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Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome

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Abstract:

Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.Key words: Spiroplasma kunkelii CR2-3x, corn stunt spiroplasma, mollicutes, genome mapping, two-dimensional pulsed-field gel electrophoresis.

Spiroplasma kunkelii (classe Mollicutes) est une bactérie sans parois d'aspect hélicoïdal qui cause la maladie du rabougrissement du maïs. Une combinaison d'analyses par enzymes de restriction, d'électrophorèse sur gel en champs pulsé (PFGE) et d'analyses par hybridation de type Southern fut employée afin de construire une carte physique et génétique du chromosome de la souche CR2-3x de S. kunkelii. L'ordre des fragments de restriction dans la carte fut déterminé en analysant la double digestion d'endonucléases réciproques, utilisant pour ce faire I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI et SalI; les fragments adjacents furent identifiés sur des gels d'électrophorèse en champs pulsé bidimensionnel. La taille du chromosome fut estimée à 1550 kb. Des paires d'oligonucléotides furent conçues afin d'amorcer l'amplification de la séquence de 26 gènes de S. kunkelii par réaction de la polymérase en chaîne (PCR). En employant les amplicons de PCR en tant que sondes, les localisations de 27 gènes putatifs à copie unique de S. kunkelii furent positionnées dans la carte par analyses d'hybridation de type Southern de fragments chromosomiques séparés par PFGE. La séquence nucléotidique de l'opéron d'ARN ribosomal unique fut déterminée et sa localisation chromosomique fut cartographiée à un segment chromosomique renfermant les sites de reconnaissance de SalI, SmaI, EagI et I-CeuI.Mots clés : Spiroplasma kunkelii CR2-3x, spiroplasmes du rabougrissement du maïs, mollicutes, électrophorèse sur gel en champ pulsé bidimensionnel.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: September 1, 2006

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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