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J Biol Chem, Vol. 275, Issue 18, 13628-13636, May 5, 2000
From the The structural organization of the
Drosophila melanogaster gene encoding mitochondrial
single-stranded DNA-binding protein (mtSSB) has been determined and its
pattern of expression evaluated during Drosophila
development. 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 the
mtSSB 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 novo
transcript 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 the
Drosophila mtSSB gene is regulated by the DRE/DREF system.
This finding represents a first link between nuclear and mitochondrial
DNA replication.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF181084.
Regulation of Mitochondrial Single-stranded DNA-binding Protein
Gene Expression Links Nuclear and Mitochondrial DNA Replication in
Drosophila*
§,
Department of Biochemistry, Michigan State
University, East Lansing, Michigan 48824-1319 and the
§ Instituto de Investigaciones Biomedicas Alberto Sols,
Universidad Autónoma de Madrid-Consejo Superior de
Investigaciones Científicas, Arturo Duperier 4, Madrid 28028, Spain
*
This work was supported in part by National Institutes of
Health Grants GM45295 and HL59656 (to L. S. K.), Grant PB94-0088 from
the Ministerio de Educación y Ciencia, Spain, and European Union
Human Capital and Mobility Program Network Grant CHRX-CT94-0494 (to
R. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
517-353-6703; Fax: 517-353-9334; E-mail: lskaguni@pilot.msu.edu.
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