Cloning of the ATP sulphurylase gene of Schizosaccharomyces pombe by functional complementation

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Abstract:

The ATP sulphurylase gene of Schizosaccharomyces pombe has been cloned by complementation of cysteine auxotrophy of a selenate-resistant mutant, which supposedly had a defect in ATP sulphurylase. A sulphate nonutilizing (cysteine auxotrophic) and selenate-resistant mutant of S. pombe was transformed with a wild-type S. pombe genomic library and sulphate-utilizing clones were isolated. The open reading frame encoding the ATP sulphurylase enzyme was found to be responsible for the restoration of sulphate assimilation. Transformants became as sensitive for selenate as the wild-type strain and produced a comparable amount of ATP sulphurylase as the prototrophic strains. The cloned ATP sulphurylase gene (sua1) proved to be an efficient selection marker in an ARS vector, when different isogenic or nonisogenic S. pombe selenate-resistant mutants were used as cloning hosts. Complementation of sua1- mutations by sua1-bearing multicopy vectors functions as a useful dual positive and negative selection marker. The cloned sua1 gene also complemented the met3 (ATP sulphurylase deficient) mutation in Saccharomyces cerevisiae.

Le gène de l’ATP sulfurylase de Schizosaccharomyces pombe a été cloné par complémentation de l’auxotrophie à la cystéine d’un mutant résistant au sélénate qui était supposément déficient en ATP sulfurylase. Un mutant de Schizosaccharomyces pombe n’utilisant pas le sulfate (auxotrophe à la cystéine) et résistant au sélénate a été transformé avec une banque génomique de S. pombe sauvage et les clones utilisant le sulfate ont été isolés. Le cadre de lecture ouvert codant l’enzyme ATP sulfurylase s’est avéré responsable du rétablissement de l’assimilation de sulfate. Les transformants étaient devenus aussi sensibles au sélénate que la souche sauvage et produisaient une quantité d’ATP sulfurylase comparable à celle des souches prototrophes. Le gène de l’ATP sulfurylase (sua1) cloné dans un vecteur ARS s’est avéré un marqueur de sélection efficace lorsque différents mutants isogéniques ou non-isogéniques de S. pombe résistant au sélénate étaient utilisés comme hôtes de clonage. La complémentation des mutations sua1- par sua1cloné dans des vecteurs à copies multiples fonctionne comme un double marqueur de sélection positif et négatif. Le gène cloné sua1 complémentait aussi la mutation met3 (déficience en ATP sulfurylase) chez Saccharomyces cerevisiae.

Document Type: Research Article

Publication date: January 1, 2008

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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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