Studies of Epstein-Barr Virus (EBV)-associated Nuclear Antigen: Solubilization from Raji Cell Chromatin with 5 M-Urea-2 M-NaCl and Fractionation on Hydroxyapatite

Brown, T. D. K.; Rickwood, D.; MacGillivray, A.J.; Klein, George

doi:10.1099/0022-1317-40-3-511

Cited by 4 publications

(2 citation statements)

References 15 publications

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“…In these studies the CP-3 protein fraction was isolated from hen oviduct chromatin with column chromatography using chromatin-hydroxylapatite resin. This method was derived from previous reports on the fractionation of chromatin proteins using hydroxylapatite and chaotropic agents (Bluthmann et al, 1975;Bloom & Anderson, 1978; Brown et al, 1978, Kilianska et al, 1980. The distinction of the method presented here was the mode of fractionating the proteins from the resin.…”

Section: Methodsmentioning

confidence: 99%

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

Spelsberg¹,

Gosse

Littlefield

et al. 1984

Biochemistry

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A specific fraction of avian oviduct chromosomal proteins can be reannealed to pure avian DNA to reconstitute nativelike specific nuclear binding sites (acceptor sites) for the oviduct progesterone receptor (PR). These specific nuclear binding sites represent the difference between the binding to the reconstituted NAP and that to pure DNA. The specific fraction of chromatin protein which contains the acceptor activity, fraction CP-3, is very tightly bound to hen DNA in a complex termed nucleoacidic protein (NAP). Removal of the CP-3 fraction from NAP results in a loss of specific PR binding sites. Resins containing chromatin adsorbed to hydroxylapatite are used as a rapid method to isolate the CP-3 fraction. Reconstitution of the CP-3 fraction to DNA by the described method involving a regressing gradient of 6-0 M guanidine hydrochloride (Gdn-HCl) results in a reconstituted NAP which displays specific PR binding sites identical with those in native (undissociated) NAP and whole chromatin. Optimal conditions and potential problems for reconstituting these nucleoproteins are described. Only partially purified receptor preparations were used in these cell-free binding analyses since they have been shown to bind with similar properties and patterns as the nuclear binding in vivo. Therefore, the binding of PR to the reconstituted NAPs was demonstrated to be receptor dependent, saturable, and of high affinity. Further, the pattern of binding to the reconstituted sites mimics those which are observed in vivo. Thus, nonfunctional receptors that cannot translocate and bind to the nuclear acceptor sites in vivo also failed to bind to the acceptor sites on the reconstituted NAPs generated by the acceptor proteins. In contrast, the binding to pure DNA does not reflect these receptor differences in receptor bindings. Specific binding of PR to reconstituted NAP can be reversed by again removing the protein fraction. Moreover, the specific binding can be destroyed by proteases and protected by protease inhibitors, indicating that acceptor activity is proteinaceous in nature. The reconstitution of the activity is both a concentration-dependent and time-dependent process. During the reconstitution, acceptor activity appears to reconstitute on the DNA when the Gdn-HCl concentration reaches 2.0 M. By use of the reconstitution method as an assay for acceptor activity, the activity in the CP-3 fraction was shown by molecular sieve chromatography to elute in a relatively broad molecular weight range between 13 000 and 25 000. The activity also focuses in isoelectric focusing resins with apparent pI's of 5.2 and 6.4.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Methodsmentioning

confidence: 99%

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

Spelsberg¹,

Gosse

Littlefield

et al. 1984

Biochemistry

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“…Chromatin isolation. Nuclei were first isolated by the technique of Brown et al (5), with minor modifications. The chromatin from these purified nuclei was prepared by the procedure of Spelsberg et al (32,33,35), with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

Evidence for two classes of chromatin-associated Epstein-Barr virus-determined nuclear antigen

Spelsberg¹,

Sculley

Pikler

et al. 1982

J Virol

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A new class of Epstein-Barr virus nuclear antigen (EBNA) was identified by the complement fixation assay. This new species of EBNA is more tightly bound to chromatin and was termed class II EBNA, as opposed to the more weakly associated species, class I EBNA. Preparations of this new antigen(s) specifically reduced absorption with the titer of anti-EBNA antibodies as determined by the anticomplement immunofluorescence assay. Therefore, the complement fixation antigens (class II EBNA) appear to be related to the classical EBNA (class I EBNA). The class I EBNA was found to focus at the same pH (4.6) as the soluble antigen found in the cytosol. The class II EBNA differed from the class I EBNA with regard to its overall charge, molecular size, antigenicity, and affinity for chromatin. The class II EBNA appeared to be a basic protein, based on its apparent pl of 9.2 and its binding to cation-exchange resins. It differed from histones with regard to its molecular size (molecular weight between 60,000 and 70,000) and its elution from hydroxylapatite chromatography. Steps were taken to prevent proteolysis and artifacts in the immunological assays and in the overall charge estimation of the new antigen by nonspecific basic histone protein-acidic protein interactions. Both class I and class II EBNA were identified by radioimmunoelectrophoresis on two-dimensional polyacrylamide gels with pI values of 5.0 and 8.5, respectively, and a molecular weight range of 60,000 to 70,000 for both. A lower-molecular-weight antigen identified by molecular sieve chromatography appeared to be due to interference by histones in the immunoassays since it was not observed by the two-dimensional gel electrophoresis. Further characterization of this class II EBNA is in progress.

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Immunology of Epstein—Barr Virus

Henle

1982

The Herpesviruses

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Studies of Epstein-Barr Virus (EBV)-associated Nuclear Antigen: Solubilization from Raji Cell Chromatin with 5 M-Urea-2 M-NaCl and Fractionation on Hydroxyapatite

Cited by 4 publications

References 15 publications

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

Evidence for two classes of chromatin-associated Epstein-Barr virus-determined nuclear antigen

Immunology of Epstein—Barr Virus

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