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D A Compton

Publications and source records attributed to D A Compton.

33 records · Page 2Linked to original sources
PubMed1995

NuMA is required for the organization of microtubules into aster-like mitotic arrays.

NuMA (Nuclear protein that associates with the Mitotic Apparatus) is a 235-kD intranuclear protein that accumulates at the pericentrosomal region of the mitotic spindle in vertebrate cells. To determine if NuMA plays an active role in organizing the microtubules at the polar region of the mitotic spindle, we have developed a cell free system for the assembly of mitotic asters derived from synchronized cultured cells. Mitotic asters assembled in this extract are composed of microtubules arranged in a radial array that contain NuMA concentrated at the central core. The organization of microtubules into asters in this cell free system is dependent on NuMA because immunodepletion of NuMA from the extract results in randomly dispersed microtubules instead of organized mitotic asters, and addition of the purified recombinant NuMA protein to the NuMA-depleted extract fully reconstitutes the organization of the microtubules into mitotic asters. Furthermore, we show that NuMA is phosphorylated upon mitotic aster assembly and that NuMA is only required in the late stages of aster assembly in this cell free system consistent with the temporal accumulation of NuMA at the polar ends of the mitotic spindle in vivo. These results, in combination with the phenotype observed in vivo after the prevention of NuMA from targeting onto the mitotic spindle by antibody microinjection, suggest that NuMA plays a functional role in the organization of the microtubules of the mitotic spindle.

Animals↗
PubMed1995

Mutation of the predicted p34cdc2 phosphorylation sites in NuMA impair the assembly of the mitotic spindle and block mitosis.

NuMA is a 236 kDa intranuclear protein that is distributed into each daughter cell during mitosis through association with the pericentrosomal region of the mitotic spindle. NuMA's interaction with the microtubules of the mitotic spindle is mediated through its 45 kDa carboxyl-terminal globular tail, and there is indirect evidence suggesting that NuMA's interaction with the mitotic spindle is controlled in a mitosis-specific manner. Consistent with this evidence is the fact that all four of the predicted p34cdc2 consensus phosphorylation sites in the NuMA protein are located in the carboxyl-terminal globular domain, and we demonstrate here that NuMA is phosphorylated in a mitosis-specific fashion in vivo. To test if the predicted p34cdc2 phosphorylation sites are necessary for NuMA's mitosis-specific interaction with the mitotic spindle, we have introduced mutations into the human NuMA cDNA that convert these predicted p34cdc2 phosphorylation sites from threonine or serine residues into alanine residues, and subsequently determined the cell cycle-dependent localization of these altered NuMA proteins following their expression in tissue culture cells. While none of these specific mutations in the NuMA sequence alters the faithful targeting of the protein into the interphase nucleus, mutation of threonine residue 2040 alone or in combination with mutations in other potential p34cdc2 phosphorylation sites abolishes NuMA's ability to associate normally with the microtubules of the mitotic spindle. Instead of binding to the mitotic spindle these mutant forms of NuMA concentrate at the plasma membrane of the mitotic cell. Cells expressing these mutant forms of NuMA have disorganized mitotic spindles, fail to complete cytokinesis normally, and assemble micronuclei in the subsequent interphase. These data suggest that NuMA's interaction with the microtubules of the mitotic spindle is controlled by cell cycle-dependent phosphorylation in addition to differential subcellular compartmentalization, and the characteristics of the dominant negative phenotype induced by these mutant forms of NuMA support a role for NuMA in the organization of the mitotic spindle apparatus.

Animals↗
PubMed1994

NuMA, a nuclear protein involved in mitosis and nuclear reformation.

NuMA, a nuclear protein that associates with the mitotic apparatus, was identified in 1980 as a high molecular weight component of the nuclear matrix with the unusual property of associating with the microtubules of the spindle apparatus during mitosis. Over the past two years, a burst of interest in this intriguing protein has led to the clear documentation of its cell cycle redistribution, determination of its primary sequence, elucidation of its cell cycle dependent targeting domains, as well as disruption of its function through antibody microinjection and expression of dominant-negative mutants. Together, these data support a central role for NuMA in both mitotic-spindle dynamics and the reformation of the daughter cell nuclei at the end of mitosis.

Animals↗
PubMed1994

Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove.

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the nuclear matrix. We have previously identified B-type lamins as MAR-binding matrix components (M. E. E. Ludérus, A. de Graaf, E. Mattia, J. L. den Blaauwen, M. A. Grande, L. de Jong, and R. van Driel, Cell 70:949-959, 1992). Here we show that A-type lamins and the structurally related proteins desmin and NuMA also specifically bind MARs in vitro. We studied the interaction between MARs and lamin polymers in molecular detail and found that the interaction is saturable, of high affinity, and evolutionarily conserved. Competition studies revealed the existence of two different types of interaction related to different structural features of MARs: one involving the minor groove of double-stranded MAR DNA and one involving single-stranded regions. We obtained similar results for the interaction of MARs with intact nuclear matrices from rat liver. A model in which the interaction of nuclear matrix proteins with single-stranded MAR regions serves to stabilize the transcriptionally active state of chromatin is discussed.

Animals↗
PubMed1993

NuMA is required for the proper completion of mitosis.

NuMA is a 236-kD intranuclear protein that during mitosis is distributed into each daughter cell by association with the pericentrosomal domain of the spindle apparatus. The NuMA polypeptide consists of globular head and tail domains separated by a discontinuous 1500 amino acid coiled-coil spacer. Expression of human NuMA lacking its globular head domain results in cells that fail to undergo cytokinesis and assemble multiple small nuclei (micronuclei) in the subsequent interphase despite the appropriate localization of the truncated NuMA to both the nucleus and spindle poles. This dominant phenotype is morphologically identical to that of the tsBN2 cell line that carries a temperature-sensitive mutation in the chromatin-binding protein RCC1. At the restrictive temperature, these cells end mitosis without completing cytokinesis followed by micronucleation in the subsequent interphase. We demonstrate that the wild-type NuMA is degraded in the latest mitotic stages in these mutant cells and that NuMA is excluded from the micronuclei that assemble post-mitotically. Elevation of NuMA levels in these mutant cells by forcing the expression of wild-type NuMA is sufficient to restore post-mitotic assembly of a single normal-sized nucleus. Expression of human NuMA lacking its globular tail domain results in NuMA that fails both to target to interphase nuclei and to bind to the mitotic spindle. In the presence of this mutant, cells transit through mitosis normally, but assemble micronuclei in each daughter cell. The sum of these findings demonstrate that NuMA function is required during mitosis for the terminal phases of chromosome separation and/or nuclear reassembly.

Animals↗
PubMed1992

Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis.

From a collection of monoclonal antibodies that specifically bind to various parts of the mitotic apparatus in human cells (1991. J. Cell Biol. 112: 1083-1097), two (1F1 and 1H1) recognize a greater than 200-kD intranuclear protein that associates with the spindle immediately upon nuclear envelope breakdown and progresses down the spindle microtubules to concentrate ultimately at the pericentrosomal region. At the completion of anaphase this protein dissociates from the spindle microtubules and is imported into the regenerating nuclei through the nuclear pores. Overlapping cDNA clones that span the entire length of the corresponding 7.2-kb mRNA reveal an encoded polypeptide of 236,278 D that is predicted to contain two globular domains separated by a discontinuous alpha-helix with characteristics for adopting a coiled-coil structure. The corresponding gene is highly conserved but neither the DNA sequence nor the predicted amino acid sequence shows significant homology to any previously reported. Since the cDNA also encodes the epitopes recognized by antibodies specific for two previously described proteins, NuMA and centrophilin, and all three show similar molecular weights and localization during the cell cycle, NuMA, centrophilin, and the 1F1/1H1 antigen represent either the same protein or a family of proteins, for which the original name, NuMA, seems most appropriate. While the function of NuMA remains uncertain, its unusual pattern of segregation at mitosis defines a novel pathway for the segregation of nuclear proteins during cell division.

Amino Acid Sequence↗
PubMed1991

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.

Anaphase↗
PubMed1991

Rapid verification of identity and content of drug formulations using mid-infrared spectroscopy.

A general method for the rapid verification of both identity and content of complete solid drug formulations has been devised. Infrared spectra for the samples were recorded using the diffuse reflectance technique, and specially written software was employed to identify the type of formulation and level of active ingredient. This software was devised to ensure reliable use when applied by those with minimal operator skills. Three differing drug tablet formulations containing simvastatin, enalapril maleate and lovastatin, as well as a capsule formulation containing finastride were studied. Adequate precision was obtained to reliably verify drug dosage levels. Near-infrared (NIR) and mid-infrared (MIR) spectrometers were evaluated for use with the method. The MIR instrument allowed sufficient resolution and spectral/structural selectivity to reliably verify correctness of either of two near derivative drugs necessarily present in the same clinical study. Drug tablet and capsule dosage levels tested ranged from 0.2 to 40 mg of drug. Approximately 1% (w/w) of the drug in the formulation was the minimum amount determined. Parameters affecting method ruggedness in routine use were optimized. Experimental addition of an extraneous material to a simvastatin formulation was easily detected and flagged by the routine test procedure. Subsequent data retrieval and searching against spectral libraries was used to demonstrate identification of the additive.

Anticholesteremic Agents↗
PubMed1991

Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds.

We describe the generation of 11 monoclonal antibodies that bind to the centromere/kinetochore region of human mitotic chromosomes. These antibodies were raised against mitotic chromosome scaffolds and screened for centromere/kinetochore binding by indirect immunofluorescence against purified chromosomes. Immunoblot analyses with these antibodies revealed that all of the antigens are greater than 200 kD and are components of nuclei, chromosomes, and/or chromosome scaffolds. Comparison of the immunolocalization of the antigens with that observed for the centromere-associated protein CENP-B revealed that each of these centromere/kinetochore proteins lies more peripherally to the DNA than does CENP-B. In cells normally progressing through the cell cycle, these antigens displayed four distinct patterns of centromere/kinetochore association, corresponding to a minimum of four novel centromere/kinetochore-associated proteins.

Animals↗
PubMed1990

A panel of restriction fragment length polymorphisms for chromosomal band 11p13.

A panel of seven chromosome 11p13 restriction fragment length polymorphisms (RFLPs) detected by five DNA probes is described. Two alleles were identified for each polymorphism, and Mendelian segregation of alleles was observed. Allele frequencies range from 0.13/0.87 to 0.44/0.56. This panel of 11p13 RFLPs will be useful for linkage studies investigating the role of 11p13 genes in Wilms' tumor, aniridia, and genitourinary anomalies. Additionally, these RFLPs will be important tools for studying tumor-specific 11p13 alterations in Wilms' tumor and other cancers.

Alleles↗
PubMed1990

Definition of the limits of the Wilms tumor locus on human chromosome 11p13.

In a previous report, we described a contiguous restriction map of chromosome band 11p13 that localized the Wilms tumor locus to a small group of NotI fragments. In an effort to identify and isolate the 11p13-associated sporadic Wilms tumor locus, we developed a panel of NotI fragment-specific DNA probes. These probes were selected from genomic libraries constructed using the Chinese hamster ovary-human somatic cell hybrid carrying only human 11p. The libraries were prepared from NotI-digested DNA after size selection by pulsed-field gel electrophoresis. The selected NotI fragments had been previously targeted on the basis of deletion mapping as having a high probability of containing the Wilms tumor locus. We used these newly identified 11p13-specific probes to improve the resolution of the restriction map spanning the Wilms tumor locus. The locus has been defined by a homozygous deletion in a sporadic Wilms tumor. Using these probes, the region of homozygous deletion in this tumor and presumably all or part of the Wilms tumor gene have been confined to two small SfiI fragments spanning less than 350 kb.

Animals↗
PubMed1988

Long range physical map of the Wilms' tumor-aniridia region on human chromosome 11.

The relationship between genetic alterations at chromosomal band 11p13 and the WAGR (Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome is not clearly understood. To aid our understanding of this relationship, we have constructed a physical map of this region of the genome using pulsed field gel electrophoresis. Fifteen newly identified 11p13-specific probes and four previously reported probes were used to subdivide 11p13 into five intervals defined by overlapping constitutional deletions from several WAGR patients. This new repertoire of DNA probes was used to construct a physical map of this region using the infrequently cutting restriction enzymes MIuI and NotI. This map spans approximately 13 Mb and encompasses deletion and translocation breakpoints associated with genitourinary abnormalities, aniridia, and Wilms' tumor. The map also makes it possible to localize the genes for Wilms' tumor (WT) and aniridia (AN2) to a small number of specific NotI restriction fragments.

Chromosome Mapping↗
PubMed1988

Lack of linkage of familial Wilms' tumour to chromosomal band 11p13.

Wilms' tumour (WT), a paediatric renal neoplasm, affect approximately 1 in 10,000 children. One or both kidneys can be affected and 5-10% of tumours are bilateral. Most tumours occur sporadically; however, around 1% of the cases are familial, with siblings or cousins most often being affected. Familial cases are more frequently bilateral, and familial and bilateral tumours are diagnosed at an earlier age. On the basis of these observations, it was proposed that the development of WT requires two mutations. In most sporadic unilateral WT, both are somatic; in familial and bilateral tumours the first is thought to be germinal. Cytogenetic and molecular studies have demonstrated germinal mutations in WT/aniridia patients and somatic mutations in sporadic WT at chromosomal band 11p13. To investigate whether familial predisposition to WT is due to a germinal 11p13 mutation, we studied a WT family with seen DNA markers that span the 11p13 region. We found that familial WT predisposition was not genetically linked to any of the 11p13 markers. This suggests that the gene involved in familial WT predisposition is outside 11p13 and is distinct from the gene involved in tumorigensis and in WT predisposition in WT/aniridia 11p13-deletion patients.

Chromosomes, Human, Pair 11↗