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.     

Subcellular localization of Bcl-2 protein in synovial sarcoma

The subcellular localization of Bcl-2 protein in surgically resected, fixed-frozen tissue specimens of seven tumors from six cases of synovial sarcoma and a synovial sarcoma cell line was examined using laser-scanning confocal microscopy and immunoelectron microscopy. Bcl-2 protein in synovial sarcoma cells was detected in the nuclear envelope, endoplasmic reticulum membrane, and mitochondrial circumference. Based on the finding of pre-embedding immunoelectron-microscopy observation, the distribution of Bcl-2 protein in such membranous organella was patchy. A computer-based image analysis failed to reveal any quantitative differences between the inner and the outer mitochondrial membranes. The tumorigenesis of synovial sarcoma is also discussed from the viewpoint of Bcl-2 overexpression.     

Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis

Zinc is an essential nutrient for all cells, but remarkably little is known regarding bacterial zinc transport and its regulation. We have identified three of the key components acting to maintain zinc homeostasis in Bacillus subtilis. Zur is a metalloregulatory protein related to the ferric uptake repressor (Fur) family of regulators and is required for the zinc-specific repression of two operons implicated in zinc uptake, yciC and ycdHIyceA. A zur mutant overexpresses the 45-kDa YciC membrane protein, and purified Zur binds specifically, and in a zinc-responsive manner, to an operator site overlapping the yciC control region. A similar operator precedes the ycdH-containing operon, which encodes an ABC transporter. Two lines of evidence suggest that the ycdH operon encodes a high-affinity zinc transporter whereas YciC may function as part of a lower-affinity pathway. First, a ycdH mutant is impaired in growth in low-zinc medium, and this growth defect is exacerbated by the additional presence of a yciC mutation. Second, mutation of ycdH, but not yciC, alters the regulation of both the yciC and ycdH operons such that much higher levels of exogenous zinc are required for repression. We conclude that Zur is a Fur-like repressor that controls the expression of two zinc homeostasis operons in response to zinc. Thus, Fur-like regulators control zinc homeostasis in addition to their previously characterized roles in regulating iron homeostasis, acid tolerance responses, and oxidative stress functions.     

A fixed distance for separation of newly replicated copies of oriC in Bacillus subtilis: implications for co-ordination of chromosome segregation and cell division

The Spo0J protein of Bacillus subtilis is required for normal chromosome segregation and forms discrete subcellular assemblies closely associated with the oriC region of the chromosome. Here we show that duplication of Spo0J foci occurs early in the DNA replication cycle and that this requires the initiation of DNA replication at oriC but not elongation beyond the nearby STer sites. Soon after duplication, sister oriC/Spo0J foci move rapidly apart to achieve a fixed separation of about 0.7 microm, reminiscent of the segregation of eukaryotic chromosomes on the mitotic spindle. The magnitude of the fixed separation distance may explain how chromosome segregation is kept in close register with cell growth and the initiation mass for DNA replication. It could also explain how segregation can proceed accurately in the absence of cell division. The kinetics of focal separation suggest that one role of Spo0J protein may be to facilitate formation of separate sister oriC complexes that can be segregated.     

Polar localization of the MinD protein of Bacillus subtilis and its role in selection of the mid-cell division site

Cell division in rod-shaped bacteria is initiated by formation of a ring of the tubulin-like protein FtsZ at mid-cell. Division site selection is controlled by a conserved division inhibitor MinCD, which prevents aberrant division at the cell poles. The Bacillus subtilis DivIVA protein controls the topological specificity of MinCD action. Here we show that DivIVA is targeted to division sites late in their assembly, after some MinCD-sensitive step requiring FtsZ and other division proteins has been passed. DivIVA then recruits MinD to the division sites preventing another division from taking place near the newly formed cell poles. Sequestration of MinD to the poles also releases the next mid-cell sites for division. Remarkably, this mechanism of DivIVA action is completely different from that of the equivalent protein MinE of Escherichia coli, even though both systems operate via the same division inhibitor MinCD.     

Ndj1p, a meiotic telomere protein required for normal chromosome synapsis and segregation in yeast

The Saccharomyces cerevisiae gene NDJ1 (nondisjunction) encodes a protein that accumulates at telomeres during meiotic prophase. Deletion of NDJ1 (ndj1Delta) caused nondisjunction, impaired distributive segregation of linear chromosomes, and disordered the distribution of telomeric Rap1p, but it did not affect distributive segregation of circular plasmids. Induction of meiotic recombination and the extent of crossing-over were largely normal in ndj1Delta cells, but formation of axial elements and synapsis were delayed. Thus, Ndj1p may stabilize homologous DNA interactions at telomeres, and possibly at other sites, and it is required for a telomere activity in distributive segregation.     

An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport

The azlB locus of Bacillus subtilis was defined previously by a mutation conferring resistance to a leucine analog, 4-azaleucine (J. B. Ward, Jr., and S. A. Zahler, J. Bacteriol. 116:727-735, 1973). In this report, azlB is shown to be the first gene of an operon apparently involved in branched-chain amino acid transport. The product of the azlB gene is an Lrp-like protein that negatively regulates expression of the azlBCDEF operon. Resistance to 4-azaleucine in azlB mutants is due to overproduction of AzlC and AzlD, two novel hydrophobic proteins.     

Subcellular distribution and phosphorylation of the nuclear localization signal binding protein, NBP60

We previously purified a nuclear localization signal binding protein, NBP60, from rat liver (1993, J. Biochem. 113, 308-313). In this study, the subcellular localization of NBP60 was examined using anti-NBP60. Most NBP60 was found to be localized in the nuclear envelope fraction of rat liver obtained on cell fractionation followed by immunoblotting. Staining of the nuclei of cultured cells by the antibody was observed on immunofluorescence microscopy. NBP60 was widely detected in rat nuclear fractions prepared from other tissues and also in nuclei of cultured cells derived from other species. It was shown by immunoelectron microscopy that most NBP60 is present in the nuclear envelope and at least some of that is present on nuclear pore complexes. Although NBP60 was localized in the nuclear envelope in interphase cells, it diffused into the cytoplasm in the mitotic phase. The purified NBP60 was highly phosphorylated by a cdc2 mitotic kinase, whereas nuclear pore proteins p144, p62, p60, and p54 were not phosphorylated by the kinase directly. NBP60 was also phosphorylated by protein kinase A, calmodulin-dependent protein kinase II, and casein kinase II. The phosphorylation of NBP60 by cdc2 kinase and/or the other kinases may be related to the change in the protein's location during the mitotic phase.     

Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement

Shigella flexneri uses elements of the host cell cytoskeleton to move within cells and from cell to cell. IcsA, an S. flexneri protein involved in this movement, was purified and studied in vitro. IcsA bound the radiolabelled ATP analog 3'(2')-O-(4-benzoyl)benzoyl-ATP and hydrolyzed ATP. In addition, the surface localization of IcsA on both extracellular and intracellular shigellae was unipolar. Further, in HeLa cells infected with shigellae, IcsA antiserum labelled the actin tail throughout its length, thereby suggesting that IcsA interacts with elements within the tail. Localization of IcsA within the tail at a distance from the bacterium would require its secretion; we demonstrate here that in vitro IcsA is secreted into the culture supernatant in a cleaved form.     

SinI modulates the activity of SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction

SinR, a 111-amino-acid DNA-binding protein, is a pleiotropic regulator of several late growth processes in Bacillus subtilis. It acts as a developmental switch, positively regulating genes for competence and motility and repressing aprE and stage II sporulation genes. It is encoded by the second gene in a two gene operon, but previous results have also indicated that these two genes are differently regulated. We show in this discussion that the product of sinI, the first open reading frame (ORF) of this operon, interferes with the function of SinR. In vivo experiments have demonstrated that overexpression of sinI results in phenotypes that are observed in cells with a null mutation of sinR. A chromosomal in-frame deletion of sinI gives rise to a phenotype associated with higher levels of SinR. Thus, SinI acts as an antagonist to SinR. In vitro experiments have shown that the interaction between these two proteins is a direct one. SinI prevents SinR from binding to its target sequence on aprE, and the two proteins form a complex that can be immunoprecipitated with antibodies to either SinR or SinI.     

Subcellular localization of lead in synaptosomes

The neurotoxicology of lead is reported to involve effects on cholinergic and monoaminergic pathways. However, no information is available on the site of action at which lead exerts neurotoxic effects. The technique of x-ray elemental analysis coupled scanning-transmission mode electron microscopy has been applied to study the in vitro interactions of lead with synaptosomes. To minimize possible translocation effects on elemental distribution, isolated synaptosomes were air dried on EM grids; no fixing or staining procedures were used. In such preparations, synaptosomes and intrasynaptosomal mitochondria were distinguishable. Lead was found only in mitochondria in association with Ca ad P. Thus, lead appears to enter isolated nerve terminals and to be sequestered in mitochondria.     

comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases

The family has increasingly been recognized as an important component in the development, maintenance, and treatment of alcoholism. Few empirical studies, however, have examined alcoholism within a family context. Questionnaire and interview data were collected from women whose husbands received inpatient treatment for alcoholism. Since wives now typically work outside the home, this article focuses on the 60 employed wives. Employment was examined as a source of stress as well as social support. The majority of working wives reported minimal negative impact of their husbands' drinking on all areas of their work functioning, with a small subset indicating impairment attributable to the drinking. These wives were very satisfied with their current positions and described work as a positive experience. However, unobtrusive measures that alcoholism in a family member intrudes into the workplace were apparent, including changing jobs, absenteeism, and discussing husbands' drinking at work. Further, these women scored closer to a sample of depressed women than a community sample on measures of physical and mental health, depressed mood, and smoking symptoms. Possible reasons for the discrepancy between subjective reports and objective indicators are discussed.     

First steps from a two-dimensional protein index towards a response-regulation map for Bacillus subtilis

Data on the identification of proteins of Bacillus subtilis on two-dimensional (2-D) gels as well as their regulation are summarized and the identification of 56 protein spots is included. The pattern of proteins synthesized in Bacillus subtilis during exponential growth, during starvation for glucose or phosphate, or after the imposition of stresses like heat shock, salt- and ethanol stress as well as oxidative stress was analyzed. N-terminal sequencing of protein spots allowed the identification of 93 proteins on 2-D gels, which are required for the synthesis of amino acids and nucleotides, the generation of ATP, for glycolyses, the pentose phosphate cycle, the citric acid cycle as well as for adaptation to a variety of stress conditions. A computer-aided analysis of the 2-D gels was used to monitor the synthesis profile of more than 130 protein spots. Proteins performing housekeeping functions during exponential growth displayed a reduced synthesis rate during stress and starvation, whereas spots induced during stress and starvation were classified as specific stress proteins induced by a single stimulus or a group of related stimuli, or as general stress proteins induced by a variety of entirely different stimuli. The analysis of mutants in global regulators was initiated in order to establish a response regulation map for B. subtilis. These investigations demonstrated that the alternative sigma factor sigma B is involved in the regulation of almost all of the general stress proteins and that the phoPR two-component system is required for the induction of a large part but not all of the proteins induced by phosphate starvation.     

Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillus actinomycetemcomitans

The subcellular locations, ultrastructure, and cytotoxic activity of the GroEL-like protein from Actinobacillus actinomycetemcomitans were investigated. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that synthesis of the GroEL-like protein is substantially increased after a thermal shock. Analysis of the purified native GroEL-like protein by transmission electron microscopy revealed the typical 14-mer cylindrical molecule, which had a diameter of about 12 nm. A. actinomycetemcomitans cells grown at 35 degreesC and heat shocked at 43 degreesC were fractionated, and fractions were separated by SDS-PAGE and analyzed by Western immunoblotting using antibodies to GroEL- and DnaK-like proteins. The GroEL-like protein was found in both the soluble and membrane fractions, whereas the DnaK-like protein was mostly found in the cytoplasm. An increase in specific proteins, including the GroEL- and DnaK-like proteins, was found in heat-shocked cells. The subcellular localization of the GroEL-like protein was examined by immunoelectron microscopy of whole cells. More GroEL-like protein was detected in stressed cells than in unstressed cells, and most of it was found not directly associated with outer membranes but rather in extracellular material. The native GroEL-like protein was assessed for cytotoxic activities. The GroEL-like protein increased the proliferation of periodontal ligament epithelial cells at concentrations between 0.4 and 1.0 microgram/ml. The number of cells in the culture decreased significantly at higher concentrations. A cell viability assay using HaCaT epithelial cells indicated that the GroEL-like protein was strongly toxic for the cells. These studies suggest the extracellular nature of the GroEL-like protein and its putative role in disease initiation.     

Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k[cat]/Km of 35000 M[-1] s[-1]), alpha-N-succinylthreonylprolyllysine 4-nitroanilide (k[cat]/Km of 18000 M[-1] s[-1]) and alpha-N-serylprolyllysine 4-nitroanilide (k[cat]/Km of 2600 m[-1] s[-1]), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 microM; k[cat] of 1.4 s[-1]; k[cat]/Km of 16000 M[-1] s[-1]). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.     

Modulation of the spc operon affects growth and protein secretion in Bacillus subtilis

A proximal segment of B. subtilis secY gene was placed under the control of the inducible spac promoter/Lac repressor system. This fusion was integrated into the chromosomal spc operon of B. subtilis via Campbell-like reciprocal recombination. The growth of the resulting strain was strongly IPTG dependent. With staphylokinase and alpha-amylase as reporter proteins it was found, that the protein secretion capacity of this strain was correlated to the conditions of repression or induction of the chromosomal spac promoter.