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Protein disulfide isomerases (PDIs) constitute a family of structurally related enzymes which catalyze disulfide bonds formation, reduction, or isomerization of newly synthesized proteins in the lumen of the endoplasmic reticulum (ER). They act also as chaperones, and are, therefore, part of a quality-control system for the correct folding of the proteins in the same subcellular compartment. While their functions in the ER have been thoroughly studied, much less is known about their roles in non-ER locations, where, however, they have been shown to be involved in important biological processes. At least three proteins of this family from higher vertebrates have been found in unusual locations (i.e., the cell surface, the extracellular space, the cytosol, and the nucleus), reached through an export mechanism which has not yet been understood. In some cases their function in the non-ER location is clearly related to their redox properties, but in most cases their mechanism of action has still to be disclosed, although their propensity to associate with other proteins or even with DNA might be the main factor responsible for their activities.

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57., ((c) 2002 Elsevier Science (USA).)

For the study of in vitro and in vivo DNA-protein interactions, cross-linking reactions driven by UV or formaldehyde have been frequently used, followed by standard protocols of immunoprecipitation and analysis of the DNA isolated from the complexes. Here we present a basically modified method to analyze the DNA-protein cross-linked complexes obtained by an alternative cross-linking reagent. The innovations presented here include cross-linking by cis-diamminedichloroplatinum II, a fast method to isolate DNA-protein complexes using gel-filtration chromatography, and a modified procedure to obtain specific immunocomplexes that can be analyzed either for DNA or for protein content. The application of this method to two nuclear proteins from chicken liver nuclei is described., ((C)2002 Elsevier Science (USA).)

Protein disulfide isomerase ERp57 is localized predominantly in the endoplasmic reticulum, but is also present in the cytosol and, according to preliminary evidence, in the nucleus of avian cells. Conclusive evidence of its nuclear localization and of its interaction with DNA in vivo in mammalian cells is provided here on the basis of DNA-protein cross-linking experiments performed with two different cross-linking agents on viable HeLa and 3T3 cells. Nuclear ERp57 could also be detected by immunofluorescence in HeLa cells, where it showed an intracellular distribution clearly different from that of an homologous protein, located exclusively in the endoplasmic reticulum. Mammalian ERp57 resembles the avian protein in its recognition of S/MAR-like DNA sequences and in its association with the nuclear matrix. It can be hypothesized that ERp57, which is known to associate with other proteins, in particular STAT3 and calreticulin, may contribute to their nuclear import, DNA binding, or other functions that they fulfil inside the nucleus., (Copyright 2002 Wiley-Liss, Inc.)

A microassay was developed to detect human herpesvirus 6 (HHV-6) binding to its cellular receptor using flow cytometry. Comparable results were obtained either by using HHV-6 preparations conjugated with fluorescein isothiocyanate or by indirect immunofluorescent labeling of membrane-bound virus using as primary antibody a monoclonal antibody specific for the HHV-6 gp60/110 envelope glycoprotein. Virus attachment to the plasma membrane was specific and saturable. As expected, among cell lines of various origin, maximum binding was detected on human T-lymphoid cells (HSB-2). Papain digestion of HSB-2 cells prevented HHV-6 attachment and reduced significantly virus infection, indicating the involvement of a protein-based receptor in the attachment step. After removal of the protease, virus receptors were resynthesized and their regeneration was prevented partially by cycloheximide, an inhibitor of protein synthesis. Unexpectedly, only high concentrations (mg/ml) of soluble heparan sulfate and heparin inhibited HHV-6 binding and infection. Under the same conditions, few micrograms (per ml) of heparin suppressed completely herpes simplex type 1 (HSV-1) attachment to the same cell line. Treatment of HSB-2 cells with heparitinase and heparinase, at doses that reduced significantly HSV-1 attachment, had little effect on HHV-6 binding to the cell membrane, indicating a different requirement of heparan sulfate-containing glycosaminoglycans for the two herpesviruses. These data suggest that protein components of the cellular membrane play an essential role in HHV-6 binding and infection while heparan sulfate-glycos-aminoglycans appear to be involved only partially in virus-receptor interaction., (Copyright 2000 Wiley-Liss, Inc.)

DNA-protein cross-linkages were formed in isolated nuclei from immature and mature chicken erythrocytes by reaction with cis-diammine dichloroplatinum. On the basis of electrophoretic behaviour, the most abundant proteins involved in the cross-linking appeared to be present also in preparations of nuclear matrix. The maturation of the erythrocyte, which is accompanied by transcriptional inactivation, leads to a decrease in the amount of DNA-interacting proteins, to a loss of proteins capable of a specific recognition of DNA sequences and, unexpectedly, to the appearence of some new DNA-protein interactions. At least three cross-linked proteins were found predominantly or exclusively in nuclei of immature cells, and three others in those of mature ones. The three DNA-bound proteins, typical of mature erythrocytes, were not found among the components of a high-salt preparation of nuclear matrix. The results obtained suggest that, in addition to the well-known histone H5 and MENT protein, these newly identified DNA-bound proteins contribute to the formation of the condensed, inactive chromatin characteristic of mature erythrocyte.

Eukaryotic nucleoli contain a large family of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs) that are involved in processing and site-specific methylation of pre-rRNA. Several proteins have been reported to be common factors of box C/D snoRNPs in lower and higher eukaryotes; nevertheless none of them has been clearly shown to directly interact with RNA. We previously identified in Xenopus laevis, by means of UV crosslinking in vivo, two proteins associated with box C/D snoRNAs, fibrillarin and p68. Here we show that fibrillarin interacts directly and specifically with the U16 box C/D snoRNA in a X. laevis oocyte nuclear extract and that it does not require p68 for binding. Specific binding is also obtained with a recombinant fibrillarin demonstrating that the protein is able to bind directly and specifically to U16 snoRNA by itself.

Protein ERp60, previously found in the internal nuclear matrix in chicken liver nuclei, is a member of the protein disulfide isomerase family. It binds DNA and double helical polynucleotides in vitro with a preferential recognition toward the matrix-associated regions of DNA and poly(dA) x poly(dT), and its binding is inhibited by distamycin. ERp60 can be cross-linked chemically to DNA in the intact nuclei, suggesting that its association with DNA is present in vivo. As a whole, these results indicate that ERp60 is a component of the subset of nuclear matrix proteins that are responsible for the attachment of DNA to the nuclear matrix and for the formation of DNA loops. A distinctive feature of this protein, which has two thioredoxin-like sites, is that its affinity to poly(dA) x poly(dT) is strongly dependent on its redox state. Only its oxidized form, in fact, does it bind poly(dA) x poly(dT). The hypothesis can be made that through the intervention of ERp60, the redox state of the nucleus influences the formation or the stability of some selected nuclear matrix-DNA interactions.

DNA-protein cross-linkages were produced in intact nuclei of chicken erythrocytes by the action of cis-diammine dichloroplatinum. The telomeric DNA-protein cross-linked complexes were then isolated by hybridization with a biotinylated oligonucleotide and selective binding on immobilized streptavidin. Two main nonhistone proteins were present in the purified complexes, migrating in SDS-gel electrophoresis with apparent molecular masses of 66 and 58 kDa, respectively. Although the identity of these two proteins is still unknown, it is significant that two proteins with similar electrophoretic behavior have been described as constituents of the human telomeric complexes. This procedure could also be applied to the isolation of DNA-protein cross-linked complexes containing any chosen DNA sequence., (Copyright 1999 Academic Press.)

Jointly developed by members of the American Society for Artificial Internal Organs and the Society of Thoracic Surgeons along with staff from the Food and Drug Administration, the National Heart, Lung and Blood Institute and other experts, this recommendation describes the reliability considerations and goals for Investigational Device Exemption and Premarket Approval submissions for long-term, mechanical circulatory support systems. The recommendation includes a definition of system failure, a discussion of an appropriate reliability model, a suggested in vitro reliability test plan, reliability considerations for animal implantation tests, in vitro and animal in vivo performance goals, the qualification of design changes during the Investigational Device Exemption clinical trial, the development of a Failure Modes Effects and Criticality Analysis, and the reliability information for surgeons and patient candidates. The document will be periodically reviewed to assess its timeliness and appropriateness within five years.

Jointly developed by members of the American Society for Artificial Internal Organs and the Society of Thoracic Surgeons along with staff from the Food and Drug Administration, the National Heart, Lung and Blood Institute and other experts, this recommendation describes the reliability considerations and goals for Investigational Device Exemption and Premarket Approval submissions for long-term, mechanical circulatory support systems. The recommendation includes a definition of system failure, a discussion of an appropriate reliability model, a suggested in vitro reliability test plan, reliability considerations for animal implantation tests, in vitro and animal in vivo performance goals, the qualification of design changes during the Investigational Device Exemption clinical trial, the development of a Failure Modes Effects and Criticality Analysis, and the reliability information for surgeons and patient candidates. The document will be periodically reviewed to assess its timeliness and appropriateness within five years.

DNA-protein cross-linkages were formed in intact nuclei of chicken erythrocytes and liver cells by the action of cis-diammine dichloroplatinum (II). Most cross-linked proteins were components of the nuclear matrix, and their heterogeneity reflected the different complexity of liver and erythrocytes matrices, respectively. Some basic proteins, including histones, were also cross-linked, particularly in erythrocyte nuclei. South-Western blotting revealed that a variety of proteins isolated from the cross-linked liver nuclei recognized DNA specifically. In this group of proteins two relatively abundant, acidic, species of 38 and 66 kDa, respectively, might represent novel DNA-binding proteins from the nuclear matrix. In the case of erythrocytes, only the basic proteins showed a DNA-recognition capacity, and among them there were some unidentified species, absent from liver. Lamin B2 was cross-linked but was unable to recognize DNA, and the same was true for other abundant, cross-linked proteins from both types of nuclei. This led to the hypothesis that for some DNA-nuclear matrix interactions the aggregation typical of matrix proteins is essential for the specificity of DNA recognition. Hybridization analysis of the DNA isolated from the cross-linked complexes showed that SARs (scaffold attachment regions) and telomeric sequences were well represented in the cross-linked fragments, that the cross-linked DNA of liver was partially different from that of erythrocytes and that two defined SAR sequences were found to be present only in the cross-linked DNA. These results are in agreement with the present views on DNA-nuclear matrix interactions, which are usually studied on isolated nuclear matrices or purified proteins. Instead, our results provide experimental evidence obtained directly from intact nuclei.

Fractionation of internal matrix proteins from chicken liver nuclei led to the isolation of a 32 kDa protein which was identified by partial amino acid sequence and immunological analysis as annexin V, an unreported nuclear matrix component. Our results showed that this protein is preferentially associated with the internal nuclear matrix fraction, since this is the only nuclear fraction where the protein can be immunodetected. Immunostaining on cultured cells also revealed a nuclear distribution with the exclusion of the nucleolar compartment and an association with cytosolic filamentous structures most likely corresponding to the cytoskeleton. Moreover, immunostaining on extracted cells to reveal the nuclear matrix showed a network-like distribution. Since annexin V has been reported as an inhibitor of protein kinase C, its nuclear localization in association with the internal matrix, which plays an important role in several nuclear processes, indicates its involvement in the regulation of signal transduction.

A 60-kDa protein was purified from chicken liver internal nuclear matrix and its nuclear localization was confirmed by immunofluorescence analysis. Structural information acquired from sequence analysis of the intact protein and of fragments obtained from enzymatic and chemical cleavages strongly suggests that it belongs to the carboxylesterases family, even if with some very peculiar features. The N-terminal sequence of the 60-kDa protein is completely different from the other carboxylesterases, but is similar to a region that is normally internal to all mammalian esterase sequences and localized after the serine residue at the active site. This suggests that the protein may be derived from a gene duplication and/or rearrangement. Since the 60-kDa protein shows a low esterase activity of about 0.2 micromol x min(-1) x mg(-1) using either p-nitrophenyl acetate or p-nitrophenyl butyrate as substrates, it is not possible to rule out that the protein shares only a sequence similarity with carboxylesterases and is not a true esterase. Otherwise it could be an esterase which has developed different properties, i.e. a special substrate specificity, the requirement of additional factors or a different stability in solution. In the latter case, this protein could be related to the physiological control of hydrolysis of exogenous and endogenous esters which can act on nuclear functions and/or metabolism.

To detect the interactions of DNA with the nuclear matrix proteins, DNA-protein cross-linkages were induced in intact nuclei from chicken liver by the use of cis-diammine dichloroplatinum. Methods have been devised for fast purification both of the proteins and of the DNA fragments involved in the cross-linked complexes. By Southern-Western blotting a number of matrix proteins isolated from the complexes have been shown to recognize specifically DNA sequences present in the cross-linked DNA fragments. This experimental approach not only allows to identify the nuclear matrix-DNA interactions existing in the nucleus before its disruption, but also provides a preparation of matrix proteins enriched in those species which are involved in such interactions and which can therefore be detected with high sensitivity.

DNA in cell nuclei is organized in large loops, which are formed by the binding of DNA to proteins present in a nuclear structure called matrix or scaffold. There is ample evidence that these interactions have not only a structural role, but also a functional one. Studies of these interactions have so far been carried out mainly in vitro. Their relevance, therefore, for the in vivo situation is not proven. We have analyzed the DNA-protein interactions directly in the intact nucleus by means of cross-linking reactions. Cross-linking by UV irradiation and by cis-diamine dichloro platinum (II) have been performed on nuclei from chicken liver and compared. The platinum complex has been found to be more efficient. The proteins complexed to DNA have been isolated and analyzed, and have been found to be enriched in species present in the nuclear scaffold, independently from its method of preparation.

The presence of glycoproteins and sugar-binding sites in the nucleus is well ascertained. In order to verify the existence of specific nuclear protein-carbohydrate interactions, oligosaccharides were released from nuclear or non-nuclear glycoproteins by Peptide-N-glycosidase F, labeled by reduction with NaB3H4 and added to whole chicken liver nuclei and to subnuclear insoluble fractions (nucleoli, nuclear matrix and nuclear envelope). The analysis of the binding of the oligosaccharides to the nuclear fractions, performed in the presence or absence of competitor sugars, suggest that some rare oligosaccharides species, present only among the nuclear carbohydrates, are specifically recognized by the nuclear matrix.

The nuclear matrix from chicken liver cells contains a small amount of glycoproteins recognized by Concanavalin A. These proteins are present not only in the peripheral matrix, but also in the internal one. In this latter localization many glycoprotein species appear, by cross-linking experiments, to be placed in the proximity of DNA. The effect of a partial enzymatic deglycosylation of matrix preparations suggests that these proteins contribute to the stabilization of the native matrix structure.

Proteins of the internal nuclear matrix from chicken liver were fractionated, by chromatographic procedures, in non denaturing conditions. At least two fractions were present with phosphatidylinositol-specific phospholipase C and three with thiol:protein-disulfide oxidoreductase activity. A 57kDa protein was isolated which copurified with both these activities. Partial amino acid sequences showed a high degree of homology with a cytosolic protein previously identified as a phospholipase C and with a microsomal protein identified as a thiol:protein-disulfide oxidoreductase. Our finding leaves the question still unanswered of the real function of this protein, which for the first time has been isolated from the nuclear matrix.

It has been observed that double stranded DNA in solution is thermally destabilized by the presence of several organic molecules, among which are alcohols and polyols. This finding, and the fact that sugar moieties are components of some nuclear proteins and DNA binding drugs, may suggest a role for oligosaccharides in DNA recognition and/or interaction. In order to investigate this possibility, the effect of several sugars and other polyols on the thermal stability and on the conformation of DNA has been studied by high sensitivity differential scanning calorimetry and circular dichroism. While addition of small size polyols does not influence appreciably the melting enthalpy, it always results in a decrease in the DNA melting temperature. The magnitude of the destabilizing effect depends on the concentration of additives, reaching 13.5 degrees C in 50% (5.4M) glycerol. It also depends on the nature of the additives, e.g., sorbitol is more efficient than inositol, while dextran has no effect on the DNA melting temperature. According to circular dichroism results, DNA undergoes a significant structural change in the presence of small sugars or polyols: a continuous loss in 'B' character is observed as the glycerol concentration is gradually increased. This conformation change appears to be related to the decrease in thermostability.

A nuclear glycoprotein with an apparent Mr of 66,000 Da has been isolated from pig kidney chromatin after extraction with urea, guanidine-HCl and 2 M NaCl, and some of its structural features have been characterized. It belongs to the group of N-glycosylated proteins, which in the nucleus has so far received little attention. From its monosaccharide composition and recognition by lectins its oligosaccharides appear to be of high mannose and/or hybrid types. Some properties of its protein moiety suggest that it has a role in the packing of the DNA loops in the condensed chromatin.

Investigation of structural features of native chromatin requires the use of intact nuclei, a turbid material which cannot be analyzed by optical methods. Differential scanning calorimetry does not require optically clear samples and has been proved by a number of authors to be a powerful tool in this field of study. By this technique, chicken erythrocyte nuclei were found to undergo at least four thermal transitions, centered at 59, 74, 88 and 98 degrees C. The highest temperature transition is strongly dependent on age and storage conditions of the nuclei. Adequate storage conditions overcame this problem and reproducible scans were obtained over a period of several months. This technical improvement has permitted the reconsideration of the occurrence of the fourth calorimetric transition, previously believed to be displayed only in replicating nuclei. Evidence gathered in the presence of perturbants and possible ligands allows the assignment of the four transitions to a nuclear protein scaffold, histones, nucleosomal DNA and a superstructured form of DNA. Moreover, it suggests that the higher-order structure is stabilized by fibronectin-like proteins.

In order to detect the nuclear matrix proteins involved in DNA binding, avoiding possible artifacts derived from the disruption of nuclei, proteins were crosslinked to DNA by the action of cis-diamminedichloroplatinum on intact chicken liver cells and analyzed by two-dimensional gel electrophoresis. At least eleven species of crosslinked proteins were found to derive from the nuclear matrix prepared from the same cell type, and five of these were found also among the proteins crosslinked to DNA in intact liver cells from ox and pig. This subset of common proteins, conserved in different animal species, is likely to have a fundamental role for the anchorage of DNA to the nuclear matrix.

The protein-DNA crosslinking capability of cis-dichloro diammineplatinum has been exploited to check the intranuclear location of N-glycosylated proteins. When intact liver cells were treated with this reagent, a number of glycoproteins, recognized by Concanavalin A, have been shown to become crosslinked to DNA; many of them have been recognized as nuclear matrix components. The recognition by this lectin was abolished by treatment with N-glycosidase F, showing the presence of N-glycosidic bonds between the sugar moiety and the protein. Most of the glycoproteins appeared to have high mannose oligosaccharide chains, but sialic acid containing oligosaccharides were also identified.

Nuclear glycoproteins recognized by Concanavalin A have been isolated from pig, rabbit and chicken tissues. Mono and bidimensional electrophoresis patterns of proteins loosely and tightly bound to DNA have been examined. The tissue specificity rather than species-specificity appears to be a quite general property of these proteins, suggesting for them a role in the mechanism of regulation of chromatin functions.

We have investigated the distribution of tissue-specific tightly bound non-histone proteins in the first and third levels of chromatin organization. The proteins of this class have been extracted from whole chromatin and chromatin fractions prepared from pig liver or kidney. The tissue-specific proteins have high molecular mass (ranging from 135 KDa to 70 KDa in liver, over 135 KDa in kidney) and in kidney a more basic isoelectric point. These proteins are mainly located outside the core particles, and are instead present only in the chromatin matrix, become more intense after extensive digestion of the matrix with DNAase I.

Microcalorimetric titrations of whole chromatin and histones with sodium dodecylsulfate were performed at pH 7 and 25 degrees C. Enthalpy variations at low detergent concentration (less than 0.02%) are much more negative for histones than for chromatin. At 0.065% sodium dodecylsulfate the difference between the two curves becomes constant and, after correction for monomerization effects, amounts to +130 kcal/mol of nucleosomal unit. Core particles show heat effects similar to those of histones. These findings suggest that the chromatin structure is not stabilized exclusively by electrostatic interactions and that hydrogen bonds responsible for the additional stability may be contributed by non histone chromatin proteins.

Non-histone proteins, tightly bound to DNA, have been extracted from whole chromatin and core particles prepared from pig liver or kidney. We have investigated by bidimensional slab gel electrophoresis the distribution of this protein class in the first level of repeating structure of chromatin. Our results reveal that non-histone proteins tightly bound to DNA are a heterogeneous protein class. Some of them, particularly in the core particles, appear to be essentially the same in both tissues, though having differences in their isoelectric point, which may be attributed to postsynthetic modifications. We have calculated that this protein class is associated to only 10% of nucleosomes, these nucleosomes having, on the average, one protein molecule for each core DNA. The tissue-specific proteins have high molecular mass (ranging from 135 kDa to 70 kDa in liver, over 135 kDa in kidney) and, in kidney, a more basic isoelectric point. These proteins are mainly located outside the core particles; they could be situated in the spacer regions and/or be involved in determining higher levels of chromatin organization.

The precursor to rat liver mitochondrial aspartate aminotransferase has been expressed in Escherichia coli JM105 using the pKK233-2 expression vector. This mammalian natural precursor has been isolated as a soluble dimeric protein. The amino-terminal sequence and the amino acid composition of the isolated protein correspond to those predicted from the inserted cDNA (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). The isolated precursor contains bound pyridoxal phosphate and shows catalytic activity with a specific activity equal to that of the mature form of the enzyme. This precursor can also be processed by mitochondria into a form with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of mature enzyme. The isolation of this precursor as a stable and catalytically active entity indicates that the presequence peptide does not necessarily interfere with much of the folding and basic structural properties of the mature protein component.

The tissue specificity of a particular class of non-histone chromatin proteins characterized by strong bonds to DNA, tightly bound non-histone chromatin proteins (t.b. NHCp), was investigated. In connection with its possible role in the cell differentiation, its distribution in chromatin fractions obtained by digestion with DNAase II and precipitation with MgCl2 was studied. It was shown by bidimensional gel electrophoresis that some proteins of this class are tissue specific for pig liver compared to kidney chromatin. For both tissues, most tissue specific proteins were found to be peculiar to the chromatin fraction that remains insoluble after prolonged digestion with DNAase II. The proteins found in this chromatin fraction are common to the nuclear matrix proteins and, considering the chromatin organization, may be involved in the attachment point for loops in the axial matrix structure. It is possible that proteins of this class, directly or indirectly, play an important role in the regulation of transcription and differentiation in eukaryotes, by modulating the supercoiled state of DNA.

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.

Total half-cystine residues in proteins of pig liver chromatin have been measured. About half of them are present in the reduced state. Thiol groups of non-histone chromatin proteins, which amount to about 40 nmol/mg of protein, are preferentially located in chromatin fragments which are more easily solubilised either by DNAse I or by DNAse II. The data obtained are compatible with an involvement of SH and SS groups in chromatin structure and function.

Core particles prepared by micrococcal nuclease digestion of pig liver chromatin have been adsorbed on hydroxyapatite and dissociated by gradual increase in ionic strength and finally by urea and guanidine. By this method non-histone proteins have been found to be associated with the core particles. Proteins tightly bound to the core particle DNA (i.e. dissociated only by urea and guanidine) have also been found: these are proteins with a limited heterogeneity, with respect to their molecular weights, since only six components are present with molecular weights ranging from 71000 to 20000. They show, furthermore, a peculiar amino acid composition. Other tightly bound proteins have been shown to be present only in the spacer regions. The existence of two different classes of tightly bound proteins probably reflects different modes of binding to the DNA, which are compatible or incompatible, respectively, with the simultaneous binding of the histone octamer.

Chromatin glycoproteins recognized by Concanavalin A have been isolated from pig liver, kidney and heart by the use of immobilized lectin. Two groups of proteins differing in affinity for DNA have been analysed. Glycoproteins are mainly present in the group of proteins which are tightly bound to DNA. Mono and bidimensional electrophoretic patterns of total tightly bound proteins reveal a similarity among the three organs examined, while the corresponding patterns of the glycoproteins are typical for each organ. The tissue specificity of chromatin glycoproteins, together with their capability to interact not only with DNA but possibly also with other nuclear components, suggest a role for these proteins in the mechanism of genome expression.