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A New Spectrin, βIV, Has a Major Truncated Isoform That Associates with Promyelocytic Leukemia Protein Nuclear Bodies and the Nuclear Matrix

2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês

10.1074/jbc.m009307200

ISSN

1083-351X

Autores

William T. Tse, Ju Tang, Ou Jin, Catherine Korsgren, Kathryn M. John, Andrew L. Kung, Babette Gwynn, Luanne L. Peters, Samuel E. Lux,

Tópico(s)

Nuclear Structure and Function

Resumo

We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10–16 of β-spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length β-spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin βIV, thus encodes a full-length minor isoform (SpβIVΣ1) and a truncated major isoform (SpβIVΣ5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpβIVΣ5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpβIVΣ5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin βIV is the first β-spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds. We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10–16 of β-spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length β-spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin βIV, thus encodes a full-length minor isoform (SpβIVΣ1) and a truncated major isoform (SpβIVΣ5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpβIVΣ5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpβIVΣ5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin βIV is the first β-spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds. promyelocytic leukemia acute promyelocytic leukemia cAMP-response element-binding protein CREB-binding protein expressed sequence tag green fluorescence protein Madin-Darby canine kidney cells mesenchymal stem cells a contig of overlapping spectrin βIV clones N164 and N155 non-repeat segment of SpβIVΣ5 nuclear mitotic apparatus protein phosphate-buffered saline polymerase chain reaction retinoic acid receptor alpha radiation hybrid reduced pigmentation mouse mutation room temperature SpβIV-specific antibody small ubiquitin-like modifier protein glutathione S-transferase base pair(s) kilobase(s) 1,4-piperazinediethanesulfonic acid Spectrin is an important component of the membrane skeleton attached to the inner leaf of the lipid bilayer of plasma membranes. First described in the erythrocyte (1Marchesi V.T. Steers Jr., E. Science. 1968; 159: 203-204Crossref PubMed Scopus (257) Google Scholar), spectrins are found in all or almost all cells (2Glenney Jr., J.R. Glenney P. Cell. 1983; 34: 503-512Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 3Burridge K. Kelly T. Mangeat P. J. Cell Biol. 1982; 95: 478-486Crossref PubMed Scopus (186) Google Scholar, 4Repasky E.A. Granger B.L. Lazarides E. Cell. 1982; 29: 821-833Abstract Full Text PDF PubMed Scopus (205) Google Scholar). In erythrocytes, an intact spectrin-based membrane skeleton is critical for the structural integrity of the plasma membrane. Defects in its components are associated with red cell fragility and premature destruction in the human diseases hereditary spherocytosis and elliptocytosis and their animal models (5Tse W.T. Lux S.E. Br. J. Haematol. 1999; 104: 2-13Crossref PubMed Scopus (246) Google Scholar). The function of a spectrin-based plasma membrane skeleton in non-erythroid tissues is less well defined, but it is hypothesized to be important in establishing and maintaining the asymmetric distribution of proteins in specialized plasma membrane domains, particularly in polarized cells (6Rodriguez-Boulan E. Nelson W.J. Science. 1989; 245: 718-725Crossref PubMed Scopus (821) Google Scholar). Recently, components of a spectrin-based membrane skeleton have also been found in several intracellular organelles. Isoforms of spectrin and ankyrin exist in Golgi membranes (7Beck K.A. Buchanan J.A. Malhotra V. Nelson W.J. J. Cell Biol. 1994; 127: 707-723Crossref PubMed Scopus (168) Google Scholar, 8Devarajan P. Stabach P.R. Mann A.S. Ardito T. Kashgarian M. Morrow J.S. J. Cell Biol. 1996; 133: 819-830Crossref PubMed Scopus (161) Google Scholar, 9Stankewich M.C. Tse W.T. Peters L.L. Ch'ng Y. John K.M. Stabach P.R. Devarajan P. Morrow J.S. Lux S.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14158-14163Crossref PubMed Scopus (117) Google Scholar), lysosomal membranes (10Hoock T.C. Peters L.L. Lux S.E. J. Cell Biol. 1997; 136: 1059-1070Crossref PubMed Scopus (67) Google Scholar), and secretory vesicles (11Malchiodi-Albedi F. Ceccarini M. Winkelmann J.C. Morrow J.S. Petrucci T.C. J. Cell Sci. 1993; 106: 67-78PubMed Google Scholar, 12Smith P.R. Bradford A.L. Joe E.H. Angelides K.J. Benos D.J. Saccomani G. Am. J. Physiol. 1993; 264: C63-C70Crossref PubMed Google Scholar, 13Corcoran S.L. Wylie P.G. Hayes N.V. Baines A.J. Thomas H.M. Biochem. Soc. Trans. 1997; 25: 483SCrossref PubMed Scopus (5) Google Scholar, 14Michaely P. Kamal A. Anderson R.G. Bennett V. J. Biol. Chem. 1999; 274: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Spectrin also associates with actin-related protein 1 (centractin), a subunit of the dynactin complex, which associates with dynein and transports vesicles along microtubules in the secretory pathway (15Holleran E.A. Tokito M.K. Karki S. Holzbaur E.L. J. Cell Biol. 1996; 135: 1815-18129Crossref PubMed Scopus (197) Google Scholar). A spectrin-based membrane skeleton attached to intracellular organelles may provide a structural framework to anchor the vesicular transport machinery (16Lippincott-Schwartz J. Curr. Opin. Cell Biol. 1998; 10: 52-59Crossref PubMed Scopus (156) Google Scholar, 17De Matteis M.A. Morrow J.S. Curr. Opin. Cell Biol. 1998; 10: 542-549Crossref PubMed Scopus (119) Google Scholar, 18De Matteis M.A. Morrow J.S. J. Cell Sci. 2000; 113: 2331-2343Crossref PubMed Google Scholar, 19Muresan V. Stankewich M.C. Steffen W. Morrow J.S. Holzbaur E.L. Schnapp B.J. Mol. Cell. 2001; 7: 173-183Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The potential role of a spectrin-based membrane skeleton in the nucleus is unclear. There are interesting recent reports indicating that spectrin αII is part of a nuclear protein complex involved in repair of DNA interstrand cross-links (20Brois D.W. McMahon L.W. Ramos N.I. Anglin L.M. Walsh C.E. Lambert M.W. Carcinogenesis. 1999; 20: 1845-1853Crossref PubMed Scopus (24) Google Scholar, 21McMahon L.W. Walsh C.E. Lambert M.W. J. Biol. Chem. 1999; 274: 32904-32908Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 22Kumaresan K.R. Lambert M.W. Carcinogenesis. 2000; 21: 741-751Crossref PubMed Scopus (50) Google Scholar). Whether αII-spectrin binds with a β-spectrin partner in the nucleus to form a membrane skeleton is unknown. We have previously described spectrin βIII (see footnote 1 for nomenclature), 1Nomenclature: β1Σ1-Spectrin is the erythroid isoform (Σ1) of erythroid (β1) spectrin. It is also called spectrinR. The gene name is SPTB (human) orSpnb1 (mouse). β1Σ2-Spectrin is the muscle isoform (Σ2) of erythroid spectrin. It contains a different C-terminal sequence. βII-Spectrin is "non-erythroid" β-spectrin. It has also been called fodrin, brain spectrin, spectrinG, or spectrin beta, non-erythroid type 1. The gene name is SPTB1(human) or Spnb2 (mouse). βIII-Spectrin has the gene namesSPTBN2 (human) or Spnb3 (mouse), respectively. Spectrin βV (gene name BSPECV) is the recently described mammalian equivalent of Drosophila beta heavy spectrin. Spectrin βIV has at least five isoforms, βIVΣ1 through βIVΣ5. Isoforms βIVΣ1 and βIVΣ5 are discussed in this report. The gene names of spectrin βIV are SPTBN3 (human) andSpnb4 (mouse). which associates with the Golgi and intracellular vesicles (9Stankewich M.C. Tse W.T. Peters L.L. Ch'ng Y. John K.M. Stabach P.R. Devarajan P. Morrow J.S. Lux S.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14158-14163Crossref PubMed Scopus (117) Google Scholar). We now identify another intracellular β-spectrin, spectrin βIV, which has a major truncated isoform (βIVΣ5) and a full-length isoform (βIVΣ1). Spectrin βIV resides in the cytoplasm, where it may attach to vesicles, and in the nucleus, where it associates with PML2 bodies and the nuclear matrix. While this manuscript was in revision, Berghs et al. (23Berghs S. Aggujaro D. Dirkx R. Maksimova E. Stabach P. Hermel J.M. Zhang J.P. Philbrick W. Slepnev V. Ort T. Solimena M. J. Cell Biol. 2000; 151: 985-1002Crossref PubMed Scopus (238) Google Scholar) independently described spectrin βIV and four of its isoforms: βIVΣ1–βIVΣ4. Their βIVΣ1 isoform corresponds to our full-length spectrin βIV, also named βIVΣ1. The 77-kDa isoform (βIVΣ5) described here is the major isoform of spectrin βIV. It was not reported by Berghs et al. and could not have been detected with the antibodies they employed (23Berghs S. Aggujaro D. Dirkx R. Maksimova E. Stabach P. Hermel J.M. Zhang J.P. Philbrick W. Slepnev V. Ort T. Solimena M. J. Cell Biol. 2000; 151: 985-1002Crossref PubMed Scopus (238) Google Scholar). Search of the GenBank™ data bases was performed using the NCBI BLAST similarity search programs. Nucleotide sequence analysis was done using the University of Wisconsin Genetics Computer Group sequence analysis programs. Screening and isolation of cDNA clones were done either by standard methods or by the GeneTrapper cDNA positive-selection method according to the manufacturer's instructions (Life Technologies, Rockville, MD). A hybridization oligonucleotide (5′-CCA ACG CCA CTG CCG CTT-3′) and human brain plasmid cDNA library (Life Technologies) were used in the GeneTrapper method. Automated nucleotide sequencing was performed in the Children's Hospital Mental Retardation Research Center DNA Sequencing Core Facility using the dideoxynucleotide termination method. Polymerase chain reaction (PCR), and anchored PCR amplifications were done using the Advantage 2 polymerase kit (CLONTECH Laboratories, Inc., Palo Alto, CA). PCR templates used were Marathon-ready cDNAs prepared from retina or brain (CLONTECH). The cloning strategy and sequences of oligonucleotide primers used in the isolation of overlapping clones that constitute the full-length spectrin βIV cDNA are available upon request. Chromosome localization of the human spectrin βIV gene utilized the Stanford G3 radiation hybrid panel (24Stewart E.A. McKusick K.B. Aggarwal A. Bajorek E. Brady S. Chu A. Fang N. Hadley D. Harris M. Hussain S. Lee R. Maratukulam A. O'Connor K. Perkins S. Piercy M. Qin F. Reif T. Sanders C. She X. Sun W.L. Tabar P. Voyticky S. Cowles S. Fan J.B. Mader C. Quackenbush J. Myers R.M. Cox D.R. Genome Res. 1997; 7: 422-433Crossref PubMed Scopus (280) Google Scholar) (Research Genetics, Huntsville, AL). Hybrid human-hamster clones were assayed for the presence of spectrin βIV gene by PCR. Using the primers 5′-CGG CTG GCA GCT GTG AAC CAG ATG GTG-3′ (forward) and 5′-AAC TGG CAC TGG GTC TCG GCT CAG GC-3′ (reverse), a 268-bp product was generated in clones that carried the spectrin βIV gene. The data were analyzed by the Stanford Human Genome Center RH Server on the Web. Chromosome localization of the mouse gene was done using the Jackson Laboratory BSS Interspecific ((C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei males) backcross panel (25Rowe L.B. Nadeau J.H. Turner R. Frankel W.N. Letts V.A. Eppig J.T. Ko M.S. Thurston S.J. Birkenmeier E.H. Mamm. Genome. 1994; 5: 253-274Crossref PubMed Scopus (628) Google Scholar). A 453-bpPstI fragment of human EST clone AA054636 detected aPstI restriction fragment length polymorphism in the mouse genome corresponding to a 3.7-kb hybridization band in C57BL/6JEi and 4.1-kb band in SPRET/Ei. The segregation pattern of the polymorphism in progeny of the cross was used to determine the map location of the gene. The typing data have been placed in the Mouse Genome Data base (accession number J:63322) and can be accessed on the Web. For Northern analysis, a 1.96-kb fragment of clone N164/N155 corresponding to bp 114–2071 was generated by PCR, subcloned, labeled, and used as a probe in hybridization with mRNA from various mouse tissues blotted on a charged nylon membrane (Multiple Tissue Northern blot, CLONTECH). High stringency hybridization and washes were performed according to the manufacturer's instructions (ExpressHyb,CLONTECH). The membranes were then exposed to Kodak X-OMAT AR film for 4 days to visualize the positive signals. For PCR analysis, complementary DNAs (1 ng) from various mouse tissues (Mouse Multiple Tissue cDNA Panel, CLONTECH; and Multiple Choice cDNAs, OriGene Technologies, Inc., Rockville, MD) were analyzed by PCR (94 °C, 40 s; 60 °C, 45 s; 72 °C, 3 min; 38 cycles) using the primers: 5′-CAA GCC CAG GTG CCC CTC-3′ (forward) and 5′-GTT GTC ATT CCA TTG AGA AG-3′ (reverse). The 107-bp product, representing the unique C-terminal sequence of spectrin βIVΣ5, was analyzed by agarose gel electrophoresis. Whole mount in situ hybridization was done according to published protocols (26Conlon R.A. Rossant J. Development. 1992; 116: 357-368Crossref PubMed Google Scholar). Briefly, day 9.5 mouse embryos were isolated, fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and bleached in methanol and hydrogen peroxide. They were then treated with proteinase K in PBS containing 0.1% Tween 20, refixed in 0.2% glutaraldehyde and 4% paraformaldehyde in PBS, and hybridized with digoxigenin-labeled RNA probes at 63 °C overnight. After high stringency washes, the embryos were incubated sequentially with alkaline phosphatase-conjugated anti-digoxigenin antibodies and 5-bromo-4-chloro-3-indoyl-phosphate chromogenic substrate for visualization of hybridization signals. The digoxigenin-labeled antisense RNA probes were generated by in vitrotranscription with T7 polymerase using as the template a 1.96-kb fragment of clone N164/N155 (nucleotides 114–2071) generated by PCR and subcloned into a pBluescript SK vector. RNA transcripts in the sense direction were generated with T3 polymerase and used as negative controls. An anti-peptide antiserum to βIV-spectrin, termed SpB4-R15, was produced by immunizing rabbits with a keyhole limpet hemocyanin-conjugated synthetic peptide (Zymed Laboratories Inc., South San Francisco, CA). The peptide sequence was RLTTPPEPRPSASS, corresponding to codons 535–548 of clone N164/N155, a region that contains little homology to spectrins βI, βII, βIII, or βV. The antibodies were affinity-purified by passing through an AminoLink Plus column (Pierce, Rockford, IL) containing a recombinant peptide of βIV-spectrin repeat 15 fused to a GST protein, and eluted with 0.1 m glycine (pH 2.5). A cDNA encoding βIV-spectrin repeat 15 was generated by PCR (forward primer 5′-GCG GGA TCC TCT CGG GAG CTT CAT AAG TTC-3′; reverse primer 5′-GCG GAA TTC GGA GCT GAC ATG CAG GCG GGC-3′) and cloned into the BamHI and EcoRI sites of a pGEX-6P-1 vector (Amersham Pharmacia Biotech, Piscataway, NJ). The recombinant GST-βIV-spectrin peptide was produced in theEscherichia coli BL21 strain and purified with glutathione-Sepharose beads according to the manufacturer's instructions (Amersham Pharmacia Biotech). For Western analysis, mouse tissues were excised and homogenized on ice with a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) in 0.32 m sucrose, 10 mm Tris (pH 8.0), 5 mm N-ethylmalemide, 2 mm EDTA, 5 μg/ml each of protease inhibitors leupeptin, pepstatin, aprotinin, and 0.4 mm diisopropyl fluorophosphate. Protein concentrations were determined by the method of Bradford with bovine serum albumin as the standard. Protein samples (35 μg) were run on a 3.5–17% non-linear gradient Laemmli SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose filter. The filter was incubated with 500 ng/ml affinity-purified SpB4-R15 antibody and goat anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA). Immunoreactive proteins were visualized with Lumi-Light Western blotting Substrate (Roche Molecular Biochemicals, Indianapolis, IN). Molecular sizes of the positive bands were determined by comparison with the molecular weight of red cell membrane proteins (27Fairbanks G. Steck T.L. Wallach D.F. Biochemistry. 1971; 10: 2606-2617Crossref PubMed Scopus (6179) Google Scholar) run in parallel. Experiments using affinity-purified antisera from two rabbits immunized with the βIV-spectrin peptide independently give similar results. The affinity-purified rabbit polyclonal SpB4-R15 antibody was used in immunofluorescence microscopy studies. Canine kidney cells (MDCK), human neuroblastoma cells (SK-N-SH), and green monkey kidney cells (COS-7) were obtained from the American Type Culture Collection, Manassas, VA. Human embryonic kidney 293T and hepatoma Hep3B cells were kind gifts from Drs. Len Zon (Children's Hospital, Boston, MA) and David Livingston (Dana-Farber Cancer Institute, Boston, MA), respectively. Human mesenchymal stem cells were isolated, characterized, and cultured as described (28Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (18235) Google Scholar). For immunofluorescence studies, cells were grown in slide chambers to subconfluency. They were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) at room temperature (RT) for 10 min and permeabilized with 0.1% Triton X-100 in PBS at RT for 10 min. Alternatively, cells were fixed and permeabilized in 100% methanol at −20 ° for 10 min. The cells were then incubated with affinity-purified SpB4-R15 antibody at 1:10 to 1:100 dilutions at RT for 30 min, rinsed with PBS, and incubated with Cy3-conjugated goat anti-rabbit IgG for 30 min at RT. All second stage antibodies were from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA. The cells were then rinsed and mounted in ProLong antifade reagent (Molecular Probes, Inc., Eugene, OR). Double immunofluorescence studies were performed by adding a second mouse antibody in the first incubation step and an fluorescein isothiocyanate-conjugated goat anti-mouse IgG in the second step. The primary antibodies used in double label experiments with the SpB4-R15 antibody included anti-PML (mouse monoclonal PG-M3; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-SUMO-1 (mouse monoclonal 21C7; Zymed Laboratories Inc.). Additional primary antibodies used to stain SpβIVΣ5-transfected cells included anti-nucleoporin p62 (mouse monoclonal 53; Transduction Laboratories, Lexington, KY), anti-CBP (rabbit polyclonal A-2; Santa Cruz Biotechnology), and anti-c-myc (mouse monoclonal 9E10, Santa Cruz Biotechnology). Secondary stage antibodies were Cy3-conjugated goat anti-mouse or anti-rabbit IgG. Antibody dilutions used were empirically determined and ranged between 1:100 and 1:1000. Fluorescence microscopy was done using a Zeiss Axioskop microscope. Microscopic images were taken with Kodak Elite ASA 400 film, digitized with a Nikon CoolScan 2000 slide scanner, and processed with the Adobe Photoshop 5.5 program on a Power Macintosh G3 computer. Confocal microscopy was performed at the Brigham and Women's Hospital Confocal Microscopy Core Facility, using a Bio-Rad MRC-1024/2P confocal microscope interfaced with a Zeiss Axiovert microscope. To make the SpβIVΣ5-green fluorescence protein (SpβIVΣ5-GFP) construct, a fragment of clone N164/N155 was generated by PCR and subcloned into a eukaryotic expression vector pcDNA4/HisMaxA (Invitrogen Corp., Carlsbad, CA) into which a cDNA for the enhanced GFP (CLONTECH) had first been inserted. The primers used in the PCR were DEL1 (forward: 5′-CTG ATG GCG CGG GAT AGC ACG CGG-3′) and DEL3 (reverse: 5′-TTC CAT TGA GAA GGG GGC TGT-3′), and the PCR product corresponded to codons 2–678 of clone N164/N155. The resulting construct encoded a fusion protein consisting of six histidines and an Xpress epitope tag, followed by the SpβIVΣ5 peptide fused in-frame to GFP. The myc-tagged human CREB-binding protein construct was a kind gift of Dr. David Housman (Massachusetts Institute of Technology, Boston, MA). These constructs were transfected into 293T, COS-7, and Hep3B cell lines using LipofectAMINE (Life Technologies) or FuGENE 6 (Roche Molecular Biochemicals) reagents. Expression of the heterologous protein was analyzed after 24–48 h. Deletion constructs of SpβIVΣ5-GFP were generated in a similar fashion, using PCR primer pairs as followed: SpβIVΣ5ΔNR-GFP (DEL1 (forward) and DEL4 (reverse: 5′-CCT GGG CTT GTC GGC TGC CCC-3′)), SpβIVΣ5ΔR16-GFP (DEL1 (forward) and DEL5 (reverse: 5′-GGA GCT GAC ATG CAG GCG GGC-3′)), SpβIVΣ5ΔR10-GFP (DEL2 (forward: 5′-AGG CCA GCA AAG CAG ACC AGC TG-3′) and DEL3 (reverse)) and SpβIVΣ5ΔR15-GFP (DEL1 (forward), DEL6 (forward: 5′-CGG GCC CAG CTG CTG GCC GCC ACA GCC GAC GCC CTG CGC TTC-3′), DEL7 (reverse: 5′-GAA GCG CAG GGC GTC GGC TGT GGC GGC CAG CAG CTG GGC CCG-3′) and DEL3 (reverse)). The deleted domains are: SpβIVΣ5ΔNR-GFP, codons 649–678; SpβIVΣ5ΔR16-GFP, codons 617–678; SpβIVΣ5ΔR10-GFP, codons 2–84; SpβIVΣ5ΔR15-GFP, codons 509–616. The deletion constructs were transfected into mammalian cell lines, and expression of the truncated peptides was analyzed as for the SpβIVΣ5-GFP construct. Preparation of nuclear matrix was performed as published previously (29He D.C. Nickerson J.A. Penman S. J. Cell Biol. 1990; 110: 569-580Crossref PubMed Scopus (369) Google Scholar). Briefly, cells grown on chamber slides were treated sequentially with cytoskeleton buffer (10 mm Pipes (pH 6.8), 100 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 1 mm EGTA, 0.5% Triton X-100, 4 mm vanadyl ribonucleoside complex, 1 mm Pefabloc) at 4 °C for 3 min, 25 units/ml DNase I in nuclease buffer (cytoskeleton buffer with 50 mm NaCl) at RT for 30 min, 0.25 m ammonium sulfate in nuclease buffer at RT for 10 min three times, high salt buffer (nuclease buffer with 2 m NaCl) at RT for 5 min three times, and then 100 μg/ml RNase A and 40 units/ml RNase T1 in nuclease buffer at RT for 60 min. The extracted cells were fixed in methanol at −20 °C for 10 min and immunofluorescence microscopy was performed. Pefabloc, DNase I, RNase A, and RNase T1 were purchased from Roche Molecular Biochemicals; vanadyl ribonucleoside complex was from Life Technologies; all other reagents were from Sigma-Aldrich, St. Louis, MO. Nucleotide sequences of αI-, αII-, βI-, βII-, and βIII-spectrins were used as query sequences to search the GenBank™ EST data base for clones that were similar but not identical to known spectrin chains. One EST clone from a human retinal library was identified that had sequence similarity to repeats 10 and 11 of β-spectrins (GenBank™ accession number AA054636). Screening of bacteriophage cDNA libraries by conventional methods using a 453-bpPstI fragment of this clone as a probe failed to yield positive clones. Subsequently, the GeneTrapper positive selection method was used (Life Technologies). Using sequence information obtained from the clone, an oligonucleotide probe was synthesized, biotinylated, and hybridized with single-stranded DNA from a human brain cDNA library. Six unique clones that contained sequences complementary to the oligonucleotide probe were recovered using streptavidin-coated paramagnetic beads. All of the clones were polyadenylated, but four appeared to be incomplete or partially spliced and are not described here. The complete coding sequence reported here is a composite of the two unique clones, N164 and N155 (Fig.1 A). Clone N164 extends from nucleotide 1–2172 of the composite sequence and is followed by a poly(A) tail. Clone N155 extends from nucleotide 10–2418 and is also polyadenylated. However, neither clone contains a consensus polyadenylation signal close to the polyadenylation site, and both are too short to account for the transcripts observed on Northern blots (described below). We suspect they are minor transcripts and that a significant portion of the 3′-untranslated repeat has not been cloned. Sequence analysis showed that clones N164 and N155 contained an identical 2034-bp open reading frame that potentially encodes a peptide 678 residues in length, with a calculated molecular weight of 77,197 Da (Fig. 1 B). The first methionine in this open reading frame (bp 128–130) was taken as the start codon. The amino acid sequence of this peptide is very similar to repeats 10–16 of other known β-spectrins, with ∼45–65% identity over the repeat domain. Analysis of the secondary structure of this spectrin-like peptide predicts the formation of multiple α-helical coils that fold into triple helical coiled-coil units, a characteristic feature found in all spectrin peptides (30Yan Y. Winograd E. Viel A. Cronin T. Harrison S.C. Branton D. Science. 1993; 262: 2027-2030Crossref PubMed Scopus (337) Google Scholar). The spectrin-like peptide represented by clone N164/N155 is predicted to have five full repetitive motifs (repeats 11–15), flanked on each side by two partial repeats (repeats 10 and 16). Partial repeat 10 consists of two coils of a triple helical coiled-coil unit (helices B and C) and partial repeat 16, a single coil (helix A) (Fig. 1 C). These coils may potentially allow head-to-tail interaction of multiple peptides to form concatemers (Fig.1 D), analogous to the way β- and α-spectrins interact to form heterotetramers. However, in the latter case β-spectrin ends with two helical coils (helices A and B) and α-spectrin starts with a single coil (helix C). The repeat domain of the spectrin-like peptide is followed by a C-terminal, non-repeat domain (NR) of 30 residues, which is rich in proline residues and dissimilar to the C termini of other β-spectrins. No similar sequence is reported in the protein data bases. To show that clones N164 and N155 are transcripts of a new spectrin-like gene and not an alternative transcript of other known β-spectrins, we determined the chromosomal location of the new spectrin-like gene. PCR primers derived from the sequence of clone N164/N155 were used to screen the Stanford G3 human-hamster radiation hybrid genomic DNA panel for the presence or absence of its gene in different cell lines (24Stewart E.A. McKusick K.B. Aggarwal A. Bajorek E. Brady S. Chu A. Fang N. Hadley D. Harris M. Hussain S. Lee R. Maratukulam A. O'Connor K. Perkins S. Piercy M. Qin F. Reif T. Sanders C. She X. Sun W.L. Tabar P. Voyticky S. Cowles S. Fan J.B. Mader C. Quackenbush J. Myers R.M. Cox D.R. Genome Res. 1997; 7: 422-433Crossref PubMed Scopus (280) Google Scholar). Tight linkage (lod score 11.25) of the gene was found to DNA marker SHGC-33106, a part of the biliverdin reductase B gene (also called NADPH-flavin reductase), which maps to chromosome 19q13.13-q13.2 (31Saito F. Yamaguchi T. Komuro A. Tobe T. Ikeuchi T. Tomita M. Nakajima H. Cytogenet. Cell Genet. 1995; 71: 179-181Crossref PubMed Scopus (16) Google Scholar). This chromosomal location differs from other human β-spectrin genes, which are located on chromosomes 2, 11, 14, and 15, indicating that clone N164/N155 is a transcript of a new β-spectrin-like gene. Of note, the chromosome location of this new gene is close to that of α-actinin 4 (32Kaplan J.M. Kim S.H. North K.N. Rennke H. Correia L.A. Tong H.Q. Mathis B.J. Rodriguez-Perez J.C. Allen P.G. Beggs A.H. Pollak M.R. Nat. Genet. 2000; 24: 251-256Crossref PubMed Scopus (1050) Google Scholar). It has been previously shown that the spectrin βI and βIII genes both localize close to an actinin gene (9Stankewich M.C. Tse W.T. Peters L.L. Ch'ng Y. John K.M. Stabach P.R. Devarajan P. Morrow J.S. Lux S.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14158-14163Crossref PubMed Scopus (117) Google Scholar, 33Youssoufian H. McAfee M. Kwiatkowski D.J. Am. J. Hum. Genet. 1990; 47: 62-71PubMed Google Scholar), suggesting that sequential duplications of a chromosome region containing a primordial β-spectrin gene and α-actinin gene may have given rise to the neighboring locations of these genes. We next mapped the location of this spectrin-like gene in the mouse. Analysis of the segregation pattern of a PstI restriction fragment polymorphism in 94 progeny of The Jackson Laboratory BSS interspecific backcross panel (25Rowe L.B. Nadeau J.H. Turner R. Frankel W.N. Letts V.A. Eppig J.T. Ko M.S. Thurston S.J. Birkenmeier E.H. Mamm. Genome. 1994; 5: 253-274Crossref PubMed Scopus (628) Google Scholar) localized the gene to mouse chromosome 7 near the centromere (7.5 centimorgans), a region of the mouse genome homologous to human chromosome 19q13.1. The location of the gene is also different from other known β-spectrin genes, which map to chromosomes 11, 12, and 19. Mouse spectrin βV has not yet been mapped. This gene location approximates the position of the spontaneous mouse mutation reduced pigmentation (rp) (34Gibb S. Hakansson E.M. Lundin L.G.

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