The presence of an antigen reactive with a human autoantibody inTrichosia pubescens (Diptera: Sciaridae) and its association with certain transcriptionally active regions of the genome

Amabis, J M; Amabis, Dulce Cabral; Kaburaki, Junichi; Stollar, B. David

doi:10.1007/bf01735325

Cited by 24 publications

(9 citation statements)

References 30 publications

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“…In vitro studies have shown that this abundant nuclear protein binds to the ends of double-stranded DNA and DNA ending in stem-loop structures, probably via the 70-kDa subunit (7,10,42). Ku protein has been found to be associated with DNase I-sensitive nucleosomes lacking H1 histone (68); Ku protein has also been found to be localized on certain transcriptionally active loci of chromosomal DNA (2,51). Several studies have demonstrated that Ku protein directly modulates RNA polymerase I-mediated transcription (15,24,25).…”

Section: Discussionmentioning

confidence: 99%

“…The Ku autoantigen is a heterodimer consisting of 70-kDa (Ku-70) and 86-kDa (Ku-80) polypeptides (41,42,50,52,69) and is an abundant nuclear protein (41,42,50,52,69). Various cellular roles for Ku have been suggested, including a role in transcription, recombination, replication, and DNA repair (2,4,9,10,13,27,28,51,68). However, until recently there is a paucity of experimental data on Ku's in vivo function.…”

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confidence: 99%

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Modulation of Thermal Induction of hsp70 Expression by Ku Autoantigen or Its Individual Subunits

Yang

Nussenzweig

et al. 1996

Molecular and Cellular Biology

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Previously, we proposed a dual control mechanism for the regulation of the heat shock response in mammalian cells: a positive control mediated by the heat shock transcription factor HSF1 and a negative control mediated by the constitutive heat shock element-binding factor (CHBF). To study the physiological role of CHBF in the regulation of heat shock response, we purified CHBF to apparent homogeneity and showed it to be identical to the Ku autoantigen, a heterodimer consisting of 70-kDa (Ku-70) and 86-kDa (Ku-80) polypeptides. To study further the functional significance of Ku/CHBF in the cellular response to heat shock, we established rodent cell lines that stably and constitutively overexpressed one or both subunits of the human Ku protein, and examined the thermal induction of hsp70 and other heat shock proteins in these Ku-overexpressing cells. We show that expression of the human Ku-70 and Ku-80 subunits jointly or of the Ku-70 subunit alone specifically inhibits heat-induced hsp70 expression. Conversely, expression of human Ku-80 alone does not have this effect. Thermal induction of other heat shock proteins in all of the Ku-overexpressing cell lines appears not to be significantly affected, nor is the state of phosphorylation or the DNA-binding ability of HSF1 affected. These findings support a model in which hsp70 expression is controlled by a second regulatory factor in addition to the positive activation of HSF1. The Ku protein, specifically the Ku-70 subunit, is involved in the regulation of hsp70 gene expression.Cells and organisms respond to heat shock and a number of other environmental stresses by rapidly increasing the level of transcription of heat shock genes and the translation of their messages, leading to an elevated cellular level of the heat shock proteins (hsps). Among these proteins, hsp70 has been implicated in playing a key role in the cellular response to heat shock. Many recent studies suggest that one of the functions of hsp70 is to protect cells from thermal damage (3,17,21,29,30,44,45,53) by maintaining the native state and proper folding of cellular proteins under physiological stress (5,8,19,56), and/or by facilitating the restoration of certain cellular functions (37,48,57).In recent years, extensive studies of the mammalian heat shock transcription factor HSF1 have implicated this protein as a key positive regulatory factor in the heat shock response (1, 33-35, 59, 67). Although the importance of HSF1 in the regulation of mammalian hsp70 expression is well-established, recent data indicate that activation of HSF1 is not sufficient for the induction of hsp70 gene expression (36,39,43). Studies from our laboratory and others suggest the existence of an additional regulatory factor or factors (18,36). Experiments with extracts of control and heat-shocked cells demonstrated that rodent cells may contain two different heat shock element (HSE)-binding factors: in addition to HSF1, there appears to be a constitutive HSE-binding factor (CHBF) (22,36).Analysis of these two factors in ...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Modulation of Thermal Induction of hsp70 Expression by Ku Autoantigen or Its Individual Subunits

Yang

Nussenzweig

et al. 1996

Molecular and Cellular Biology

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“…Slide preparation, immunostaining reaction and analysis procedures are described in Gorab et al (1996) and Amabis et al (1990), respectively. Slides from a number of different developmental stages of Trichosia were evaluated by phase contrast and epifluorescence microscopy and microphotography.…”

Section: Methodsmentioning

confidence: 99%

Antibodies against the D-domain of a Chironomus ecdysone receptor protein react with DNA puff sites in Trichosia pubescens

et al. 1997

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An antiserum (called AScE/D) against the semiconserved D-domain of a Chironomus tentans ecdysone receptor protein (cEcR) gave indirect immunofluorescence signals at DNA puff sites in Trichosia pubescens. The signals varied in maximum intensity at different DNA puff sites. Control experiments using the secondary rhodamine-labeled anti-rabbit IgG alone, preimmune serum, affinity purified AScE/D (called pABcE/D) and AScE/D preabsorbed with expressing bacterial extract or highly purified bacterially expressed cEcR indicated that the signals obtained at these chromosomal sites were likely to be due to specific interaction between an endogenous sciarid EcR and antibodies against cEcR. This conclusion was supported by observation of signals at certain Ec-inducible primary RNA puff sites. AScE/D signals began to appear at DNA puff sites during L3, the stage when amplification initiates, but at most sites their mean intensity was low and not statistically significant. Sites with AScE/D signals of significant mean intensity at this stage already showed evidence of transcription. The number and strength of transcription signals increased during L4. Comparison of the developmental course of signals for AScE/D, DNA synthesis, RNA presence/synthesis, and puff size for several DNA puffs during late larval- prepupal development showed a closer relationship of AScE/D signals with the initiation of RNA synthesis than with the initiation of DNA synthesis. Therefore, although we cannot absolutely eliminate a direct involvement of EcR in the amplification process at some sites, this investigation gives stronger support for its direct involvement in transcription. Since AScE/D signals are observed at DNA puff sites from the time the latter begin amplification/transcription through their regression, it appears that Ec and EcR are necessary as a sustained stimulus at these regions.

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“…In these studies, additional controls reinforced the antibody specificity (Rudkin and Stollar 1977b;Alcover et al 1982;Büsen et al 1982;Díez and Barettino 1984;Amabis et al 1990;Testillano et al 1994;Gorab 2003;Stocker and Gorab 2003).…”

Section: Introductionmentioning

confidence: 96%

Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster

et al. 2009

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Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson-Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.

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The presence of an antigen reactive with a human autoantibody inTrichosia pubescens (Diptera: Sciaridae) and its association with certain transcriptionally active regions of the genome

Cited by 24 publications

References 30 publications

Modulation of Thermal Induction of hsp70 Expression by Ku Autoantigen or Its Individual Subunits

Modulation of Thermal Induction of hsp70 Expression by Ku Autoantigen or Its Individual Subunits

Antibodies against the D-domain of a Chironomus ecdysone receptor protein react with DNA puff sites in Trichosia pubescens

Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster

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