The centrosomal protein centrosomin A and the nuclear protein centrosomin B derive from one gene by post-transcriptional processes involving RNA editing.

The identification of a gene encoding concomitantly a nuclear protein and an intrinsic centrosomal protein further emphasizes the close and presumably developmental relationship between the cell nucleus and the centrosome. Screening of a murine RNA-based cDNA library with an antiserum to a centrosomal protein and rescreening with the insert of an initial clone released two complete cDNAs (1.2 kbp and 2.2 kpb) coding for proteins with notable characteristics. The amino-terminal sections of centrosomin A (276 amino acid residues, molecular mass 34.5 kDa) and of centrosomin B (447 amino acid residues, molecular mass 54.8 kDa) are identical over 272 amino acid residues. The carboxy-terminal section of the larger protein comprises additional 175 amino acid residues including nuclear location signals. The mRNAs encoding centrosomin A and B derive from a single gene. Chromogenomic DNA as template and primer pairs complementary to the sequence which is identical in centrosomin A and B cDNAs results in amplification of only one DNA fragment. Moreover, one exon of the genomic sequence and the centrosomin B-encoding cDNA sequence include a G which is deleted in the centrosomin A-encoding cDNA. Accordingly, the two mRNAs are the products of either alternative splicing or alternative polyadenylation in combination with RNA editing. The recombinantly expressed chimeric protein consisting of centrosomin A and the green fluorescent protein from Aequorea victoria accumulates in centrosomes while the corresponding fusion protein with the centrosomin B sequence is transported into nuclei.

Molecular cloning of a cDNA encoding an antigen which is salt-stably attached to centrosomes.

A monoclonal antibody (MAB 2A8) was used for expression-cloning of a complete cDNA (1133/5) to a mRNA (3 kb) encoding a murine 76 kDa polypeptide. The N-terminal section of the polypeptide is composed of domains capable to form alpha-helical coiled-coils. Its C-terminus is proline-rich and has characteristics of the Src homology region 3 (SH3). Affinity-purified antibodies to a recombinant section of the protein show that the antigen is salt-stably associated with the centrosome throughout the cell cycle.

High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei.

Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller, S.A., Dykes, D.D. and Polesky, H.F., Nucleic Acids Res., 16, 1215, 1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56 degrees C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptides are not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 +/- 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10(-4) M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin-depleted nuclei.

cDNA-derived molecular characteristics and antibodies to a new centrosome-associated and G2/M phase-prevalent protein.

Differential screening of a murine RNA-based cDNA library with cell cycle phase-specific transcripts released a cDNA clone (lambda CCD41) to a mRNA (1.349 kb) which, according to the mode of its detection, increases as expected during the cell cycle. The molecular characteristics of the protein (27 x 10(3) M(r)) encoded by this mRNA were deduced from the cDNA sequence and antibodies were prepared against the recombinant protein. Immunofluorescence studies performed with PtK2 cells revealed that the amount of the antigen specified by the CCD41 sequence increases during the cell cycle out of proportion with the DNA content. In G1 phase cells, the antigen is exclusively located at the site of the centrosome. During cell cycle progression the antigen becomes also detectable in perinuclear vesicles that increase in number and size, reaching a maximum in G2 phase cells. The centrosomal location of the CCD41 antigen was investigated in relation to another centrosomal antigen, centrosomin A. Since the latter antigen is detected by a monoclonal antibody reacting specifically and permanently with the centrosomes in PtK2 cells throughout the cell cycle it was possible to investigate the relative positions of the two proteins at the site of the centrosome and to add new information about the general architecture of the organelle and its changes during the cell cycle. While the centrosomin A antibody detects the pronounced cell cycle stage-dependent shape changes of the centrosome, the CCD41-encoded protein appears to be localized as a compact structure inside the centrosome. Its epitopes are exposed throughout the cell cycle except during a brief period immediately after the formation of the daughter centrosome.

Detection of the 125-kDa nuclear protein mitotin in centrosomes, the poles of the mitotic spindle, and the midbody.

Mitotin is a nuclear protein detectable in all proliferating cells investigated so far, including human and plant cells. In interphase cells the protein is localized mainly in the nucleoplasm. In G2/M phase it displays a characteristic redistribution and a marked increase which initiated the name mitotin. This study presents the precise localization of mitotin in cytoplasmic structures in two cell types, the potoroo rat kangaroo PtK2 cell and the human lung cancer EPLC 65 cell. In addition to its nuclear localization the antigen is detectable in centrosomes, in the poles of the mitotic spindle, and along spindle fibers. During the last mitotic stages, cytokinesis and reconstitution of nuclei, mitotin displays a rapid decrease and another redistribution. A significant amount of the antigen is retained in the bridge connecting the dividing cells, the midbody.

Murine cDNAs coding for the centrosomal antigen centrosomin A.

Screening of an induced Ehrlich ascites cell-derived lambda gt11 cDNA library with an antibody (GP1), immunoreacting specifically with centrosomal antigen(s) of interphase and mitotic cells of different species, released a partial cDNA clone (lambda P10A) encoding the carboxy-terminal section of a centrosome-specific antigen. This specificity of the clone lambda P10A could be verified by lacZ-directed antigen expression from Escherichia coli Y1089 lysogenized with the recombinant phage lambda P10A and subsequent production of centrosome-specific antibodies by means of the recombinant antigen. Using the lambda P10A insert as a probe, two types of cDNA clones were identified in a lambda gt10 cDNA library by plaque-hybridization. The inserts of PN1 type clones were 1.2 kb (kilobases) and those of PN5 type clones were 2.2 kb in length. The DNA sequence of a PN1 type clone revealed its full-length cDNA nature. The open reading frame of PN1 encodes a rather hydrophilic and highly charged 34.5 x 10(3) Mr polypeptide comprising short but apparently significant strings of 100% sequence identity with the major nuclear lamina polypeptides lamins A/C and lamin B. Restriction enzyme mapping of PN1 and PN5 inserts, cross-hybridization experiments and comparison of overlapping DNA sequences indicate that the 1.2 kb and 2.2 kb cDNAs code for the same 34.5 x 10(3) Mr polypeptide, termed centrosomin A. Western blots of Ehrlich ascites cell proteins show a second, larger GP1 antigen (centrosomin B) whose cDNA has not been cloned. It remains to be investigated whether centrosomin B is encoded by a second mRNA or whether it reflects an oligomeric or a postranslationally modified form of centrosomin A.