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Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis
Author(s) -
Irene Carmela,
Maciariello Clelia,
Cioci Francesco,
Camilloni Giorgio,
Newlon Carol S.,
Fabiani Lucia
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03914.x
Subject(s) - kluyveromyces lactis , biology , autonomously replicating sequence , kluyveromyces , saccharomyces cerevisiae , genetics , consensus sequence , plasmid , conserved sequence , origin of replication , sequence (biology) , sequence analysis , peptide sequence , yeast , gene
Summary The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101 , has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae . A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.