Identification of a phosphoprotein in the nuclear matrix by monoclonal antibodies

Halikowski, M. J.; Liew, Choong‐Chin

doi:10.1042/bj2410693

Cited by 8 publications

(6 citation statements)

References 33 publications

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“…Halikowski and Liew (51) have also identified a presumptive chromatin protein(s) termed B2 that migrated in the same general position as this basic cluster. B2, which is also a highly phosphorylated protein, was shown to be also associated with the nuclear matrix (52).…”

Section: Discussionmentioning

confidence: 99%

Nuclear matrins: identification of the major nuclear matrix proteins.

Nakayasu

Berezney

1991

Proc. Natl. Acad. Sci. U.S.A.

182

115

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A preparative two-dimensional polyacrylamide gel system was used to separate and purify the majorCoomassie blue-stained proteins from the isolated rat liver nuclear matrix. Approximately 12 major proteins were consistently found. Of these, 5 proteins represented identified proteins, including nuclear lamins A, B, and C, the nucleolar protein B-23, and residual components of core heterogeneous nuclear ribonucleoproteins. The remaining eight major proteins termed the nuclear matrins consisted of matrin 3 (125 kDa, slightly acidic), matrin 4 (105 kDa, basic), matrins D-G (60-75 kDa, basic), and matrins 12 and 13 (42-48 kDa, acidic). Peptide mapping and two-dimensional immunoblot studies indicate that matrins D-G compose two pairs of related proteins (matrins D/E and F/G) and that none of the matrins resemble the nuclear lamins or any of the other major proteins detected on our two-dimensional gels. Subfractionation immunoblot experiments demonstrated the nearly exclusive localization of matrins F/G and other matrins to the nuclear matrix fraction of the cell. These results were further supported by indirect immunofluorescence microscopy that showed a strictly interior nuclear localization of the matrins in intact cells in contrast to the peripherally located nuclear lamins. We conclude that the nuclear matrins are a major class of proteins of the nuclear matrix interior and are distinct from the nuclear lamins.There is a growing awareness that many of the important answers to questions concerning the expression and regulation of the eukaryotic genome will require an understanding of the higher-order arrangement and function of the genetic components as they interact within the complex threedimensional architecture of the cell nucleus (1-13). The nuclear matrix, a residual nuclear structure that has been isolated from a wide variety of eukaryotic cells throughout the phylogenetic scale (1-3, 6, 7, 13-16), offers a potentially valuable in vitro approach for studying nuclear processes in relation to nuclear structure. Indeed, numerous studies have implicated the matrix as a site of organization for virtually all known nuclear processes (1-11, 13, 16), such as, DNA loop attachment, DNA replication, transcription, RNA splicing and transport, hormone receptor function, carcinogen binding, oncogene proteins, viral proteins, and protein phosphorylation. Despite this progress, our knowledge of the proteins that compose this proteinaceous nucleoskeletal structure is still in its infancy. In this study we have used high-resolution preparative PAGE to identify and purify many of the major Coomassie blue-stained nuclear matrix proteins. A class of nuclear matrix proteins termed the nuclear matrins is identified and characterized by peptide maps, polyclonal antibodies generated against the individual purified matrins, and indirect immunofluorescence microscopy.

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Section: Discussionmentioning

confidence: 99%

Nuclear matrins: identification of the major nuclear matrix proteins.

Nakayasu

Berezney

1991

Proc. Natl. Acad. Sci. U.S.A.

182

115

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“…Monoclonal antibodies have been produced to this protein and have been used in Western blot analyses. We have also shown that the antigen is present in the nuclear matrix [12]. This nuclear antigen has a molucular weight of 68 kDa and exhibits heterogeneity due to a p1 ranging from pH 6.5 to 8.2.…”

Section: Discussionmentioning

confidence: 86%

A nuclear protein associated with actively transcribed nucleosomes exhibits Zn²⁺‐binding activity

Liew

Chen

1989

FEBS Letters

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The presence of the zinc-finger motif in nuclear protein structure often confers specific DNA-binding capabilities and, consequently, may implicate such proteins as being involved in gene regulation. We measured the zinc-binding capacity of nuclear proteins by using a zinc (Z$+) blotting technique. This assay revealed that some non-histone proteins are selectively associated with this metal ion. Of particular interest, a nuclear protein, B, (Mr 68000; p1 7.3), associated with actively transcribed nucleosomes and possibly involved with premessenger splicing, exhibits Znz'-binding activity. Moreover, this activity can be achieved with as little as 2 pg of nuclear protein and the zinc-binding capacity is, on average, 2 ions per molecule of the protein B,.Nuclear phosphoprotein complex; Binding domain, Zn2+

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“…Western blotting conditions were also described previously [8,15] and utilized '251-protein A and autoradiographic detection system. …”

Section: Methodsmentioning

confidence: 99%

“…Characterization of monoclonal antibodies and the radioimmunoassay were as described previously [7]. Western blotting conditions were also described previously [8,15] and utilized '251-protein A and autoradiographic detection system.…”

Section: Methodsmentioning

confidence: 99%

“…A nuclear phosphoprotein has been previously identified within operationally defined transcriptionally-active chromatin and shown to become more abundant following 16 h of liver regeneration [4-61. This purified protein has been used for preparing monoclonal antibodies [7]. Using these antibodies for two-dimensional PAGE Western blotting, this phosphoprotein was also identified among proteins which comprise the operationally defined nuclear matrix fraction [8]. Moreover, electron microscopy of immunogoldlabeled thin sections revealed a preponderance of this nuclear protein in the vicinity of the nuclear envelope [9].…”

Section: Introductionmentioning

confidence: 99%

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Immunological evidence for the role of phosphoprotein p68/pI=7.3 in premessenger RNA splicing

Liew

Smith

1989

FEBS Letters

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A 68 kDa (pl= 7.3) nuclear phosphoprotein has been previously characterized as a component of transcriptionally active chromatin. Two-dimensional PAGE Western blotting and radioimmunoassay with monoclonal antibodies have identified this protein in nuclear extracts used for in vitro RNA splicing. In vitro splicing activity could be quantitatively inhibited by preincubating nuclear extracts with the antibodies, but the assembly of 60 S spliceosomes could not.

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Identification of a phosphoprotein in the nuclear matrix by monoclonal antibodies

Cited by 8 publications

References 33 publications

Nuclear matrins: identification of the major nuclear matrix proteins.

Nuclear matrins: identification of the major nuclear matrix proteins.

A nuclear protein associated with actively transcribed nucleosomes exhibits Zn²⁺‐binding activity

Immunological evidence for the role of phosphoprotein p68/pI=7.3 in premessenger RNA splicing

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Identification of a phosphoprotein in the nuclear matrix by monoclonal antibodies

Cited by 8 publications

References 33 publications

Nuclear matrins: identification of the major nuclear matrix proteins.

Nuclear matrins: identification of the major nuclear matrix proteins.

A nuclear protein associated with actively transcribed nucleosomes exhibits Zn2+‐binding activity

Immunological evidence for the role of phosphoprotein p68/pI=7.3 in premessenger RNA splicing

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A nuclear protein associated with actively transcribed nucleosomes exhibits Zn²⁺‐binding activity