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Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic gene disruption and complementation analysis. | LitMetric

AI Article Synopsis

  • - The researchers created a comprehensive cosmid library of Mesorhizobium loti MAFF303099, consisting of 480 clones that collectively cover nearly the entire genome (99.6%) with an average insert size of 26.9 kbp and overlaps of 11.1 kbp.
  • - The genome includes one chromosome (7,036,071 bp) and two plasmids, with the chromosome being mostly covered (99.68%) by 445 clones, while all of the larger plasmid (pMLa) is covered and most of the smaller plasmid (pMLb) is also included (98.85%).
  • - The study also involved creating deletion mutants to investigate gene functions,

Article Abstract

For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobium loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host-range vector. The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively. The genome of M. loti consists of a single chromosome and two plasmids. The chromosome (7,036,071 bp) was covered 99.68% by 445 clones with four gaps, although two clones were unstable in E. coli. The larger plasmid pMLa (351,911 bp) was completely covered by 23 clones, while the smaller pMLb (208,315 bp) was covered 98.85% by 12 clones with two gaps. We have also made ancillary plasmids to facilitate the construction of deletion mutants using derivatives of the library clones. As a pilot experiment to uncover regions which contain novel symbiotic genes, 13 deletion mutants were constructed to lack in total 180.5 kbp of the genome. All the mutants formed apparently normal nodules and supported symbiotic nitrogen fixation, however, one mutant that lacked a 5.3 kbp chromosomal region, 4,551,930-4,557,222, did not produce normal exopolysaccharides as judged by fluorescence on medium containing Calcofluor. The results supported the effectiveness of the approach to detect gene functions.

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Source
http://dx.doi.org/10.1093/pcp/pcf175DOI Listing

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