A Comprehensive Genome-Wide Map of Autonomously Replicating Sequences in a Naive Genome
(A) Site-directed mutagenesis was used to replace trinucleotides in KlARS515. The mutant plasmids were tested for ARS activity. The mutated bases are underlined, and ARS function is indicated on the left and by the text color of the mutated nucleotides. The predicted KlACS motif is highlighted in blue. Mutants that did not affect function (+) are highlighted in black, while mutants affecting ARS function (+/− and −) are colored in red. Mutants that completely destroyed ARS function are indicated as (−), mutants that support the growth of minute colonies but contain dead cells on restreaking are indicated as (+/−) and mutants that had no effect are indicated as (+). Numbers on the right of the sequences are shown for reference. (B) Pictures of K. lactis transformed with mutant ARS plasmids on selective medium plates after 6 days of growth at 30°C. Plasmids with ARS activity comparable to the wild type plasmid (ARS+) are shown on the top row. Plasmids with weak ARS activity (ARS+/−) are shown in the middle row. Plasmids which show no visible ARS activity (ARS-) are on the bottom row. Numbers above the pictures correspond to mutant constructs from Figure 4A. Multiple clones of all plasmids were tested. Representative plasmids are shown. (C) ARS sequences of KlARS618, KlARS612 and KlARS524 were mutagenized to test the necessity of the predicted KlACS motif for ARS function. In all cases (6 total, see Table S1) the predicted mutation destroyed KlARS function. Each of the full KlARS sequences is 451bp. (D) DNA flanking the KlACS motif was truncated and the resulting sequences were tested for ARS function. The number of shortened KlARSs that retained function is shown on the right and Table S1.