell proliferation [232, 318, 372, 375, 376, 420], progression through the cell cycle [35, 132, 374], response to various pharmacologic agents [323], viral infection and transformation [78, 218, 219, 235, 319, 383, 455], cellular aging [376] and a broad range of other biological phenomena. In many biological situations steroid hormones play an important role in modifying structural and functional properties of eukaryotic cells. Such cellular modifications which are intimately involved with growth, development, differentiation and neoplasia are often associated with changes in gene readout. Evidence that hormone-mediated modifications in gene expression are at least in part regulated at the transcriptional level can be gleaned from quantitative changes in the availability of DNA as template for RNA transcription [160, 184, 279, 400] and from activation of the transcription of defined messenger RNA sequences; e.g. progesteroneinduced activation of avidin messenger RNA transcription in chick oviduct [254] and estrogeninduced activation of ovalbumin messenger RNA

358

GARY S. STEIN et aL

transcription [253, 318, 417]. It is now well accepted that steroid hormones initially form complexes with receptor proteins residing in the cytoplasm and that these hormone-receptor complexes are translocated to the nucleus where they interact with the genome [280]. Several lines of evidence suggest that nonhistone chromosomal proteins play an essential role in hormone-induced modifications in gene expression, perhaps by functioning as genome-associated accepter macromolecules. Such a point of view is supported by (a) the preferential binding of the hormone-receptor complexes in nuclei of target, compared with nontarget tissues [280], and (b) chromatin reconstitution studies which have shown that a specific class of nonhistone chromosomal proteins present in target tissues interacts with hormone-receptor complexes [280, 365]. Since hormone-responsive biological systems offer considerable promise for focusing on basic problems of regulatory processes, this is an area which is currently being intensively investigated. The selective affinity of proteins for binding to DNA, presumably by recognizing and interacting with particular nucleotide sequences, has provided further evidence for specificity of nonhistone chromosomal proteins. A subclass of the nonhistone chromosomal proteins has been shown by its selective binding to homologous D N A to exhibit species specificity. Several procedures have been employed for assaying protein-DNA interactions. Kleinsmith and coworkers, utilizing DNA--cellulose chromatography, a technique originally developed by Alberts [6] for isolation of DNA-binding proteins from bacteriophage T4, demonstrated that at physiologic ionic strength 1-2 ~ of the purified nonhistone chromosomal phospholarotein fraction from rat liver chromatin binds to homologous DNA [210]. Sucrose gradient sedimentation was used in Allfrey's laboratory to show that species-specific nonhistone chromosomal proteins bind only to homologous DNAs and result in changes in transcriptional properties of the genome [409]. Nitrocellulose membrane filtration techniques have also been utilized [290]. Recently, DNA-affinity columns containing moderately reiterated and highly reiterated DNA have been employed by Allfrey and coworkers to isolate nonhistone chromosomal protein fractions which preferentially interact with specific regions of the genome [9]. It is anticipated that in the near future specific genes, isolated from DNA fractionated by restriction enzyme cleavage, will be employed as highly defined genetic sequences. DNA-atiinity methods undoubtedly provide a powerful method for fractionation and purification of chromosomal proteins. However, caution should be exercised in

assuming that the types of protein-DNA interactions which are observed by these affinity methods are those which occur in the nuclei of intact cells. Many of the nonhistone chromosomal proteins undergo phosphorylation, specifically at the serine and threonine residues, and phosphorylation of defined molecular weight classes of these proteins has been correlated with modifications in gene activity in numerous biological systems [208, 209, 388]. These findings suggest that phosphorylation of nonhistone chromosomal proteins may be an important aspect of the mechanism by which genetic sequences are regulated. Phosphorylation of nonhistoric chromosomal proteins, as previously mentioned, is catalyzed by both cyclic AMP-dependent and cyclic AMP-independent [205] protein kinases. An important question remaining to be resolved which relates to nonhistone chromosomal protein phosphorylation and genetic control is whether specificity resides with particular nonhistone chromosomal proteins which are phosphorylated or with the protein kinases which catalyze the phosphorylation reactions. Recent studies which have shown differences between nonhistone chromosomal protein kinases of normal and neoplastic tissues suggest that regulation of nonhistone chromosomal protein phosphorylation may at least in part reside with the phosphorylating enzymes [41 l]. The quantitative and qualitative variations in nonhistone chromosomal proteins just discussed indeed suggest a regulatory role for these macromolecules. However, the evidence is of a correlative nature and therefore circumstantial. In vitro transcription studies and analysis of RNAs synthesized under cell-free conditions have permitted a more direct assessment of the involvement of nonhistone chromosomal proteins in the control of gene readout.

Nonhistone chromosomal proteins and regulation of specific genes Initial attempts at directly examining the role of nonhistone chromosomal proteins in the regulation of transcription were made by assessing the effects of nonhistone chromosomal proteins on DNAdependent RNA synthesis. Results from several laboratories indicated that nonhistone chromosomal proteins, when added to histone-DNA complexes or to various chromatin preparations, stimulated the availability of DNA as template for transcription of RNA [194, 226, 362, 363, 433 and reviewed in 380]. While these early results were encouraging they are somewhat inconclusive due to formation of nonspecific aggregates. Further evidence for a direct involvement of nonhistone chromosomal proteins in transcriptional control has been provided by chro-

Chromosomal proteins and gene expression matin reconstitution experiments where the aggregation problem can be effectively overcome. The isolated eukaryotic genome (chromatin) can be dissociafed and fractionated into its principal components--DNA, histones and nonhistone chromosomal proteins--and then utilizing a technique developed by Bonnet and coworkers [30, 173], chromatin can be reconstituted with selected genome components. Essentially DNA, histones, and nonhistone chromosomal proteins are combined in high salt-urea, and the salt is progressively removed by stepwise dialysis, followed by removal of the urea. Several lines of evidence support the fidelity of chromatin reconstituted by the procedure [30, 294, 379, 381]. However, the choice of tissues or cells and the methods for preparation of nuclei and chromatin are extremely important. In some situations utilization of protease inhibitors appears to be essential. The similarity of native and reconstituted chromatin preparations is suggested by quantitative and qualitative evaluations of the chromosomal protein, assessment of transcriptional properties and utilization of probes for structural integrity. Gilmour and Paul demonstrated that when DNA and histories from thymus and bone marrow were pooled and reconstituted with nonhistone chromosomal proteins prepared only from bone marrow, the RNA made from this reconstituted chromatin, assayed by competition hybridization analysis, resembled that normally transcribed from bone marrow chromatin. In contrast, when DNA and histories from thymus and bone marrow were pooled and reconstituted with thymus nonhistone chromosomal proteins, the RNA synthesized from such reconstituted chromatin resembled that normally transcribed from thymus chromatin [134]. Similar results were obtained by Hnilica and coworkers using other tissues [361,364]. These results thus pointed to the conclusion that the presence of tissue-specific nonhistone chromosomal proteins determines the pattern of genes which will be transcribed in a given tissue. Chromatin reconstitution studies have also provided evidence that nonhistone chromosomal proteins are responsible for the cell cycle stage specific variations in transcription from chromatin during the S-phase and mitotic periods of the cell cycle in continuously dividing HeLa S~ cells [379]. Similar studies have shown that nonhistone chromosomal proteins are involved with the activation of chromatin transcription following stimulation of non-dividing cells to proliferate [377]. Recent findings also suggest that certain nonhistone chromosomal proteins exhibit an inhibitory effect on transcription [217, 435]. It is therefore possible that components of the nonhistone chromosomal proteins may exert both negative and

359

positive influences on the outflow of genetic information. Although the regulation of specific genes could not be assayed by the methods employed in the studies just described, a regulatory function of the nonhistone chromosomal proteins was clearly indicated. During the past several years all-labeled, single stranded DNAs complementary to defined messenger RNAs (cDNAs) have been synthesized in vitro utilizing the enzyme reverse transcriptase (RNAdependent DNA polymerase). These cDNAs have been effectively employed as high resolution probes for identification and quantitation of specific RNA sequences isolated from intact cells and amongst in vitro chromatin transcripts. Results from several laboratories indicate that nonhistone chromosomal proteins associated with chromatin of erythropoietic tissues are responsible for the tissue-specific transcription of giobin genes [23, 72, 292, 418]. In these results it was shown that when chromatin from tissues which do not transcribe giobin genes is dissociated and subsequently reconstituted in the presence of nonhistone chromosomal proteins from erythropoietic tissues (fetal liver, bone marrow or DMSO-induced Friend cells), these reconstituted chromatin preparations exhibit transcription of globin mRNA sequences. Globin mRNA sequences were detected by hybridization with a 3H-labeled giobin eDNA. Chromatin reconstitution studies have also provided evidence that nonhistone chromosomal proteins associated with the genome during the period of the cell cycle when DNA replication occurs dictate the transient availability of histone genes for transcription at this time. When chromatin from cells in the prereplicative (GI) phase of the cell cycle, which does not transcribe histone genes, is dissociated and then reconstituted in the presence of nonhistone chromosomal proteins from S-phase cells, such reconstituted chromatin transcribes histone mRNA sequences. Activation of histone gene transcription by S-phase nonhistone chromosomal proteins has been demonstrated in continuously dividing HeLa cells [288, 386, 390] as well as in WI-38 human diploid fibroblasts following stimulation to proliferate [183]. The presence of histone mRNA sequences in chromatin transcripts was based on formation of $I nuclease-resistant, TCA-precipitable hybrids with single stranded DNA complementary to polyadenylated histone mRNAs. A similar approach has been pursued by O'Malley and coworkers to establish that nonhistone chromosomal proteins play a principal role in the steroid hormone-induced activation of ovalbumin gene transcription [416]. In evaluating results from transcription studies

360

GARY S. STEXNet al.

utilizing native and reconstituted chromatin the following crucial points must be considered. R N A associated with chromatin preparation may provide a background level of R N A sequences which can int,~rfere with quantitation of results from transcription experiments. The level of such endogenous R N A appears to depend on the particular genetic sequences in question and the method of chromatin preparation. Assessment of R N A s transcribed in vitro can be facilitated by carrying out transcription in the presence of mercurated ribonucleoside triphosphates. The mercurated R N A s can subsequently be separated from non-mercurated endogenous sequences. Chromatin-associated R N A s may also co-fractionate with nonhistone chromosomal proteins and thus interfere with interpretation of results from reconstitution experiments. Nucleic acids can be separated from chromosomal proteins by buoyant density centrifugation in cesium chloride-urea and the purified proteins can then be tested for their ability to render specific genes transcribable. However, the limitation o f such an approach is that small stretches o f nucleic acid which might be covalently bound to chromosomal proteins would not be removed. Although some of the early chromatin studies did not take the above-mentioned factors into account, later studies employing these necessary controls substantiate the contention that components of the nonhistone chromosomal proteins are involved in the regulation of several defined genes [380]. While indeed the techniques of in vitro transcription and reconstitution of chromatin may have limitations, they provide the most effective system presently available for evaluating the contribution of genomc-associated macromolecules to expression of genetic sequences. It should additionally be noted that to date most chromatin transcription studies, including those just described for elucidation of globin and histone gene transcription, have been executed utilizing bacterial R N A polymerase. While these studies have demonstrated a role for nonhistoric chromosomal proteins in dictating the availability of genes for transcription in chromatin, it is quite possible that there is an additional level of regulation which exists in the intact cell and which can be recognized by the appropriate homologous eukaryotic R N A polymerase. Nonhistone molecules

chromosomal

protet~s

as

regulatory

In the preceding discussion we have attempted to review progress which has been made during the past several years toward elucidating the role of chromosomal proteins in determining the structural and functional properties of the genome. However, we

are only at the threshold of understanding the nature of eukaryotic, regulatory macromolecules and their mode of action. With regard to the regulation of gene expression, histories appear to act as nonspecific repressors of DNA-dependent R N A synthesis. In contrast, amongst the complex and heterogeneous nonhistone chromosomal proteins are components which appear to regulate the transcription of specific genetic sequences. Yet, many perplexing problems concerning the nonhistone chromosomal proteins remain to be resolved. The specific nonhistone chromosomal proteins responsible for rendering particular genetic sequences transcribable must be identified. This is an especially difficult problem since to date the nature o f t b e regulatory proteins is an enigma. It is generally assumed that specific regulatory proteins comprise only a very small percentage of the nonhistone chromosomal proteins. However, experimental evidence to support this assumption is lacking. It is also reasonable to construct a viable model for gene regulation in which multiple copies of regulatory proteins are associated with the genome, with only a limited number of these proteins existing in a "functional interaction". An important concept which should be considered is that a single protein may regulate several genes--particularly in situations where cellular events are functionally interrelated or coupled. F o r example, one may envision several genes involved with genome replication being controlled by a single regulatory protein. A similar situation may exist with hormone-stimulated processes. It is not clear whether regulatory Proteins should comprise a subset of the nonhistone chromosomal proteins with common characteristics such as molecular weight, charge or structure. In this regard it will be interesting to establish whether similar types of proteins control "single copy genes" such as globin genes, as opposed to "reiterated genes" such as historic and ribosomal genes. A basic question to be answered is whether activation of genes is brought about by newly synthesized nonhistone chromosomal proteins or by modifications of pre-existing genome-associated nonhistoric chromosomal proteins. Alternatively, proteins residing in the cytoplasm or in the nucleoplasm may be modified in such a manner that they become associated with the genome and thereby render genes transcribable. Allfrey and coworkers have observed that activation of lymphocytes by rnitogenic agents results in accumulation of pre-existing cytoplasmic proteins in the nucleus [188]. A nucleoplasmic pool of nonhistone chromosomal proteins has also been reported [391]. These latter two observations are consistent with the possibility that alterations in

Chromosomal proteins and gene expression gene readout involve recruitment of proteins from the cytoplasm or nucleoplasm and their subsequent association with the genome. However, other studies suggest that protein synthesis is required for activation of transcription in human diploid fibroblasts following stimulation to proliferate [320]. In addition to numerous correlations between post-translational modifications of nonhistone chromosomal proteins and changes in gene readout, recent studies suggest that the phosphate groups on nonhistone chromosomal proteins are important in rendering histone genes transcribable [211,412]. Elucidating the manner in which nonhistone chromosomal proteins are associated with other genome components should significantly enhance our understanding of the mechanisms by which regulatory proteins interact with defined regions of the genome to render specific genes transcribable. While tenaciously bound as well as readily dissociable nonhistone chromosomal proteins have been purported to be the subclass of nonhistone chromosomal proteins which contains regulatory macromolecules, direct experimental evidence to permit distinguishing between these two alternatives is at present limited. It is also presently unclear if nonhistoric chromosomal proteins interact directly with D N A or with histone-DNA complexes. In addition to understanding the mechanism by which nonhistone chromosomal proteins activate transcription of specific genes, the mechanism by which transcription is "turned off", too, must be accounted for. One may envision inactivation of a gene or set of genes via degradation of the activator protein or proteins. Proteases which may utilize nonhistone chromosomal proteins as substrates have been shown to be associated with chromatin. An alternative mechanism for inactivation of regulatory proteins may involve acetate and/or phosphate groups added post-translationally to nonhistone chromosomal proteins. One can speculate that repression of genetic sequences may be brought about by removal of such moieties from nonhistone chromosomal protein molecules. Deacetylases as well as phosphatases have been identified within the nucleus, lending credence to such speculation. As fractionation and characterization of the nonhistone chromosomal proteins progress, the functional properties of eukaryotic gene regulators may become more apparent. GENE REGULATION AND NEOPLASIA It has been postulated that cancer is a disease of gene regulation [83,247, 437]. This would imply that the abnormalities seen in malignancy are due to the

361

malfunctioning of the very complex and, up to now, undefined mechanisms which dictate the gene expression of a given cell at the particular time of its life cycle. The malfunctioning of these mechanisms would allow for the derepression or repression of genes in a manner that is prohibited in the normal differentiated cell. Although the causative factors may be quite varied and unrelated, the manifestations of the disease would be solely due to the disruption of these regulating mechanisms. If cancer can then be explained by the untimely or abnormal expression of "'normal" genes (genes that are expressed in some cell type and contribute to the natural development or sustenance of the organism), then one would expect that all aspects of cancer cells would be exhibited by some cell at some time during the life cycle of the organism. To substantiate this postulate let us briefly examine the major characteristics and macromolecules exhibited by neoplastic cells and briefly review the evidence that these properties are found in some types of "normal" cells. The major distinguishable features of cancer cells; invasiveness, metastasis, rapid cell growth, and escape from the host immune system, are also the properties of many embryonic cells. Invasiveness is the prime characteristic of the trophoblast [203], and tumor cells are also similar to trophoblasts in the fact that both invade non-decidualized tissue but not deciduai tissue [444]. In addition a specific protease, plasminogen activator, which has recently been correlated with the invasibility of cells, has been demonstrated in both trophoblast and malignant cells [301, 345]. During embryogenesis many cells dissociate themselves from surrounding cells, migrate through tissues, and establish themselves in a new position. This is an analogous situation to the property of metastasis in tumor cells [83]. Alterations accompanying transformation include changes in the cell surface. These changes can be shown by the property of neoplastic cells to bind lectins, such as Concanavalin A or wheat germ agglutinin, to a much greater extent than do normal adult cells [50]. After agglutination, they have been shown to exhibit a more normal growth pattern in culture [50]. Embryonic cells are also agglutinated by Concanavalin A and by wheat germ agglutinin after proteolytic treatment to uncover the binding sites [262]. Tumors have long been known to produce a factor which stimulates the host to provide a blood supply [114, 115, 145]. This angiogenesis factor is also found in the placenta [113]. A characteristic of some human tumors that can sometimes lead to its detection is Radiogallium uptake and, although the mechanisms of localization arc unknown, this phenomenon has

362

GARY S. STEIN e t al. TABLE 2. COMMON COMPONENTS TO NORMAL AND NEOPLASTIC CELLS

Component

Neoplastic tissue or cell of origin

Normal tissue or cell of origin

Reference

KgOENZYMES

Thymidine kinase

HeLa cells Human fetal cell KB cells Human spleen Human fibroblasts transformed with SV40 Human rhabdomyosarcoma Wilm's tumor Bladder adenocarcinoma

Alcohol dehydrogenase (fast migrating)

Rat hepatoma

Aldolase C

Rat fetal liver Fast growing hepatomas Human rhabdomyosarcomas Yoshida ascites hepatoma Zajdela ascites hepatoma

Hexokinase Type H

Uterine carcinomas

Fetal tissue, intestine, heart, skeletal muscle

[200]

Uridine kinase (low molecular weight form)

Novikoff ascites hepatoma

Fetal liver

[222]

Carbamyl phosphate synthetase TyPe II

Ehirlch ascites carcinomas Hepatomas

Fetal liver

[155, 156, 252, 452]

Branched-chain amino acid transferascs Type I & II

Yoshida ascites hepatoma

Type I-fetal liver Type III-rat brain

Branched-chain amino acid transferases Type HI

3' MeDAB primary tumors Rat brain Morris hepatomas Rat brain Chemical carcinogen Rat ovary Spontaneously transformed Rat placenta cells Treated rat hepatocytes

Glycogen synthetase (muscle form)

Ascites hepatomas AH 66F and AH 130

Muscle

[329, 3301

Phosphorylase (muscle type)

Poorly differentiated hepatomas

Muscle

[3311 [332]

Alkaline phosl~hatase ("Regan" form)

Human bronchogenic Placenta carcinoma Human lung, gastrointestinal tract Genital tract tumors Human hepatocellular

Fructose-I 6-diphosphatase (muscle type)

Rapidly growing hepatomas Muscle of several species Ehrlich ascites

Glutaminase (Kidney type)

AH- 130 hepatoma Novikoff hepatoma LC 18 hepatoma Mammary carcinomas

Rat stomach Rat fetal liver

Kidney Fetal liver

[190, 368, 404]

[34] [337, 338, 339 340, 398]

[274] [2751 [177, 274, 275]

[108, 109, 110, I 11,396]

[333, 3341 [169, 197, 212, 237]

Chromosomal proteins and gene expression

363

TABLE 2--continued

Component

Neoplastic tissue or cell of origin

Normal tissue or cell of origin

Reference

FETALANTIGENSAND ANALOGS c~-Fetoprotein

Human hepatocellular carcinoma

Fetal liver

[I, 2, 136, 242, 304]

Carcinoembryonic antigen (CEA)

Human gastrointestinal tumors

Fetus

[135, 137, 261,429]

c~2 H-globulin

Serum of children with tumors (81%)

Fetal organ

Fetal hemoglobin

Leukemia and other hematological diseases

Fetus

[256]

Intestinal antigen

Gastric neoplasia

Fetal stomach and intestine

[2641

Fetal sulfoglycoprotein

Gastric cancer

Fetus

Nonspecific fetal antigens

Rat-human sarcoma HSI Chemically induced mouse

Fetal tissues

[157, 158, 159]

[54, 55, 56, 295, 302, 350, 414, 430]

sarcomas

Mice-3-MCA sarcomas Mice-polyoma tumor Coiorectal carcinomas Germinal cell tumors of testis

[49]

Embryonic tissues Fetal tissues

Plasminogen activator

SV40 transformed rat embryo cells

Mouse embryo

//-onco fetal antigen

Colon carcinoma Breast carcinoma Melanoma Endometrial carcinoma

Fetal organs

[1201

Placental antigens (PA-1)

SV40 transformed rabbit kidney cells (TRK-1)

Placenta

[69]

6S DNA polymerase

3' mDAB induced hepatomas Morris hepatomas

Rat embryos Neonatal liver Regenerating liver

[75]

Nuclear protein DNA antigens

Embryonic tissues

[73]

Isoferritins

3' mDAB induced hepatomas Malignant tissues

Early fetal tissues

[14]

Tissue polypeptide antigen

Malignant tissues

Placenta

Mouse plasma cell tumors Morris hepatomas Ehrlich ascites tumor Mouse fibrosarcomas Reuber hepatoma cells

Fetal liver undifferentiated stem cell

[301, 345]

[37, 242]

RNA SPECIES

tRNAs tRNA methyltransferases

[22, 38, 41, 51, 52, 122, 138, 146, 162, 198, 199, 317, [441, 446]

364

GARY S. STEm et al.

minute amounts of aFP, has led to the use of ,~-fetoprotein as a diagnostic tool. This tumor associated antigen can be detected in greater than 70~o of patients with hepatocellular carcinoma by methods of radioimmunoassay and greater than 6 0 ~ of patients with teratocarcinomas [143]. Recent studies have been initiated towards examining a-fetoprotein and its expression at the molecular level. Researchers have reported the isolation of the messenger RNA for ~-fetoprotein and report both poly A plus and poly A minus forms [402]. Other investigators have determined the partial sequence [92, 105, 153]. Examining the cancer cell on a molecular level, of the ,~-fetoprotein molecule [325]. Studies involving many macromolecules have been identified in the relationship of a-fetoprotein to the cell cycle and tumors which are characteristic of other adult cell cell proliferation [356], and the relationship of types or of fetal tissue. Included in these macro-- a-fetoprotein to hormone binding are also being molecules are cellular isoenzymes [87, 437]. Many currently examined [425]. Investigations have produced the detection of fetal isoenzymes have been shown to be produced many other onco-fetal antigens. By use of radioby tumors [83, 336], and stomach, brain and hauscle immunoassay techniques, detection of a ferroprotein isoenzymes have been identified in many hepatomas [336]. The research on isoenzymes is so extensive (a~H globulin), normally found in fetal organs and in that it is presented here in chart form (Table 2) and fetal serum, has been found in greater than 8 0 ~ of is necessarily incomplete. However, sufficient evi- children with tumors [49]. Another antigen that may dence is presented to substantiate the idea" that be of diagnostic value is the carcinoembryonic tumor cells exhibit the resurgence of fetal molecular antigen (CEA) found in human fetal gut and in a forms of enzymes and the expression of beteroiogous high percentage of human colon cancers. Additional cellular isoenzymes. An example of such an iso- examples of embryonic analogs and antigens are enzyme that has received attention in regard to its presented in Table 2. Also listed are certain forms of diagnostic capabilities is the "Regan" form of R N A species which are common to embryonic and alkaline phosphatase [108, 109, 110, 111, 396]. tumor tissues. An outstanding example of expression in neoplasia This isoenzyme, found to be distinguishable from the placental isoenzyme, has been found in of normally repressed genetic information is the patients with cancer of various organs, includ- observation of the decondensation and reactivation ing lung, gastrointestinal tract and genitals. The of the normally condensed X chromosomes in frequency of occurence of the "Regan" form of human female cervical and gastric carcinomas [410]. alakline phosphatase in cancer seems to be about This may be a relapse to a fetal state since it is known that both X chromosomes must be active at 10%. Many embryonic and fetal analogs of adult some time during embryogenesis in order to have proteins have been shown in neoplastic tissues [2, normal development of the fetus [240]. Also illustrat75], an example being the appearance of fetal ing the property of malignant cells to express hemoglobin found in leukemia and a few other genetic information normally repressed prior to the hematological diseases [256, 347, 348]. There is also onset of malignancy is the production of hormones a rapidly increasing number of different embryonic by nonendocrine tumors [46, 47, 99, 102, 140, 241, and fetal antigens found to be associated with neo- 243, 249, 326, 360]. The most common of these plastic tissue [I00, 136, 228, 454, 112, 447, 73, 53]. tumors are bronchiogenic carcinomas which secrete The most studied example is ~-fetoprotein pro- ACTH. However, tumors of the thymus, kidney and duced by hepatocellular carcinomas and embryonal pancreas have also been shown to synthesize a cell carcinomas of testis and ovaries which was first variety of endocrine hormones. The above discussions lead one to the conclusions described by Abelev e t al. [3]. The development of techniques for the detection of a-fetoprotein, in- that the normal cell contains all the necessary cluding enzyme immunoassay, radioimmunoassay information for the phenotypic expression of (RiA) [161], double antibody R I A [31, 204], im- malignancy and even if one cannot fully accept that munoperoxidase [i 54] and immunofluorescent the manifestations of cancer are entirely the result probes [154, 436, 270] and counter immunoelectro- of abnormalities in the control of genetic regulation, phoresis [213], which allow for the detection of one must certainly concede that malignant cells show also been observed in embryonic tissues [96, 283]. Tumors containing many tumor-specific and fetal antigens have the ability to escape destruction by the host immune response. Since this is also a characteristic of fetal tissues [176, 341,423] which are known to induce antibodies in the mother [193], a common escape mechanism has been suggested for both fetal and neoplastic tissues [83, 193, 349]. An excellent system for the possible study of the correlation between embryonic gene expression and malignant gene expression is the study of teratocarcinomas

Chromosomal proteins and gene expression the derepression of fetal genes and express genes that are normally only expressed in other cell types. These conclusions intimately relate the control of genetic expression with the disease state of malignancy and strongly indicate that further study of the regulating mechanisms in eukaryotes will elucidate the mechanisms of neoplastic transformation.

Mechanisms by which normal control of genetic expression may be heritably altered The molecular mechanisms by which chemical carcinogens and radiation induced cancer are not well understood, but speculation and correlative evidence [27] have suggested modifications leading to mutation of the cellular DNA. This modification could be either direct, or indirect, such as alterations in molecules that decrease the fidelity of copying DNA or interfere with DNA repair mechanisms. Utilizing the final active metabolite (ultimate carcinogen) of many chemical carcinogens, a good correlation has been established between mutagenesis and carcinogenesis [11, 12, 95, 163, 281]. The following are means by which mutations could result in heritable alterations in the control of genetic expression. (I) Mutations of cellular DNA could conceivably occur in a portion of DNA coding for a regulatory protein. If the mutation occurred at the regulatory or RNA polymerase binding site, the gene could be rendered untranscribable. If the mutation occurred in the structural portion, the result could be the production of a defective regulatory molecule. (2) If, as has been considered, regulatory molecules act by direct binding to specific DNA sequences thereby altering the transcriptional ability of the sequence, mutation of DNA could result in the inability of a regulatory molecule to recognize its binding site or alter the stability of such binding. (3) Mutation of DNA could possibly result in the production of defective molecules, such as histones or post-synthetic modifiers, that play an intermediary role in the relationship of a regulatory molecule and the genetic material. Chromosomal aberrations such as deletions, translocations or multiplicity of genetic material are caused by many cancer-inducing agents such as radiation, certain mutagens and viruses [32, 107, 168, 265, 266, 343, 370, 428]. Many persons with non-malignant diseases associated with gross chromosomal abnormalities (Fanconi's anemia, Down's syndrome, Bloom's syndrome, ataxiatelangiectasia, X0, etc.) have been found to have a much higher incidence of cancer, thereby suggesting a possible correlation between chromosomal abnormalities and

365

malignant diseases [15, 116, 130, 257, 258, 270, 453]. A definite correlation has been established between the transiocation of a portion of chromosome 22 to chromosome 9 and chronic myelogenous leukemia [179, 271,272, 308, 321]. Chromosomal aberrations could result in alterations of the control of genetic regulation by the loss of genes coding for regulatory proteins or the translocation of structural genes separating them from the DNA sequences at which regulation of the gene takes place. Translocation of DNA within the structural portion of a gene coding for a regulatory molecule could result in the production of an incomplete regulatory molecule. Also, the translocation and interjection of genetic material between a gene-regulatory or RNA polymerase attachment site and the structural portion could adversely affect the regulation of the gene. Any abnormalities that result in a modified chromatin structure also may be possibly related to malignancy. Recent work on xeroderma pigmentosum (XP) patients, who have an extremely high incidence of skin cancer, has shown that the structure of chromatin is at least partially responsible for the deficiencies in D N A repair in these patients [82, 85]. Introduction of new genetic material into the cell, as in the case of viral episomes or integrated viral DNA, has been extensively evaluated as a possible cause of malignancy [28, 174, 175, 407, 408, 415]. In human malignancy, the Epstein Barr Virus (EBV) and its relationship to Burkitts lymphoma and nasopharyngeal carcinoma has received considerable attention. Supporting a corelationship between EBV and malignancy are the findings of Klein and coworkers of EBV DNA and EBV associated nuclear antigens in human tumor cells [196, 206, 207, 236, 269, 457]. It is possible that included in the integrated viral genome is the genetic information for a new regulatory protein, or an antagonist or modifier of host regulatory molecules which results in altered genetic expression of the host DNA. Additionally there exists the possibility that the viral genome may be integrated in the host DNA in a manner that sterically interferes with the regulatory and structural segments of a gene. As has been alluded to in the previous discussion, a number of mechanisms that are consistent with the current concepts for the etiology of cancer and which can heritably alter the mechanisms of normal control of genetic expression in eukaryotic cells are theoretically possible. Obviously, more information as to the actual molecules involved in gene regulation and their mechanisms of action are required before more sophisticated and accurate mechanisms for their alterations can be postulated.

366

GARY S. STEIN et al.

been reported in rat hepatomas [378]. Some incidences of increased phosphorylation of HI historic If cancer is the biological situation where normal have been reported in rat hepatoma [20, 74]. Howcontrol mechanisms for genetic expression in ever, Balhorn et al. [18] observed that increased eukaryotic cells are disastrously altered, then the phosphorylation of H1 historic in a variety of logical question that comes to mind is: can one tumors could be directly correlated to the degree of observe any alterations accompanying malignancy cell proliferation associated with the tumor. Since in the molecules that are involved in the regulation H l phosphorylation has been previously associated of genetic expression ? Having previously alluded to with cellular replication [19, 21,346] it is reasonable to suspect that increased H1 phosphorylation in the roles that nuclear proteins play in this respect, tumors is related only to the increase in cell prolet us examine the alterations in these proteins accompanying malignant stages. However, caution liferation and is not unique in malignant disease. should be exercised in interpreting results from such The relationship of these alterations in histones to studies since neoplastic-associated modifications in the neoplastic transformation is not apparent at this time; however, it can be speculated that modificanuclear proteins may in part reflect changes in the proliferative states of the cells in vivo as well as in tions of histones could result in a destabilization of D N A - h i s t o n e complexes or a favoring of histonevitro. nonhistone protein complexes allowing for increased The histone fraction of the nuclear proteins, as availability of transcribable genes. Also it should be has been discussed earlier, may be involved in nonspecific repression of genetic information and also noted that carcinogens have been shown to bind in structural aspects of the genome. In neoplastic histones as well as nonhistone chromosomal proteins cells, differences in histones have been found by and D N A by many investigators [24, 191,312, 342, 352], but no evidence exists that any carcinogen chromatographic and immunological techniques, and in post-translational modifications between their bound nuclear protein is functionally modified. The normal counterparts. Preparations of HI histones specificity and functional significance of this binding contain a number of closely related lysine-rich has not been resolved. Since components of the nonhistone chromosomal proteins and can be resolved by chromatographic elution into several subfractions that are character- proteins (NHCP) are felt to be the specific regulators of gene expression in mammalian cells, interest in istic of the source of H1 histones [55, 56, 90, 201, the alterations of nuclear proteins in malignant 287, 307, 354]. Using these techniques, Kincade [202] tissues has been directed towards the nonhistone and Sluyser and Bustin [353] found that HI histone subfractions from rat hepatoma have tumor-specific proteins. Parameters of these proteins that have been features in addition to features that are common to examined in neoplastic cells are N H C P : D N A ratios, both normal and malignant tissues. The latter fractionation and comparison by one and two diresearchers report that a subfraction o f HI found in mentional polyacrylamide gel electrophoresis [54, normal rat liver is either absent or appears sub- 282, 449], immunological competence of N H C P D N A complexes [431], and post-transcriptional stantially diminished in hepatoma cells. These modifications such as phosphorylation and researchers also found that subfractions of H1 from rat hepatoma and rat liver are immunologically thiolation. N H C P : D N A ratios have been shown to be sigspecific using microcomplement fixation techniques. However, differences do not seem to be unique to all nificantly increased in hepatomas [13, 67, 72, 378] tumors since Hohman et al. [167] could not detect and human leukemia cells [91]. When further any chromatographic differences in HI histone examined by one or two dimensional polyacrylamide gel electrophoresis, the nonhistone chromosomal fractions between mouse mammary tumors and proteins showed qualitative and quantitative normal mammary gland tissues. Increases in certain histone methylation enzymes have been found in differences from their normal counterparts in experiNovikoff hepatomas [286]. However, certain car- mental rat hepatomas [72, 282, 310, 378, 443, 450] cinogens have been shown to inhibit histone methyla- and neoplastic mammary cells of CaH and Fisher tion by methyltransferases [63]. Some investigators mice [192]. The changes included proteins found in also suggest the possibility of increased histone- normal liver and absent in hepatomas and proteins found in the hepatomas that could not be demondirected protease activity in cancer cells [106]. strated in normal liver [443]. In other neoplastic D N A : h i s t o n e ratios appear to be consistent with normal controls in human leukemia [91] and ex- systems [449, 451] such as intestinal epithelial cells perimental rat bepatomas [378]; however, differences [38, 39] and human lymphocytes [448] differences in the nonhistone chromosomal proteins of tumor and in the relative quantities of histone fractions have Changes in nuclear proteins in malignant cells in vivo

Chromosomal proteins and gene expression normal tissues have also been found. In contrast, no differences could be found in nonhistone chromosomal proteins examined by polyacrylamide gel electrophoresis between normal and azo dye-induced hepatomas [74] and between normal and nitrosamine-treated rat livers [148]. That the N H C P fractions of certain tumor cells are not identical to their normal counterparts is demonstrated by immunological experiments [53, 447]. Busch and coworkers reported finding a nuclear antigen in Novikoff hepatoma cells which formed precipitin bands in an immunoprecipitin assay with chromatin proteins from Novikoff hepatoma, Walker 256 carcinosarcoma and fetal rat liver. However, no precipitin bands were formed when chromatin proteins of normal or regenerating rat liver were utilized [53, 447]. Wakabayoshi & Hnilica [73, 431] reported that antiserum made from Novikoff hepatoma D N A : N H C P complexes will only slightly react with D N A : N H C P complexes from rat liver and calf thymus. D N A : N H C P complexes from other malignant tissues (AS-30A hepatoma, Walker carcinosarcoma) cross react with anti-hepatoma D N A : N H C P to a greater degree than normal rat liver D N A : N H C P complexes. This suggests that more similarity exists between N H C P of tumors of different origin than between N H C P of malignant and normal counterparts of the same cell type. Other investigators [65] have immunologically examined tissues of tumor-bearing dogs and have reported similar results. Also demonstrating differences in the nonhistone chromosomal protein components of normal and neoplastic tissues is the work of Kostraba & Wang [216]. These researchers demonstrated by R N A D N A hybridization experiments, the presence of R N A species similar to those synthesized from Walker tumor when Walker tumor, nonhistone chromosomal proteins were used to activate (in vitro) transcription of rat liver chromatin. Conversely, R N A species similar to those synthesized from rat liver chromatin were demonstrated when N H C P from normal rat liver were used to activate in vitro transcription of Walker tumor chromatin. This work suggests that the nonhistone chromosomal proteins are at least partially responsible for the transcriptional differences between rat liver and Walker tumor. Phosphorylation of nonhistone chromosomal proteins has been strongly implicated in the role of genetic regulation for reasons that have been previously discussed. Hence alterations in phosphorylation in malignancy are especially interesting and have been studied by many researchers [10, 72, 74, 101]. Nonhistone chromosomal protein phosphory-

367

iation, specific activity with respect to sap, and nuclear protein kinase activity were found by Chiu et al. [72, 74] to increase concomitantly with template activity in rat liver chromatin during N, Ndimethyl-p-(m-tolylazo)aniline(3'-MDAB) administration. Maximal protein kinase activity was observed 28 days after the continued introduction of the azo dye into the diet and at this time changes of the phosphorylation pattern of N H C P were evident when examined by polyacrylamide gel electrophoresis. This is in good agreement with the report of Ahmed [5] who noted a maximal incorporation of 32p 26 days after azo dye administration. Granner et al. [144] also reported on significant increases in protein kinase activity in HTC hepatoma cells. In addition to reports of increased total nuclear protein kinase activity in malignant cells [5, 74, 104, 144, 411] some investigators have subdivided the nuclear protein kinase activities by chromatographic techniques and have attempted to further characterize these enzymes in neoplastic cells [104, 411]. Farron-Furstenthal [104] reported the separation by D E A E cellulose of hepatoma nuclear protein kinase activity into five subfractions as compared to 3 subfractions from normal liver nuclei. Thomson et al. [411] observed an additional protein kinase subfraction common to Novikoff hepatoma, Ehrlich ascites and Walker tumor nuclei separated by phosphocellulose chromatography. This tumor-associated enzyme fraction had the unique property of preferring Mn ~+ to Mg ~+ for optimal kinase activity. This enzyme fraction was also responsible for the phosphorylation of a single protein band (when phosphoproteins were examined by polyacrylamide gel electrophoresis) that was found only in the neoplastic tissues, indicating that tumor cells possess not only a unique protein kinase but also a unique phosphoprotein that is the substrate for the enzyme.

Changes in nuclear proteins in transformed cells

Since transformation of eukaryotic cells by viruses has been related to the etiology of cancer [28, 174, 175, 407, 408, 415] studies of viral transformed cells in culture offer a system for studying biochemical and molecular alterations that may elucidate the events happening in vivo during malignant transformation. The study of ceils in culture offers distinct advantages. Unlike in vivo systems where the tissues studied may consist of several cell types, cell cultures allow for the study of homogenous cell populations. Also, the environment of cells in culture can be completely controlled, and the introduction of virus into the cell environment allows for the study of the transformation process at all stages. Viral trans-

368

GARY S. STEINet al.

formation of eukaryotic cells is known to produce morphological and biochemical changes which reflect alterations in gene expression [33, 263, 406]. A striking example of abnormal expression is the activation of embryonic globin genes by Rous Sarcoma virus in chicken fibroblasts [152]. It is therefore reasonable that changes in the nuclear proteins can be observed during viral transformation. Evidence that substantial alterations exist between chromatins from normal and transformed cells is reported by Lin et al. [235]. These investigators detected differences in the C D spectra of chromatins from normal and SV40-transformed WI-38 fibreblasts and observed that these differences could be abolished by prior washing of the chromatins with 0.25 M NaCI. Since only nonhistone chromosomal proteins are removed by 0.25 M NaC1, it was coneluded that the differences in the two chromatins can be attributed to the nonhistone chromosomal proteins. Differences in chromatin from normal and SV40-transformed fibroblasts were also determined by immunological techniques [455]. Antiserum made from WI-38 fibroblast chromatin reacted only weakly to SV40-transformed WI-38 fibroblasts (2RA cell) chromatin. An analogous result was found when reacting 2RA chromatin antiserum with WI-38 fibroblast chromatin. When the histone fractions of the chromatins were used as the antigen, the antiserum reacted equally well with the histones from WI-38 or 2RA cells. However, when nonhistone chromosomal protein fractions (NHCP) were used as the antigen, the heterologous N H C P fraction reacted much less than the original antigen. These differences could be explained by either quantitative or qualitative differences in the N H C P fraction of the two chromatins, or in a different association or arrangement of the N H C P in chromatin. More detailed studies of nonhistone chromosomal proteins o f normal and transformed cells have provided further evidence for alterations in this class of nuclear proteins accompanying viral transformation. Variations in the rates of synthesis of nonhistone chromosomal proteins in normal compared with SV40-transformed fibroblasts have been observed [139, 178, 220, 227, 319], especially associated with nonhistone proteins of high molecular weight ( < 100,000 daltons). This is further supported by reports of variations in nonhistone chromosomal protein synthesis after Rous Sarcoma virus infection of chick embryo fibroblasts [383]. Evidence that the mechanisms for N H C P synthesis in transformed cells may not be analogous to normal WI-38 fibroblasts is reported by Cholon et al. [78]. These investigators reported that the inhibition of low molecular weight nonhistone chromosomal protein synthesis in WI-38

fibroblasts by aminonucleoside cannot be demonstrated in SV40-transformed WI-38 fibroblasts suggesting that N H C P synthesis is in some way protected in the transformed cell. Differences in the composition of N H C P in normal and transformed cells as determined by examination of these proteins by the techniques of polyacrylamide gel electrophore is have been reported by many investigators [219, 220]. Also, evidence of increased turnover of these proteins has been established in SV40-transformed cells [219]. Recent reports of the post-translational modification of nonhistone chromosomal proteins in normal and transformed cells have exhibited marked variations [219, 300]. Increases in the phosphorylation of most molecular weight classes of nonhistone chromosomal proteins in SV40-transformed WI-38 cells have been observed [304], with some investigators reporting a ten-fold increase in a~p incorporation into nonhistone chromosomal phosphoproteins of SV40-transformed as compared with normal WI-38 fibroblasts. This striking increase could not be explained by an increased rate of synthesis of N H C P or an increased rate of phosphate transport [304]. Comparison of phosphorylation patterns of N H C P by polyacrylamide gel electrophoresis have shown that there are specific effects of phosphorylation on individual protein species associated with viral transformation [300]. Alterations of the nuclear histones associated with viral transformation have not been reported to be as dramatic as those associated with the N H C P fraction. Studying normal and SV40-transformed WI-38 fibroblasts, Krause et aL [220] reported that although all five major histone fractions were present in both cell populations, variations were found i n the relative amounts, rates of synthesis and binding of histone fractions. This included an increase in the amount of histone H2a, an increase in the synthesis of histone HI and a decrease in the synthesis of histone H4 in transformed cells. Nishimura et aL [267] reported the presence of two new peaks of histones on polyacrylamide gels in BHK cells after infection with HSV-2, a virus associated with human cervical carcinoma [309]. It should be also noted that histones are known to be contained within some D N A tumor viruses [117, 297], and there is evidence that these viral-associated histones are more highly acetylated than host cell histones [335]. That acetylation of histones may be a significant factor in viral transformation is proposed by the work of Schaffhausen & Benjamin [335], who have correlated deficiency of histone acetylation with loss of transforming abilities of polyoma virus. The significance of these observations is not known at this time.

Chromosomal proteins and gene expression

PROSPECTS While an important role for chromosomal proteins in dictating structural and functional properties of the eukaryotic genome is apparent, much remains to be resolved before gene regulation in eukaryotic cells can be understood. Predicated on recent developmerits in the area of chromosomal protein research, conceptual as well as technological, it is reasonable

369

to anticipate that significant progress can be expected towards comprehending the manner in which expression of specific genes is governed. Resolution of this problem will undoubtedly provide insight into the cellular and molecular basis of a broad spectrum of disease processes, particularly neoplasia, where aberrations in gene expression appear to be a primary component of the etiology.

REFERENCES 1. 2. 3. 4. 5. 6.

7. 8. 9.

10. 11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23.

ABELEVG. I. (1965) Antigenic structure of chemically-induced hepatomas. Progr. exp. Tumor Res. 7, 104. ABELEVG. I. (1971) ~-Fetoprotein in ontogenesis and its association with malignant tumors. Adv. Cancer Res. 14, 295. ABELEVG. I., PEROVAS. D., KHRAMKOVAN. I., POS~IKOV^ Z. A. & IRLln I. S. (1963) Production of embryonal a-globulin by transplantable mouse hepatomas. Transplantation 1, 174. ADLERK., BEYREUTHERK., F A N ~ O E., GE~SLERN., GRONEnBORNB., KLEMMA,, MtmLL~-HILL B., PFAHLM. & SCHMrrz A. (1972) How lac repressor binds to DNA. Nature 237, 322. AHMEDK. (1974) Increased phosphorylation of nuclear phosphoproteins in precancerous liver. Res. Commun. Chem. Path. Pharm. 9, 771. ALBERTSB. M., AMODIOF. J., JENKINSM., GUTMANNE. D. & FERRISF. J. (1968) Studies with DNA-cellulose chromatography--I. DNA-binding proteins from Escherichia coB. Cold Spring Harb. Syrup. quant. Biol. 33, 289. ALL~EYV. G. (1966) Control mechanisms in ribonucleic acid synthesis. Cancer Res. 26, 2026. ALLFREYV. G., FAULKNERR. & MIRSKYA. E. (1964) Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. hath. Acad. Sci. 51, 786. ALLFREYV. G., InOUEA. & JOHNSOnE. M. (1975) Use of DNA columns to separate and characterize nuclear nonhistone proteins. In Chromosomal Proteins and their Role in the Regulation of Gene Expression (STEIN G. & KLEraSMrm L., Eds.), p. 265. Academic Press, New York. ALLFREYV. G., Lrrrau V. C. & MmsKV A. E. (1963) On the role of histones in regulating ribonucleic acid synthesis in the cell nucleus. Proc. hath. Acad. Sci. 49, 414. AMESB. N., DURSTONW. E., YAMASAKIE. & LEE F. D. (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. natn. Acad. Sci. 70, 2281. AMESB. N., LEE F. D. & Dtmsxon W. E. (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. natn. Acad. Sci. 70, 782. ARNOLDE. A., BUKSASM. M. & YOUNGK. E. (1973) A comparative study of some properties of chromatin from two minimal deviation hepatomas. Cancer Res. 33, 1169. AROSlOP., ADELMANT., YOKOTAM. & DRYSDALEJ. W. (1976) A comparative study of isoferritins in normal and malignant tissues. In Onco-Developmental Gene Expression (FIsHmANW. H. & SELL S., Eds.), p. 743. Academic Press, New York. ALmR~ACHA. D. & WOLMAN S. R. (1976) Susceptibility of Fanconi's anaemia fihroblasts to chromosome damage by carcinogens. Nature 261, 494. AXELK., C'ED/d~H. & FELSE~ELD G. (1975) The structure of the globin genes in chromatin. Biochemistry 14, 2489. B~L~On~R.., BALHORNM. J~ CHALKLEYR. (1972) Lysinv-rich histone phosphorylation and hyperplasia in the developing rat. Dee. Biol. 29, 199. BALHORNR., BALHORNIV[., MORRISH. P. & C H A L ~ R.. (1972) Comparative high-resolution electrophoresis of tumor histones: variation in phosphorylation as a function of cell replication rate. Cancer Res. 32, 1775. BJo.~OnN 1~., BORDWELLJ., SELLERSL., GRANNERD. K. ~" CHALKLEYl~. (1972) I-Iistone phosphorylation and DNA synthesis are linked in synchronous cultures of HTC cells. Biochem. Biophys. Res. Commun. 46, 1326. BALHO~ R., ~ K L ~ f R. & GaA~n,mR D. (1972) Lysine-rich histone phosphorylation. A positive correlation with cell replication. Biochemistry 11, 1094. B^LHOnNR., REIKEW. O. & CHALKLEYR. (1971) Rapid electrophoretic analysis for histone phosphorylation A reinvestigation of phosphorylation of lysine-richhistone during rat liver regeneration. Biochemistry 10, 3952. BALIOAB. S., BOOK E., WE~srEra I. B. & SS_~nVASXNP. R. (1969) Differences in the transfer RNAs of normal liver and Novikoff hepatoma. Proc. natn. Acad. Sci. 62, 899. BAng~rr T., M . ~ a y ~ D., tDO~YN P. & GOULD H. (1974) Nonhistone proteins control gent expression in reconstituted chromatin. Proc. natn. Acnd. Sci. 71, 5057.

370 24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34. 35. 36. 37. 38. 39. 40.

41. 42. 43. 44. 45. 46. 47.

48. 49. 50. 51.

52.

GARY S. STEIN et aL BARRYE. J., OVECHr,.AC. A. & GUTMANN H. R. (1968) Interaction of aromatic amines with rat liver proteins in vivo---II. Binding of N-2-fluorenylacetamide-9-~4C to nuclear proteins. J. biol. Chem. 243, 51. BARTLEYJ. ,~' CHALKLEYR. (1970) Further studies of a thymus nucleohistone-associated protease. J. biol. Chem. 245, 4286. BASEROAg. (1974) Nonhistone chromosomal proteins in normal and abnormal growth. Life Sci. 15, 1057. BECKERF. F. 0975) Cancer: d Comprehensive Treatise, Vol. 1. Etiology: Chemical and Physical Carcinogenesis. Plenum, New York. BECKER F. F. (1975) Cancer: A Comprehensive Treatise, Vol. 2. Etiology: Viral Carcinogenesis. Plenum, New York. BECKWITHJ. g. & ZIPSER D. 0970) The Lactose Operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N e w York. BEKI-IORI,, K U N G G. & BONNER J. 0969) Sequence-specificinteraction of D N A and chromosomal proteins. J. molec. Biol. 39, 351. BEt.ANGERL., DAGUILLARDJ., WAITHE W. I. & DUFOUR D. (1976) Studies on the effect of alpha-fetoprotein on immune status of children with tyrosinosis and on mitogen-induced rat and human lymphocyte tra n sformation in vitro.'In Onco-Developmental Gene Expression (FlSHMAN W. H. & SELL S., Eds.), p. 329. Academic Press, New York. BENDERM. A., GRIOOS H. G. & BEDFORDJ. S. (1974) Mechanisms of chromosomal aberration production--III. Chemicals and ionizing radiation. Murat. Res. 23, 197. BENJAMINT. L. (1972) Physiological and genetic studies of polyoma virus. Curt. Topics in Microbiol. & [mmunol. 59, 107. BERTOLOrrI R. & WEISS M. C. (1972) Expression of differentiated functions in hepatoma cell hybrids. VI. Extinction and re-expression of liver alcohol dehydrogenase. Biochimie 54, 195. BHORJEEJ. & PEDERSOS T. (1972) Nonhistone chromosomal proteins in synchronized HeLa cells. Proc. natn. Acad. Sci. 69, 3345. BIRNIEG. D., RICKWOODD. &; HELL A. (1973) Buoyant densities and hydration of nucleic acids, proteins and nucleoprotein complexes in metrizamide. Biochim. Biophys. Acta 331,283. BJ6RS:LUNDB. (1976) Tissue polypeptide antigen in cancer and other conditions. In Onco-Developmental Gene Expression (FISHMANW. H. & SELL S., Eds.), p. 501. Academic Press, New York. BOFFA L. C. ,qr ALLFREY V. G. (1976) Characteristic complements of nuclear nonhistone proteins in colonic epithelial tumors. Cancer Res. 36, 2678. BOFFAL. C., VIDAL[ G. & ALLFREY V. G. 0976) Changes in nuclear nonhistone protein composition during normal differentiation and carcinogenesis of intestinal epithelial cells. Expl Cell Res. 98, 396. KucmNo Y. & BORERE. (1976) Similar patterns of t R N A structures and t R N A methyltransferases in embryonic and tumor tissue. In Onco-Developmental Gene Expression (FISHMAN W. H. & SELL S., Eds.), p. 95. Academic Press, New York. BOREI¢E. & KERN S. J. (1972) Atypical transfer RNAs and their origin in neoplastic cells. Adv. Cancer Res. 15, 163. BORU~4T. W., GABRIELL!F., AJIRO K., 7WEIKLERA. & BAGLIONIC. (1975) Further evidence for transcriptional and translational control of historic messenger RNA during the HeLa $3 cycle. Cell 4, 59. BORUNT. W., PEARSOND. &; PAIl( W. K. (1972) Studies of histone methylation during the HeLa $3 cell cycle. J. biol. Chem. 247, 4288. BORtm T. W., SCHARFF M. D. & RoaaINs E. (1967) Rapidly labeled, polyribosome-associated R N A having the properties of histone messenger. Proc. natn. Acad. Sci. 58, 1977. BORtm T. W. & STEIN G. S. (1972) The synthesis of acidic chromosomal proteins during the cell cycle of HeLa Ss cellswII. The kinetics of residual protein synthesis and transport, jr. Cell Biol. 52, 308. BOWERB. F. & GORDON G. S. 0965) Hormonal effects of nonendocrine tumors. Ann. Rev. Med. 16, 83. BRAUNSTEtNG. D., BRIDSON W. E., GLASS A., HULL E. W. & MCINTIRE K. R. (1972) In vivo and in vitro production of human chorionic gonadotropin and alpha-fetoprotein by a virilizing hepatoblastoma. J. clin. Endocrinol. Metab. 35, 857. BmorrrWlZLL M. D., LEECrt C. E., O'FARR~LL M. K., WHISH W. J. & SHALL S. (1975) Poly(ADP ribose) polymerase in Physarum polycephalum. Biochem. J. 147, 119. Btr~E D., RaMaAtrr C., LEMERLEJ., SCrtWEISGUTHO. & BURTIN P. (1970) Presence of a ferroprotein of tissular origin, the asH globulin, in sera of children with tumors. Int. J. Cancer 5, 85. " BURGERM. M. & NOONAN K. D. (1970) Restoration of normal growth by covering of agglutinin sites on tumor cell surface. Nature 228, 512. BOSCHG. I., YEOMAN L. C., TAYLOR C. W. & BUSCH H. (1974) Modified two dimensional polyacrylamide gel electrophoresis systems for higher molecular weight nonhistone chromatin proteins from normal rat liver and Novikoff hepatoma ascites cells. Physiol. chem. Phys. 6, 1. Busch H. (1974) Messenger R N A and other high molecular RNA. In Molecular Biology of Cancer (Busch H. Ed.), p. 88. Academic Press, New York.

Chromosonal proteins and gene expression 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63.

64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85.

371

BUSCHH. (1962) Metabolism of R N A in tumors and other tissues. In Biochemistry of the Cancer Cell (Bosch H., Ed.), p. 113. Academic Press, New York. Busch H., YEOMAN L. C., Busch R. K., JORDONJ. J., RAO M. R. A., TAYLORC. W. & Wu B. C. (1976) Antigenically active Novikoff hepatoma chromatin protein in cancer cell. Cancer Res. 36, 3399. BUSTI~ M. & COLE R. D. (1968) Species and organ specificity in very lysine-rich histones. J. biol. Chem. 243, 4500. BUSTINM. & COLE R. D. (1969) Bisection of a lysine-rich histone by N-bromosuccinimide. J. biol. Chem. 244, 5291. BUTLERW. B. & MUELLER G. C. (1973) Control of histone synthesis in HeLa cells. Biochim. Biophys. Acta 294, 481. BUTTLEG. A. H., EPERON J. L. & KOVACSE. (1962) An antigen in malignant and in embryonic tissues. Nature 194, 780. BUTTLEG. A. H., EPEgSON J. & MENZIESD. H. (1964) Induced tumor resistance in rats. Lancet ii, 12. BtrrrL~ G. A. H. & FRAV~ A. (I 967) Effect of previous injection of homologous embryonic tissue on the growth of certain transplantable mouse tumors. Nature 215, 1495. BYVOETP. (1966) Metabolic integrity of deoxyribonucleichistones. J. molec. Biol. 17, 311. BYVOETP. (197 i) Uptake of label into methylated amino acids from rat tissue histones after in vivo administration of [Me-14C] methionine. Biochim. Biophys. Acta 238, 375a. BYVOETP. & BAXTERC. S. (1975) Histone methylation, a functional enigma. In Ghromosomal Proteins and their Role in the Regulation of Gene Expression (Sa'~n~ G. & KLE~SMrr8 L., Eds.), p. 127. Academic Press, New York. CAMERONI. ~ J ETER J. R. (Eds.) (1974) Acidic Proteins of the Nucleus. Academic Press, New York. CARLOD. J., BIGLEY N. J. & VAN WINKLE Q. (1970) Immunochemical differences in deoxyribonucleoproteins from normal and malignant canine tissues. Immunology 19, 879. CARTER D. B. & CHAE C.-B. (1976) Chromatin bound protease: degradation of chromosomal proteins under chromatin dissociation conditions. Biochemistry 15, 180. CHAEC.-B., SMITH M. C. & MORRISH. P. (1974) Chromosomal nonhistone proteins of rat hepatomas and normal rat liver. Biochem. Biophys. Res. Commun. 60, 1468. CHAMaON P., WEILL J. D. & MANDEL P. (1963) Nicotinamide mononucleotide activation of a new D N A dependent polyadenylic acid synthesizing nuclear enzyme. Biochem. Biophys. Res. Commun. U , 39. CHANGC. H., N1SHIYAMAT., ANGELUS D. & CONNERYC. (1976) Isoimmunogenic placental proteins. In OneoDevelopmental Gene Expression (FtsHMAN W. H. & SELL S., Eds.), p. 235. Academic Press, New York. CHANGL. M. S. (1973) Low molecular weight deoxyribonucleic acid polymerase from calf thymus chromatin--I. Preparation of homogeneous enzyme. J. biol. Chem. 248, 3789. CHENC., SMITHD. L. BRUEGGERB., HALPERN R. M. & SMITH R. A. (1974) Occurence and distribution of acidlabile histone phosphates in regenerating rat liver. Biochemistry 13, 3785. CHIu J.-F., BRADEW. P., THOMSONJ. A., TSAI Y.-H. & HNILICA L. S. (1975) Nonhistone protein phosphorylation in normal and neoplastic rat liver chromatin. Expl Cell Res. 91, 200. Cmu J.-F., CHYT1L F. & HNILICA L. S. (1976) Onco-fetal antigens in chromatin of malignant cells. In OncoDevelopmental Gene Expression. (PtSHMAN W. H. & SELL S, Eds.), p. 271. Academic Press, New York. CHIU J.-F., CRADDOCK C., GETZ S. & HNILICA L. S. (1973) Nonhistone chromatin protein phosphorylation during azo-dye carcinogenesis. FEBS Lett. 33, 247. CHIu J.-F. & HNILICA L. S. (1976) D N A polymerases in developing and neoplastic tissues. In Onto-Developmental Gene Expression (FIsI-IA~ W. H. & SELL S., Eds.), p. 65. Academic Press, New York. Cmu J.-F., TSA1 Y.-H., SAKUMAK. 3k HNILICA L. S. (1975) Regulation of in vitro m R N A transcription by a fraction of chromosomal proteins. J. biol. Chem. 250, 9431. C m u N. & BASERGA R. (1975) Changes in template activity and structure of nuclei from WI-38 cells in the prereplicative phase. Biochemistry 14, 3126. CHOLONJ. J. & STUDZINSKIG. A. (1974) Effect of aminonucleoside on serum stimulation of nonhistone nuclear protein and D N A synthesis in normal and SV-40 transformed human fibroblasts. Cancer Res. 34, 588. CnONG M. T., GARRARD W. T. & BONNER J. (1974) Purification and properties of a neutral protease from rat liver chromatin. Biochemistry 13, 5128. CLARKR. J. & FELSENFELDG. (1971) Structure of chromatin. Nature, New Biol. 229, 101. CLARKg. J. d~. FELSENFELDG. (1974) Chemical probes of chromatin structure. Biochemistry 13, 3622. CLEAVERJ. E., Boo'rSMA D. & FRIEDBERYE. (1975) Human diseases with genetically altered D N A repair processes. Genetics 79 (suppl. 215), 225. COGGINJ. H. & ANDERSONN. G. (1974) Cancer, differentiation and embryonic antigens: some central problems. Adv. Cancer Res. 19, 105. COMBD. G., SARKARN. & PINZINO C. J. (1966) The methylation of lysine residues in protein. J. biol. Chem. 241, 1857. Correspondent. Mammalian D N A repair. (1976) Nature 261, 456.

372 86. 87. 88. 89.

90. 91. 92. 93.

94. 95. 96.

97. 98. 99. 100. 101. 102. 103. 104. 105.

106. 107. 108. 109. 110.

111, 112. 113. 114. 115. 116.

GARY S. STERNet aL Cox R. F. (1973) Transcription of high-molecular weight R N A from hen oviduct chromatin by bacterial and endogenous form-B R N A polymerases. Fur. J. Biochem. 39, 49. CRIss W. E. (1971) A review of isozymes in cancer. Cancer Res. 31, 1523. DALY M., ALLFREY V. (3. & MIP,SKY A. E. (1952) Uptake of glycine-16N by components of cell nuclei. J. gen. Physiol. 36, 173. DELANOER. J., FAMBROUO8D. M., SMITH E. L. & BONNER J. (1969) Calf and pea histone IV--IlL Complete amino acid sequence of pea seedling histone; IV. Comparison with the homologous calf thymus histone. J. biol. Chem. 244, 5669. DELANGER. J. & SMITH E. L. (1971) Histones: structure and function. Ann. Rev. Biochem. 40, 279. DESAXL. S., WULFF U. C. & FOLEY G. E. (1975) Properties of chromosomal proteins of human leukemic cells. Bioehimie 57, 315. DEXTERD. L., BuC-CARoN M.-H., JAKOB H., NtCOLAS J. F. & GACHEUN G. (1974) Teratocarcinorrte de la souris: etude des antig6nes de la surface de cellules ~. potentialit6s multiples. Ann. Microbiol. 125(B), 347. DINOMANC. & SPORN M. (1964) Studies on chromatin--I. Isolation and characterization of nuclear complexes of deoxyribonucleic acid, ribonucleic acid, and protein from embryonic and adult tissues of the chicken. J. biol. Chem. 239, 3483. DOUVAS A. S., FIARPaNGTONC. A. & BONNER J. (1975) Major nonhistone proteins of rat liver chromatin: preliminary identification of myosin, actin, tubulin, and tropomyosin. Proc. natn. Acad. Sci. 72, 3902. DUNCAN M. E. & BROOKES P. (1973) The induction of azaguanine-resistant mutants in culture of Chinese hamster ceils by reactive derivatives of carcinogenic hydrocarbons. Mutat. Res. 21, 107. EDWARDSC. L. & HAYESR. L. (1972) Localization of tumors with radioisotopes in clinical uses of radionuclides: critical comparison with other techniques. A E C Syrup. Set. 27, Goswitz A. F., Andrews G. A. & Viamonte M. U.S. Atomic Energy Commission, p. 618. ELOrNS. & BONNER J. (1970) Limited heterogeneity of the major nonhistone chromosomal proteins. Biochemistry 9, 4440. ELGINS. C. R. & WEIN'rRAUBH. (1975) Chromosomal proteins and chromatin structure. Ann. Rev. Biochem. 44, 725. ELmL L. P. (1968) Non-endocrine secreting neoplasms: clinical manifestations. Cancer Bull. 20, 37. EvELEiorl J. W., ANDERSONN. G. & COGGIN J. H., Jr. (Eds.) (1972) In Embryonic and Fetal Antigens in Cancer Vol. 2, p. 133. Springfield, Va: U.S. Dept. Commerce. EZRAILSONE. (3., OLSON M. O. J., (3UETZONK. A. & BUSCHH. (1976) Phosphorylation of nonhistone chromatin proteins in normal and regenerating rat liver, Novikoff hepatoma and rat heart. FEBS Lett. 62, 69. FAIMANC., COLWELLJ. A., RYAN R. J., HER~HMANJ. M. & SHIELDST. W. (1967) Gonadotropin secretion from a bronchogenic carcinoma. New Engl. J. Med. 277, 1395. FARQUHARM. & MCCARTHYB. (1973) Histone m R N A in eggs and embryos of Strongylocentrotus purpuratus. Biochem. Biophys. Res. Commun. 53, 515. FARRoN-FtraSTENrr~L F. (1975) Protein kinases in hepatoma, and adult fetal liver of the rat--I. Subcellular distribution. Biochem. Biophys. Res. Commun. 67, 307. FAUVER. M., JAKOB H., (3AILLARDJ. A. & JACOB F. (1974) Immunologie prolif6ration et diff6renciation de ceilules primitives de t6ratocarcinome chez des souris allogen(~s trait6es par un endotoxine. C. r. Acad. Sci. Paris 278, 1919. FISH R. (3., PRrrCHARD J. A. V. & DEELEY T. J. (1974) Human peripheral lymphocytes and cancer. In vitro studies of the basic protein, histone F2A2 fraction. Br. J. Cancer 30, 222. FISHBEIN L., FLAMM W. (3. & FLAK G. L. (1970) Chemical Mutagens, Environmental Effects on Biological Systems. Academic Press, New York. FISHMANW. H. (1969) Immunologic and biochemical approaches to alkaline phosphatase isoenzyme analysis: the regan isoenzyme. Ann. IV. Y. Acad. Sci. 166, 745, FISHMANW. H., INGLIS N. R. & GREEN S. (1971) Regan isoenzyme: a carcinoplacental antigen. Cancer Res. 31, 1054. FISHMANW. H., INGLIS N. R., GREEN S., ANSTISSC. L., Gosh N. K., i~EIF A. E., RUSTIGIANR., KRNAT M. J. & STOLBACH L. L. (1968) Immunology and biochemistry of regan isoenzyme of alkaline phosphatase in human cancer. Nature 219, 697. FISHMANW. H., INGLIS N. I., STOLBACHL. L. & KRANT M. J. (1968) A serum alkaline phosphatase isoenzyme of human neoplastic cell origin. Cancer Res. 28, 150. FISrIMAN W. H. & SINGER R. M. Onco-trophoblast proteins and neoplastic transformation. Cancer Res. (in press). FOLKMANJ. (1972) Anti-angiogenesis: new concept for therapy of solid tumors. Ann. Surg. 175, 409. FOLKMANJ. (1974) Tumor angiogenesis. Adv. Cancer Res. 19, 331. FOLKMANJ. (1976) The vascularization of tumors. Scient. Am. 234, 58. FRAUMENIJ. F. (1969) Constitutional disorders of man predisposing to leukemia and lymphoma. Nam. Cancer Inst. Monogr. 32, 221.

Chromosomal proteins and gene expression 117. 118.

373

FREARSONP. M. & CRAWFORDL. V. (1972) Polyoma virus basic proteins. J. gen. Virol. 14, 141. FRENSTERJ. H., ALLFREY V. G. & MIRSKY A. E. (1963) Repressed and active chromatin isolated from interphase lymphocytes. Proc. natn. Acad. Sci. 50, 1026. 119. FRIEDMANM., SHULL K. H. Pc FARBERE. (1969) Highly selective in vivo ethylation of rat liver nuclear protein. Biochem. Biophys. Res. Commun. 34, 857. 120. FRITSCHE R. & MACrl J. P. (1975) Identification of a new oncofetal antigen associated with several types of human carcinomas. Nature 258, 734. 121. GABRIELLIF. & BAGLION! C. (1975) Maternal messenger R N A and historic synthesis in embryos of the surf clam, Spisula solidissima. Dev. Biol. 43, 254. 122. GALLOR. C. & PFSrKA S. (1970) Transfer R N A species in normal and leukemic human lympboblasts. J. molec. Biol. 52, 195. 123. GALLWITZD. (1975) Kinetics of inactivation of histone m R N A in the cytoplasm after inhibition of D N A replication in synchronized HeLa cells. Nature 257, 247. 124. GALLWITZD. d~. BREINDL M. (1972) Synthesis of histones in a rabbit reticulocyte cell-free system directed by a polyribosomal R N A fraction from synchronized HeLa cells. Bioehem. Biophys. Res. Commun. 47, 1106. 125. GALLWITZ D. & SURFS I. (1972) Histone acetylation. Purification and properties of three histone-sl~,cific acetyltransferases from rat thymus nuclei: Biochim. Biophys. Acta 263, 315. 126. GANGULYA., DAS A., MONDAL H., MANDAL R. K. & BISWAS B. B. (1973) Molecular weight and subunit structure of R N A polymerase I and initiation factor from chromatin of plant cell nuclei. FEBS Lett. 34, 27. 127. GAREL A. & AXEL R. (1976) Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei. Proc. natn. Acad. Sci. 73, 3966. 128. GARRELSJ. I., ELGIN S. C. R. & BONN-ERJ. (1972) A histone protease of rat liver chromatin. Biochem. Biophys. Res. Commun. 46, 545. 129. GAZIEVA. I. & KUZIN A. M. (1973) The localization of D N A ligase in the ehromatin of animal cells. Fur. J. Biochem. 37, 7. 130. GERMAN J. (1972) Genes which increase chromosomal instability in somatic ceils and predispose to cancer. Prog. reed. Genet. 8, 61. 131. GERMONDJ. E., HIRT B., OtroEr P., GROSS-BELLARDM. & Cr~Mm3N P. (1975) Folding of the D N A double helix in chromatin-like structures from simian virus 40. Proe. natn. Acad. SeL 72, 1843. 132. GERNERE. W. & HUMPHREYR. M. (1973) The ceil-cycle phase synthesis of nonhistone proteins in mammalian cells. Biochim. Biophys. Acta 331, 117. 133. GmnEg'r W. & Mi2ELLER-HILLB. (1966) Isolation of the lac repressor. Proc. natn. Acad. Sci. $6, 1891. 134. GILMOUR R. S. & PAUL J. (1970) Role of nonhistone components in determining organ specificity of rabbit chromatins. FEBS Lett. 9, 242. 135. GOLD P. (1967) Circulating antibodies against carcinoembryonic antigens of the human digestive system. Cancer 20, 1663. 136. GOLD P. & FREEDMANS. O. (1965) Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J. exp. Med. 121, 439. 137. GOLD P. & FREEDMANS. O. (1965) Specific carcinoembryonic antigens of the human digestive system. J. exp. Med. 122, 467. 138. GONANOP., PIRRO G. 8~: SILVE'I'I'IS. (1973) Fetal liver t R N A ~,n~ in rat he~atoma. Nature, New Biol. 242, 236. 139. GONAZLEZ C. A. & R U S K . R. (1975) Nonhistone chromosomal proteins from virus-transformed and untransformed 3T3 mouse fibroblasts. Biochim. Biophys. Acta 395, 361. 140. GOODALLC. M. (1969) On para-endocrine cancer syndromes. Int. J. Cancer 4, 1. 141. GOTT~EELD J. M., GARRAD W. T., BADI G., WILSON R. F. & BON/qER J. (1974) Partial purification of the template-active fraction of chromatin: a preliminary report. Proc. natn. Acad. Sci. 71, 2193. 142. GO'ITFSFELDJ., KENT D., ROSS M. & BONNER (1975) Chromatin structure and transcriptional activity. In Chromosal Proteins and their Role in the Regulation o f Gene Expression (S~rN G. & KLEINSMrrH L., Eds.), p. 227. Academic Press, New York. 143. GoussEv A. I., ENGELHARDTN. V., MASSAYEFFR., CAMAINR. & BAs'rERISB. (1971) Immunofluorescent study of alpha-fetoprotein in liver and liver tumors--II. Localization of alpha-fetoprotein in the tissues of patients with primary liver cancer. Int. J. Cancer 7, 207. 144. GRANNERD. K. (1972) Protein kinase: altered regulation in a hepatoma cell line deficient in adenosine Y,5'cyclic monophosphate-binding protein. Biochem. Biophys. Res. Commun. 46, 1516. 145. GREENBLAT'rM. & SHUBIK P. (1968) Tumor angiogenesis: transfilter diffusion studies in the hamster by the transparent chamber technique. J. natn. Cancer Inst. 41, 111. 146. GgrFFI~ A. C. (1969) Aminoacyl synthetase and transfer ribonucleic acid patterns in normal and neoplastic tissues. Adv. Enzyme Regulation 7, 361. 147. GgI~rHr~ J. D. (1975) Chromatin structure: deduced from a minichromosome. Science 187, 1202. 148. GRONOW M. (1971) Changes in rat liver nonhistone nuclear proteins during nitrosamine carcinogenesis. Biochem. 3". 124, 49.

374 149. 150. 151. 152. 153. 154.

155.

156. 157. 158. 159. 160. 161.

162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175.

176. 177.

178.

GARY S. SIEIN et al. GRONOW M. & THACKRAHT. (1973) The nonhistone nuclear proteins of some rat tissues. Archs Biochem. Biophys. 158, 377. GROSSP. R. & COUSn~F.AUG. H. (1964) Macromolecule synthesis and the influence of actinomycin on early development. Expl Cell Res. 33, 368. GROSSK. W., JACOBS-LOR£NAM., BAGLIONIC. & GROSS P. (1973) Cell-free translation of maternal messenger R N A from sea urchin eggs. Prec. hath. Acad, Sci. 70, 2614. GROUDINE M. & WErNraAUB H. (1975) Rous sarcoma virus activates embryonic globin genes in chicken fibroblasts. Prec. hath. Acad. SoL "/2, 4464. GUENETJ. L., JAKOB H., NICOLAS J. F. & JACOB F. (1974) T6ratocarcinome de la souris: etude cytog6n6tique de cellules ~t potentialites multiples. Ann. Microbiol. 125(A), 135. GUILLOUZOA., FELOMANNG. & BELANGER L. (1976) Localization of alpha-fetoprotein-containing liver cells in tyrosinosis and in newborn rat. In Once-Developmental Gene Expression (FISHMANW. H. & SELL S., Eds.), p. 647. Academic Press, New York. HAGERS. E. & JONES M. E. (1967) Initial steps in pyrimidine synthesis in Ehrlich ascites carcinoma in vitro--IL The synthesis of carbamyl phosphate by a soluble glutamine--dependent carbamyl phosphate synthetase. J. bioL Chem. 242, 5667. HAGERS. E. & JONES M. E. (1967) A glutamine-dependent enzyme for the synthesis of carbamyl phosphate for pyrimidine biosynthesis in fetal rat liver. J. biol. Chem. 242, 5674. H~KKINENI., JgaVt O. & GR6NROOS J. (1968) Suiphoglycoprotein antigens in the human alimentary canal and gastric cancer. An immunohistoiogical study. Int. J. Cancer 3, 572. H.~KKINENI., KORrIONEN L. K. & SAXON L. (1968) The time of appearance and distribution of sulphoglycoprotein antigens in the human fetal alimentary canal. Int. J. Cancer 3, 582. I-'L~KKINENI. & VIKARI S. (1969) Occurence of fetal sulphoglycoprotein antigen in the gastric juice of patients with gastric diseases. Ann. Sure. 169, 277. HAMILTONT. H. (1968) Control by estrogen of genetic transcription and translation. Science 161, 649. HANCOCK R. L., FORRESTERP. I., LORSCHEXDERF. L., LAX P. C. W. & HAY D. M. (1976) Ethionine-induced activation of embryonic genes. In Once-Developmental Gene Expression (FlSHMAN W. H. & SELL S., Eds.), p. 247. Academic Press, New York. HEADYJ. E. & KERR S. J. (1975) Alteration of glycine N-methyltransferase activity in fetal, adult and tumor tissues. Cancer Res. 35, 640. HEIDELBERGERC. (1973) Current trends in chemical carcinogenesis. Fed. Prec. 32, 2154. HERSHEYA. D. & DOVE W. (1971) Introduction to lambda. In The Bacteriophage Lambda, Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. HEwtsrl D. & BURGOYNE L. (1973) Chromatin sub-structure. The digestion of chromatin D N A at regularly spaced sites by a nuclear deoxyribonuclease. Biochem. Biophys. Res. Commun. 52, 504. HNILICAL. S. (1972) The Structure and Biological Function ofHistones. C. R. C. Press, Cleveland, Ohio. HOHMANN P., COLE g. D. & BERN H. A. (1971) Comparison of lysine-rich histones in various normal and neoplastic mouse tissues, d. hath. Cancer Inst. 47, 337. HOLMBEROM. & JONASSONJ. (1974) Synergistic effect of X-ray and UV irradiation on the frequence of chromosome breakage in human lymphocytes. Mutat. Res. 23, 213. HOROWtTZ M. L. & KNOX W. E. (1968) A phosphate activated glutaminase in rat liver different from that in kidney and other tissues. Enzymol. Biol. Clin. 9, 241. HOWK R. & WANG T. Y. (1969) D N A polymerase from rat liver chromosomal proteins--I. Partial purification and general characteristics. Archs Biochem. Biophys. 133, 238. HOWK R. & WANG T. Y. (1970) D N A polymerase from rat liver chromosomal proteins. Alteration of template specificity and alkaline deoxyribonuclease activity. Fur. J. Biochem. 13, 455. HUANG R. C. & BONNER J. (1962) Histone, a suppressor of chromosomal RNA synthesis. Prec. natn. Acad. Sci. 48, 1216. HUANG R. C., BONNER J. & MURRAY K. (1964) Physical and biological properties of soluble nucleohistones. d. melee. BioL 8, 54. HUEBNERR. J. & TODAROG. J. (1969) Ocongenes of RNA tumor viruses as determinants of cancer. Prec. hath. Mead. Sci. 64, 1087. HUEBNER R. J., TODARO G. J., SARMA P. S., HARTLEYJ. W., FREEMANA. E., PETERS R. L., WHITMIREC. E., MEIRER H. & GILDEN R. V. (1970) Defectiveness, rescue, stimulation oncogenic viruses. Int. Symp. Tumor Viruses, 2nd Paris No. 183, p. 33. HULKAJ. F. & MOHR K. (1969) Plhcental hormones and graft rejection. Am. J. Obstet. GynecoL 104, 889. ICHIHARA A., GOTO M. & TOMITA Y. (1976) Gene expression of branched chain amino acid transaminase isozymes during cellular differentiation and carcinogenesis. In Once.Developmental Gene Expression (FISHMAN W. H. & SELL S., Eds.), p. 723. Academic Press, New York. IIDAH. & ODA K. (1975) Stimulation of nonhistone chromosomal protein synthesis in simian virus 40-infected simian cells. J. ViroL 15, 471.

Chromosomal proteins and gene expression 179.

375

ISHIHARAT., KUHNO S.-I. & KUMATORI T. (1974) Ph translocation involving chromosomes 21 and 22. Br. J. Cancer 29, 340. 180. JACKSONW., GRANNER D. d~. CHALKLEY R. (1975) The deposition of histones onto replicating chromosomes. In Chromosomal Proteins and their Role in the Regulation o f Gene Expression (STEIN G. & KLEINSMITHL., Eds.), p. 93. Academic Press, New York. 181. JACKSON V., G.ZANNER D. & CHALKLEY R. (1975) D~position of histories onto replicating chromosomes. Proc. natn. Acad. Sci. 72, 4440. 182. JACOBS-LORENAM., BAGLIONt C. & B3RUN T. W. (1972) Translatioa of m~-ssenger R N A for histones from HeLa cells by a cell-free extract from mouse ascites tumor. Proc. natn. Acad. Sci. 69, 2095. 183. JANSiNGR. L., STEIN J. L., STEIN G. S. (1977) A:tivation of histone g~ne transcriptioa by nonhistone chromosomal proteins in WI-38 human diploid fibroblasts. Proc. natn. Acad. Sci. 74, 173. 184. JENSEN E. V. & DESOMBRE E. R. (1972) Mechanism of action of female sex hormones. Ann. Rev. Biochem. 41,203. 185. JOBEA. & BOURGEOISS. (1972) Lac repressor--operator interaction--Vl. The natural inducer of the iac operon. J. molec. Biol. 69, 397. 186. JOCKUSCHB. M., BECKER M., HINDENNACHI. & JOCKUSCHH. (1974) Slime mold actin: homology to vertebrate actin and presence in the nucleus. Expl Cell Res. 89, 241. 187. JOHNSON A. W. & HNIUCA L. S. (1971) Cytoplasmic and nuclear basic protein synthesis during early sea urchin development. Biochim. Biophys. Acta 246, ]41. 188. JOHNSONE. M., KARN J. & ALLFREY V. G. (1974) Early nuclear events in the induction of lymphocyte proliferation by mitogens. J. biol. Chem. 249, 4990. 189. JOHNSONJ. D., DOUVAS A. S. & BONNER J. (1974) Chromosomal proteins. Int. Rev. Cytol. (Suppl.) 4, 273. 190. JONESO. W., TAYLORA. & STAFFORDM. A. (1972) Fetal gene expression in human tumor ceUs. J. olin. Invest. 51, 47a. 191. JUNGMAN R. A. & SCHWEPPE J. S. (1972) Binding of chemical carcinogens to nuclear proteins of rat liver. Cancer Res. 32, 952. 192. KADOHAMAN. & TURKINGTONR. W. (1973) Altered populations of acidic chromatin proteins in breast cancer cells. Cancer Res. 33, 1194. 193. KALISSN. (1968) Transfer from mother to offspring of antifetal antibody induced in the mouse by muitiparity. Proc. Soc. exp. Biol. Med. 129, 83. 194. KAMIYAMAM. & WANG T. (1971) Activated transcription from rat liver chromatin by nonhistone proteins. Biochim. Biophys. Acta 228, 563. 195. KARN J., JOHNSON E. M., VIDALI G. & ALLVREYV. G. (1974) Differential phosphorylation and turnover of nuclear acidic proteins during the cell cycle of synchronized HeLa cells. J. biol. Chem. 249, 667. 196. KASCHKA-DIERICHC., LINDAHLT., ADAMSA., BORNKAMMG. W., BJURSELLG. ~e KLEIN G. Intracellular forms of Epstein-Barr Virus D N A in human tumor cells in vivo. Nature (in press). 197. KATSUNUMAN., KATSUNUMAT., TOMINO I. & MATSUDA Y. (1968) Regulation of glutaminase activity and differentiation of the isozyme during development. Adv. Enzyme Regal. 6, 227. 198. TREWYN R. W. & KERR S. J. (1976) Regulation of t R N A methyltransferase activity in onco-developmental systems. In Onco-Developmental Gene Expression (FISHMANW. H. & SELL S., Eds.), p. 101. Academic Press, New York. 199. KERR S. J. & BOREK E. (1973) Regulation of the t R N A methyltransferases in normal and neoplastic tissues. Adv. Enzyme Regulation 11, 63. 200. KmucHi Y., SATO S. & SUGIMImA T. (1972) Hexokinase isozyme patterns of human uterine tumors. Cancer 30,444. 201. KINCADEJ. M. (1969) Qualitative species differences and quantitative tissue differences in the distribution of lysine-rich histones. J. biol. Chem. 244, 3375. 202. KINCADEJ. M. (1971) Differences in the quantitative distribution oflysine-rich histones in neoplastic and normal tissues. Proc. Soc. exp. Biol. Med. N.Y. 13/, 1131. 203. KIRBYn . R. S. • COWELLT. P. (1968) Trophoblast host interactions. In EpitheliaI-Mesenehymal Interactions, (FLEISHMAJER R. & GILLINGHAMR. E., Eds.), p. 64. Williams & Wilkins, Baltimore, Md. 204. KIRKPArmCK A., WEPS~C H. T. & NAKAMImA R. (1976) Double antibody radioimmunoassay for the determination of human alpha-fetoprotein in serum: its optimization in clinical application. In Onco-Developmental Gene Expression (FISHMAN W, H. & SELL S., Eds.), p. 695. Academic Press, New York. 205. KISH V. & KLEINSMITHL. J. (1974) Nuclear protein kinases. Evidence for their heterogeneity, tissue specificity, substrate speciflcities, and differential responses to cyclic adenosine 3'-5'-monophosphate.J. biol. Chem. 249, 750. 206. KLEING. (1974) Studies on the Epstein-Barr Virus genome and the EBV-determined nuclear antigen in human malignant disease. Cold Spring Harbor Symp. quant. Biol. 39, 783. 207. KLEIN G., GIOVANELLAB. C., LINOAr~LT., FL~LKOW P. J., Sn~Grt S. & S ' r ~ L ~ J. (1974) Direct evidence for the presence of Epstein-Barr Virus D N A and nuclear antigen in malignant epithelial cells from patients with poorly differentiated carcinoma of thenasopharynx. Proc. Bath. Acad. ScL 71,4737.

376

GARY S. STEIN et al.

KLEINSMITHL. J. (1974) Acidic nuclear phosphoproteins. In Acidic Proteins o f the Nucleus (CAMERONI: R. ¢~ JETER J. R., Eds.), p. 103. Academic Press, New York. 209. KLEINSMITHL. J. (1975) Phosphorylation of nonhiston¢ proteins in the regulation of chromosome structure and function. J. Cell Physiol. 85, 459. 210. KL~INSMn'H L. J., HEIDEMAJ. & CARROLL A. (1970) Specific binding of rat liver nuclear proteins to DNA. Nature 226, 1025. 211. KLEINSMITHL. J., STEIN J. L. & STEIN G. S. (1976) Dephosphorylation of nonhistone proteins specifically alters the pattern of gene transcription in reconstituted chromatin. Prec. HatH. Acad. Sci. 73, 1174. 212. KNOXW. E., LtNDER M. & FRIEDELLG. H. (1970) A series of transplantable rat mammary tumors with graded differentiation, growth rate, and glutaminase content. Cancer Res. 30, 283. 213. KOHN J. (1976) AIpha-fetoprotein in testicular tumors. In Once-Developmental Gene Expression (F~sHMAN W. H. & SELL S., Eds.), p. 387. Academic Press, New York. 214. KORNaERG R. (1974) Chromatin structure: a repeating unit of histories and DNA. Science 184, 868. 215. KORNBE~G R. & THOMASJ. (1974) Chromatin structure: oligomers of the histones. Science 184, 865. 216. Kos'rRAaA N. C. & WANG T. Y. (1972) Transcriptional transformation of Walker tumor chromatin by nonhistone proteins. Cancer Res. 32, 2348. 217. KOSTRAaA N. C. & WANG T. Y. (1975) Inhibition of transcription in vitro by a nonhiston¢ protein isolated from Ehrlich ascites tumor chromatin. J. biol. Chem. 250, 8938. 218. KRAUSEM. O., KLEINSMITH L. J. & S1"EING. S. (1975) Properties of the genome in normal and SV-40 transformed WI-38 human diploid fibroblasts--I. Composition and metabolism of nonhiston¢ chromosomal proteins. Expl Cell Res. 92, 164. 219. KRAUSEM. O., KLEINSMITtt L. J. & STEIN G. S. (1975) Properties of the genome in normal and SV-40 transformed human diploid fibroblasts--III. Turnover of nonhistone chromosomal proteins and their phosphate groups. Life Sci. 16, 1047. 220. KRAUSEM. O. & S~IN G. S. (1974) Modifications in the chromosomal proteins of SV-40 transformed Wl-38 human diploid fibroblasts. Biochem. Biophys. Res. Commun. 59, 796. 221. KRIEGERD. E., LEVINE R., MERRIFIELDR. B., VIDALIG. & ALLFREYV. G. (1974) Chemical studies of histone aeetylation, substrate specificity of a histone deacetylase from calf thymus nuclei. J. biol. Chem. 249, 332. 222. KRYSTALG. & WEnS T. C. (1971) Multiple forms of uridine kinase in normal and neoplastic rat lever. Biochem. J. 12,4, 943. 223. LAcY E. & AX~L R. (1975) Analysis of D N A of isolated chromatin suhunits. Prec. HatH. Acad. Sci. 72, 3978. 224. LANOANT. A. (1968) Histone phosphorylation: stimulation by adenosine 3'-5'-monophosphate. Science 162, 579. 225. LANOANT. A. & HOHMANN P. (1975) Analysis of specific phosphorylation sites in lysine-rich (HI) histone: an approach to the determination of structural chromosomal protein function. In Chromosomal Proteins and their Role in the Regulation o f Gene Expression (STEIN G. & Kt.EINSMITH L., Eds.), p. 113. Academic Press, New York. 226. LANGANT. A. & SMrrH L. K. (1966) The effect of a phosphoprotein preparation from liver nuclei on the inhibition of RNA synthesis by histones. Fed. Prec. 25, 778 abstract No. 3335. 227. L~DINKO L. (1973) Nuclear acidic protein changes in adenovirus-infected human embryo kidney cultures. Virology 54, 294. 228. LE GERFO P., KRUPEY J. & HANSEN H. J. (1971) Demonstration of an antigen common to several varieties of neoplasia. New Engl. J. Med. 285, 138. 229. LENT~R D. & LI~ZlUSA. G. (1972) Mouse-myeloma R N A polymerase B. Template specificities and the role of a transcription-stimulating factor. Ezir. J. Biochem. 30, 278. 230. LES'rouRt3V.ONW. M., FORER A., Y'ANO Y. Z., BER'rRAM 1. S. & RUSCH H. P. (1975) Contractile proteins. Major components of nuclear and chromosomal nonhistone proteins. Biochim. Biophys. Acta 379, 529. 231. LESTOUROEONW. M., WRAY W. & RUSCH H. P. (1973) Functional homologies of acidic chromatin proteins in higher and lower eukaryotes. Expl Cell Res. 79, 487. 232. LEW R., LEVY S., ROSENnERGS. & SIMPSON R. (1973) Selective stimulation of nonhistone chromatin protein synthesis in lymphoid cells by phytohemagglutinin. Biochemistry 12, 224. 233. LI, H. J. (1975) A model for chromatin structure. Nucleic Acids Res. 2, 1275. 234. Llnnv P. R. (1968) Histone aeetylation by cell-free preparations from rat uterus; stimulation by estradiol-17~. Biochem. Biophys. Res. Commun. 31, 59. 235. LIN J. C., NICOLINI C. & BASERGAR. (1974) A comparative study of some properties of chromatin from normal diploid and SV-40 transformed human fibroblasts. Biochemistry 13, 4127. 236. LINDAHLT., KLEIN G., REEDMANB. M., JOHANgSONB. ,R, SINGH S. (1974) Relationship between Epstein-Barr Virus (EBV) D N A and the EBV-determined nuclear antigen (EBNA) in Burkitt lymphoma biopsies and other lymphoproliferative malignancies. Int. J. Cancer 13, 764. 237. LINDER-HOROWrrz M., KNOX W. E. & MORRm H. P. (1969) Glutaminase activities and growth rates of rat hepatomas. Cancer Res. 29, 1195. 208.

Chromosomal proteins and gene expression

377

238. LOEB L. A. (1970) Molecular association of D N A polymerase with chromatin in sea urchin embryos. Nature 226, 447. 239. L O u m A. J. (1974) The organization of proteins in polyoma and cellularchromatin. Cold Spring Harbor Syrup. quant. Biol. 39, 259. 240. LYON M. F. (1961) Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 190, 372. 241. LIPSETT M. B. (1968) Hormonal syndromes associated with neoplasia. In Advances in Metabolic Disorders (LEVrNE R. & Lurr R., Eds.), Vol. 3, p. Il I. Academic Press, N e w York. 242. Lt~lNG B., REDELIUS P., W m L V N D B. & BJORKLVND B. (I976) Biochemistry of TPA. In Onco-Developmental Gene Expression (FIsHMAN W. H. & SELL S., Eds.), p. 773. Academic Press, N e w York. 243. LYSSETr M. B., ODELL W. D., ROSENeUaO L. E. & W A L D M A N N T. A. (1964) Humoral syndromes associated with nonendocrine tumors. Ann. intern. IVied.61, 733. 244. M c C A R r ~ : B. J., NxsmtmA J. T., DOENECKE D., N ~ S a R D. S. & JOW~SON C. B.'(1973) Transcription and chromatin structure. Cold Spring Harbor Syrup. quant. Biol. 38, 763. 245. M A c G I L U V R A Y A. J., CARROLL D. & PAUL J. (1971) The heterogeneity of the nonhistone chromatin proteins from mouse tissues.F E B S Lett. 13, 204. 246. M A C G I L U V R A Y A. J., PAUL J. & THRELFALL G. (1972) Transcriptional regulation in eukaryotic cells. Adv. Cancer Res. 15, 93. 247. MARKERT C. L. (1968) Neoplasia: a disease of cell differentiation.Cancer Res. 28, 1908. 248. M A R K S D. B., PAre W. K. & BORt~ T. W. (1973) The relationship of histone phosphorylation to deoxyribonucleic acid replication and mitosis during the HeLa $3 cellcycle../,biol. Chem. 248, 5660. 249. MARKS L. J., RUSSFIELDA. B. & ROSENBAUMD. L. (1963) Corticotropin-secreting carcinoma. J. Am. reed. Assoc. 183, 115. 250. MARUSHIGEK. & BONNER J. (1971) Fractionation of liver chromatin. Proe. natn. Acad. Sci. 68, 2941. 251. MASSEYEFFR. (1972) Human alpha-fetoprotein. Pathal. Biol. 20, 703. 252. MAYFmLDE. D., LYMAN K. & BRESNICK E. (1967) Incorporation of bicarbonate-l'C into pyrimidine and into ribonucleic acid of the Novikoff ascites tumor cell. Cancer Res. 2/, 476. 253. MEANSA. K., COMSTOCKJ. P., ROSEN~LD G. S. & O'MALL~Y B. W. (1972) Ovalbumin messenger R N A ofchick oviduct: partial characterization, estrogen dependence, and translation in vitro. Proe. natn. Aead. Sci. 69, 1146. 254. MEANSA. R. & O'MALLEY B. W. (1971) Protein biosynthesis on chick oviduct polyribosomes--II. Regulation by progesterone. Biochemistry 10, 1570. 255. MESELSONM. & STAttL F. (1958) The replication of D N A in Eachericbia coll. Proc. natn. Acad. Sci. 44, 671. 256. MILLERD. R. (1969) Raised fetal hemoglobin in childhood leukemia. Br. J. Heamat. 17, 103. 257. MILLERR. W. (1971) Neoplasia and Down's Syndrome. Ann. N . Y . Acad. Sci. 1"/1, 637. 258. MILLERR. W. & TODARO G. J. (1969) Viral transformation of cells from persons at high risk of cancer. Lancet i, 81. 259. MIYAKAWAN., UEDA K. & HAYAISHI O. (1972) Association of poly ADP-ribose glycohydrolase with liver chromatin. Biochem. Biophys. Res. Commun. 49, 239. 260. MONDALH., GANGULY A., DAS A., MANOAL R. K. & BmWAS B. B. (1972) Ribonucleic acid polymerase from eukaryotic cells. Effect of factors and rifampicin on the activity of R N A polymerase from chromatin o f coconut nuclei. Fur. d. Biochem. 28, 143. 261. MOORET. L., KUPCmK H. Z., MAROON N. & ZAMCHECKN. (1971) Carcinoembryonic antigen assay in cancer of the colon and pancreas and other digestive tract disorders. Am..I. dig. Dis. 16, 1. 262. MOSCONAA. A. (1971) Embryonic and neoplastic cell surfaces: availability of receptors for concanavalin A and wheat germ agglutinin. Science 171, 905. 263. Moscovlcl C. & VOlT P. K. (1968) Effects of genetic cellular resistance on cell transformation and virus replication in chicken hematopoietic cell cultures infected with avian myeioblastosis virus (BAI-A). Virology 35, 487. 264. NAIRN R. C., FOTI-mRGILLJ. E., McE~rrEOART M. G. & RICHMOND H. G. (1962) Loss of gastro-intestinal specific antigen in neoplasia. Br. reed. 1. 1, 1791. 265. NICHOLSW. W. (1966) The role of viruses in the etiology of chromosomal abnormalities. Am. ,1. hum. Genct. 18, 81. 266. NICHOLSW. W., LEVANA., CORIELLL. L., GOLDNERH. & AHLSrROMC. G. (1964) Chromosome abnormalities in vitro in human leukocytes associated with Schmidt-Ruppin Rous sarcoma virus. Science 146, 248. 267. NlSmMURAC., NAKASUJIK. & NISHIOORXH. (1974) Early increase of new histones in B H K cells after Herpes simplex virus infection. Kitasato Arehs exp. Med. 47, 103. 268. NOLL M. (1974) Subunit structure of chromatin. Nature 251, 249. 269. NONOYAMAM., HUANG C. H., PAGANOJ. S., KLEI~ G. & SINGH S. (1973) D N A of Epstein-Barr Virus detected in tissue of Burkitts lymphoma and nasopharyngeal carcinoma. Proe. hath. Acad. Sci. 70, 3265. 270. NOWELLP. C. (1975) In Cancer, Vol. I. Etiology: Chemical and Physical Carcinogenesis ('BECKERF. F., Ed.). Plenum, New York.

378 271. 272. 273. 274. 275. 276. 277. 278. 279.

280.

281. 282.

283. 284. 285. 286. 287. 288. 289. 290.

291. 292. 293.

294. 295. 296. 297. 298.

299. 300.

GARY S. STEIN eta/. NOWELLP. C. "g" HUNGERFORDD. A- (1960) A minute chromosome in human chronic granulocytic leukemia. Science 132, 1497. NOWELLP. C. & HUNGERFORDD. A. (1961) Chromosome studies in human leukemia--II. Chronic granulocytic leukemia. J. hath. Cancer Inst. 27, 1013. O'CONNOR P. J. (1969) An alkaline deoxyribonuclease from rat liver nonhistone chromatin proteins. Biochem. Biophys. Res. Commun. 35, 805. OGAWA K. & [CHIHARA A. (1972) Isozyme patterns of branched-chain amino acid transaminase in various rat hepatomas. Cancer Res. 32, 1257. OOAWAK., YOKOJIMAA. & Icmr~gA A, (1970) Transaminase of branched-chain amino acids--VII. Comparative studies on isozymes of aseites hepatoma and various normal tissues of rat. J. Biochem. 68, 901. OgrrA K., Ga~NSTEIN M., KLAmeR M. & FARSeR E. (1974) Localization of a-fetoprotein by immunofluorescence in hyperplastic nodules during hepatocareinogenesis induced by 2-acetylaminofluorene. Cancer Res. 34, 2758. OLINSA. & OLINS D. (1974) Spheroid chromatin units (v bodies). Science 183, 330. OLSON M. O. J., EZgAILSON E. G., Gumrzow K. & BuscH K. (1975) Localization and phosphorylation of nucleolar and extranucleolar nonhistone proteins of Novikoff hepatoma ascites cells. J. molec. Biol. 97, 61 I. O'MALLEY B. W., SheRMAN M. g., Totrr D. O., SeEt,SB~-ROT. C., SCrmADEg W. T. & STEGGLESA. W. (1971) A specific oviduct target-tissue receptor for progesterone. Identification, characterization, partial purification, intercompartmental transfer kinetics and specific interaction with the genome. Adv. Biosci. 7, 213. O'MALLEY B. W., S~Lsnego T. C., SCHaT,DeR W. T., CaVTtL F. & STEOOLeS A. W. (1972) Mechanisms of interaction of a hormone-receptor complex with the genome of a eukaryotic target cell. Nature 235, 141. eNG. T.-M. & DESERReS F. J. (1972) Mutagenicity of chemical carcinogens in Neurospora crassa. Cancer Res. 32, 1890. ORRICg L. P-,., OLSON M. O. J. & BUSCH H. (1973) Comparison of nucleolar proteins of normal rat liver and Novikoff hepatoma ascites cells by two dimensional polyacrylamide gel electrophoresis. Prec. natn. Acad. Sci. 70, 1316. OTTEN J. A., TVNOALL R. L., ESTES P. C., GUDe W. D. & SWARTZENDRUDERD. C. (1973) Localization of gallium-67 during embryogenesis. Prec. Soc. exp. Biol. Med. 142, 92. OUDET P., GROSa-BeLLARD M. & CHAMBON P. (1975) Electron microscopic and biochemical evidence that chromatin structure is a repeating unit. Cell 4, 281. PAll
Chromosomal proteins and gene expression 301.

302. 303. 304. 305. 306. 307. 308. 309. 310, 311. 312. 313. 314. 315. 316. 317. 318. 319. 320. 321. 322. 323. 324. 325.

326. 327. 328. 329. 330. 331.

379

POLLACKR., RISSERR., CONLONS. & RIFKn~ D. (1974) Plasminogen activator production accompanies loss of anchorage regulation in transformation of primary rat embryo cells by simian virus 40. Proc. natn. Acad. Sci" 71, 4792. PREHNR. T. (1967) The significance of tumor-distinctive histocompatibility antigens. In Cross-Reacting Antigens and Neoantigens (TRENTIN J. J., Ed.), p. 105. Williams & Wilkins, Baltimore, Md. PTASHNEM. (1967) Isolation of the A-phage repressor. Proc. hath. Acad. Sci. 57, 306. PUMOD. E., STEIN G. S. & KLEINSMITHL. J. (1975) Stimulated phosphorylation of nonhistone phosphoproteins in SV-40 transformed WI-38 human diploid fibroblasts. Biochim. Biophys. Acta 402, 125. RACEY L. A. & BYVOET P. (1971) Histone acetyitransferase in chromatin. Evidence for in vitro enzymatic transfer of acetate from acetylcoenzyme A to histories. Expl Cell Res. 64, 366. RACEY L. A. & BYVOET P. (1972) Histone acetyltransferase in chromatin. Extraction of transferase activity from chromatin. Expl Cell Res. 73, 329. RALL S. C. & COLE R. D. (1971) Amino acid sequence and sequence variability of the amino-terminal regions of lysine-rich histones. J. biol. Chem. 246, 7175. RAPOSA T., NATAR~OANA. T. & Gr, ANSER~ I. (1974) Identification of Ph chromosome and associated translocation in chronic myelogenous leukemia by Hoechst 33258. J. hath. Cancer Inst. 52, 1935. RAPP F. 0974) Herpesviruses and cancer. Adv. Cancer Res. 19, 265. REECX G. R. & MORXIS H. P. (1974) Nonhistone chromatin proteins of transplantable rat hepatomas and kidney tumors. Proc. Am. Ass. Cancer Res. 15, 29. REECKG. R., SIMPSON R. T. & SOSER H. A. (1972) Resolution of a spectrum ofeucleoprotein species in sonicated chromatin. Proc. hath. Acad. Sci. 69, 2317. REES K. R. & VARCOE J. S. (1967) The interaction of tritiated p
380

332. 333. 334. 335. 336. 337. 338. 339. 340. 341. 342. 343.

344. 345. 346. 347.

348. 349. 350. 351. 352. 353. 354. 355.

356.

357. 358.

359. 360. 361.

GARY S. STEIN e t o2.

SATOK., MO~dS H. P. & WEINHOUSES. (1973) Characterization of glycogen synthetases and phosphorylases in transplantable rat hepatomas. Cancer Res. 33, 724. SArO K. & Tstnra S. (1968) Fructose 1,6-diphosphatase of mouse Ehrlich ascites tumor and its comparison with the enzymes of liver and skeletal muscle of the mouse. 8iochim. Biophys. Acta 159, 130. SATOK. & TstnKl S. (1969) Fructose 1,6-diphosphate of rat tissues and transplantable rat hepatomas. Archs Biochem. Biophy$. 129, 173. SCrIAFFr~US~N B. S. & BENJAMINT. L. (1976) Deficiency in histone acetylation in nontransforming host range mutants of polyoma virus. Prec. hath. Acad. Aci. 73, 1092. S c ~ p l ~ F. (1973) Isozymes and cancer. Adv. Cancer Res. 18, 77. SCrO~PIRAF., HATZ~LD A. & REUBER M. D. (1971) Fetal pattern of aldolase in transplantable hepatomas. Cancer Res. 31, 1224. SCHAPmA F. & Joslr~wlcz A. (1970) Anomalies de type foetal des isozymes de I'adolase et de la lacticodehydrogenase dans hepatomes ascitiques. C. r. See. Biol. 164, 31. SCHAPraAF., MICr~AU C. & JUNmN C. (1972) Foetal enzyme pattern of two alveolar rhabdomyosarcomas. Rev. Ear. Etud. din. Biol. 17, 896. SCrIApmA F., R_m.~R M. D. & I'IATZrELD A. (1970) Resurgence of two fetal-type of aldolases (A and C) in some fast-growing hepatomas. Bioehem. Biophys. Res. Commun. 40, 321. Scrm~ir~GEt~ M. (1962) Uterus of rodents as site for manifestation of transplantation immunity against transplantable tumors. J. ham. Cancer Inst. 2& 927. Scrlwm, PE J. S., KOT E. & Jtn,~GM^N R. A. (1971) Effects of gonadectomy on the uptake of polycyclic hydrocarbons by rat liver nuclei, D N A and histones. Prec. Soc. exp. Biol. Med. 138, 167. SEARLEA. G., FORD C. E., EVANS E. P., BEECHEYC. V., BURTF-NSrlAWM. D. & CLEGG H. M. (1974) The induction of transiocations in mouse spermatozoa--l. Kinetics of dose response with acute X-irradiation. Murat. Res. 22, 157. SrmL'roN K. R. & NEELIN J. M. (1971) Nuclear residual proteins from goose erythroid cells and liver. Biochemistry 10, 2342. SHERMANM. I. Trophoblast and embryoblast cultures and teratocarcinoma. Cancer Res. (in press). SrmRODD., Jom~soN G. & CHALKLEYR. (1970) Phosphorylation of mouse ascites tumor cell lysine-rich histone. Biochemistry 9, 4611. Sn~GEt~ K., Cm~RNOr'r A. I. & SINGER L. 0951) Studies on abnormal hemoglobins--I. Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali denaturation. Blood. 6, 413. SINGERK., CHERNoa'r A. I. & StNOER L. (1951) Studies on abnormal hemoglobins--II. Their identification by means of the method of fractional denaturation. Blood 6, 429. Sir~Kovlcs J. G., DISAIA P. J. & RtrrLED~E F. N. (1970) Tumor immunology and evolution of the placenta. •Lancet ii, 1190. S J t m ~ H. O. (1976) Embryonai antigens in rat colorectal carcinomas. In Once-Developmental Gene Expression (FfSHMAN W. H. & SELL S., Eds.). Academic Press, New York. SKOULTCrn A. & GROSS P. R. (1973) Maternal histone messenger R N A : detection by molecular hybridization. Prec. hath. Acad. Sei. 70, 2840. SLt.rYsI~RM. (1968) Interactions of carcinogens with histones in vitro. Biochim. Biophys. Acta 154, 606. SLtrCstR M. & BUST~ M. (1974) Immunological speeificities of lysine-rich histones from tumors. J. biol. Chem. 249, 2507. SLUVSERM. & HERMrS Y. (1973) A rat-specific lysine-rich historic. Biochim. Biophys. Acta 295, 605. SMrrH D. L., C H ~ C. C., BRUEC,t~EI~B. B., HOL'rZ S. L., HALP~mq R. M. & SMn-H R. A. (1974) Characterization of protein kinases forming acid-labile histone phosphates in Walker-256 carcinosarcoma cell nuclei. Biochemistry 13, 3780. SrmJOCt.ER E. A., KoPtrrz M. & SELL S. (1976) a-fetoprotein responses following phenobarbital stimulation of rat liver. In Onco-Develoomental Gene Expression (FIsnMAN W. H. & SELL S., Eds.), p. 253. Academic Press, New York. SMUtSON M., HENRIKSEN O. & RIOEAU C. (1971) Activity of polyadenosine diphosphoribose polymerase during the human cell cycle. Biochem. Biophys. Res. Commun. 43, 1266. SOLLNER-WEaa B. & F~LSENn~LD G. (1975) Protein interaction with D N A in chromatin. In Chromosomal Proteins and their Role in the Regulation o f Gene Expression (STEIN G. & KLEIr~SM~THL., Eds.), p. 213. Academic Press, New York. SPALtm,~t~J., KAnWAR^ K. & MU~t.LEa G. (1966) The metabolism of basic proteins in HeLa cell nuclei. Prec. hath. Aead. Sci. 56, i 535. SPARAGANAM., PmLLIPS G., HOr'FMANC. & KUCERA L. (1971) Ectopic growth hormone syndrome associated with lung cancer. Metabolism 20, 730. SPELSaEaG R. C. & H~ILICA L. (1970) Deoxyribonucleoproteins and the tissue-specific restriction of the deoxyribonucleic acid in ehromatin. Bioehem. J. 120, 435.

Chromosomal proteins and gene expression 362. 363. 364. 365. 366. 367. 368. 369. 370. 371. 372. 373. 374.

375. 376. 377. 378. 379. 380. 381. 382. 383. 384.

385. 386. 387. 388. 389. 390.

391.

381

SPELSBERGT. C. & HNILICA L. S. (1971) Proteins of chromatin in template restriction--1. R N A synthesis in vitro. Biochim. Biophys. Aeta 228, 202. SPELSBERGT. C. X, HNILICA L. S. (1971) Proteins of chromatin in template restriction--II. Specificity of R N A synthesis. Biochim. Biophys. Acta 228, 212. SPELSBERGT. C. & HNILICAL. S. (1971) Proteins of chromatin in template restriction--Ill. The macromolecules in specific restriction of the chromatin DNA. Biochim. Biophys. Acta 228, 550. SPELSBERGT. C., STEGGLES A. W. ~g" O'MALLEY B. W. (1971) Progesteron-binding components of chick oviduct--III. Chromatin acceptor sites. J. biol. Chem. 246, 4188. SPELSBERGT. C., WILHELMJ. A. ~ HNIL1CA L. S. (1972) Nuclear proteins in genetic restriction--II. The nonhistone proteins in chromatin. Subcell. Biochem. 1, 107. SRIVASTAVAB. 1. S. (1972) Association of terminal deoxynucleotidyl-transferase activity with chromatin from plant tissue. Biochem. Biophys. Res. Commun. 48, 270. STAFFORDM. A. & JONES O. W. (1972) The presence of "fetal" thymidine kinase in human tumors. Biochim. Biophys. Acta 277, 439. STEDM^WE. & STEDMANE. (1943) Probable function of histone as a regulator of mitosis. Nature 152, 556. STEENISH. V., TUSCANYR. & LEION B. (1974) The dis*.ribution of X-ray induced chromosomal abnormalities in rat kangaroo (Potorous tridoctylis) cells in vitro. Mutat. Res. 23, 223. STEIN G. & BASERGA R. (1970) Continued synthesis of nonhistone chromosomal proteins during mitosis. Biochem. Biophys. Res. Commun. 41, 715. STEING. S. & BASERGA R. (1970) The synthesis of acidic nuclear proteins in the prereplieative phase of the isoproterenol-stimulated salivary gland. J. biol. Chem. 245, 6097. STEING. S. & BASERGAR. (1972) Nuclear proteins and the cell cycle. Adv. Cancer Res. 15, 287. STEIwG. S. & BORUN T. W. (1972) The synthesis of acidic chromosomal proteins during the cell cycle of HeLa $3 cells--I. The accelerated accumulation of acidic residual nuclear proteins before the initiation of D N A replication. J. Cell Biol. 52, 292. STEING. S. & BUR'rNER D. E. (1974) Utilization of pre-existing messenger RNAs for nonhistone chromosomal protein synthesis in WI-38 human diploid fibroblasts. Expl Cell Res. 88, 319. STEING. S. & BUgTNER D. E. (1975) Gene activation in human diploid cells. Age-dependent modifications in the stability of messenger RNAs for nonhistone chromosomal proteins. Bioehim. Biophys. Acta 390, 56. STEING. S., CSAUD.ffR1 S. C. & BASERGAR. (1974) Gene activation in WI-38 fibroblasts stimulated to proliferate. Role of nonhistone chromosomal proteins. J. biol. Chem. 249, 3918. STEnqG. S., CRmS W. E. & MORRISH. P. (1974) Properties of the genome in experimental hepatomas: variations in the composition of chromatin. Life Sci. 14, 95. STEING. S. & FARBERJ. (1972) Role of nonhistone chromosomal proteins in the restriction of mitotic chromatin template activity. Proc. natn. Acad. Sci. 69, 2918. STEm G. S. & KLEINSM1THL. J. (Eds.). (1975) Chromosomal Proteins and their Role in the Regulation of Gene Expression. Academic Press, New York. STERNG. S., MANS R. J., GABn^Y E. J., STEIN J. L., DAVIS J. & ADAWADr~R P. D. (1975) Evidence for fidelity of chromatin reconstitution. Biochemistry 14, 1859. STEING. S. & MAT'nt'EWSD. E. (1973) Nonhistone chromosomal protein synthesis: utilization of pre-existing and newly transcribed messenger RNAs. Science 181, 71. STEI~ G. S., Moscovicl G., MoscovicI C. & Mows M. (1974) Acidic nuclear protein synthesis in R.ous sarcoma virus infected chick embryo fibroblasts. FEBS Lett. 38, 295. STEr~ G. S., PARK W. D., STEIN J. L. & I.,mnV.nMAWM. W. (1976) Synthesis of nuclear proteins during D N A repair synthesis in human diploid fibroblasts damaged with ultraviolet radiation of N-acetoxy~-acetylaaminofluorene. Proc. hath. Aead. Sci. 73, 1466. STEING. S., PARK W. D., THRALL C. L., MAWSR. J. & STEIw J. L. (1975) Cell cycle stage-specific transcription of histone genes. Biochem. Biophys. Res. Commun. 63, 945. STERNG., PARK W., THRALLC., MAWS R. & STEIN J. L. (1975) Regulation of cell cycle stage-specific transcription of histone genes from chromatin by nonhistone chromosomal proteins. Nature 257, 764. STEING. S., ROBERTS R. M., DAVIS J. L., HEAD W. J., STEIN J. L., THRALL C. L., VAN VEEN J. & WELCH D. (1975) Are glycoproteins and glycosaminoglyeans components of the eucaryotic genome? Nature 258, 639. STEING. S., SPELSnEROT. C. & KLEINsMrrH L. J. (1974) Nonhistone chromosomal proteins and gene regulation. Science 183, 817. STEING. S., STEIN J. L. & KLEINsMrrH L. J. (1975) Chromosomal proteins and gene regulation. Scient. Am. 232, 46. STERNG., STEIN J., THRALL C. & PARK W. (1975) Regulation of histone gene transcription during the cell cycle by nonhistone chromosomal proteins. In Chromosomal Proteins and their Role in the Regulation of Gene Expression (STEIN G. & KLEINSMITHL., Eds.), p. 1. Academic Press, New York. STERNG. S. & THRALL C. L. (1973) Evidence for the presence of nonhistone chromosomal proteins in the nucleoplasm of HeLa Ss cells. FEBS Left. 32, 41.

382 392. 393. 394. 395. 396. 397. 398. 399. 400. 401. 402.

403. 404. 405. 406. 407. 408. 409.

410. 411. 412.

413. 414. 415. 416. 417.

418. 419. 420. 421.

GARY S. STEIN et al. STEIN G. S. & THRALL C. L. (1973) Uncoupling of nonhistone chromosomal protein synthesis and D N A replication in human diploid WI-38 fibroblasts. FEBS Lett. 34, 35. STEtN J. L., THRALL C. L., PARK W. D., MANS R. J. & STE~N G. S. (1975) Hybridization analysis of histone messenger R N A : association with polyribosomes during the cell cycle. Science 189, 557. STELLWAGENR. & COLE R. (1969) Chromosomal proteins. Ann. Rev. Biochem. 38, 951. STELLWAGEN R. & COLE R. (1969) Histone biosynthesis in the mammary gland during development and lactation. J. biol. Chem. 244, 4878. STOLBACH L. L., KRANT M. J. & FISHMAN W. H. (1969) Ectopic production of an alkaline phosphatase isoenzyme in patients with cancer. New Engl. J. Med. 281, 757. SUGIMURAT. (1973) Poly(adenosine diphosphate ribose). Prog. nucleic Acid Res. molec. Biol. 13, 127. StJGIMURA T., SATO S. & KAWABE S. (1970) The presence of aldolase C in rat hepatoma. Biochem. Biophys. Res. Commun. 39, 626. SURIAS. & LIEW C. C. (1974) Isolation of nuclear acidic proteins from rat tissues. Characterization of acetylated liver nuclear acidic proteins. Biochem. J. 137, 355. SWANECKG. E., CHU L. L. H. & EDELMAN I. S. (1970) Stereospecific binding of aldosterone to renal chromatin. J. biol. Chem. 245, 5382. TAKEOA M,, YAMAMURA H. & OHGA Y. (1971) Phosphoprotein kinases associated with rat liver chromatin. Biochem. Biophys. Res. Commun. 42, 103. TAMAOKI T., MIURA K., LINT. & BANKS P. (1976) Developmental changes in ~-fetoprotein and albumin messenger RNAs. In Onco-Developmental Gene Expression (FISHMAN W. H. & SELL S., Eds.), p. 115. Academic Press, New York. TATA J. R. & BAKER B. (1974) Sub-nuclear fractionation--l. Procedure and characterization of fractions--II. Intranuclear compartmentation of transcription in vivo and in vitro. Expl Cell Res. 83, 111, 125. TAYLOR A. T., STAFFORD M. A. & JONES O. W. (1972) Properties of thymidine kinase partially purified from human fetal and adult tissue. J. biol. Chem. 247, 1930. TAYLOR J. H., WOODS P. S. & HUOrtES W. L. (1957) The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine. Proc. hath. Acad. Sci. 43, 122. TEMIN H. M. (1971) Mechanism of cell transformation by R N A tumor viruses. Ann. Rev. Microbiol. 25, 609. TEMIN H. M. (1971) RNA-directed D N A polymerase systems: not necessarily transforming Rous viruses. J. natn. Cancer Inst. 48, 118 i. TEMIN H. M. (1974) On the origin of RNA tumor viruses. Ann. Rev. Genet. 8, 155. TENG C. S., TENG C. T. & ALLFREY V. G. (1971) Studies of nuclear acidic proteins: evidence for their phosphorylation, tissue specificity, selective binding to deoxyribonucleic acid and stimulatory effects on transscription. J. biol. Chem. 246, 3597. TEPLITZ R. L., BARR K. J. & LAURE H. J. (1972) Karyological and biochemical evidence for chromosomal dedifferentiation in neoplasia. In vitro %, 195. THOMSON J. A., CHtU J.-F. & HNILICA L. S. (1975) Nuclear phosphoprotein kinase activities in normal and neoplastic tissues. Biochim. Biophys. Acta 407, 114. THOMSONJ. A., STEIN J. L., KLEINSMITH L. J. & STEIN G. S. (1976) Activation of in vitro histone gene transcription from HeLa Sn chromatin by S phase nonhistone chromosomal phosphoproteins. Science 194, 628. TIDWELL T., ALLFREY V. G. & MmSKY A. E. (1968) The methylation of histones during regeneration of the liver. J. biol. Chem. 243, 707. TtNG R. C. (1968) Failure to induce transplantation resistance against polyoma tumor cells with syngeneic embryonic tissues. Nature 217, 858. TODARO G. J. & HUERNER R. J. (1972) N. A. S. Symposium: New evidence as the basis for increased efforts in cancer research. Proc. hath. Acad. Sci. 69, 1009. TSAI S. Y., HARRIS S. E., TSAr M.-J. & O'MALLEY B. W. (1976) Effect of estrogen on gene expression in chick oviduct. Proc. natn. Acad. Sci. 73, 4713. TSAI S. Y., TSAI M.-J., SCI-{WARTZ R.., KALIM M., CLARK J. l"I. & O'MALLEY B. W. (1975) Effects of estrogen on gene expression in chick oviduct: nuclear receptor levels and initiation of transcription. Proc. natn. Acad. Sci. 72, 4228. TsAI Y. H., CHILI J. F., SAKUMA K. & HNILICA L. S. (1974) Regulation of chicken globin m R N A transcription in vitro. J. Cell Biol. 63, 353a No. 705. TSANEV T. & RUSSEV G. (1974) Distribution of newly synthesized histone during D N A replication. Ettr. J. Biochem. 43, 257. TSUBOlA. & BASERGA R. (1972) Synthesis of nuclear acidic proteins in density inhibited fibroblasts stimulated to proliferate. J. cell. Physiol. 77, 107. TSURUOT., TOMITA Y'., SATOH & UKITA T. (1972) D N A polymerases of ascites hepatoma cells--II. Purification and properties of D N A polymerases from nuclear membrane-chromatin fraction. Biochem. Biophys. Res. Commun. 48, 776.

Chromosomal proteins and gene expression 422. 423. 424. 425. 426. 427. 428. 429. 430.

431. 432. 433. 434. 435.

436.

437. 438. 439. 440. 441.

442.

443.

444. 445. 446. 447.

448. 449.

383

TsuRuo T. & UKITA T. (1974) Purification and further characterization of three D N A polymerases of rat ascites hepatoma cells. Biochim. Biophys. Acta 353, 146. TUFrEY M., BISHUN N. P. & BARNES R. D. (1969) Porosity of the mouse placenta to maternal cells. Nature 221, 1029. URBANCZYKJ. & STUDZINSKI G. P. (1974) Chromatin-associated D N A endonuclease activities in HeLa cells. Biochem. Biophys. Res. Commun. 59, 616. URIEL J., BOUILLON D., AUSSEL C. & DOPIERS M. (1976) a-fetoprotein: the major high-affinity estrogen binder in rat uterine cytosols. Proc. hath. Acad. Sci. 73, 1452. VIDALIG., BOFEA L. C. & ALLFREV V. G. (1972) Properties of an acidic histone-binding protein fraction from cell nuclei. Selective precipitation and deacetylation of histone F~I and F3. J. biol. Chem. 247, 7365. VIDALIG., BOFFA L., LITTAU V. C., ALLFREY K. M. & ALLFREY V. G. (1973) Changes in nuclear acidic protein . complement of red blood cells during embryonic development. J. biol. Chem. 248, 4065. VOGELF. & ROHRBORN G. (1970,~ Chemical Mutagenesis in Mammals and Man. Springer, New York. VON KLEIST S. & BUR'rIN P. (1969) Isolation of a fetal antigen from human colonic'tumors. Cancer Res. 29, t961. WAHRENB., ESPOSTI P., GADLER H. & ALPERT E. 0976) Marker antigens and immunization with heterologous A F P in germinal tumors of the testis. In Onco-Developmental Gene Expression (FISHMAN W. H. & SELL S., Eds.), p. 147. Academic Press, New York. WAKABAYASHIK. & HNILICA L. S. (1973) The immunospecificity of nonhistone protein complexes with DNA. Nature, New Biol. 242, 153. WANG T. Y. (1968) Isolation of a terminal DNA-nucleotidyl transferase from calf thymus nonhistone chromatin proteins. Archs Biochem. Biophys. 127, 235. WANG T. Y. (1968) Restoration of histone-inhibited DNA-dependent R N A synthesis by acidic chromatin proteins. Expl Cell Res. 53, 288. WANG T. Y. (1971) Tissue specificity of nonhistone chromosomal proteins. Expl Cell Res. 69, 217. WANG T. Y. & KOSTRABA N. (1975) Stimulation and inhibition of transcription in vitro by nonhistone chromosomal proteins. In Chromosomal Proteins and their Role in the Regulation o f Gene Expression (STEIN G. & KLEINSMITH L., Eds.), p. 35. Academic Press, New York. WATABE H., LEFFERT H. & SELL S. (1976) Developmental and maturational changes in a-fetoprotein and albumin production by cultured fetal rat hepatocytes in vitro. In Onco-Developmental Gene Expression (FISHMAN W. H. & SELL S., Eds.), p. 123. Academic Press, New York. WEINHOUSE S. (1972) Glycolysis, respiration, and anomalous gene expression in experimental hepatomas: G. H. A. CIower Memorial Lecture. Cancer Res. 32, 2007. WEINTRAUBH. & GROUDINE M. (1976) Chromosomal subunits in active genes have an altered conformation. Science 193, 848. WEINTRAUBH. & VAN LENTE E. (1974) Dissection of chromosome structure with trypsin and nucleases. Proc. natn. Acad. Sci. 71, 4249. WEISS S. B. (1960) Enzymatic incorporation of ribonucleoside triphosphates into the interpolynucleotide linkages of ribonucleic acid. Proc. hath. Acad. Sci. 46, 1020. WIKMANJ., QUAGLIAROTTI G., HOWARD E., CHOI Y. C. & BUSCH H. (1970) A comparison of a2p distribution in oligonucleotides of ribosomal 28S R N A from normal liver and Novikoff hepatoma ascites cells. Cancer Res. 30, 2749. WILHELMJ., ANSEV1N A. T., JOHNSON A. W. & HNIL1CA L. S. (1972) Problems of chromatin in genetic restrict i o n - I V . Comparison of histone and nonhistone proteins of rat liver nucleolar and extranucleolar chromatin. Biochim. Biophys. Acta 272, 220. WILSON B., LEE M. A., VIDALI G. & ALLFREY V G. (1975) Fractionation of nuclei and analysis of nuclear proteins of rat liver and Morris hepatoma 7777. Cancer Res. 35, 2954. WILSON I. B. (1963) A tumor tissue analogue of the implanting mouse embryo. Proc. zool. Soc. Lond. 141, 137. YAMADAN. & SUGIMtrRA T. (1973) Effect of deoxyribonucleic acid and histone on the number and length of poly(adenosine diphosphate-ribose). Biochemistry 12, 3303. YANG W.-K. (1971) Isoaccepting transfer RNAs in mammalian differentiated cells and tumor tissues. Cancer Res. 31,639. YEOMANL. C., JORDON J. J., BUSCH R. K., TAYLOR C. W., SAVAGEH . . E & BUSCH H. (1976) A fetal protein in the chromatin of Novikoff bepatoma and Walker 256 carcinosarcoma tumor that is absent from normal and regenerating rat liver. Proc. ham. Acad. Sci. 73, 3258. YEOMAN L. C., SUaER S., TAYLOR C. W., FUNBACr~ D. J., FALLETrA J. M., JORDON J. J. & BUSCH H. (1976) Differences in chromatin proteins of resting and growing h u m a n lymphocytes. Expl Cell Res. 100, 47. YEOMAN L. C., TAYLOR C. W. & BUSCH H. (1974) Two dimensional gel electrophoresis of acid-extractable nuclear proteins of regenerating and thioacetamide-treated rat liver, Morris 9618 A h e p a t o m a and Walker 256 carcinosarcoma. Cancer Res. 34, 424.

384

GARY S. STEIN et al.

450. YEOMANL. C., TAYLORC. W., JORDONJ. J. & BUSCHH. (1973) Two dimensional polyacrylamide gel electrophoresis of chromatin proteins of normal rat liver and Novikoff hepatoma ascites cells, Biochem. Biophys. Res. Commun. $3, 1067. 451. YEOMANL. C., TAYLORC. W., JORDONJ. J. & BUSCHH. (1975) Differences in chromatin proteins of growing and nongrowing tissues. Expl Cell Res. 91,207. 452. Ytp M. C, M. & KNOX W. E. (1970) Glutamine-dependent carbamyl phosphate synthetase. Properties and distribution in normal and neoplastic rat tissues. J. biol. Chem. 245, 2199. 453. YotmG D. (1971) The susceptibility to SV-40 virus transformation of fibroblasts obtained from patients with Down's Syndrome. Fur. J. Cancer 7, 337. 454. ZAMCHECKN., MOORET. L., DHAR P. & KUPCmK H. (1972) Immunologic diagnosis and prognosis of human digestive-tract cancer: careinoembryonic antigens. New Engl. J. Med. 286, 83. 455. ZARDI L., LIN J. C. & BASEROAR. (1973) Immunospecificity to nonhistone chromosomal proteins of antichromatin antibodies. Nature, New Biol. 245, 211. 456. ZORNETZERM. & STEING. S. (1975) Gene expression in mouse neuroblastoma cells: properties of the genome. Proc. natn. Acad. Sci. 72, 3119. 457. ZtJR I-IAus~N H., SCHULT~-HoL'r~r~SENH., KLEIN G., HENLE W., HENLE G., CLIFFORDP. & SANTESSOr~L. (1970) EBV DNA in biopsies of Burkitt tumors and anaplastic carcinomas of the nasopharynx. Nature 228, 1056.