Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum |
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Authors: | G Ozcengiz J-H Kim W R Lin E Ozcengiz D Westenberg L R Lynd A L Demain |
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Institution: | (1) Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, US;(2) Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA, US;(3) Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA, US |
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Abstract: | Cloning of Clostridium thermocellum acetate kinase (ack) and/or phosphotransacetylase (pta) genes in Escherichia coli by functional complementation of ack and/or pta mutants was complicated by an alternative acetate assimilation pathway involving acetyl-CoA synthetase (ACS). In addition
to the problems encountered with the complementation approach, cloning of these genes was not readily achieved using heterologous
probing with corresponding genes from Escherichia coli and Methanosarcina thermophila due to the lack of sufficient homology. The use of a PCR-based approach, on the other hand, yielded a specific C. thermocellum gene fragment which showed significant sequence identity to the ack gene for which primers were designed. The subcloned ack fragment was then successfully used as a probe for the isolation of the corresponding gene and restriction analysis of that
region.
Received 22 January 1998/ Accepted in revised form 31 August 1998 |
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Keywords: | : Clostridium thermocellum acetate kinase phosphotransacetylase thermophilic bacteria PCR gene cloning |
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