Nucleic Acids Research, 1994, Vol. 22, No. 21 4432-4440
© 1994
MOLECULAR BIOLOGY |
Identification of DNA-binding proteins that recognize a Conserved Type I repeat sequence in the replication origin region of Tetrahymena rDNA
Department of Zoology and Genetics, Singal Transduction Training Group Ames, IA 50011, USA 1Molecular, Cellular and Developmental Biology Program Ames, IA 50011, USA 2Interdepartmental Genetics Program, 2114 Molecular Biology Building, lowa State University Ames, IA 50011, USA
*To whom correspondence should be addressed
Received July 15, 1994. Revised September 16, 1994. Accepted September 16, 1994.
An origin of DNA replication has been mapped within the 5' non-transcribed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila. Mutations in 33 nt conserved AT-rich Type I repeat sequences located in the origin region cause defects in the replication and/or maintenance of amplified rDNA in vivo. Fe(ll)EDTA cleavage footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by proteins in extracts of Tetrahymena cells. Two classes of proteins that bound the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays. One of these, ds-TIBF, bound preferentially to duplex DNA and exhibited only moderate specificity for Type I repeat sequences. In contrast, a single-stranded DNA-binding protein, ssATIBF, specifically recognized the A-rich strand of the Type I repeat sequence. Deletion of the 5' or 3' borders of the conserved sequence significantly reduced binding of ssA-TIBF. The binding properties of ssATIBF, coupled with genetic evidence that Type I sequences function as c/s-acting rDNA replication control elements in vivo, suggest a possible role for ssA-TIBF in rDNA replication in Tetrahymena.
+Present addresses: Human Gene Therapy Research Institute, 1415 Woodland Avenue, Suite 218, Des Moines, IA 50309
Molecular Biology Program, College of Medicine, Univesity of lowa, lowa City, IA 52242, USA
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