Selection of homeotic proteins for binding to a human DNA replication origin
Identifieur interne : 001852 ( Istex/Corpus ); précédent : 001851; suivant : 001853Selection of homeotic proteins for binding to a human DNA replication origin
Auteurs : Elisa De Stanchina ; Davide Gabellini ; Paolo Norio ; Mauro Giacca ; Fiorenzo A. Peverali ; Silvano Riva ; Arturo Falaschi ; Giuseppe BiamontiSource :
- Journal of Molecular Biology [ 0022-2836 ] ; 2000.
English descriptors
- KwdEn :
- Teeft :
- Abdurashidova, Academic press, Accession, Accession number, Activation, Activation domain, Amino, Amino acid residues, Assay, Biamonti, Binding site, Binding sites, Biol, Catalogue number, Catase activity, Cdna, Cdna probes, Cell biol, Cell cycle, Cell extracts, Cell lines, Chloramphenicol acetyl transferase, Chromatin structure, Clone, Codon, Cold spring harbor, Cold spring harbor laboratory, Cold spring harbor laboratory press, Coli, Developmental regulators, Different portions, Dimitrova, Donaldson blow, Dutta bell, Electrophoretic mobility shift assay, Emsa, Escherichia coli, Eukaryotic, Eukaryotic cells, Footprinting, Footprinting experiments, Fusion protein, Genbank, Genbank accession number, Gene, Genetic analysis, Genomic, Genomic clone, Giacca, Hela, Hela cells, High level, Homeobox, Homeodomain, Homeodomain proteins, Homeogene family, Homeoproteins, Homeotic genes, Human lamin, Hybrid protein, Hybrid proteins, Kpni site, Lacz gene, Lamin, Mammalian cells, Marahrens stillman, Metazoan cells, Minutes incubation, Molar, Natl acad, Onehybrid screening, Open reading frame, Origin activation, Origin area, Origin figure, Origin recognition, Origin region, Origin sequence, Other hand, Other proteins, Partial cdna, Plasmid, Possible role, Ppv1, Ppv1 gene, Ppv1 gene promoter, Proliferative stimuli, Promoter, Protein, Rabbit polyclonal, Recombinant protein, Recombinant proteins, Replication, Replication origin, Replication origins, Reporter gene, Riva, Room temperature, Saccharomyces cerevisiae, Same extracts, Same orientation, Same unlabelled oligo, Serum starvation, Simian virus, Single intron, Standard methods, Stillman, Stimulatory effect, Total proteins, Transcription, Transcription factor, Transcription factors, Transcriptional, Trends biochem, Untranslated region, Ura3 locus, Vivo, Vivo footprinting analysis, Yeast, Yeast cells, Yeast origins, Yeast replication origins, Yeast strain.
Abstract
Abstract: We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.
Url:
DOI: 10.1006/jmbi.2000.3782
Links to Exploration step
ISTEX:0CCB1215FCD742084270E7AD438918B3D149F1B4Le document en format XML
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Peverali</name><affiliation><mods:affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</mods:affiliation></affiliation></author><author><name sortKey="Riva, Silvano" sort="Riva, Silvano" uniqKey="Riva S" first="Silvano" last="Riva">Silvano Riva</name><affiliation><mods:affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</mods:affiliation></affiliation></author><author><name sortKey="Falaschi, Arturo" sort="Falaschi, Arturo" uniqKey="Falaschi A" first="Arturo" last="Falaschi">Arturo Falaschi</name><affiliation><mods:affiliation>International Center for Genetic Engineering and Biotechnology, Padriciano 99 34012 Trieste, Italy</mods:affiliation></affiliation></author><author><name sortKey="Biamonti, Giuseppe" sort="Biamonti, Giuseppe" uniqKey="Biamonti G" first="Giuseppe" last="Biamonti">Giuseppe Biamonti</name><affiliation><mods:affiliation>E-mail: biamonti@igbe.pv.cnr.it</mods:affiliation></affiliation><affiliation><mods:affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="ISSN">0022-2836</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">299</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="667">667</biblScope><biblScope unit="page" to="680">680</biblScope></imprint><idno type="ISSN">0022-2836</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-2836</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>3′-UTR</term><term>CAT</term><term>DNA replication origin</term><term>EMSA</term><term>Lamin 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methods</term><term>Stillman</term><term>Stimulatory effect</term><term>Total proteins</term><term>Transcription</term><term>Transcription factor</term><term>Transcription factors</term><term>Transcriptional</term><term>Trends biochem</term><term>Untranslated region</term><term>Ura3 locus</term><term>Vivo</term><term>Vivo footprinting analysis</term><term>Yeast</term><term>Yeast cells</term><term>Yeast origins</term><term>Yeast replication origins</term><term>Yeast strain</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>lamin</json:string><json:string>cdna</json:string><json:string>replication</json:string><json:string>biol</json:string><json:string>clone</json:string><json:string>ppv1</json:string><json:string>biamonti</json:string><json:string>homeoproteins</json:string><json:string>footprinting</json:string><json:string>codon</json:string><json:string>plasmid</json:string><json:string>abdurashidova</json:string><json:string>homeodomain</json:string><json:string>transcriptional</json:string><json:string>genbank</json:string><json:string>hela</json:string><json:string>homeobox</json:string><json:string>emsa</json:string><json:string>accession</json:string><json:string>giacca</json:string><json:string>assay</json:string><json:string>origin 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family</json:string><json:string>single intron</json:string><json:string>yeast replication origins</json:string><json:string>trends biochem</json:string><json:string>homeodomain proteins</json:string><json:string>cell biol</json:string><json:string>cold spring harbor laboratory press</json:string></teeft></keywords><author><json:item><name>Elisa de Stanchina</name><affiliations><json:string>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</json:string></affiliations></json:item><json:item><name>Davide Gabellini</name><affiliations><json:string>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</json:string></affiliations></json:item><json:item><name>Paolo Norio</name><affiliations><json:string>International Center for Genetic Engineering and Biotechnology, Padriciano 99 34012 Trieste, Italy</json:string></affiliations></json:item><json:item><name>Mauro Giacca</name><affiliations><json:string>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</json:string></affiliations></json:item><json:item><name>Fiorenzo A Peverali</name><affiliations><json:string>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</json:string></affiliations></json:item><json:item><name>Silvano Riva</name><affiliations><json:string>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</json:string></affiliations></json:item><json:item><name>Arturo Falaschi</name><affiliations><json:string>International Center for Genetic Engineering and Biotechnology, Padriciano 99 34012 Trieste, Italy</json:string></affiliations></json:item><json:item><name>Giuseppe Biamonti</name><affiliations><json:string>E-mail: biamonti@igbe.pv.cnr.it</json:string><json:string>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Regular article</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DNA replication origin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>homeoproteins</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>one-hybrid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Lamin B2</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ORC : origin recognition complex</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>3′-UTR : 3′ untranslated region</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ORF : open reading frame</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>EMSA : electrophoretic mobility shift assay</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>CAT : chloramphenicol acetyl transferase</value></json:item></subject><arkIstex>ark:/67375/6H6-V2HQS6HC-C</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.</abstract><qualityIndicators><score>9.088</score><pdfWordCount>8339</pdfWordCount><pdfCharCount>49322</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>14</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>174</abstractWordCount><abstractCharCount>1127</abstractCharCount><keywordCount>10</keywordCount></qualityIndicators><title>Selection of homeotic proteins for binding to a human DNA replication origin</title><pmid><json:string>10835276</json:string></pmid><pii><json:string>S0022-2836(00)93782-3</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Molecular Biology</title><language><json:string>unknown</json:string></language><publicationDate>2000</publicationDate><issn><json:string>0022-2836</json:string></issn><pii><json:string>S0022-2836(00)X0192-1</json:string></pii><volume>299</volume><issue>3</issue><pages><first>667</first><last>680</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>2000</json:string><json:string>2400</json:string><json:string>1998</json:string><json:string>2200</json:string><json:string>1896</json:string><json:string>3993</json:string><json:string>3921</json:string></date><geogName></geogName><orgName><json:string>Medical Center, Durham</json:string><json:string>Jackson Immuno Research, Inc</json:string><json:string>Clontech</json:string><json:string>Cold Spring Harbor Laboratory, Cold Spring Harbor, NY</json:string><json:string>Italy We</json:string><json:string>Duke University</json:string><json:string>Howard Hughes Medical Institute, University of Massachusetts Medical School</json:string><json:string>MURST</json:string></orgName><orgName_funder><json:string>MURST</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>E. Boncinelli</json:string><json:string>Silvano Riva</json:string><json:string>Multiple</json:string><json:string>H. P. Bogerd</json:string><json:string>Northern</json:string><json:string>Paolo Norio</json:string><json:string>E. de Stanchina</json:string><json:string>A. Peverali</json:string><json:string>Via Abbiategrasso</json:string><json:string>Davide Gabellini</json:string><json:string>Dr Scott</json:string><json:string>G. Draetta</json:string><json:string>Arturo Falaschi</json:string><json:string>P. Norio</json:string><json:string>D. Gabellini</json:string><json:string>Elisa de Stanchina</json:string><json:string>Giuseppe Biamonti</json:string><json:string>J.J. Li</json:string><json:string>Mauro Giacca</json:string></persName><placeName><json:string>San Raffaele</json:string><json:string>Cos</json:string><json:string>Germany</json:string><json:string>UK</json:string><json:string>Trieste</json:string><json:string>MA</json:string><json:string>Italy</json:string><json:string>Sweden</json:string><json:string>Worcester</json:string><json:string>Netherlands</json:string></placeName><ref_url><json:string>http://www.idealibrary.com</json:string></ref_url><ref_bibl><json:string>Abdurashidova et al., 1998</json:string><json:string>Hatton et al., 1988</json:string><json:string>Holmquist, 1989</json:string><json:string>Stornaiuolo et al., 1990</json:string><json:string>Abdurashidova et al., 2000</json:string><json:string>Gorman et al. 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Yaniv</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Sequence of human HOXC10 and HOXC13 proteins. (a) ORF of the human HoxC10 cDNA along with the deduced amino acid sequence indicated in the one-letter code. The homeobox region is boxed. Numbers refer to the nucleotides of the ORF. (b) Sequence of HoxC13 cDNA numbered from the first nucleotide of the start codon. The start and the stop codons are both underlined and the homeobox is boxed. The ORF is in capital letters while the upstream region, the intron and the 3′-UTR region up to the poly(A) tail are in lower-case letters. Only a portion of the intron is sequenced and it is not considered in numbering. The 5′-end of the partial cDNA isolated in the one-hybrid screening (clone 134) is indicated by an arrow. (c) Alignment of the four paralogous sequences at position 13 as determined by means of the FASTA3 program. Identities are boxed and the homeobox is indicated by an arrow underneath.</note><note type="content">Figure 2: Expression of HOXC10 and HOXC13 in E. coli. The entire HoxC10 cDNA was expressed in E. coli as a His-tagged protein. The C-terminal portion of HOXC13 (amino acid residues 135 to 330) was expressed in E. coli as a GST fusion. Proteins were purified as detailed in Materials and Methods. An aliquot (1 μg) of the purified proteins was subjected to SDS-10 % PAGE and revealed by Coomassie staining.</note><note type="content">Figure 3: EMSA analysis of HOXC10 and HOXC13 binding to the 74 bp box. Double-stranded oligonucleotides corresponding to positions 3921–3993, 3921–3954 and 3953–3993 of the Lamin B2 gene were used in band-shift assay with purified His-tagged-HOXC10 or GST-HOXC13 fusion as described in Materials and Methods. The end-labelled oligonucleotide (0.2–0.5 ng) was incubated in a 50 μl reaction with 20 ng of recombinant protein. After 30 minutes incubation, complexes were resolved by 6 % PAGE. Autoradiograms of the dried gels are shown. (a) The 3921–3993 region was assayed with His-tagged HOXC10. Binding specificity was assessed by adding the indicated molar excess of specific (the same unlabelled oligo) or of non-specific competitor. (b) As in (a) except that the GST-HOXC13 fusion protein was used. (c) and (d) Binding of His-tagged HOXC13 to 3921–3954 and 3953–3993 regions, respectively. The indicated molar excess of specific competitor was added to the assay. (e) and (f) Binding of GST-HOXC10 fusion to 3921–3954 and 3953–3993 regions, respectively. The indicated molar excess of specific competitor was added to the assay.</note><note type="content">Figure 4: In vitro footprinting analysis of the interaction between the GST-HOXC13 fusion protein and the Lamin B2 origin. An end-labelled fragment, covering positions 3795 to 4074 of the Lamin B2 gene, was incubated with 10 ng of purified GST-HOXC13 and subjected to controlled digestion with DNase I. The digestion products were then resolved through a denaturing 6 % polyacrylamide gel. An autoradiogram of the gel is shown. Binding of HOXC13 to both strands has been analysed. Numbers on the left refer to the Lamin B2 gene positions. The protected area is schematically represented on the right and compared to the in vivo protected area described elsewhere (Abdurashidova et al., 1998). The sequence used as a probe in the one-hybrid screening is shown at the bottom of the Figure. The in vitro protected nucleotides are boxed. The grey boxes evidence the almost perfect direct repeat in the origin region.</note><note type="content">Figure 5: CAT-assay analysis of the interaction of HOXC10 and HOXC13 with the Lamin B2 origin. (a) Schematic representation of the different reporter constructs described in the text. Numbers, below the scale, refer to the Lamin B2 gene sequence position (GenBank accession number: M94363). The 3′-end of the Lamin B2 and of the 5′-end of the ppv1 gene transcripts are shown along with the position of the Lamin B2 origin. The grey box represents the 74 bp sequence protected in vivo, while the black box indicates the 20 bp sequence corresponding to the downstream binding site of GST-HOXC13 in vitro (see Figure 4). (b) The indicated constructs were cotransfected with plasmids expressing the entire HOXC10, or a portion of HOXC13 from Ser135 to the stop codon, or the same region of HOXC13 fused to the activation domain of the transcription factor VP16. As a control the various CAT plasmids were cotransfected with the sole pCR 3.1 vector. After 48 hours, transfected cells were harvested and the CATase activity was quantified. The stimulatory effect due to the overexpression of the different homeoproteins was calculated as a ratio with the CATase activity measured by cotransfecting the different CAT plasmids with pCR 3.1 vector. Each value is the mean of at least five independent experiments, each consisting of three identical cotransfections. The calculated standard deviation is shown.</note><note type="content">Figure 6: Expression of HoxC10 and HoxC13 genes in different human tissues and cell lines. Autoradiograms of commercial Northern blots of poly(A)+ RNAs, extracted from different adult or foetal tissues, hybridised to HoxC10 or HoxC13 cDNA probes ((a) and (b), respectively). Molecular mass markers are indicated on the left. (c) Total RNA (10 μg) was extracted from HeLa S3, U937 and K562 cells and analysed by Northern blotting with HoxC10 and HoxC13 cDNA probes.</note><note type="content">Figure 7: HOXC10 protein is not present in quiescent or in differentiated murine muscular C2C12 cells. About 50 μg of total proteins from exponentially growing (G) or serum-arrested (Q) C2C12 myoblasts or from differentiated (D) C2C12 myotubes were fractionated on SDS-10 % PAGE and analysed by Western blot to assess, with specific antibodies, the steady-state levels of HOXC10, cyclin A and MyoD (see Materials and Methods). The expression levels of cyclin A and MyoD are shown as controls of differentiation and/or serum starvation (see the text). The actin level is shown as a relative measure of the amount of loaded proteins.</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Selection of homeotic proteins for binding to a human DNA replication origin</title><author xml:id="author-0000"><persName><forename type="first">Elisa</forename><surname>de Stanchina</surname></persName><note type="biography">Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</note><affiliation>Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</affiliation><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Davide</forename><surname>Gabellini</surname></persName><note type="biography">Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</note><affiliation>Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</affiliation><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Paolo</forename><surname>Norio</surname></persName><note type="biography">Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</note><affiliation>Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</affiliation><affiliation>International Center for Genetic Engineering and Biotechnology, Padriciano 99 34012 Trieste, Italy</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Mauro</forename><surname>Giacca</surname></persName><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Fiorenzo A</forename><surname>Peverali</surname></persName><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Silvano</forename><surname>Riva</surname></persName><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation></author><author xml:id="author-0006"><persName><forename type="first">Arturo</forename><surname>Falaschi</surname></persName><affiliation>International Center for Genetic Engineering and Biotechnology, Padriciano 99 34012 Trieste, Italy</affiliation></author><author xml:id="author-0007"><persName><forename type="first">Giuseppe</forename><surname>Biamonti</surname></persName><email>biamonti@igbe.pv.cnr.it</email><note type="correspondence"><p>Corresponding author</p></note><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation></author><idno type="istex">0CCB1215FCD742084270E7AD438918B3D149F1B4</idno><idno type="ark">ark:/67375/6H6-V2HQS6HC-C</idno><idno type="DOI">10.1006/jmbi.2000.3782</idno><idno type="PII">S0022-2836(00)93782-3</idno></analytic><monogr><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="pISSN">0022-2836</idno><idno type="PII">S0022-2836(00)X0192-1</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">299</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="667">667</biblScope><biblScope unit="page" to="680">680</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2000</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.</p></abstract><textClass><keywords scheme="keyword"><list><head>article-category</head><item><term>Regular article</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>DNA replication origin</term></item><item><term>homeoproteins</term></item><item><term>one-hybrid</term></item><item><term>Lamin B2</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>ORC</term><term>origin recognition complex</term></item><item><term>3′-UTR</term><term>3′ untranslated region</term></item><item><term>ORF</term><term>open reading frame</term></item><item><term>EMSA</term><term>electrophoretic mobility shift assay</term></item><item><term>CAT</term><term>chloramphenicol acetyl transferase</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2000-04-05">Modified</change><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-V2HQS6HC-C/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="GR1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="GR2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="GR3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="GR4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="GR5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="GR6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="GR7"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YJMBI</jid><aid>93782</aid><ce:pii>S0022-2836(00)93782-3</ce:pii><ce:doi>10.1006/jmbi.2000.3782</ce:doi><ce:copyright type="full-transfer" year="2000">Academic Press</ce:copyright><ce:doctopics><ce:doctopic><ce:text>Regular article</ce:text></ce:doctopic></ce:doctopics></item-info><head><ce:dochead><ce:textfn>Regular article</ce:textfn></ce:dochead><ce:title>Selection of homeotic proteins for binding to a human DNA replication origin<ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref><ce:footnote id="FN1"><ce:label>1</ce:label><ce:note-para><ce:bold><ce:italic>Edited by M. Yaniv</ce:italic></ce:bold></ce:note-para></ce:footnote></ce:title><ce:author-group><ce:author><ce:given-name>Elisa</ce:given-name><ce:surname>de Stanchina</ce:surname><ce:cross-ref refid="FN2"><ce:sup>2</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Davide</ce:given-name><ce:surname>Gabellini</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref><ce:cross-ref refid="FN2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Paolo</ce:given-name><ce:surname>Norio</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref><ce:cross-ref refid="FN2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Mauro</ce:given-name><ce:surname>Giacca</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Fiorenzo A</ce:given-name><ce:surname>Peverali</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Silvano</ce:given-name><ce:surname>Riva</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Arturo</ce:given-name><ce:surname>Falaschi</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Giuseppe</ce:given-name><ce:surname>Biamonti</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:e-address>biamonti@igbe.pv.cnr.it</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>1</ce:label><ce:textfn>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>2</ce:label><ce:textfn>International Center for Genetic Engineering and Biotechnology, Padriciano 99 34012 Trieste, Italy</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Corresponding author</ce:text></ce:correspondence><ce:footnote id="FN2"><ce:label>2</ce:label><ce:note-para>Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</ce:note-para></ce:footnote></ce:author-group><ce:date-received day="28" month="2" year="2000"></ce:date-received><ce:date-revised day="5" month="4" year="2000"></ce:date-revised><ce:date-accepted day="11" month="4" year="2000"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>We have previously shown that a cell cycle-dependent nucleoprotein complex assembles <ce:italic>in vivo</ce:italic> on a 74 bp sequence within the human DNA replication origin associated to the <ce:italic>Lamin B2</ce:italic> gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the <ce:italic>Abdominal-B</ce:italic> gene of <ce:italic>Drosophila melanogaster</ce:italic> and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the <ce:italic>HoxC13</ce:italic> gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and <ce:italic>in vitro</ce:italic> footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>DNA replication origin</ce:text></ce:keyword><ce:keyword><ce:text>homeoproteins</ce:text></ce:keyword><ce:keyword><ce:text>one-hybrid</ce:text></ce:keyword><ce:keyword><ce:text>Lamin B2</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>ORC</ce:text><ce:keyword><ce:text>origin recognition complex</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>3′-UTR</ce:text><ce:keyword><ce:text>3′ untranslated region</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>ORF</ce:text><ce:keyword><ce:text>open reading frame</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>EMSA</ce:text><ce:keyword><ce:text>electrophoretic mobility shift assay</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>CAT</ce:text><ce:keyword><ce:text>chloramphenicol acetyl transferase</ce:text></ce:keyword></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Selection of homeotic proteins for binding to a human DNA replication origin</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Selection of homeotic proteins for binding to a human DNA replication origin</title></titleInfo><name type="personal"><namePart type="given">Elisa</namePart><namePart type="family">de Stanchina</namePart><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation><description>Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Davide</namePart><namePart type="family">Gabellini</namePart><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation><description>Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Paolo</namePart><namePart type="family">Norio</namePart><affiliation>International Center for Genetic Engineering and Biotechnology, Padriciano 99 34012 Trieste, Italy</affiliation><description>Present addresses: E. de Stanchina, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; D. Gabellini, Howard Hughes Medical Institute, University of Massachusetts Medical School, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA; P. Norio, Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461, USA.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Mauro</namePart><namePart type="family">Giacca</namePart><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Fiorenzo A</namePart><namePart type="family">Peverali</namePart><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Silvano</namePart><namePart type="family">Riva</namePart><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Arturo</namePart><namePart type="family">Falaschi</namePart><affiliation>International Center for Genetic Engineering and Biotechnology, Padriciano 99 34012 Trieste, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Giuseppe</namePart><namePart type="family">Biamonti</namePart><affiliation>E-mail: biamonti@igbe.pv.cnr.it</affiliation><affiliation>Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207 27100, Pavia, Italy</affiliation><description>Corresponding author</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><dateModified encoding="w3cdtf">2000-04-05</dateModified><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.</abstract><note type="footnote">Edited by M. Yaniv</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Sequence of human HOXC10 and HOXC13 proteins. (a) ORF of the human HoxC10 cDNA along with the deduced amino acid sequence indicated in the one-letter code. The homeobox region is boxed. Numbers refer to the nucleotides of the ORF. (b) Sequence of HoxC13 cDNA numbered from the first nucleotide of the start codon. The start and the stop codons are both underlined and the homeobox is boxed. The ORF is in capital letters while the upstream region, the intron and the 3′-UTR region up to the poly(A) tail are in lower-case letters. Only a portion of the intron is sequenced and it is not considered in numbering. The 5′-end of the partial cDNA isolated in the one-hybrid screening (clone 134) is indicated by an arrow. (c) Alignment of the four paralogous sequences at position 13 as determined by means of the FASTA3 program. Identities are boxed and the homeobox is indicated by an arrow underneath.</note><note type="content">Figure 2: Expression of HOXC10 and HOXC13 in E. coli. The entire HoxC10 cDNA was expressed in E. coli as a His-tagged protein. The C-terminal portion of HOXC13 (amino acid residues 135 to 330) was expressed in E. coli as a GST fusion. Proteins were purified as detailed in Materials and Methods. An aliquot (1 μg) of the purified proteins was subjected to SDS-10 % PAGE and revealed by Coomassie staining.</note><note type="content">Figure 3: EMSA analysis of HOXC10 and HOXC13 binding to the 74 bp box. Double-stranded oligonucleotides corresponding to positions 3921–3993, 3921–3954 and 3953–3993 of the Lamin B2 gene were used in band-shift assay with purified His-tagged-HOXC10 or GST-HOXC13 fusion as described in Materials and Methods. The end-labelled oligonucleotide (0.2–0.5 ng) was incubated in a 50 μl reaction with 20 ng of recombinant protein. After 30 minutes incubation, complexes were resolved by 6 % PAGE. Autoradiograms of the dried gels are shown. (a) The 3921–3993 region was assayed with His-tagged HOXC10. Binding specificity was assessed by adding the indicated molar excess of specific (the same unlabelled oligo) or of non-specific competitor. (b) As in (a) except that the GST-HOXC13 fusion protein was used. (c) and (d) Binding of His-tagged HOXC13 to 3921–3954 and 3953–3993 regions, respectively. The indicated molar excess of specific competitor was added to the assay. (e) and (f) Binding of GST-HOXC10 fusion to 3921–3954 and 3953–3993 regions, respectively. The indicated molar excess of specific competitor was added to the assay.</note><note type="content">Figure 4: In vitro footprinting analysis of the interaction between the GST-HOXC13 fusion protein and the Lamin B2 origin. An end-labelled fragment, covering positions 3795 to 4074 of the Lamin B2 gene, was incubated with 10 ng of purified GST-HOXC13 and subjected to controlled digestion with DNase I. The digestion products were then resolved through a denaturing 6 % polyacrylamide gel. An autoradiogram of the gel is shown. Binding of HOXC13 to both strands has been analysed. Numbers on the left refer to the Lamin B2 gene positions. The protected area is schematically represented on the right and compared to the in vivo protected area described elsewhere (Abdurashidova et al., 1998). The sequence used as a probe in the one-hybrid screening is shown at the bottom of the Figure. The in vitro protected nucleotides are boxed. The grey boxes evidence the almost perfect direct repeat in the origin region.</note><note type="content">Figure 5: CAT-assay analysis of the interaction of HOXC10 and HOXC13 with the Lamin B2 origin. (a) Schematic representation of the different reporter constructs described in the text. Numbers, below the scale, refer to the Lamin B2 gene sequence position (GenBank accession number: M94363). The 3′-end of the Lamin B2 and of the 5′-end of the ppv1 gene transcripts are shown along with the position of the Lamin B2 origin. The grey box represents the 74 bp sequence protected in vivo, while the black box indicates the 20 bp sequence corresponding to the downstream binding site of GST-HOXC13 in vitro (see Figure 4). (b) The indicated constructs were cotransfected with plasmids expressing the entire HOXC10, or a portion of HOXC13 from Ser135 to the stop codon, or the same region of HOXC13 fused to the activation domain of the transcription factor VP16. As a control the various CAT plasmids were cotransfected with the sole pCR 3.1 vector. After 48 hours, transfected cells were harvested and the CATase activity was quantified. The stimulatory effect due to the overexpression of the different homeoproteins was calculated as a ratio with the CATase activity measured by cotransfecting the different CAT plasmids with pCR 3.1 vector. Each value is the mean of at least five independent experiments, each consisting of three identical cotransfections. The calculated standard deviation is shown.</note><note type="content">Figure 6: Expression of HoxC10 and HoxC13 genes in different human tissues and cell lines. Autoradiograms of commercial Northern blots of poly(A)+ RNAs, extracted from different adult or foetal tissues, hybridised to HoxC10 or HoxC13 cDNA probes ((a) and (b), respectively). Molecular mass markers are indicated on the left. (c) Total RNA (10 μg) was extracted from HeLa S3, U937 and K562 cells and analysed by Northern blotting with HoxC10 and HoxC13 cDNA probes.</note><note type="content">Figure 7: HOXC10 protein is not present in quiescent or in differentiated murine muscular C2C12 cells. About 50 μg of total proteins from exponentially growing (G) or serum-arrested (Q) C2C12 myoblasts or from differentiated (D) C2C12 myotubes were fractionated on SDS-10 % PAGE and analysed by Western blot to assess, with specific antibodies, the steady-state levels of HOXC10, cyclin A and MyoD (see Materials and Methods). The expression levels of cyclin A and MyoD are shown as controls of differentiation and/or serum starvation (see the text). The actin level is shown as a relative measure of the amount of loaded proteins.</note><subject><genre>article-category</genre><topic>Regular article</topic></subject><subject lang="en"><genre>Keywords</genre><topic>DNA replication origin</topic><topic>homeoproteins</topic><topic>one-hybrid</topic><topic>Lamin B2</topic></subject><subject lang="en"><genre>Abbreviations</genre><topic>ORC : origin recognition complex</topic><topic>3′-UTR : 3′ untranslated region</topic><topic>ORF : open reading frame</topic><topic>EMSA : electrophoretic mobility shift assay</topic><topic>CAT : chloramphenicol acetyl transferase</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>YJMBI</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued></originInfo><identifier type="ISSN">0022-2836</identifier><identifier type="PII">S0022-2836(00)X0192-1</identifier><part><date>2000</date><detail type="volume"><number>299</number><caption>vol.</caption></detail><detail type="issue"><number>3</number><caption>no.</caption></detail><extent unit="issue-pages"><start>551</start><end>842</end></extent><extent unit="pages"><start>667</start><end>680</end></extent></part></relatedItem><identifier type="istex">0CCB1215FCD742084270E7AD438918B3D149F1B4</identifier><identifier type="ark">ark:/67375/6H6-V2HQS6HC-C</identifier><identifier type="DOI">10.1006/jmbi.2000.3782</identifier><identifier type="PII">S0022-2836(00)93782-3</identifier><accessCondition type="use and reproduction" contentType="copyright">©2000 Academic Press</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Academic Press, ©2000</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-V2HQS6HC-C/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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