Chromosome-associated Proteins Research Articles

Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation.

Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg extracts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, we show here that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. We propose that RCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.

Identification of ZFR, an ancient and highly conserved murine chromosome-associated zinc finger protein.

In a screen for RNA binding proteins expressed during murine spermatogenesis, we cloned a novel, ancient zinc finger protein possessing a region common to a small class of RNA binding proteins. Zfr (zinc finger RNA binding) encodes a protein of 1052 amino acids with three widely spaced Cys2His2 zinc fingers. Outside of the zinc fingers, ZFR shares a region that is highly conserved between several RNA binding proteins containing copies of the double-stranded RNA binding motif. By northern blotting, Zfr is expressed at highest levels within the testis, ovary and brain. Immunohistochemistry and confocal microscopy were used to show that ZFR is highly expressed during meiosis I in males and females and is chromosome associated. Zfr is also expressed in Sertoli cells in the testis and granulosa cells in the ovary where it is localized to the nucleus. Using fluorescent in situ hybridization we mapped Zfr to chromosome 15 region A. ZFR appears to be an ancient protein, as apparent homologs exist in invertebrates (D. melanogaster) nematodes (C. elegans) and humans (H. sapiens).

Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics.

The structural maintenance of chromosomes (SMC) family member proteins previously were shown to play a critical role in mitotic chromosome condensation and segregation in yeast and Xenopus. Other family members were demonstrated to be required for DNA repair in yeast and mammals. Although several different SMC proteins were identified in different organisms, little is known about the SMC proteins in humans. Here, we report the identification of four human SMC proteins that form two distinct heterodimeric complexes in the cell, the human chromosome-associated protein (hCAP)-C and hCAP-E protein complex (hCAP-C/hCAP-E), and the human SMC1 (hSMC1) and hSMC3 protein complex (hSMC1/hSMC3). The hCAP-C/hCAP-E complex is the human ortholog of the Xenopus chromosome-associated protein (XCAP)-C/XCAP-E complex required for mitotic chromosome condensation. We found that a second complex, hSMC1/hSMC3, is required for metaphase progression in mitotic cells. Punctate vs. diffuse distribution patterns of the hCAP-C/hCAP-E and hSMC1/hSMC3 complexes in the interphase nucleus indicate independent behaviors of the two complexes during the cell cycle. These results suggest that two distinct classes of SMC protein complexes are involved in different aspects of mitotic chromosome organization in human cells.

Molecular cloning of metaphase chromosome protein 1 (MCP1), a novel human autoantigen that associates with condensed chromosomes during mitosis.

Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human chromosome-associated proteins. Previous immunolocalization studies in human and murine tissue culture cells showed that some of these monoclonal antibodies recognize nuclear antigens that associate with condensed chromosomes during mitosis. One antibody was selected for screening a human HeLa S3 cDNA expression library, and cDNAs that code for an antigen of 31-33 kDa were isolated. Immunological, biochemical and cell fractionation data indicate that the 31- to 33-kDa antigen corresponds to the chromosome-associated protein recognized by the original monoclonal antibody. Sequence analysis shows that we isolated a novel human gene. Immunolocalization to human tissue culture cells shows that during interphase the antigen is dispersed in the nucleus and that during mitosis it associates exclusively with condensed chromosomes. A similar pattern of localization was also observed in mouse fibroblasts, suggesting that the antigen is conserved among different species. Finally, we show that part of the antigen remains bound to the scaffold/matrix component, even after high salt extraction.

A chromatin-associated kinesin-related protein required for normal mitotic chromosome segregation in Drosophila.

The tiovivo (tio) gene of Drosophila encodes a kinesin-related protein, KLP38B, that colocalizes with condensed chromatin during cell division. Wild-type function of the tio gene product KLP38B is required for normal chromosome segregation during mitosis. Mitotic cells in tio larval brains displayed circular mitotic figures, increased ploidy, and abnormal anaphase figures. KLP38B mRNA is maternally provided and expressed in cells about to undergo division. We propose that KLP38B, perhaps redundantly with other chromosome-associated microtubule motor proteins, contributes to interactions between chromosome arms and microtubules important for establishing bipolar attachment of chromosomes and assembly of stable bipolar spindles.

Chromatid Segregation at Anaphase Requires the barren Product, a Novel Chromosome-Associated Protein That Interacts with Topoisomerase II

We have isolated a Drosophila gene, barren ( barr), required for sister-chromatid segregation in mitosis. barr encodes a novel protein that is present in proliferating cells and has homologs in yeast and human. Mitotic defects in barr embryos become apparent during cycle 16, resulting in a loss of PNS and CNS neurons. Centromeres move apart at the metaphase–anaphase transition and Cyclin B is degraded, but sister chromatids remain connected, resulting in chromatin bridging. This phenotype is similar to that described in TOP2 mutants in yeast. Barren protein localizes to chromatin throughout mitosis. Colocalization and biochemical experiments indicate that Barren associates with Topoisomerase II throughout mitosis and alters the activity of Topoisomerase II. We propose that this association is required for proper chromosomal segregation by facilitating the decatenation of chromatids at anaphase.

Preparation of insect chromosomes for immunolabeling.

We describe a method for isolating chromosomes from testes of the desert locust, Schistocerca gregaria, and their subsequent incubation with antibodies directed against chromosomal proteins. The procedure involves hypotonic pretreatment of the germ cells, centrifugation onto coverslips in a cytocentrifuge and immunolabeling, while still unfixed, using a chromatin-stabilizing buffer. In the present case, an antibody specific for the acetylated isoforms of histone H4 was tested. After the antibody treatment, the preparations are fixed using formaldehyde, stained with a DNA-specific fluorescent dye and mounted. Analysis of the preparations revealed good preservation of chromosome structure in prophase spermatogonia and late prophase I spermatocytes. Fully condensed chromosomes were not observed and are probably lost during preparation. The bright fluorescence of the autosomes indicates that the reaction between the antibody against acetylated histone H4 and its chromosomal antigen is not impeded. In contrast, the X univalent remained unstained with the exception of a small terminal band. Thus, cytospin preparations of locust germ cells allow high resolution immunolabeling with antibodies against chromosome-associated proteins.

Isolation of chromosome-associated proteins from Drosophila melanogaster that bind a human centromeric DNA sequence.

The molecular mechanism involved in packaging centromeric heterochromatin is still poorly understood. CENP-B, a centromeric protein present in human cells, is though to be involved in this process. This is a DNA-binding protein that localizes to the central domain of the centromere of human and mouse chromosomes due to its association with the 17-bp CENP-B box sequence. We have designed a biochemical approach to search for functional homologues of CENP-B in Drosophila melanogaster. This strategy relies upon the use of DNA fragments containing the CENP-B box to identify proteins that specifically bind this sequence. Three polypeptides were isolated by nuclear protein extraction, followed by sequential ion exchange columns and DNA affinity chromatography. All three proteins are present in the complex formed after gel retardation with the human alphoid satellite DNA that contains the CENP-B box. Footprinting analysis reveals that the complex occupies both strands of the CENP-B box, although it is still unclear which of the polypeptides actually makes contact with the DNA. Localization of fluorescein-labeled proteins after microinjection into early Drosophila embryos shows that they associate with condensed chromosomes. Immunostaining of embryos with a polyclonal serum made against all three polypeptides also shows chromosomal localization throughout mitosis. During metaphase and anaphase the antigens appear to localize preferentially to centromeric heterochromatin. Immunostaining of neuroblasts chromosome spreads confirmed these results, though some staining of chromosomal arms is also observed. The data strongly suggests that the polypeptides we have identified are chromosomal binding proteins that accumulate mainly at the centromeric heterochromatin. Furthermore, DNA binding assays clearly indicate that they have a high specific affinity for the human CENP-B box. This would suggest that at least one of the three proteins isolated might be a functional homologue of the human CENP-B.

Genetic effects from exposure to hazardous agents.

Mammalian germ cell stages exhibit differences in DNA synthesis activity, capability to repair DNA damage, and chromosome-associated proteins. The sensitivity to mutation induction may be influenced by such factors as the accessibility of DNA to chemical mutagens, the interval between DNA damage induction and the next round of DNA replication, and the repair of DNA damage. Such qualitative and quantitative differences indicate the complexities of mutation induction in vivo and emphasize that no single in vitro test system can adequately represent the in vivo situation. Therefore, germ-cell mutagenesis in humans can most adequately be represented by an in vivo mammalian germ-cell test system. Information regarding the mechanisms of mutation induction in germ cells of the mouse, appropriate mutation test systems available in the mouse, as well as principles of chemical mutagenesis in the mouse and their implications for an adequate human genetic risk estimation will be discussed.

Mutants of the microtubule motor protein, nonclaret disjunctional, affect spindle structure and chromosome movement in meiosis and mitosis.

The Drosophila microtubule motor protein, nonclaret disjunctional (ncd), is required for proper chromosome distribution in meiosis and mitosis. We have examined the meiotic and mitotic divisions in wild-type Drosophila oocytes and early embryos, and the effects of three ncd mutants (cand, ncd and ncdD) on spindle structure and chromosome movement. The ncd mutants cause abnormalities in spindle structure early in meiosis I, and abnormal chromosome configurations throughout meiosis I and II. Defective divisions continue in early embryos of the motor null mutant, cand, with abnormal early mitotic spindles. The effects of mutants on spindle structure suggest that ncd is required for proper meiotic spindle assembly, and may play a role in forming or maintaining spindle poles in meiosis. The disruption of normal meiotic and mitotic chromosome distribution by ncd mutants can be attributed to its role as a spindle motor, although a role for ncd as a chromosome-associated motor protein is not excluded. The ncd motor protein functions not only in meiosis, but also performs an active role in the early mitotic divisions of the embryo.

Association of ubiquitin-activating enzyme with HeLa cell chromosomes during mitosis.

Ubiquitin-activating enzyme (E1) is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. Antibodies raised against E1 were affinity purified and used for immunostaining HeLa cells. Condensed chromosomes in mitotic cells were found to be strongly immunoreactive. Chromosomes from metaphase-arrested HeLa cells were isolated and chromosome-associated proteins were analyzed by Western blotting. E1 was detected in fractions containing isolated chromosomes. These results suggest that E1 is associated with condensed chromosomes during mitosis.

Interaction of a folded chromosome-associated protein with single-stranded DNA-binding protein of Escherichia coli, identified by affinity chromatography.

A single-stranded DNA-binding protein (SSB) affinity column was prepared by optimizing the coupling of Escherichia coli single-stranded DNA-binding protein to Affi-Gel 10. The bound SSB retained its ability to specifically bind single-stranded DNA. When nuclease-treated cell extracts were incubated with the SSB beads overnight at 4 degrees C, a major protein of Mr = 25,000 was bound. At shorter incubation times, two additional proteins of Mr = 32,000 and 36,000 were also detected. In the absence of nuclease treatment, eight additional proteins ranging from Mr = 14,000 to 160,000 also bound to the affinity column. The major Mr = 25,000 protein has been shown to be a folded chromosome-associated protein. Its binding to SSB is strongly enhanced by the addition of DNA polymerase III or DNA polymerase III holoenzyme.

A conserved family of nuclear proteins containing structural elements of the finger protein encoded by Krüppel, a Drosophila segmentation gene

Krüppel (Kr), a segmentation gene of Drosophila, encodes a protein sharing structural features of the DNA-binding “finger motif” of TFIIIA, a Xenopus transcription factor. Low-stringency hybridization of the Kr finger coding sequence revealed multiple copies of homologous DNA sequences in the genomes of Drosophila and other eukaryotes. Molecular analysis of one Kr-homologous DNA clone identified a developmentally regulated gene. Its product, a finger protein, relates to Kr by the invariant positioning of crucial amino acid residues within the finger repeats and by a stretch of seven amino acids connecting the finger loops, the “H/C link.” This H/C link is conserved in several nuclear and chromosome-associated proteins of Drosophila and other eukaryotic organisms including mammals. Our results demonstrate a new subfamily of evolutionarily conserved nuclear and possibly DNA-binding proteins that again relate to a Drosophila segmentation gene as in the case of the homeo domain.

Effects of 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor

The phorbol ester tumor promotor 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (125I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent Ki of 11-26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of 3H-choline and 32 P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth fact (NGF) which, in contrast to the former agents, is a differentiating stimulus for PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of 125I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.

Two-dimensional electrophoretic analyses of proteins synthesized during differentiation of 3T3-L1 preadipocytes.

The changes in protein composition and cell surface proteins that occur during the adipocyte conversion of 3T3-L1 preadipocytes were monitored by two-dimensional polyacrylamide gel electrophoresis folowing incubation of cells with [35S]methionine for periods of 3 and 24 h. Alterations in the biosynthesis of more than 30 cytoplasmic proteins, 9 non-histone, chromosome-associated proteins, and 24 membrane proteins, were detected. Although the methodological limitations of the electrophoretic systems employed result in an underestimate of the total number of differences, the alterations observed exceed the enzyme changes known to occur during differentiation of these cells. One major alteration occurring during differentiation is a decrease in the content of a protein whose position following two-dimensional electrophoresis tentatively identified it as actin. A fall in actin content accompanying adipocyte conversion was confirmed by direct analysis of the DNase 1 inhibitory activity in homogenates prepared from cells during the course of differentiation. Studies of cell surface proteins by lactoperoxidase-catalyzed iodination reveal a number of changes during differentiation including an increase in a polypeptide(s) in the molecular weight range of 16,500 to 18,500, a decrease in at least four proteins of molecular weights greater than 100,000, and in a protein of molecular weight 95,000.

Effects of administration of thioacetamide on chromosome-associated proteins in rat liver

This report deals with the isolation and analysis of liver cell nuclei from rats receiving thioacetamide daily. After 11 days of treatment, there is a decrease in the bound saline-soluble protein, no change in the phosphate-soluble protein, and a substantial increase in the histone fraction, these protein fractions being extracted in that order from the isolated nuclei. There is also an increase in the residue protein remaining after these extractions.

Effects of administration of thioacetamide or testosterone on chromosome-associated proteins in rat liver and kidney

This report deals with the isolation and analysis of liver cell nuclei from rats receiving thioacetamide daily, and of kidney cell nuclei from rats in which the testosterone balance had been altered in various ways. The results indicate that in the enlargement of liver cell nuclei produced by thioacetamide administration, there is a large increase in nuclear RNA and a large increase in “residue” protein. In the enlargement of kidney cell nuclei produced by testosterone administration, there is no increase in nuclear RNA, and there is a slight decrease in “residue” protein per milligram of DNA. Neither treatment produced any quantitative change in the capacity of the chromosomes to bind the “soluble” (saline-extractable) proteins. The values for nuclear RNA, “residue” protein, and “soluble” protein per milligram of DNA are all considerably higher for liver cell nuclei than the corresponding values for kidney cell nuclei.