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Telomere-Binding Protein from the Nuclear Envelope of Oocytes of the Frog Rana temporaria

E. A. Bugaeva1 and O. I. Podgornaya1,2

1Institute of Cytology, Russian Academy of Sciences, Tikhoretskii pr. 4, St. Petersburg, 194064 Russia; fax: (812) 520-9703; E-mail: podg@osint.stud.pu.ru

2To whom correspondence should be addressed.

Submitted July 7, 1997.
The identification of telomere-binding protein on the nuclear envelope (NE) of the frog oocyte is reported here. Nuclei and the NE were isolated manually and extracted, and the extracts were used to search for DNA--protein specific interactions. Fragment of the Tetrahymena telomere sequence (T2G4)130 from the plasmid YAC4 were used as a labeled probe. DNA--protein complexes revealed by gel shift assay were cut out of the gel and injected into a guinea pig. The antibodies obtained have common antigenic determinants with keratins; this was shown on Western-blot under conditions of competitive binding. Antibodies were purified on an affinity column with keratins attached and used in the following work. Antibodies recognize one protein with molecular weight 70 kD among the NE proteins; no signal was obtained to proteins isolated from the inner part of the oocyte, but two polypeptides of 70 and 120 kD were detected in the proteins from frog liver nuclei. The 70-kD protein is situated on the NE in a distinct pattern that looks like a network, probably of double-dots as was shown by immunofluorescence of the NE. Electron-microscope immunogold staining showed that the protein is localized in the outer surface of the oocyte NE within cup-like structures attached to the membrane. Combined in situ hybridization using the mammalian telomeric DNA sequence (T2AG3)135 and immunocytochemistry using antibodies showed them to be colocalized in the frog blood cells, so telomere sequence coexists with the protein in the interphase nuclei. Most of the telomeres are fused in highly differentiated blood cells though a double dot signal is visible. The signal appears to be localized in the outer part of the nuclei. The existence of membrane telomere-binding protein allows a discussion of the attachment of the telemeters to the membrane.
KEY WORDS: telomere-binding protein, oocyte, nuclear membrane.