Characterization of a family of nuclear and chromosomal proteins identified by a monoclonal antibody.
A monoclonal antibody (3C5) isolated from a mouse immunized with human chromatin stained the nuclei of all cultured cell types tested by indirect immunofluorescence. Experiments with HeLa and PtK1 cells demonstrated striking cell-cycle-related changes in the staining properties of the target antigen. A rapid increase in nuclear fluorescence was seen in prophase, with antigen located between the condensing chromosomes. In metaphase and anaphase cells antigen was present throughout the cytoplasm with the chromosomes apparently unstained. However, isolated metaphase chromosomes showed intense, peripheral staining. In telophase cells immunofluorescent staining was most intense among the decondensing chromosomes and by early G1 staining was predominantly nuclear. Nuclear fluorescence faded as cells progressed through interphase. By protein blotting and immunostaining, 3C5 recognized protein bands with subunit molecular weights of 130, 73, 50, 38, 32 and 22 to 25 kDa. These bands were present in all human and rodent cultured cell types tested. All bands were extracted by 6 M urea or 1% sodium dodecyl sulfate (SDS) but not by Triton X-100. Our results provide evidence against the involvement of a common carbohydrate moiety, in vitro proteolysis or non-specific cross reaction in this multi-banded pattern. The same family of proteins was detected in mitotic and interphase cells, suggesting that the changes in immunofluorescent staining through mitosis are due to changes in antigen accessibility. Subcellular fractionation experiments showed that all major bands were present in the nuclear fraction. Only two (50 and 32 kDa) were detected also in the post-nuclear membrane fraction and none were present in the soluble cytoplasmic fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
B M
BM
Turner
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S
Davies
W G
WG
Whitfield
D000818:Animals; D000911:Antibodies, Monoclonal; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D004173:Dipodomys; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000032:analysis / Q000166:cytology / Q000276:immunology; D006801:Humans; D007962:Leukocytes / Q000032:analysis* / Q000166:cytology / Q000276:immunology; D008677:Metaphase; D051379:Mice; D008807:Mice, Inbred BALB C; D008938:Mitosis; D008970:Molecular Weight; D013194:Staining and Labeling; D013347:Subcellular Fractions / Q000032:analysis
D000911:Antibodies, Monoclonal; D002868:Chromosomal Proteins, Non-Histone