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PubMed1990

Estrogen regulates the synthesis of epidermal growth factor in mouse uterine epithelial cells.

Immunocytochemical analyses, using several mouse epidermal growth factor (EGF) polyclonal antibodies, detected immunoreactivity only in uterine luminal and glandular epithelia on late proestrus, estrus, and early on day 1 of pregnancy, but not late on day 1. This immunoreactivity was not detected in the ovariectomized uterus, but after estrogen stimulation it was detected first in the luminal epithelium between 12-24 h and then also in the glandular epithelium by 48 h. After 72 h of estrogen withdrawal, EGF immunoreactivity was no longer detected. This response was specific for estrogen and did not occur after progesterone injection (2 mg/day for 4 days). Using antipeptide antibodies specific for prepro-EGF, no immunoreactivity was detected in the ovariectomized uterus, weak reactivity was detected in the estrogenized uterus and submandibular gland, and strong reactivity was detected in the kidney. Northern blot analysis of uterine RNA failed to detect the expected 4.8-kilobase prepro-EGF mRNA, but, instead, a rare transcript of 2.4 kilobases was detected, which suggests that EGF mRNA is alternately processed in the uterus. The presence of an EGF-coding uterine transcript was further documented by hybridization of an EGF-coding region-specific oligodeoxyribonucleotide (oligo) to polymerase chain reaction-amplified uterine cDNA. In situ hybridization, using a prepro-EGF cRNA probe as well as an EGF-coding region-specific oligo, showed hybridization that colocalized with the EGF immunostaining (epithelia) and was absent from non-EGF-immunoreactive cells. Pulse labeling experiments coupled with immunoaffinity chromatography showed that estrogen induced an increase in the relative rate of synthesis of an acid-soluble immunoreactive protein which was the same size as authentic EGF. Furthermore, analysis of acid-soluble uterine proteins fractionated by DEAE-cellulose chromatography demonstrated a single coincident peak of antigenic activity and receptor-binding activity which coeluted from the column with authentic EGF. Electron microscopy localized EGF immunoreactivity to the Golgi of luminal epithelial cells. Taken together these results suggest that estrogen regulates expression of the EGF gene specifically in uterine epithelial cells. Increased expression of this gene results in an increase in the relative rate of synthesis of this protein and the accumulation of mature EGF.

Animals↗
PubMed2006

TRPC3 channels colocalize with Na+/Ca2+ exchanger and Na+ pump in axial component of transverse-axial tubular system of rat ventricle.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable, nonselective cation channels activated after stimulation of G protein-coupled membrane receptors linked to phospholipase C (PLC). Although the PLC/inositol phosphate signaling pathway is known to exist in heart, expression and subcellular distribution of TRPC channel proteins in ventricular myocardium have not been evaluated. Of the six members of the TRPC channel family examined here, only TRPC3 was found by Western blot analysis of membrane proteins from rodent or canine ventricle. Likewise, only TRPC3 was observed in immunofluorescence analysis of thin sections from rat ventricle. TRPC3 was also the only family member observed in neonatal rat ventricular myocytes in culture. In longitudinal sections of rat ventricle, TRPC3 was predominantly localized to the intercalated disk region of the myocyte. However, transverse sections through heart muscle or single isolated adult myocytes revealed TRPC3-specific labeling in a vast network of intracellular membranes, where it colocalized with the Na(+)-K(+)-ATPase (NKA) pump and the Na(+)/Ca(2+) exchanger (NCX) but not with the ryanodine receptor or the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Reciprocal immunoprecipitation assays from rat or canine ventricle showed that TRPC3 associates with NKA and NCX but not with the plasmalemmal Ca(2+)-ATPase pump. Immunoprecipitations from Sf9 insect cells heterologously expressing TRPC3, NKA, and NCX in various combinations revealed that NKA and NCX interact and that TRPC3 and NCX interact, but that TRPC3 does not directly associate with NKA. Together, these results suggest that TRPC3 is localized in the ventricular myocyte to the axial component of the transverse-axial tubular system, where it exists in a signaling complex that includes NCX and NKA.

Animals↗
PubMed1996

Stereological analysis of galanin and CGRP synapses in the dorsal horn of neuropathic primates.

The present study investigates the nature of the galanin (GAL) increase and the calcitonin gene-related peptide (CGRP) decrease in the dorsal horn following peripheral nerve injury. These two peptides are known to colocalize in primary afferent terminals. Primates which had a tight ligation of the L7 spinal nerve demonstrated a variety of neuropathic symptoms 2 weeks postsurgery, including mechanical and cold allodynia, and heat hyperalgesia. Computer-enhanced image analyses of L7 spinal cord sections demonstrated an increase in GAL immunostaining and a decrease in CGRP immunostaining in the experimental compared to the control dorsal horn. Stereological analyses demonstrated that neither the numbers of GAL-labeled synapses nor the numbers or diameters of the dense-core vesicles in each GAL terminal changed after the lesion. However, there was a significant increase in the number of GAL-labeled glial cell bodies and processes on the experimental side, which accounted for the increased staining density observed at the light microscopic level. In contrast, the number of CGRP-labeled terminals was decreased on the experimental side, accounting for the decreased staining density seen at the light level. Thus, the decrease in number of CGRP synapses combined with the stable number of GAL synapses suggests that many GAL terminals no longer colocalize with CGRP after peripheral nerve lesion. This may indicate increased antinociceptive activity after nerve lesions. If so, there is less of a morphologic and more of a functional and chemical plasticity for GAL than may be presently envisioned. The possible role of GAL in neuropathic pain is discussed.

Animals↗
PubMed1996

Synaptic distribution of the AMPA-GluR2 subunit and its colocalization with calcium-binding proteins in rat cerebral cortex: an immunohistochemical study using a GluR2-specific monoclonal antibody.

Due to its role as the dominant AMPA receptor subunit in respect to regulation of calcium permeability, information on the neuronal localization of GluR2 is of particular importance, yet has been hampered by the lack of a GluR2-specific antibody. Monoclonal antibodies were raised against the putative N-terminal portion (amino acids 175--430) of GluR2, using the fusion protein linked to trpE as an antigen. Western blot analysis and immunocytochemistry of transiently transfected human embryonic kidney 293 cells unambiguously confirmed the specificity of monoclonal antibody 6C4 for GluR2, which did not recognize or cross-react with any other AMPA/Kainate GluR subunits expressed. 6C4 was used in immunohistochemical studies to characterize the regional, cellular, and subcellular distribution of the GluR2 subunit at the light and electron microscopic levels in rat hippocampus and somatosensory cortex and in colocalization studies with the three calcium-binding proteins: parvalbumin, calbindin, and calretinin. GluR2 was widely distributed in both pyramidal cells and interneurons. Asymmetric synapses were labeled on both spines and small dendritic shafts. In contrast to previous reports, our double labeling studies using monoclonal antibody 6C4 with polyclonal antisera against calcium-binding proteins demonstrated that 84--97% of parvalbumin and calbindin-immunoreactive and 45--66% of the calretinin-immunoreactive interneurons in CA1 and somatosensory cortex also contain GluR2. These data have important implications regarding heterogeneity in calcium permeability of AMPA receptors across cell types in neocortex and hippocampus, as well as for differential vulnerability to excitotoxic injury.

Animals↗
PubMed2006

Impaired recognition of apoptotic neutrophils by the C1q/calreticulin and CD91 pathway in systemic lupus erythematosus.

OBJECTIVE: A deficiency in a subcomponent of C1q can result in increased susceptibility to autoimmune diseases such as systemic lupus erythematosus (SLE). The monocyte endocytic receptor CD91 is implicated in the endocytosis of apoptotic neutrophils via interactions with C1q and calreticulin. In this clinical study, we studied the binding of C1q to leukocytes and determined whether C1q bound specifically to calreticulin and CD91 on cells undergoing apoptosis in SLE. METHODS: Proximal antibody phage display, calreticulin-transfected cells, and immunocytochemical and confocal techniques were used in a comprehensive analysis of direct binding of C1q to apoptotic neutrophils that were obtained from healthy individuals and from patients with SLE. In addition, apoptotic cellular systems were assessed in vitro. RESULTS: C1q appeared to colocalize to apoptotic blebs on the surface of leukocytes in association with both calreticulin and CD91, as determined by phage display and transfected cell studies. However, C1q did not bind to apoptotic cells isolated from SLE patients, despite the positivity of the cells for both calreticulin and CD91. Surface expression of calreticulin decreased on neutrophils as they aged, but increased on monocytes. In an apoptotic phagocytic assay, the addition of C1q and calreticulin significantly enhanced the phagocytosis of apoptotic cell debris by monocyte-derived cells. CONCLUSION: These observations indicate that neutrophils from SLE patients have a reduced ability to be recognized and removed by the C1q/calreticulin/CD91-mediated apoptotic pathway, despite the presence of main apoptotic recognition partners. This suggests that an additional component, as yet unidentified, acts as a C1q binding partner on apoptotic cells, and this component may be lacking in cells isolated from SLE patients.

Adolescent↗
PubMed1993

Differential regulation of substance P and somatostatin in Martinotti cells of the developing cat visual cortex.

In order to determine their morphological development and ontogenetic fate, Martinotti neurons immunoreactive for substance P and somatostatin have been analysed in the cat visual cortex. Martinotti neurons are located in layers V and VI. They are multipolar to bitufted, and most dendrites remain in layers V and VI. Their typical features is the ascending axon, which emerges from an apical dendrite or from the upper pole of the soma. A number of collaterals branch off in layer V, forming a local terminal plexus. The axon then branches into 2-8 collaterals, which ascend as a bundle to layers III and II, where a second terminal plexus is formed. Some collaterals ascend to layer I where they adopt a horizontal course. Horizontal collaterals in the terminal layers V, III, II, and in layer I may reach up to 400 microns in length. Martinotti neurons begin to differentiate perinatally. The quantitative analysis reveals that the initial time course of differentiation of Martinotti cells is very similar in material stained for substance P and for somatostatin. Double immunofluorescence then confirms that the two peptides are colocalized in Martinotti cells of layers V and VI during the early postnatal period. Further, substance P is colocalized with GABA. Substance P expression in Martinotti cells can be observed only in the immature visual cortex. After postnatal day 15, the Martinotti neuron system becomes less and less detectable by substance P immunoreactivity. It declines to virtually undetectable levels after the third postnatal month. The adult visual cortex is almost devoid of substance P-immunoreactive cell bodies, processes and axon terminals. In situ hybridization confirms this finding, revealing beta-preprotachykinin mRNA-expressing cell bodies in layers V and IV at postnatal day (P)6 and P12, but not in the adult cortex. This suggests a downregulation of the substance P expression at the transcriptional level. In contrast, somatostatin-immunoreactive Martinotti cells, most of which have coexpressed substance P during early postnatal life, can still be observed in the adult cortex. Thus, the Martinotti neurons constitute a persisting cell type, although many individual neurons of this type disappear during the second postnatal month by degeneration and cell death. In summary, while somatostatin is permanently expressed in Martinotti neurons in the cat visual cortex, substance P peptide and mRNA are transiently expressed during an early postnatal period, and apparently are downregulated later in development.

Afferent Pathways↗
PubMed2001

Two distinct populations of tumor necrosis factor-stimulated gene-6 protein in the extracellular matrix of expanded mouse cumulus cell-oocyte complexes.

After the luteinizing hormone surge, the cumulus cell-oocyte complexes (COCs) in the preovulatory follicles produce a viscoelastic extracellular matrix, a process that requires the synthesis of hyaluronan as well as the incorporation of some components of the inter-alpha-trypsin inhibitor (IalphaI) family. In this study we report, that a hyaluronan-binding protein, the translated product of tumor necrosis factor-stimulated gene-6 (TSG-6), is also specifically accumulated in this matrix. TSG-6 mRNA expression is quickly upregulated and peaks at approximately 1500 copies/cell 4 h after the ovulatory stimuli as assessed by quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry reveals the colocalization of the TSG-6 protein and hyaluronan around the cumulus and granulosa cells. The TSG-6 protein exists in two distinct populations in the COC matrix as demonstrated by Western-blot analysis. One population is a monomer that is anchored to the matrix by a noncovalent interaction. The second population is a covalent complex with either of the heavy chains of IalphaI and is bound to hyaluronan through a strong interaction that is resistant to denaturing conditions. The specific incorporation of the TSG-6 protein into the COC matrix suggests a structural role for this molecule.

Alpha-Globulins↗
PubMed1999

A family of mammalian proteins homologous to yeast Sec24p.

The Sec23p/Sec24p complex is a component of yeast coat protein II (COPII), the coat protein complex responsible for vesicle budding from the endoplasmic reticulum (ER). Database searches and molecular cloning reveal that four different mammalian Sec24p-like proteins exist, all with about 20% amino acid identity with the yeast Sec24p. Sec24A and Sec24B share about 50% amino acid identity. Sec24D is cloned by screening a human pancreas of cDNA library with an expressed sequence tag (EST) fragment that is homologous to, but distinct from, Sec24A and Sec24B. Sec24D shares about 50% amino acid identity with the gene product of KIAA0079, which we have designated as Sec24C. These mammalian Sec24s appear to form two subclasses based on homology. Sec24A/B and Sec24C/D share about 20% identity with each other and with the yeast Sec24p. The Sec24 sequences also share weak but significant homology to the mammalian Sec23A and Sec23B. Northern blot analysis revealed that Sec24C is ubiquitously expressed. Although Sec24D transcripts are detectable in all tissues examined, they are selectively enriched in certain tissues, particularly placenta and pancreas. myc-tagged Sec24C and sec24D colocalized with Sec13, another COPII component. This colocalization suggests that Sec24C and Sec24D are indeed associated with COPII structures on membranes of the ER-Golgi boundary. The existence of at least four forms of Sec24 in mammalian cells suggest that multiple forms of COPII complex may be involved in ER export.

Amino Acid Sequence↗
PubMed2005

Proteomic analysis of TRPC5- and TRPC6-binding partners reveals interaction with the plasmalemmal Na(+)/K(+)-ATPase.

Mammalian transient receptor potential canonical (TRPC) genes encode a family of nonselective cation channels that are activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. In Drosophila photoreceptor cells, TRP channels are found in large, multimolecular signaling complexes in association with the PDZ-containing scaffolding protein, INAD. A similar mammalian TRPC "signalplex" has been proposed, but has yet to be defined. In the present study, affinity-purified polyclonal antibodies against TRPC5 and TRPC6 were used to immunoprecipitate signalplex components from rat brain lysates. Immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and sequenced by mass spectrometry. Proteins identified in the immunoprecipitates included cytoskeletal proteins spectrin, myosin, actin, drebrin, tubulin, and neurabin; endocytic vesicle-associated proteins clathrin, dynamin and AP-2; and the plasmalemmal Na(+)/K(+)-ATPase (NKA) pump. Several of these interactions were confirmed by reciprocal immunoprecipitation followed by Western blot analysis. In lysates from rat kidney, TRPC6, but not TRPC3, was found to coimmunoprecipitate with the NKA pump. Likewise, TRPC6, stably expressed in human embryonic kidney (HEK) cells, coimmunoprecipitated with endogenous NKA and colocalized with the pump to the plasmalemma when examined by immunofluorescence microscopy. Cell surface biotinylation experiments in intact HEK cells, confirmed that both the Na(+) pump and TRPC6 were present in the surface membrane and appeared to interact. Lastly, TRPC6 coimmunoprecipitated with the NKA pump when the proteins were coexpressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. These observations suggest that TRPC6 and the Na(+) pump are part of a functional complex that may be involved in ion transport and homeostasis in both the brain and kidney.

Animals↗
PubMed1990

Comparative studies of hepatitis B virus precore and core particles.

Hepatitis B virus core antigen gene expresses two cocarboxy-terminal proteins, termed precore and core proteins. Both precore and core proteins can form nucleocapsid-like particles. In order to understand the mechanism that leads to the formation of the nucleocapsid, we have expressed precore and core protein sequences in COS cells, a monkey kidney cell line, and compared the properties of these two particles. Our results show that core protein can form particles with various densities and they are present mostly in the cytosol. Precore protein, on the other hand, forms particles with one predominant density, and a majority of these particles are present in the lumen of the endoplasmic reticulum (ER). Furthermore, our results show that, when coexpressed in the same cells, core protein and the ER-associated surface antigens (envelope protein) show colocalization, indicating interaction between these two viral structural proteins.

Animals↗
PubMed2005

The KLP-6 kinesin is required for male mating behaviors and polycystin localization in Caenorhabditis elegans.

BACKGROUND: Male mating behavior of the nematode Caenorhabditis elegans offers an intriguing model to study the genetics of sensory behavior, cilia function, and autosomal dominant polycystic kidney disease (ADPKD). The C. elegans polycystins LOV-1 and PKD-2 act in male-specific sensory cilia required for response and vulva-location mating behaviors. RESULTS: Here, we identify and characterize a new mating mutant, sy511. sy511 behavioral phenotypes were mapped to a mutation in the klp-6 locus, a gene encoding a member of the kinesin-3 family (previously known as the UNC-104/Kif1A family). KLP-6 has a single homolog of unknown function in vertebrate genomes, including fish, chicken, mouse, rat, and human. We show that KLP-6 expresses exclusively in sensory neurons with exposed ciliated endings and colocalizes with the polycystins in cilia of male-specific neurons. Cilia of klp-6 mutants appear normal, suggesting a defect in sensory neuron function but not development. KLP-6 structure-function analysis reveals that the putative cargo binding domain directs the motor to cilia. Consistent with a motor-cargo association between KLP-6 and the polycystins, klp-6 is required for PKD-2 localization and function within cilia. Genetically, we find klp-6 regulates behavior through polycystin-dependent and -independent pathways. CONCLUSION: Multiple ciliary transport pathways dependent on kinesin-II, OSM-3, and KLP-6 may act sequentially to build cilia and localize sensory ciliary membrane proteins such as the polycystins. We propose that KLP-6 and the polycystins function as an evolutionarily conserved ciliary unit. KLP-6 promises new routes to understanding cilia function, behavior, and ADPKD.

Animals↗
PubMed2001

Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of SCA7.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder caused by a CAG repeat expansion. To determine the mechanism of neurotoxicity, we produced transgenic mice and observed a cone-rod dystrophy. Nuclear inclusions were present, suggesting that the disease pathway involves the nucleus. When yeast two-hybrid assays indicated that cone-rod homeobox protein (CRX) interacts with ataxin-7, we performed further studies to assess this interaction. We found that ataxin-7 and CRX colocalize and coimmunoprecipitate. We observed that polyglutamine-expanded ataxin-7 can dramatically suppress CRX transactivation. In SCA7 transgenic mice, electrophoretic mobility shift assays indicated reduced CRX binding activity, while RT-PCR analysis detected reductions in CRX-regulated genes. Our results suggest that CRX transcription interference accounts for the retinal degeneration in SCA7 and thus may provide an explanation for how cell-type specificity is achieved in this polyglutamine repeat disease.

Age Factors↗
PubMed2003

UV-C-induced DNA damage leads to p53-dependent nuclear trafficking of PML.

The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein that localizes to distinct domains in the nucleus, described as PML nuclear bodies (PML-NBs). Recent findings indicate that PML regulates the p53 response to oncogenic signals. Here, we define a p53-dependent role for PML in response to DNA damage. We exposed cells to ultraviolet light (UV-C) and imaged the nuclear distribution of PML, p53, and the BLM helicase by confocal microscopy. After DNA damage, PML partially relocated out of the PML-NBs, and colocalized with BLM and p53 at sites of DNA repair. In addition, using the isogenic HCT116 cell lines (p53+/+ and -/-), we show that the redistribution of PML was dependent on functional p53. Western analysis revealed that the level of PML protein remained unaltered after UV-C treatment. These results are consistent with the hypothesis that PML, in conjunction with p53 and BLM, contributes to the cellular response to UV-C-induced DNA damage and its repair.

Cell Line↗
PubMed2005

Statistical coevolution analysis and molecular dynamics: identification of amino acid pairs essential for catalysis.

Molecular dynamics (MD) simulations of HhaI DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA.MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 A and form a clear focal point centered near the active site. We suggest that these distal anti-correlated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA.MD approach.

Binding Sites↗
PubMed2003

Coincidence of synteny breakpoints with malignancy-related deletions on human chromosome 3.

We have found previously that during tumor growth intact human chromosome 3 transferred into tumor cells regularly looses certain 3p regions, among them the approximately 1.4-Mb common eliminated region 1 (CER1) at 3p21.3. Fluorescence in situ hybridization analysis of 12 mouse orthologous loci revealed that CER1 splits into two segments in mouse and therefore contains a murine/human conservation breakpoint region (CBR). Several breaks occurred in tumors within the region surrounding the CBR, and this sequence has features that characterize unstable chromosomal regions: deletions in yeast artificial chromosome clones, late replication, gene and segment duplications, and pseudogene insertions. Sequence analysis of the entire 3p12-22 revealed that other cancer-associated deletions (regions eliminated from monochromosomal hybrids carrying an intact chromosome 3 during tumor growth and homozygous deletions found in human tumors) colocalized nonrandomly with murine/human CBRs and were characterized by an increased number of local gene duplications and murine/human conservation mismatches (single genes that do not match into the conserved chromosomal segment). The CBR within CER1 contains a simple tandem TATAGA repeat capable of forming a 40-bp-long secondary hairpin-like structure. This repeat is nonrandomly localized within the other tumor-associated deletions and in the vicinity of 3p12-22 CBRs.

Animals↗
PubMed2006

High expression of a cytokeratin-associated protein in many cancers.

We have described previously a cDNA library made from membrane-bound polysomal mRNA prepared from breast and prostate cancer cell lines. The library is highly enriched for cDNAs encoding membrane proteins, secreted proteins, and cytokeratins. To characterize this library, 25,277 cDNA clones were sequenced and aligned with various databases; 1,439 clones did not align with known genes. From this set of clones we identified a previously uncharacterized gene encoding a 334-aa protein. Although protein structural motif prediction programs indicate that the gene encodes a membrane protein comprising a signal sequence, a series of leucine-rich repeats, and a single transmembrane domain with a cytoplasmic tail, confocal microscopy of MCF7 breast cancer cells demonstrates that the protein is not directly associated with the plasma membrane or intracellular membranes but instead colocalizes with intermediate filaments and cytokeratins within the cell. Immunofluorescence studies also show that protein expression is increased greatly in mitotic MCF7 cells, and immunohistochemistry demonstrates its expression in human breast cancer cells. Analysis of mRNA levels in 25 different normal tissues by RT-PCR shows that this gene is expressed highly in normal prostate and salivary gland, very weakly in colon, pancreas, and intestine, and not at all in other tissues. RT-PCR studies on human cancer samples show that the RNA is expressed highly in many cancer cell lines and cancer specimens, including 26 of 33 human breast cancers, 3 of 3 prostate cancers, 3 of 3 colon cancers, and 3 of 3 pancreatic cancers. We name the protein CAPC, cytokeratin-associated protein in cancer.

Amino Acid Sequence↗
PubMed2002

Higher order arrangement of the eukaryotic nuclear bodies.

The nuclei of eukaryotic cells consist of discrete substructures. These substructures include the nuclear bodies, which have been implicated in a number of biological processes such as transcription and splicing. However, for most nuclear bodies, the details of involvement in these processes in relation to their three-dimensional distributions in the nucleus are still unclear. Through the analysis of TDP, a protein functional in both transcriptional repression and alternative splicing, we have identified a new category of nuclear bodies within which the TDP molecules reside. Remarkably, the TDP bodies (TBs) colocalize or overlap with several different types of nuclear bodies previously suggested to function in transcription or splicing. Of these nuclear bodies, the Gemini of coiled bodies (GEM) seems to associate with TB through the interaction between survival motor neuron (SMN) protein and TDP. Furthermore, TB sometimes appears to be the bridge of two or more of these other nuclear bodies. Our data suggest the existence of a hierarchy and possibly functional arrangement of the nuclear bodies within the eukaryotic nuclei.

Amino Acid Sequence↗
PubMed1999

Persistence of an alternate chromatin structure at silenced loci in vitro.

In Saccharomyces cerevisiae, transcriptional repression at the HM mating-type loci and telomeres results from the formation of a heterochromatin-like structure. Silencing requires at least three Sir proteins (Sir2p-4p), which are recruited to chromatin by silencers at the HM loci and TG1-3 tracts at telomeres. Sir proteins and telomeres colocalize at the nuclear periphery, suggesting that this subnuclear position may also contribute to transcriptional repression. To evaluate the contribution of nuclear context to silencing, we developed methodology to isolate silent chromatin for analysis in vitro. Site-specific recombination was used in vivo to produce DNA rings from the silent HMR locus, and differential centrifugation was used to isolate the rings from whole-cell lysate. The partially purified rings retained many of the intracellular hallmarks of transcriptionally repressed domains. Specifically, rings from repressed strains were resistant to restriction endonuclease digestion, bore an altered DNA topology, and were associated with Sir3p. The recombination approach also was used to form rings from HMR that lacked silencers. Despite the uncoupling of these cis-acting regulatory elements, similar but nonidentical results were obtained. We conclude that an alternate chromatin structure at silent loci can persist in vitro in the absence of silencers and nuclear compartmentalization.

Chromatin↗