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Autonomously replicating sequences from Gigaspora rosea

ARS sequences trigger the replication of the transformation vector, making unnecessary the integration of the plasmid in the genome.
In order to isolate ARS from AM fungi we used the integrating vector YIp5. Since Yip5 lacks a yeast replication origin, it can transform S cerevisiae to uracyl prototrophy only via integration at the chromosomal ura3 locus. Such integration by homologous recombination in S cerevisiae is highly inefficient. This vector gives therefore very low transformation rate unless its replication is supported by an autonomously replicating sequence.

Gigaspora rosea DNA was extracted from surface-sterilized spores and partially digested with Sau3A (2U) for 20 min. Random Sau3A fragments were cloned into the dephosphorilated BamHI site of YIp5. The ligation reaction was used to transform an ura3-1 S. cerevisiae strain (BMA64N) to uracyl prototrophy. Eight transformants were obtained and the recombinant plasmid from two out of these eight transformants successfully rescued in E. coli and the insert sequenced.

The two ARS sequences, named ARSGR2 and ARSGR6, were 1436 and 1663 bp long, respectively. They revealed an extremely high AT content (72-75%). Three elements with 10/11 nucleotide homology (two in the forward direction, one in the reverse direction) to the 11-bp ARS consensus sequences (ACS) defined for yeast vector replication (5’-(A/T)TTTA(T/C)(A/G)TTT(A/T)-3’) were found in ARSGR2. Five elements with 10/11 nucleotide homology to the S. cerevisiae ARS consensus element were found in the ARSGR6 sequence. No G. rosea ARS element showed a perfect sequence match to the S cerevisiae ACS element.

Overall, the results indicated that a non-strict match of the ACS element does not interfere with ARS function and contradict a previous study (Van Houten & Newlon, Mol Cell Biol 10, 3917, 1990) indicating that any one of numerous single point mutations in the ACS abolish ARS function. Possibly, the presence of multiple ARS elements in ARSGR2 and ARSGR6 compensate for the lack of a perfect sequence match of these elements to the S cerevisiae ACS element.

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Roberta BERGERO, (Post-doctoral scientist)
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