Lead Review Article

August 7992:217-223

Trace Elernent-Gene Interactions John K. Chesters, Ph.D.

Iron Probably the best understood examples of the regulation of expression of trace element-related transport and storage proteins relate to iron and control of the synthesis of ferritin and the plasma-

membrane receptor for transferrin. The balance between these proteins regulates iron availability, since the transferrin receptor is required for the uptake of iron, and ferritin is necessary for storage of any iron temporarily in excess of requirements. The latter is important because excess free ionic iron, a catalyst in the generation of free radicals, is generally highly toxic to living systems.’ Expression of these iron-regulatory proteins is controlled not at the level of the gene per se, but by varying the efficiency of utilization of their mRNAs.* This is achieved in an integrated manner through the mediation of an iron-dependent protein (IRE-BP), which binds to regions of the mRNAs for each regulatory protein. At the binding sites, basepairing causes the mRNA to fold back on itself, forming a “stem-loop’’ structure, the iron-response element (IRE), which is recognized by IRE-BP (Figure l).3 The affinity of IRE-BP for IRES decreases as iron availability increases. This provides the basis of an elegant control mechanism. In the case of the mRNA for ferritin, the IRE is located upstream of the protein-coding region and passage of the translation complex is blocked if the IRE has IRE-BP bound to it. Thus synthesis of ferritin is inhibited when low availability of iron increases the affinity of IRE-BP for IRE stem loops. Furthermore, recent evidence has indicated that when not masked by IRE-BP, the IRE not only permits ferritin synthesis, but actually enhances translation of its mRNA.4 In the case of the transferrin receptor mRNA, this control is neatly complemented by a related mechanism. Once again the affinity of IREBP for the stem loops is enhanced by low iron availability, but in this case multiple copies of the IRE are located downstream of the coding region and binding of IRE-BP helps to stabilize the mRNA. Thus a shortage of iron tends to facilitate binding of IRE-BP and, by stabilizing transferrin receptor mRNA, to increase levels of the receptor and, hence, iron uptake.

Dr. Chesters is a Group Leader in the Division of Biochemical Sciences of the Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, U.K.

Copper Evidence for direct involvement of copper in regulation of its uptake and storage is sparse. Copper is

f o r many of the genes encoding proteins involved in the transport, storage, and function of the trace elements, expression is regulated by the availability of the elements concerned. This control is exercised through a variety of mechanisms, including metal-activated transcription factors, modified usage of stop codons, and use of secondary structure within m R NA to regulate its translation and stability. Two widely represented groups of transcription factors, often classed as zinc-finger proteins, depend on constituent zinc ions for their activity. In addition, the sensitivity of growth and fetal development to the lack of zinc is hypothesized to relate to a requirement for the element during certain critical alterations in gene expression. The evidence for this and possible underlying mechanisms is examined.

Broadly interpreted interactions between trace elements and gene expression could include effects mediated by vitamin B,, (of which cobalt is an integral component), or by the iodine in thyroid hormones. These aspects, widely covered in reviews of the functions of vitamins and hormones, are not considered in the present paper. These aspects aside, it becomes apparent that zinc predominates in trace element gene interactions. The expression of a number of proteins involved in the transport, storage, and function of trace elements is influenced by the availability of the elements themselves, and these will be considered before attention is concentrated on the importance of zinc in this field.

Proteins Involved in the Transport, Storage, and Function of Trace Elements

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217

regulating synthesis of the yeast metallothioneinlike protein. lo

FERRlTlN

5' Coding Inactive

Coding Active

TRANSFERRIN RECEPTOR

00Q mRNA mRNA Stable Unstable Figure 1. Regulation of iron-dependent mRNA.

associated with two main proteins in plasma: albumin and ceruloplasmin. While the major coppertransport protein may be albumin, it has been postulated that ceruloplasmin may also act in this role.5 Both ceruloplasmin and a second major copper protein, copper-zinc superoxide dismutase (Cu-Zn SOD), have been used as indicators of copper status in animals because their tissue concentrations respond to variations in the amount of dietary copper.6 There have been suggestions that the concentration of ceruloplasmin mRNA is responsive to the concentration of dietary copper.' However, in isolated hepatocytes, neither increasing nor decreasing the intracellular concentration of copper influenced ceruloplasmin mRNA concentrations.* Furthermore, ceruloplasmin mRNA and apoprotein concentrations in liver have recently been shown to be unaffected by a dietary deficiency of copper sufficiently severe to eliminate virtually all ceruloplasmin-oxidase activity.' This suggests that the lack of copper prevents only the final activation of the protein's enzymatic capacity. Interestingly, studies with the yeast Saccharomyces cerevisiae have indicated that synthesis of Cu-Zn SOD is at least partially regulated at the transcriptional level by the presence in its promoter of a metal-response element (MRE-see below) similar to the MRE218

Selenium Genetic control of the incorporation of selenocysteine into selenoproteins involves a unique use of the codon U(T)GA. l 1 This codon normally acts as a stop codon but has now been shown to mark the site of insertion of a selenocysteine residue in each selenoprotein examined thus far. Evidence regarding the determinants of the translation of this codon is beginning to emerge, in particular from studies of bacterial systems.'* It is now clear that in both eukaryotes and bacteria, insertion of selenocysteine is mediated by an unusual tRNA, which initially becomes charged with a serine residue that is then converted to selenocysteine while still attached to the tRNA. In Escherichia coli, three additional gene products appear to be required for incorporation of selenocysteine into protein. Two of these are enzymes that catalyze conversion of the serine residue to selenocysteine, and the third appears to act as a translation factor ensuring correct interpretation of the UGA codon. It seems likely that, as with the regulation of the iron-related mRNAs discussed earlier, here also secondary structure within the mRNA may be important. A putative stem-loop has been found immediately downstream of the insertion site for selenocysteine in formate dehydrogenase. Mutations within this region caused the UGA codon to revert to its chain-terminating function. In addition to this regulation of selenium incorporation, there appears to be a mechanism by which the concentration of mRNA for the major selenoprotein glutathione peroxidase is linked with dietary selenium content. Although one group has reported increased glutathione peroxidase mRNA in the liver of seleniumdeficient rats,13 others have indicated that its mRNA concentration is decreased by the deficiency. 14,15 Further studies16 indicated that transcription of the glutathione peroxidase gene was not affected by selenium deficiency, indicating that the alterations in mRNA content probably reflect changes in the stability of its mRNA. It is interesting, therefore, to speculate that selenocysteinetRNA or its translational cofactor may stabilize the mRNA by binding to it in a manner analogous to that of the IRE-BP/IRE interaction referred to in the discussion of iron. Metallothionein Metallothioneins are small ( M , = 6,500) cysteinerich proteins that can bind many different divalent metal ions. Their biologic role(s) are still the subject of debate, but they undoubtedly act as a store for metals such as zinc. Their concentration within tisNutrition Reviews, Vol. 50, No. 8

sues is highly dependent on metal-ion availability; much of the regulation occurs at the level of transcription. The promoter structure of mouse metallothionein I has been most thoroughly investigated and is known to contain multiple MREs responsible for the transcriptional sensitivity of the gene to divalent metal ions.” The MREs have also been shown to confer metal responsiveness on foreign reporter genes when inserted in either orientation and either upstream or downstream of the coding region. l7 Thus they have the properties of transcriptional enhancers. There are multiple MRE sequences with varying potencies, and although a single MRE may be sufficient to confer metal-responsiveness, multiple copies can have additive effects on transcription. Uncertainties still remain regarding the precise role of these domains in the activation of transcription, but it is clear that proteins (metal-responsive transcription factors, MRTFs) exist that, upon interacting with one of the inducing metal ions, bind to the MREs and facilitate transcription of the metallothionein gene.” A single MRE can confer responsiveness to several different metal ions, but it is not yet clear whether this results from interaction with relatively few MRTFs with broad metal-ion specificity or with a family of similar MRTFs, each of which is metal-specific. Speculation on the nature and function of these proteins has been fueled by observations on the control of the metallothionein-like proteins found in yeasts. Here the ready availability of genetic analysis has yielded a far greater understanding of the regulatory mechanisms, but there are sufficiently large differences between the mammalian and fungal systems to cast doubt on interchangeability of the results. Yeast equivalents of the MRTFs have been found to contain multiple domains, including a cysteine-rich metal-binding region involved in binding to DNA and a carboxy-terminal domain that is essential for transcriptional activation. lo This division of function between different regions of these proteins is similar to that observed in many mammalian transcription factors, and may be common to the mammalian MRTFs. However, there are important differences between the mammalian and fungal proteins. So far, the yeast proteins seem to respond only to copper (and, to a lesser extent, to silver), unlike the mammalian proteins that are most sensitive to zinc and cadmium. Only further experimentation will reveal the true extent of similarity between these systems. “Zinc-Finger” Proteins

Investigations during the last decade have led to recognition of a number of generic classes of tranNutrition Reviews, Vol. 50, No. 8

scriptional regulators, including those based on helix-turn-helix, leucine-zipper, and “zinc-finger” (Zn-finger) transcription motifs. Some estimates suggest that the number of Zn-finger proteins in the human genome may considerably exceed 100; thus they would rival the Zn-containing enzymes, at least in numerical importance. l9 The first Zn-finger protein to be recognized was TFIIIA, a transcription factor involved in 5s ribosomal RNA synthesis. Since most of the early studies centered on this protein, its structure was originally used as a model for the group, but two aspects make this no longer advisable. First, unlike virtually all other Zn-finger proteins, TFIIIA acts as a transcription factor for RNA polymerase I11 and binds within the transcribed region of the gene. Most other Zn-finger proteins relate to genes transcribed by RNA polymerase I1 and bind to a promoter lying outside the coding region. Second, it is now clear that the Znfinger proteins include at least two distinctly different groups of transcription factors, the C2-H2 and C2-C2 series. There also appears to be a third group of zinc-containing transcription factors, exemplified by GAL4,20but these have a totally different structure, seem to be restricted to fungi, and will not be considered further in this review. Proteins of the C2-H2 Zn-finger series characteristically contain multiple repeats of the “finger,” with close similarity between the repeats within the same protein. Structural investigations of the fingers have modified the original concept of a series of polypeptide “loops .” Considerable evidence now indicates that each “loop” contains two distinct zones-a hairpin structure consisting of two antiparallel beta sheets and a C-terminal alpha helical region (Figure 2). These structures are held together by a zinc ion, tetrahedrally coordinated to a cysteine residue in each of the beta sheets and to two histidines in the helix, which faces inward in the major groove of the DNA molecule.21The zinc ions are essential to maintain DNA-binding capacity and the Zn-fingers are generally arranged in tandem repeats. The latter are frequently located toward the C-terminal portion of the protein, while regions mediating transcriptional activation lie outside the DNA-binding zone. Less is known of the structure and function of these regions, but in the case of the transcription factor Spl , transcriptional activation has been shown to involve interaction with the TATA box-binding factor TFIID.22 The C2-C2 Zn-finger proteins, although superficially similar to those described earlier, are significantly different. These proteins act as nuclear receptors for the steroid and thyroid hormones and for retinoids, and invariably contain only two nonidentical fingers.23 Both fingers contain one tetrahedrally coordinated zinc ion, but in the C2-C2 pro219

Beta-

\

Plane of Symmetry

Alpha-

Figure 2. Diagrammatic representation of a C2-H2 Znfinger.

teins all the coordination is provided by cysteine residues and each protein contains an extra cysteine in the C-terminal finger. The proteins contain three domains, an N-terminal zone responsible for transcriptional activation, a central DNA-binding region containing the Zn-fingers, and a C-terminal portion containing a hormone-binding site that determines the receptor’s ~pecificity.~~ Additionally, the C-terminal domain contains a dimerization region that is critical to the function of these receptors. The hormone-recognition sites on the DNA (HREs) have palindromic sequences to which the steroid receptors bind in pairs. Both the sequence of the two halves of the HREs and the spacing between them contribute to a specificity of binding, which allows the dimerized receptor to distinguish between estrogen and glucocorticoid HREs differing only in sequence (not in spacing) and between thyroid and estrogen receptors differing in spacing but not in sequence (Figure 3). As mentioned previously, more than 100 putative Zn-finger proteins have been identified in the human genome. Since all those whose function has so far been identified are transcriptional control factors, their potential contribution to the control of gene expression is substantial. Furthermore, since in each case examined the zinc ions are essential for their function, they present many opportunities for metabolic derangements in zinc-deficient animals. The only C2-H2 finger protein known to the author to have been investigated in zinc-deficient animals is TFIIIA. Here, there was no evidence for functional inadequacy in rats whose growth had been fully inhibited by dietary zinc d e f i ~ i e n c yHow.~~ ever, restriction of zinc availability by the use of chelators in cell culture, which probably resulted in a more severe deficiency than that obtained in the rats, did result in the expected impairment of 5s 220

GRE

A G U C A X X X T G U C T

ERE

A G U C A X X X T G U C T

TRE

A G U C A T G U C T

Figure 3. Palindromic base sequences of the hormone recognition elements (HREs) for glucocorticoid (GRE), estrogen (ERE), and thyroid (TRE) hormones. Underlined letters denote base differences. Note the lack of “spacer” bases in TRE.

ribosomal RNA synthesis. It appears that retention of zinc by TFIIIA is sufficiently strong to prevent its inactivation in vivo, but it is entirely possible that the structure of one or more of the other Znfinger proteins is such that its zinc ions are not retained once the supply of zinc is decreased and that this underlies certain of the clinical effects of zinc deficiency. Zinc and Gene Expression

In the young animal, a reduction in growth rate is generally the first sign of zinc deficiency; a dietary deficiency of zinc imposed on pregnant animals has repeatedly been shown to induce fetal abnormalities.25,26Although about 100 enzymes are known, none of them has been implicated in these manifestations of zinc deficiency. Instead, present evidence has led to the conclusion that the primary requirement for zinc relates to a role in gene e x p r e ~ s i o n . ~ ~ Some of this evidence is reviewed below and possible underlying mechanisms are discussed. The impact of an inadequate zinc supply has been investigated both in animals fed zinc-deficient diets and in cell cultures where zinc availability was decreased by the addition of chelating agents. In both circumstances, the lack of zinc resulted in decreased thymidine incorporation, which has been taken to indicate impaired DNA synthesis. In culture,” in the developing fetus,28and in wound healing,29the loss of thymidine incorporation was accompanied by a similar decrease in thymidine kinase activity. However, there is no evidence to suggest that thymidine kinase contains zinc, and in both the fetus and culture, DNA polymerase activity was reduced to a comparable e ~ t e n t . ~ Fur’’~~ thermore, an induced lack of zinc in regenerating Nutrition Reviews, Vol. 50, No. 8

liver resulted in a parallel reduction in thymidine incorporation and in the proportion of cells labeled.30These observations suggest that low zinc availability results in a failure to induce the group of enzymes required for DNA synthesis in individual cells rather than in an overall decrease in activity of a particular zinc enzyme. The hypothesis that the lack of zinc primarily restricts gene expression rather than enzyme activation was strengthened by the observation that the zinc-dependent loss of thymidine kinase activity in fibroblasts treated with a chelator was accompanied by a corresponding reduction in the concentration of the enzyme's mRNA. Thus the impact of the lack of zinc on thymidine kinase occurred at a pretranslational step31 (see Table 1). Since RNA polymerases are thought to contain zinc, this loss of thymidine kinase mRNA could have resulted from a general impairment of mRNA synthesis, but the concentration of mRNA for the constitutive ribosomal protein S6 was not affected. This suggested that only those proteins whose synthesis required a change in gene expression were affected by the lack of zinc. A similar situation has since been observed when myoblasts were induced to differentiate in the absence of adequate zinc. Once again the concentration of mRNA for an induced protein, creatine kinase, was reduced by the lack of zinc, but mRNA for S6 was relatively ~ n a f f e c t e d . ~ ~ A role for zinc in alterations of gene expression could also underlie another characteristic sign of zinc deficiency, namely, the incidence of parakeratosis in epidermis, esophagus, and buccal epithelium. This is characterized by retention of nuclei and poor differentiation of the cells as they move away from the basal germinative layer. In the esophagus, at least, this was associated with an apparently elevated rate of cell division despite a significantly lower tissue zinc c ~ n c e n t r a t i o n Such .~~ observations are hard to reconcile with a requirement for zinc for DNA synthesis per se but are compatible with a need for zinc during the changes Table 1. Effects of Zinc Availability on the Ratio of the Thymidine Kinase (TK) mRNA to Ribosomal S6 Protein mRNA in 3T3 Cells Stimulated from Quiescence and on the Ratio of Creatine Kinase (CK) mRNA to S6 mRNA in Differentiating Chick Myoblastsa mRNA Ratio Cultural Conditions

TWS6

CK/S6

Control Zinc-depleted Zinc-repleted * Based on refs. 29 and 30.

0.50 0.03 0.49

1.40 0.15 1.20

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in genetic expression that should accompany differentiation of these cells as they migrate from the basal layer of cells that are programmed to divide continually. If zinc is required to facilitate changes in gene expression, what are its possible roles? One role that is immediately apparent is as a component of Zn-finger proteins required to enhance transcription of specific genes. In this context, the transcription factor, Spl, raised initial interest because it belongs to the class of C2-H2 proteins and contains three Zn-fingers per mole. However, recent studies indicate that, while production of thymidine kinase mRNA was inhibited by the lack of zinc when under the control of the thymidine kinase promoter, it was not affected when the latter was replaced by the SV40 early promoter (Chesters, unpublished observation). Since the SV40 promoter is known to contain several binding sites for Spl , it seems unlikely that the lack of zinc, which was sufficient to inhibit DNA synthesis, had any marked effect on Spl activity. Furthermore, as mentioned earlier, dietary zinc deficiency did not limit TFIIIA activity sufficiently to impair 5s rRNA synthesis in v ~ v o . ~ ~ These observations suggest that Zn-fingers may be able to retain their zinc even when its availability is lowered by dietary deficiency. However, in cell culture, TFIIIA activity was inhibited by a lack of zinc induced by addition of a ~ h e l a t o r and , ~ ~a recent estimation of the zinc-binding affinity of a synthetic Zn-finger peptide suggested a K, of 3 x lop9 m ~ l / L The . ~ ~former suggests that Zn-finger proteins can be induced to release their zinc in living systems. The latter suggests that the concentrations of zinc required to saturate a Zn-finger are comparable to the free zinc ion concentrations estimated to be present in normal plasma.35As yet, too little information is available to permit firm conclusions, but these observations suggest that variations in the local environment around the zinc ions caused by differences in amino acid sequence between different Zn-finger transcription factors could be such that certain of them might lose, or fail to gain, the zinc necessary for their function under conditions of zinc depletion. A second possible route by which changes in zinc availability could influence gene expression is through alterations in chromatin structure. Gene unmasking is thought to involve a decondensation of chromatin with removal of at least the H1 histones, and these changes render active chromatin more susceptive to nuclease attack.36Furthermore, zinc deficiency has been reported to increase the resistance of chromatin to nuclease attack and to alter the proportions of histone H1 subforms present in the chromatin.37Earlier studies had indicated that zinc ions facilitated the initial phase of 221

temperature-induced chromatin dissociation, which was probably associated with removal of H1 histone,38 and that phosphorylation of lysine-rich histones by rat liver nucleoli in vitro was specifically stimulated by addition of zinc ions.39 These observations, while still fragmentary in nature, may nevertheless point toward a biologically significant role of zinc ions in facilitating H1 histone removal from chromatin, thereby exposing a different set of genes for subsequent expression. Summary

The information presented here suggests that, while many trace elements may regulate the expression of the proteins involved in their transport, storage, and function, zinc may well play a more ubiquitous role in facilitating_ and controlling_ gene expression. _ 1. Halliwell B, Gutteridge JMC. The importance of free radicals and catalytic metal ions in human diseases. Mol Aspects Med 1985;8:89-193 2. Klausner RD, Harford JB. Cis-trans models for post-transcriptional gene regulation. Science

1989;246:870-2 3. Leibold EA, Laudano A, Yu Y. Structural requirements of iron-responsive elements for binding of the protein involved in both transferrin receptor and ferritin messenger RNA post-transcriptional regulation. Nucleic Acids Res 1990;18:1819-24 4. Dix DJ, Lin PN, Kimata Y, Theil EC. The iron regulatory region of ferritin messenger RNA is also a positive control element for iron-independent translation. Biochemistry 1992;31:2818-22 5. Harris ED, Stevens MD. Receptors for ceruloplasmin in aortic cell membranes. In: Mills CF, Bremner 00, Chesters JK, eds. Trace elements in animals and man. Slough, UK: Commonwealth Agricultural Bureau, 1985:32&3 6. Suttle NF. Problems in the diagnosis and anticipation of trace element deficiencies in grazing livestock. Vet Rec 1986;119:14&52 7. Danks DM, Mercer JFB. Metallothionein and ceruloplasmin genes. In: Hurley LS, Keen CL, Lonnerdal B, Rucker RB, eds. Trace elements in man and animals-6. New York: Plenum Press, 1988:287-91 8. McArdle HJ, Mercer JFB, Sargeson AM, Danks DM. Effects of cellular copper content on copper uptake and metallothionein and ceruloplasmin mRNA levels in mouse hepatocytes. J Nutr 1990; 120:1370-5 9. Gitlin JD, Schroeder JJ, Leeambrose LM, Cousins RJ. Mechanisms of caeruloplasmin biosynthesis in normal and copper-deficient rats. Biochem J 1992;282:835-9 10. Thiele DJ. Metal-regulated transcription in eukaryotes. Nucleic Acid Res 1992;20:1183-91 11. Chambers I, Harrison PR. A new puzzle in selenoprotein biosynthesis: selenocysteine seems to be 222

encoded by a stop codon UGA. Trends Biochem Sci 1987;12:255-6 12. Bock A, Forchhammer K, Heider J, et al. Selenocysteine: the 21st amino acid. Mol Microbiol 1991;

5:515-20 13. Li N-Q, Reddy PS, Thyagaraju K,et al. Elevation of rat liver mRNA for Se-dependent glutathione peroxidase by selenium deficiency. J Biol Chem 1990;

265:108-13 14. Saedi MS, Smith CG, Frampton J, et al. Effect of selenium status on mRNA levels for glutathione peroxidase in rat liver. Biochem Biophys Res Commun 1988;153:855-61 15. Toyoda H, Himeno S , lmura N. The regulation of glutathione peroxidase gene expression; implications for species differences and the effect of dietary selenium manipulation. In: Wendel A, ed. Selenium in biology and medicine. Berlin: Springer-Verlag, 19895-7 16. Sunde RA. Molecular biology of selenoproteins. Annu Rev Nutr 1990;10:451-74 17. Stuart GW, Searle PF, Chen HY, et al. A 12 basepair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci USA 1984;81:7318-22 18. Struhl K. Helix-turn-helix, zinc-finger, and leucinezipper motifs for eukaryotic transcriptional regulatory proteins. Trends Biochem Sci 1989;14:137-

40 19. Berg JM. Zinc finger domains-hypotheses

and current knowledge. Annu Rev Biophys Biophys Chem 1990;19:405-21 20. Berg JM. Zinc fingers and other metal-binding domains-elements for interactions between macromolecules. J Biol Chem 1990;265:6513-6 21. Elbaradi T, Pieler T. Zinc finger proteins-what we know and what we would like to know. Mech Dev

1991;35:155-69 22. Smale ST, Schmidt MC, Berk AJ, Baltimore D. Transcriptional activation by Spl as directed through tata or initiator-specific requirement for mammalian transcription factor-lld. Proc Natl Acad Sci USA 1990;87:4509-13 23. Schwabe JWR, Rhodes D. Beyond zinc fingerssteroid hormone receptors have a novel structural motif for DNA recognition. Trends Biochem Sci

1991;16:291-7 24. Chesters JK, Petrie L, Boyne R, Allen G. The role of zinc in regulating ribosomal RNA synthesis in vivo and in vitro. J Trace Elem Exp Med 1988;l:

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polymerase activity in normal and Zn-deficient developing rat embryos. Proc SOC Exp Biol Med

1978;159:39-43 29. Prasad AS. Zinc deficiency in human subjects. In: Prasad AS, ed. Clinical, biochemical and nutritional aspects of trace elements. New York: A.R. Liss, 19823-62 30. Fujioka M, Lieberman I. A Zn++ requirement for synthesis of deoxyribonucleic acid by rat liver. J Biol Chem 1964;239:1164-7 31. Chesters JK, Petrie L, Travis AJ. A requirement for Zn2+ for the induction of thymidine kinase but not ornithine decarboxylase in 3T3 cells stimulated from quiescence. Biochem J 1990;272:525-7 32. Petrie L, Chesters JK, Franklin M. Inhibition of myoblast differentiation by lack of zinc. Biochem J

1991 ;276:109-11 33. Chen S-Y. Autoradiographic study of cell proliferation in acanthotic buccal epithelium of Zndeficient rabbits. Arch Oral Biol 1986;31:535-9 34. Berg JM, Merkle DL. On the metal ion specificity

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3759-61 35. May PM, Linder PW, Williams DR. Computer simulation of metal-ion equilibria in biofluids: models for the low molecular weight complex distribution of Ca (11) Mg (11) Mn (11) Fe (11) Cu (11) Zn (11) and Pb (11) ions in human blood plasma. J Chem SOC(Dalton) 1977588-95 36. Laybourn PJ, Kadonaga JT. Role of nucleosomal cores and histone-H1 in regulation of transcription by RNA polymerase-ll. Science 1991 ;254:23&

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