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Novel features of ARS selection in budding yeast Lachancea kluyveri.

Liachko I, Tanaka E, Cox K, Chung SC, Yang L, Seher A, Hallas L, Cha E, Kang G, Pace H, Barrow J, Inada M, Tye BK, Keich U - BMC Genomics (2011)

Bottom Line: Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition.Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts.Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Mathematics and Statistics, University of Sydney, Sydney, Australia. bt16@cornell.edu

ABSTRACT

Background: The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined.

Results: In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis.

Conclusions: Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

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Related in: MedlinePlus

Screen to isolate L. kluyveri ARSs. (A) L. kluyveri genomic DNA was fragmented with MboI and ligated into the pIL07 vector. The resultant libraries were transformed into L. kluyveri strain FM628 and ARS plasmids were isolated from resulting colonies. (B) Representative colony sizes of plasmids showing ARS activity or the lack thereof.
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Figure 1: Screen to isolate L. kluyveri ARSs. (A) L. kluyveri genomic DNA was fragmented with MboI and ligated into the pIL07 vector. The resultant libraries were transformed into L. kluyveri strain FM628 and ARS plasmids were isolated from resulting colonies. (B) Representative colony sizes of plasmids showing ARS activity or the lack thereof.

Mentions: In order to identify functional ARSs in L. kluyveri, we performed a random ARS screen as described previously [7,20] (Figure 1A). Genomic DNA from L. kluyveri was digested to completion with MboI (a 4-cutter restriction enzyme) and ligated into an ARS-less vector bearing a URA3 marker. Ligation mixtures were transformed into E. coli and the resultant colonies were pooled to construct genomic plasmid libraries. These genomic libraries were transformed into L. kluyveri and plated on selective media lacking uracil. Cells bearing ARS plasmids are able to propagate the plasmids and form colonies, while non-replicating plasmids do not yield colonies (Figure 1B). Plasmids from robust yeast colonies were isolated, sequenced, and transformed back into L. kluyveri to confirm ARS function. Using this approach we isolated 221 plasmids, from which we selected 84 as unique, non-overlapping sequences that unambiguously mapped to a single locus in L. kluyveri. From the set of 84 unique LkARSs, 69 localize to a single intergene, 10 overlap two consecutive intergenes (the insert spanning an entire gene), and 5 lie entirely within the ORF of an annotated gene (each fragment lies within a different gene). Two LkARSs are subtelomeric and 14 overlap with at least a single tRNA gene.


Novel features of ARS selection in budding yeast Lachancea kluyveri.

Liachko I, Tanaka E, Cox K, Chung SC, Yang L, Seher A, Hallas L, Cha E, Kang G, Pace H, Barrow J, Inada M, Tye BK, Keich U - BMC Genomics (2011)

Screen to isolate L. kluyveri ARSs. (A) L. kluyveri genomic DNA was fragmented with MboI and ligated into the pIL07 vector. The resultant libraries were transformed into L. kluyveri strain FM628 and ARS plasmids were isolated from resulting colonies. (B) Representative colony sizes of plasmids showing ARS activity or the lack thereof.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3306766&req=5

Figure 1: Screen to isolate L. kluyveri ARSs. (A) L. kluyveri genomic DNA was fragmented with MboI and ligated into the pIL07 vector. The resultant libraries were transformed into L. kluyveri strain FM628 and ARS plasmids were isolated from resulting colonies. (B) Representative colony sizes of plasmids showing ARS activity or the lack thereof.
Mentions: In order to identify functional ARSs in L. kluyveri, we performed a random ARS screen as described previously [7,20] (Figure 1A). Genomic DNA from L. kluyveri was digested to completion with MboI (a 4-cutter restriction enzyme) and ligated into an ARS-less vector bearing a URA3 marker. Ligation mixtures were transformed into E. coli and the resultant colonies were pooled to construct genomic plasmid libraries. These genomic libraries were transformed into L. kluyveri and plated on selective media lacking uracil. Cells bearing ARS plasmids are able to propagate the plasmids and form colonies, while non-replicating plasmids do not yield colonies (Figure 1B). Plasmids from robust yeast colonies were isolated, sequenced, and transformed back into L. kluyveri to confirm ARS function. Using this approach we isolated 221 plasmids, from which we selected 84 as unique, non-overlapping sequences that unambiguously mapped to a single locus in L. kluyveri. From the set of 84 unique LkARSs, 69 localize to a single intergene, 10 overlap two consecutive intergenes (the insert spanning an entire gene), and 5 lie entirely within the ORF of an annotated gene (each fragment lies within a different gene). Two LkARSs are subtelomeric and 14 overlap with at least a single tRNA gene.

Bottom Line: Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition.Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts.Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Mathematics and Statistics, University of Sydney, Sydney, Australia. bt16@cornell.edu

ABSTRACT

Background: The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined.

Results: In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis.

Conclusions: Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

Show MeSH
Related in: MedlinePlus