FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2115121837> ?p ?o ?g. }

• W2115121837 endingPage "13636" @default.
• W2115121837 startingPage "13628" @default.
• W2115121837 abstract "The structural organization of theDrosophila melanogaster gene encoding mitochondrial single-stranded DNA-binding protein (mtSSB) has been determined and its pattern of expression evaluated during Drosophiladevelopment. The D. melanogaster mtSSB gene contains four exons and three small introns. The transcriptional initiation site is located 22 nucleotides upstream from the initiator translation codon in adults, whereas several initiation sites are found in embryos. No consensus TATA or CAAT sequences are located at canonical positions, although an AT-rich sequence was identified flanking the major transcriptional initiation site. Northern analyses indicated that themtSSB transcript is present at variable levels throughout development. In situ hybridization analysis shows that maternally deposited mtSSB mRNA is distributed homogeneously in the early embryo, whereas de novotranscript is produced specifically at an elevated level in the developing midgut. Transfection assays in cultured Schneider cells with promoter region deletion constructs revealed that the proximal 230 nucleotides contain cis-acting elements required for efficient gene expression. Putative transcription factor binding sites clustered within this region include two Drosophila DNA replication-related elements (DRE) and a single putative E2F binding site. Deletion and base substitution mutagenesis of the DRE sites demonstrated that they are required for efficient promoter activity, and gel electrophoretic mobility shift analyses showed that DRE binding factor (DREF) binds to these sites. Our data suggest strongly that theDrosophila mtSSB gene is regulated by the DRE/DREF system. This finding represents a first link between nuclear and mitochondrial DNA replication. The structural organization of theDrosophila melanogaster gene encoding mitochondrial single-stranded DNA-binding protein (mtSSB) has been determined and its pattern of expression evaluated during Drosophiladevelopment. The D. melanogaster mtSSB gene contains four exons and three small introns. The transcriptional initiation site is located 22 nucleotides upstream from the initiator translation codon in adults, whereas several initiation sites are found in embryos. No consensus TATA or CAAT sequences are located at canonical positions, although an AT-rich sequence was identified flanking the major transcriptional initiation site. Northern analyses indicated that themtSSB transcript is present at variable levels throughout development. In situ hybridization analysis shows that maternally deposited mtSSB mRNA is distributed homogeneously in the early embryo, whereas de novotranscript is produced specifically at an elevated level in the developing midgut. Transfection assays in cultured Schneider cells with promoter region deletion constructs revealed that the proximal 230 nucleotides contain cis-acting elements required for efficient gene expression. Putative transcription factor binding sites clustered within this region include two Drosophila DNA replication-related elements (DRE) and a single putative E2F binding site. Deletion and base substitution mutagenesis of the DRE sites demonstrated that they are required for efficient promoter activity, and gel electrophoretic mobility shift analyses showed that DRE binding factor (DREF) binds to these sites. Our data suggest strongly that theDrosophila mtSSB gene is regulated by the DRE/DREF system. This finding represents a first link between nuclear and mitochondrial DNA replication. mitochondrial single-stranded DNA-binding protein avian myeloblastosis virus polymerase chain reaction kilobase pairs DNA replication-related element(s) DRE-binding factor base pair(s) Rous sarcoma virus β-galactosidase Animal mitochondria are essential energy-producing organelles that contain multiple copies of their small double-stranded DNA genome (1.Shadel G.S. Clayton D.A. Annu. Rev. Biochem. 1997; 66: 409-435Crossref PubMed Scopus (814) Google Scholar). Despite its limited coding capacity, mtDNA is critical because it encodes 13 polypeptides that are essential components of mitochondrial respiratory complexes. Biogenesis of mitochondria requires expression and duplication of the mtDNA genome and relies heavily on the nuclear genome, which provides all of the protein components required for these processes and also those involved in their regulation. Mitochondrial DNA polymerase (DNA polymerase γ) and mitochondrial single-stranded DNA-binding protein (mtSSB)1are key nuclear encoded components of the mtDNA replication apparatus: DNA polymerase γ is the replicative DNA polymerase (2.Bertazzoni U. Scovassi A.I. Brun G.M. Eur. J. Biochem. 1977; 81: 237-248Crossref PubMed Scopus (91) Google Scholar, 3.Bolden A. Noy G.P. Weissbach A. J. Biol. Chem. 1977; 252: 3351-3356Abstract Full Text PDF PubMed Google Scholar, 4.Hubscher U. Kuenzle C.C. Spadari S. Eur. J. Biochem. 1977; 81: 249-258Crossref PubMed Scopus (67) Google Scholar), and mtSSB functions in helix destabilization (5.Van Tuyle G.C. Pavco P.A. J. Biol. Chem. 1981; 256: 12772-12779Abstract Full Text PDF PubMed Google Scholar, 6.Van Tuyle G.C. Pavco P.A. J. Cell Biol. 1985; 100: 251-257Crossref PubMed Scopus (45) Google Scholar) and in enhancing both the activity and processivity of DNA polymerase γ (7.Thommes P. Farr C.L. Marton R.F. Kaguni L.S. Cotterill S. J. Biol. Chem. 1995; 270: 21137-21143Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8.Farr C.L. Wang Y. Kaguni L.S. J. Biol. Chem. 1999; 274: 14779-14785Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In vivo, mtSSB is associated with the mitochondrial nucleoid (9.Barat M. Rickwood D. Dufresne C. Mounolou J.-C. Exp. Cell Res. 1985; 157: 207-217Crossref PubMed Scopus (19) Google Scholar) and is concentrated within the perinuclear mitochondria that constitute active sites of mtDNA replication (10.Schultz R.A. Swoap S.J. McDaniel L.D. Zhang B. Koon E.C. Garry D.J. Li K. Williams R.S. J. Biol. Chem. 1998; 273: 3447-3451Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 11.Davis A.F. Clayton D.A. J. Cell Biol. 1996; 135: 883-893Crossref PubMed Scopus (199) Google Scholar). Its role is critical in replication because deletion of the yeast protein causes loss of mtDNA (12.Van Dyck E. Foury F. Stillman B. Brill S.J. EMBO J. 1992; 11: 3421-3430Crossref PubMed Scopus (147) Google Scholar). mtSSB shares both structural and functional similarities withEscherichia coli SSB (13.Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (445) Google Scholar, 14.Lohman T.M. Ferrari M.E. Annu. Rev. Biochem. 1994; 63: 527-570Crossref PubMed Scopus (528) Google Scholar). Both are homotetrameric proteins composed of 13–16-kDa polypeptides with similar DNA binding and replication properties. mtSSB has been purified from several sources including rat (15.Pavco P.A. Van Tuyle G.C. J. Cell Biol. 1985; 100: 258-264Crossref PubMed Scopus (32) Google Scholar, 16.Hoke G.D. Pavco P.A. Ledwith B.J. Van Tuyle G.C. Arch. Biochem. Biophys. 1990; 282: 116-124Crossref PubMed Scopus (45) Google Scholar), Xenopus (17.Mignotte B. Barat M. Mounolou J.C. Nucleic Acids Res. 1985; 13: 1703-1716Crossref PubMed Scopus (51) Google Scholar), yeast (12.Van Dyck E. Foury F. Stillman B. Brill S.J. EMBO J. 1992; 11: 3421-3430Crossref PubMed Scopus (147) Google Scholar), and Drosophila (7.Thommes P. Farr C.L. Marton R.F. Kaguni L.S. Cotterill S. J. Biol. Chem. 1995; 270: 21137-21143Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). cDNAs have been cloned and characterized from Xenopus (18.Ghrir R. Lecaer J.P. Dufresne C. Gueride M. Arch. Biochem. Biophys. 1991; 291: 395-400Crossref PubMed Scopus (25) Google Scholar, 19.Tiranti V. Barat-Gueride B. Bijl J. DiDonato S. Zeviani M. Nucleic Acids Res. 1991; 19: 4291Crossref PubMed Scopus (27) Google Scholar), yeast (12.Van Dyck E. Foury F. Stillman B. Brill S.J. EMBO J. 1992; 11: 3421-3430Crossref PubMed Scopus (147) Google Scholar), rat and man (20.Tiranti V. Rocchi M. DiDonato S. Zeviani M. Gene (Amst.). 1993; 126: 219-225Crossref PubMed Scopus (93) Google Scholar), Drosophila (21.Stroumbakis N.D. Li Z. Tolias P.P. Gene (Amst.). 1994; 143: 171-177Crossref PubMed Scopus (67) Google Scholar), and mouse (22.Li K. Williams R.S. J. Biol. Chem. 1997; 272: 8686-8694Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Comparatively little is known about the structure of the mtSSB gene and its regulation. mtSSB genes in Xenopus (23.Champagne A. Dufresne C. Viney L. Gueride M. Gene (Amst.). 1997; 184: 65-71Crossref PubMed Scopus (4) Google Scholar) and rat (24.Gupta S. Van Tuyle G.C. Gene (Amst.). 1998; 212: 269-278Crossref PubMed Scopus (8) Google Scholar) have been isolated and their promoters shown to share some regulatory elements including those that bind nuclear respiratory factors and the Sp1 transcription factor. mtSSB gene expression correlates with the mtDNA level in mammalian tissues and is up-regulated during mitochondrial biogenesis (10.Schultz R.A. Swoap S.J. McDaniel L.D. Zhang B. Koon E.C. Garry D.J. Li K. Williams R.S. J. Biol. Chem. 1998; 273: 3447-3451Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), but the molecular mechanisms orchestrating this regulation are unknown. To understand the molecular mechanism of mtSSB expression, we have cloned and determined the organization of the Drosophila mtSSB gene. We have carried out a functional analysis of the promoter region and discovered a link between mtSSBexpression and that of several nuclear replication genes. The use ofDrosophila as an animal model also gave us the opportunity to study the expression of mtSSB during development and spatial distribution of its transcript during embryogenesis. This opens several new avenues for evaluation of the in vivo regulation of the mtSSB gene. Unlabeled deoxyribonucleoside triphosphates were purchased from Amersham Pharmacia Biotech. [α-32P]dATP and [γ-32P]ATP were purchased from ICN Biochemicals. Plasmid DNAs were prepared by standard laboratory methods. Synthetic oligodeoxynucleotides were synthesized in an Applied Biosystems model 477 oligonucleotide synthesizer. E. coliNM621 (25.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) was used for screening a λEMBL3 genomic DNA library fromDrosophila melanogaster. E. coli DH5α (25.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) was used to subclone the mtSSB gene for DNA sequence analysis. Schneider S2 cells, cell culture medium, and fetal bovine serum were purchased from Life Technologies, Inc. Penicillin G and streptomycin sulfate were from Sigma. E. coli DNA polymerase I and its Klenow fragment were purchased from New England Biolabs. T4 polynucleotide kinase, RNase-free DNase I, T7 RNA polymerase, and T3 RNA polymerase were from Roche Molecular Biochemicals. T4 DNA ligase and avian myeloblastosis virus (AMV) reverse transcriptase were from Life Technologies, Inc. and Promega, respectively. Isopropyl-1-thio-β-d-galactopyranoside, 5-bromo-4-chloro-3-indolyl-β-d-galactoside, nitro blue tetrazolium, and 5-bromo-4-chloro-3-indolyl phosphate were purchased from Research Organics, Inc.o-Nitrophenyl-β-d-galactoside and RNase inhibitor were from Roche Molecular Biochemicals. A λEMBL3D. melanogaster genomic DNA library was screened usingD. melanogaster mtSSB cDNA as a probe. The probe was amplified by PCR using as a template pET-11a containing themtSSB cDNA and the primers 5′-GCCGGCACATATGGCAACAACAACAACGG-3′ (corresponding to positions 46 to 74, numbered as described (21.Stroumbakis N.D. Li Z. Tolias P.P. Gene (Amst.). 1994; 143: 171-177Crossref PubMed Scopus (67) Google Scholar)) and 5′-GTTTGGTCATATGGGCTTAAATTTTAGTT-3′ (corresponding to positions 456 to 428). The PCR-amplified DNA fragment was purified by gel electrophoresis and 5′-end labeled with [γ-32P]ATP using T4 polynucleotide kinase. Approximately 5 × 105 plaques were transferred to Zeta-Probe filters (Bio-Rad), hybridized at 65 °C with the radiolabeled probe in ZAP buffer (0.25 m phosphate buffer, pH 7.2, 7% SDS), washed in 0.5% SDS, 1 × SSC (0.15m NaCl, 0.015 m sodium citrate) at 65 °C, and the filters were autoradiographed with intensifying screens at −70 °C. Several positive plaques were subjected to two additional rounds of screening, and three of them were amplified and analyzed further. Phage DNAs were analyzed by restriction endonuclease digestion and Southern blot analysis, which showed that two were identical, and the third contained an overlapping phage DNA fragment from the same genomic region. A 3.5-kb SalI fragment that hybridized strongly with the probe was cloned into pUC1193. The recombinant fragment in the resultant plasmid, pUC3.5-SalI, containing the coding sequence and 1.7 kb of the 5′-flanking region, was sequenced in its entirety on both DNA strands by automated fluorescent DNA sequencing using the Applied Biosystems Catalyst 800 for Taqcycle sequencing and a model 373 DNA sequencer for the analysis of products. The complete sequence was assembled using the Sequencer version 2.1.1 software package. In Southern analyses, genomic DNA (10 μg) was digested with EcoRI,BamHI, XbaI, and HindIII, electrophoresed in a 0.8% agarose gel, and transferred to Zeta-Probe filters. The filters were probed with a radiolabeled 3.5-kbSalI fragment from the mtSSB genomic DNA clone and washed in 0.5% SDS, 1 × SSC at 65 °C. Filters were autoradiographed with intensifying screens at −70 °C. In Northern analyses, total RNA (10 μg) extracted as described fromD. melanogaster (Oregon R) at various stages (26.Yamaguchi M. Hayashi Y. Matsukage A. J. Biol. Chem. 1995; 270: 25159-25165Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) was electrophoresed in 1.2% agarose, 1.8 m formaldehyde gels and blotted to Zeta-Probe filters. The filters were probed in ZAP buffer at 65 °C using the radiolabeled mtSSB cDNA clone as probe, washed in 0.5% SDS, 1 × SSC at 65 °C, and autoradiographed with intensifying screens at −70 °C. Exposure times were adjusted to yield a linear response, and the data were quantitated by densitometric scanning. The data were normalized to total RNA content determined by ethidium bromide staining and quantitation. In situ hybridization toyw67 embryos was carried out as described by Tautz and Pfeifle (27.Tautz D. Pfeifle C. Chromosoma (Berl.). 1989; 98: 81-85Crossref PubMed Scopus (2090) Google Scholar). Staged embryos were dechorionated in bleach for 2.5 min and transferred to a tube containing 3 ml of fixing buffer (phosphate-buffered saline, pH 8, 66 mm EGTA), 1 ml of 37% formaldehyde, and 4 ml of heptane. After vigorous shaking for 25 min, the lower aqueous phase was aspirated, and methanol was added to remove the vitelline membrane, followed by vigorous agitation for 60 s. The solution was then aspirated, and the embryos were washed several times with methanol and stored at −20 °C. Prehybridization and hybridization treatments followed by staining and mounting were performed as described (27.Tautz D. Pfeifle C. Chromosoma (Berl.). 1989; 98: 81-85Crossref PubMed Scopus (2090) Google Scholar). Antisense and sensemtSSB riboprobes were prepared by in vitrotranscription using as template pBluescript containing themtSSB cDNA linearized by digestion with SalI (antisense) or SacI (sense), and digoxigenin-labeled UTP (Roche Molecular Biochemicals). The transcription reaction (10 μl) containing 1 × transcription buffer and digoxigenin RNA labeling mix (Roche Molecular Biochemicals), DNA (1 μg), 40 units of RNase inhibitor, and 20 units of T7 RNA polymerase (antisense) or T3 RNA polymerase (sense) was incubated for 2 h at 37 °C. Water (15 μl) was added followed by 25 μl of 2 × carbonate buffer (200 mm NaCO3, pH 10.2), and the RNA was partially hydrolyzed to shorten the probe length by heating for 40 min at 65 °C. The reaction was terminated with the addition of 0.1m NaOAc, pH 6.0 (50 μl). The RNA probe was precipitated by the addition of LiCl to 0.4 m, 100 μg of E. coli tRNA, and 2 volumes of ethanol, dissolved in 150 μl of hybridization buffer and stored at −20 °C. The riboprobe was heated for 3 min at 80 °C before use. Hybridization was carried out overnight at 55 °C in a buffer containing 50% deionized formamide, 5 × SSC, 100 μg/ml sonicated salmon sperm DNA, 50 μg/ml heparin, and 0.1% Tween 80. The digoxigenin-labeled mtSSBprobes were detected using digoxigenin-UTP antibody (Roche Molecular Biochemicals) coupled to alkaline phosphatase, and the reaction was visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. The oligonucleotide 5′-CGCCAACTCGTCCCAAAATG-3′ (5 pmol), complementary to nucleotide positions 274 to 255 (see Fig. 1), was 5′-end labeled with T4 polynucleotide kinase and annealed to 30 μg of total RNA isolated from 0–20-h embryos and adults, by heating the reaction mixture for 20 min at 65 °C in 10 μl of 10 mm Tris-HCl, pH 8.5, 120 mm KCl. The extension reaction was performed in buffer containing 10 mm Tris-HCl, pH 8.5, 120 mm KCl, 10 mm MgCl2, and 5 mmdithiothreitol, in the presence of 0.8 mm dNTPs, 20 units of AMV reverse transcriptase, and 40 units of RNase inhibitor. After incubation for 2 h at 42 °C, the RNA·DNA hybrid was ethanol precipitated and resuspended in loading buffer (98% formamide, 0.5% SDS, 25 mm EDTA, 0.02% xylene cyanol, 0.02% bromphenol blue), heated for 10 min at 70 °C, and electrophoresed in a 6% polyacrylamide, 7 m urea sequencing gel. The product sizes were determined by comparison with a DNA sequencing ladder generated with the same oligonucleotide primer and mtSSB cDNA cloned in pBluescript as the substrate. A set of 5′- and 3′-end deletion mutants was fused to the luciferase reporter gene in the vector pXp1. A DNA fragment containing the upstream region from positions −1141 to +22 of the mtSSB gene was generated by PCR using as a template pUC3.5-SalI and the primers 5′-GTGTCGTCATCCTCATCG-3′ (lsk 120; corresponding to positions −1197 to −1180, see Fig. 1) and 5′-AATAAGCTTGCGCCTTGTGT-GTTGCATCTT-3′ (lsk 117; corresponding to positions 22 to 2, and containing a HindIII cleavage site at its 5′-end). The PCR product was purified by gel electrophoresis, digested with SacI and HindIII, and cloned into pBluescript to generate pBS mtSSB-S/H. A set of deletion fragment derivatives of plasmid pBS mtSSB-S/H was then generated by digestion with AccI, EcoRI, orClaI. The ends of the linear DNAs were rendered blunt ended by end filling with DNA polymerase I Klenow fragment and then cleaved with SacI and HindIII, gel purified, and cloned into pXp1. DNA sequence analysis of the various plasmid constructs was performed to confirm their structure and sequence integrity. Four oligonucleotides were used as primers for PCR-based mutagenesis reactions (base substitution mutations in the DRE sequences are underlined): −23mut, 5′-TATCATCGATACATtagTATTTTATTAATACAATC-3′; −23mutc, 5′-AATAAAATActaATGTATCGATGATATTTACGATA-3′; −31mut, 5′-TATCGTAAATATCATtagTACATCGATATTTTATTAAT-3′; and −31mutc, 5′-ATGTActaATGATATTTACGATATGTAAACTTTTA-3′. To construct the plasmid containing base substitution mutations in the DRE −23 site, two PCRs were performed using as template pBS mtSSB-S/H. The first reaction used the primers 5′-GTCACAGTGGCGCCCTCAGCA-3′ (lsk 116; corresponding to positions −470 to −450, see Fig. 1) and −23mutc (corresponding to positions −9 to −43); the second reaction used primers lsk 117 and −23mut (corresponding to positions −34 to +1). The two PCR products were purified by gel electrophoresis and then used as template for a subsequent PCR with primers lsk 117 and lsk 116. The resulting PCR product was digested with EcoRI, rendered blunt ended, cleaved with HindIII, and then purified by gel electrophoresis and cloned into pXp1 to generate pxp mtSSB-E/DRE-23*. The construct pxp mtSSB-E/DRE-31* was generated in the same way but using the primers −31mut (corresponding to positions −43 to −6) and −31mutc (corresponding to positions −21 to −55) instead of −23mut and −23mutc. DRE binding site deletion mutants were generated as follows. pBS mtSSB-S/H was digested with ClaI, and the linearized plasmid was purified by gel electrophoresis. The ClaI ends were either self-ligated to generate pBS mtSSB-S/H/ΔDRE containing only one DRE site, or they were rendered blunt ended to generate a 2-bp insertional mutation at the center of the DRE site following self-ligation, generating pBS mtSSB-S/H/ΔDRE+DRE* containing no DRE sites. Both plasmids were digested with EcoRI, and the resulting fragments were rendered blunt ended and then cleaved withHindIII, gel purified, and cloned into pXp1 to generate pxp mtSSB-E/ΔDRE and pxp mtSSB-E/ΔDRE+DRE*. DNA sequence analysis of the various plasmid constructs was performed to confirm their structure and sequence integrity. Transient transfection in Schneider S2 cells was performed as described by Soeller et al. (28.Soeller W.C. Oh C.E. Kornberg T.B. Mol. Cell. Biol. 1993; 13: 7961-7970Crossref PubMed Scopus (146) Google Scholar) with some modifications. Streptomycin (100 μg/ml) and penicillin G (100 IU/ml) were added to the medium. Transfection reactions contained the control vector pRSV-βGAL (15 μg) with the various pXp1 constructs (5 μg). After transfection, cells were incubated for 24 h at 25 °C, washed twice with phosphate-buffered saline, resuspended in fresh medium (5 ml), and incubated for 48 h at 25 °C. To prepare extracts, cells were harvested by centrifugation, washed with phosphate-buffered saline followed by TEN buffer (40 mm Tris-HCl, pH 7.5, 1 mm EDTA, 150 mm NaCl), resuspended in 67 mm Tris-HCl, pH 7.5 (0.1 ml), and lysed by four cycles of freezing for 60 s at −70 °C followed by heating for 60 s at 37 °C. Cell debris was removed by centrifugation at 16,000 × g. To normalize the luciferase activity among transfection reactions, the β-galactosidase activity of the cotransfected plasmid was measured. To do so, the cell lysates (30 μl) were incubated in 0.1m sodium phosphate, pH 7.5, 1 mmMgCl2, 45 mm β-mercaptoethanol, and 0.88 mg/ml o-nitrophenyl-β-d-galactoside at 37 °C, and stopped by the addition of 0.5 ml of 1 mNa2CO3. Promega substrate was then used for luciferase assays according to the manufacturer's instructions. All transient transfection assays were performed at least three times. dref cDNA was obtained by reverse transcription PCR. Total RNA from D. melanogaster was extracted using Trizol (Life Technologies, Inc.) and treated for 30 min with RNase-free DNase I (1 unit/μg of RNA). RNA (1 μg) was heated for 5 min at 70 °C and then used in a reverse transcription reaction containing oligo(dT) (1 μg) as primer, 1 mm dNTPs, 0.5 mm dithiothreitol, 40 units of RNase inhibitor, 1 × AMV reverse transcriptase buffer (Promega) and 40 units of AMV reverse transcriptase (Promega). The reaction was incubated for 1 h at 42 °C and then heated for 5 min at 95 °C and chilled on ice. Amplification of the dref cDNA (5 μl) was carried out by PCR using the primers 5′-AAGTACGAGGATGTGTCGCAG-3′ (corresponding to positions 52 to 72, numbered as in GenBank AB010823) and 5′-CGAATTGCCTAGGACTTGACG-3′ (corresponding to positions 783 to 763). A D. melanogaster cDNA library prepared in λ-ZAP (Stratagene) was screened under high stringency conditions using radiolabeled dref cDNA as a probe. Several positive phages were purified and their corresponding pBluescript clones recovered using the R408 helper phage according to the manufacturer's instructions (Stratagene). DNA sequence analysis of the plasmids was performed to confirm their structure and sequence integrity. Radiolabeled DREF protein was produced by coupled in vitrotranscription and translation of pBluescript containing the full-lengthdref cDNA as template and the rabbit reticulocyte lysate system (Promega) according to the manufacturer's instructions. For preparation of DREF antiserum, the DREF cDNA was subcloned for bacterial overexpression. Rabbit antiserum was generated using gel-purified protein as described by Wang et al. (29.Wang Y. Farr C.L. Kaguni L.S. J. Biol. Chem. 1997; 272: 13640-13646Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In gel mobility shift assays, recombinant DREF protein or nuclear extracts from Schneider cells (5 μg of total protein) were incubated for 30 min on ice in the presence of a 5′-end-labeled double-stranded oligonucleotide (0.03 pmol) containing the DRE sequence (5′-GATCCGTAAATATCATCGATACATCGATATTTTATTAA-3′, mtSSB-DRE) and 0.5 μg of poly(dI-dC) (Amersham Pharmacia Biotech), in 20 μl of 20 mm HEPES, pH 8.0, 200 mm KCl, 5 mmMgCl2, 20% glycerol, 1 mm dithiothreitol, 0.2 mm EDTA. The following competitor oligonucleotides were included in the reaction mixtures where indicated in the figure legends: mtSSB-DRE, as above; mut −23, an oligonucleotide containing three base substitutions within the DRE site at position −23, 5′-GATCCGTAAATATCATCGATACATtagTATTTTATTAA-3′; mut −31, an oligonucleotide containing three base substitutions within the DRE site at position −31, 5′-GATCCGTAAATATCATtagTACATCGATATTTTATTA-3′; mut −23/−31, an oligonucleotide containing two 3-base substitutions within the two DRE sites, 5′- GATCCGTAAATATCATtagTACATtagTATTTTATTAA-3′. In supershift assays, rabbit antiserum against recombinant DREF protein (2 μl of a 1:100 dilution) was added after 15 min of incubation, and the samples were incubated for an additional 15 min. After incubation the samples were electrophoresed in a 6% native polyacrylamide gel in 0.5 × TBE, and the gel was dried and autoradiographed. A genomic DNA clone of D. melanogaster mtSSB was isolated by screening a λEMBL3 genomic library under high stringency conditions using the full-length cDNA (21.Stroumbakis N.D. Li Z. Tolias P.P. Gene (Amst.). 1994; 143: 171-177Crossref PubMed Scopus (67) Google Scholar) as a probe. Several independent positive clones were identified and plaque purified, and the largest of ∼13 kilobase pairs in length was examined by restriction endonuclease digestion and Southern blot hybridization. Restriction fragments hybridizing to the probe were cloned into pUC1193 and sequenced. The complete nucleotide sequence of the D. melanogaster mtSSBgene is shown in Fig. 1. Comparison of the genomic DNA sequence with that of the cDNA established its organization. The mtSSB gene contains four exons and three small introns. The nucleotide sequence of the exons is 100% identical to that of the corresponding regions of the cDNA (21.Stroumbakis N.D. Li Z. Tolias P.P. Gene (Amst.). 1994; 143: 171-177Crossref PubMed Scopus (67) Google Scholar). All intron/exon boundaries contain conserved GT and AG sequences at their donor and acceptor sites, respectively. The mitochondrial presequence of 16 amino acids (7.Thommes P. Farr C.L. Marton R.F. Kaguni L.S. Cotterill S. J. Biol. Chem. 1995; 270: 21137-21143Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) is interrupted by the first intron, immediately after the translational initiator ATG codon. The sequence flanking the initiator codon (GCGC) does not match the consensus sequence forDrosophila initiator codons (C/A AA A/C (30.Cavener D.R. Ray S.C. Nucleic Acids Res. 1991; 19: 3185-3192Crossref PubMed Scopus (526) Google Scholar)); this lack of conservation has been found in other Drosophila genes (31.Cavener D.R. Nucleic Acids Res. 1987; 15: 1353-1361Crossref PubMed Scopus (743) Google Scholar). A Southern analysis was performed to examine the possibility of multiple mtSSB genes in the haploid genome. TotalDrosophila DNA was digested with restriction endonucleasesEcoRV and BamHI and subjected to Southern blot analysis using a 3.5-bp SalI fragment from the genomic DNA clone as a probe. The sizes of fragments detected corresponded to those expected from the restriction map of the genomic clone (data not shown), indicating that the mtSSB gene is present inD. melanogaster in a single copy. To initiate our studies of the transcriptional regulation of Drosophila mtSSB, we examined the developmental pattern of gene expression by Northern and RNA in situ hybridization analyses. Northern analysis was performed using total RNA derived from various embryonic, larval, and pupal stages, and from adult flies. We detected a single transcript of 0.5 kb in length, corresponding in size to that expected from the single copy gene (Fig. 2). ThemtSSB transcript is abundant in the earliest embryos, most likely because of active ovarian transcription that results in deposition of a high level of maternal transcript in the oocyte. The steady-state level decreases slightly during early embryonic development and increases in later embryos (9–12 h) when mtDNA replication resumes (32.Rubenstein J.L. Brutlag D. Clayton D.A. Cell. 1977; 12: 471-482Abstract Full Text PDF PubMed Scopus (37) Google Scholar). An elevated transcript level was also observed in the larval stages, where active cell proliferation occurs in various tissues such as the digestive tract. In contrast, the pupal stages show a relatively low level of the mtSSB transcript which increases in adults, consistent with a high level in eggs. The spatial and temporal expression pattern of the Drosophila mtSSB gene during embryogenesis was explored by whole-mountin situ hybridization using antisense RNA as a probe. This revealed that mtSSB mRNA is distributed uniformly throughout the early embryo (Fig.3 A); the mRNA is of maternal origin because zygotic transcription begins only 1.5–2 h after egg deposition. The maternal mRNA disappears by cellular blastoderm (Fig. 3 B), and by early stage 12, high levels ofmtSSB expression occur in the anterior and posterior midgut primordia (Fig. 3 C). Dur" @default.
• W2115121837 created "2016-06-24" @default.
• W2115121837 creator A5028183256 @default.
• W2115121837 creator A5029674346 @default.
• W2115121837 creator A5060648910 @default.
• W2115121837 creator A5075116498 @default.
• W2115121837 date "2000-05-01" @default.
• W2115121837 modified "2023-09-29" @default.
• W2115121837 title "Regulation of Mitochondrial Single-stranded DNA-binding Protein Gene Expression Links Nuclear and Mitochondrial DNA Replication inDrosophila" @default.
• W2115121837 cites W10054795 @default.
• W2115121837 cites W1484257762 @default.
• W2115121837 cites W1485248300 @default.
• W2115121837 cites W1506451090 @default.
• W2115121837 cites W1515500852 @default.
• W2115121837 cites W154063391 @default.
• W2115121837 cites W1541993086 @default.
• W2115121837 cites W1567457876 @default.
• W2115121837 cites W1573917001 @default.
• W2115121837 cites W1579310140 @default.
• W2115121837 cites W1709924179 @default.
• W2115121837 cites W1963882282 @default.
• W2115121837 cites W1967421126 @default.
• W2115121837 cites W1967530571 @default.
• W2115121837 cites W1971567974 @default.
• W2115121837 cites W1981095308 @default.
• W2115121837 cites W1988608516 @default.
• W2115121837 cites W1990017624 @default.
• W2115121837 cites W1992265722 @default.
• W2115121837 cites W1992357330 @default.
• W2115121837 cites W1994352764 @default.
• W2115121837 cites W1995395798 @default.
• W2115121837 cites W2002842695 @default.
• W2115121837 cites W2003217244 @default.
• W2115121837 cites W2005928903 @default.
• W2115121837 cites W2007848978 @default.
• W2115121837 cites W2008444931 @default.
• W2115121837 cites W2009469731 @default.
• W2115121837 cites W2010236625 @default.
• W2115121837 cites W2010463336 @default.
• W2115121837 cites W2014908429 @default.
• W2115121837 cites W2017201927 @default.
• W2115121837 cites W2022236187 @default.
• W2115121837 cites W2022412306 @default.
• W2115121837 cites W2026020843 @default.
• W2115121837 cites W2041495357 @default.
• W2115121837 cites W2042789770 @default.
• W2115121837 cites W2044651119 @default.
• W2115121837 cites W2046573194 @default.
• W2115121837 cites W2052632710 @default.
• W2115121837 cites W2055595737 @default.
• W2115121837 cites W2067405008 @default.
• W2115121837 cites W2078707642 @default.
• W2115121837 cites W2081729236 @default.
• W2115121837 cites W2098992864 @default.
• W2115121837 cites W2102807244 @default.
• W2115121837 cites W2107177579 @default.
• W2115121837 cites W2107308666 @default.
• W2115121837 cites W2116541356 @default.
• W2115121837 cites W2149682982 @default.
• W2115121837 cites W2153436893 @default.
• W2115121837 cites W2178168667 @default.
• W2115121837 cites W2191115168 @default.
• W2115121837 cites W4233723707 @default.
• W2115121837 doi "https://doi.org/10.1074/jbc.275.18.13628" @default.
• W2115121837 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10788480" @default.
• W2115121837 hasPublicationYear "2000" @default.
• W2115121837 type Work @default.
• W2115121837 sameAs 2115121837 @default.
• W2115121837 citedByCount "36" @default.
• W2115121837 countsByYear W21151218372012 @default.
• W2115121837 countsByYear W21151218372013 @default.
• W2115121837 countsByYear W21151218372017 @default.
• W2115121837 crossrefType "journal-article" @default.
• W2115121837 hasAuthorship W2115121837A5028183256 @default.
• W2115121837 hasAuthorship W2115121837A5029674346 @default.
• W2115121837 hasAuthorship W2115121837A5060648910 @default.
• W2115121837 hasAuthorship W2115121837A5075116498 @default.
• W2115121837 hasConcept C104317684 @default.
• W2115121837 hasConcept C113271649 @default.
• W2115121837 hasConcept C115960442 @default.
• W2115121837 hasConcept C153911025 @default.
• W2115121837 hasConcept C24586158 @default.
• W2115121837 hasConcept C54355233 @default.
• W2115121837 hasConcept C552990157 @default.
• W2115121837 hasConcept C73573662 @default.
• W2115121837 hasConcept C86339819 @default.
• W2115121837 hasConcept C86803240 @default.
• W2115121837 hasConcept C94966510 @default.
• W2115121837 hasConcept C95444343 @default.
• W2115121837 hasConceptScore W2115121837C104317684 @default.
• W2115121837 hasConceptScore W2115121837C113271649 @default.
• W2115121837 hasConceptScore W2115121837C115960442 @default.
• W2115121837 hasConceptScore W2115121837C153911025 @default.
• W2115121837 hasConceptScore W2115121837C24586158 @default.
• W2115121837 hasConceptScore W2115121837C54355233 @default.
• W2115121837 hasConceptScore W2115121837C552990157 @default.
• W2115121837 hasConceptScore W2115121837C73573662 @default.
• W2115121837 hasConceptScore W2115121837C86339819 @default.

• next