Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis.
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.
Irene
Carmela
C
Dipartimento Biologia Cellulare e dello Sviluppo, Universita La Sapienza, Piazzale A Moro, 5, Rome Italy.
Maciariello
Clelia
C
Cioci
Francesco
F
Camilloni
Giorgio
G
Newlon
Carol S
CS
Fabiani
Lucia
L
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D015825:Chromosomes, Fungal / Q000378:metabolism*; D018983:DNA Footprinting; D004261:DNA Replication; D007716:Kluyveromyces / Q000235:genetics* / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D046911:Macromolecular Substances; D000255:Adenosine Triphosphate