Isolation of maltose-regulated genes from the hyperthermophilic archaeum, Pyrococcus furiosus, by subtractive hybridization.
The hyperthermophilic archaeum, Pyrococcus furiosus, utilizes maltose as a preferred carbon source for growth. 32P-labeled complementary DNA (cDNA) probes representing maltose-regulated genes were obtained by a subtractive hybridization procedure that minimized retrieval of ribosomal RNA (rRNA) sequences during screening. Genomic DNA clones were isolated by positive hybridization to these probes. Genes whose expression varied both in the level of transcription, relative to rRNA, as well as in the degree of regulation were obtained; the extent of regulation varied over a wide range, from as little as fivefold to as high as 50-100-fold. DNA sequence analysis of several of these regulated genes indicated that the subtraction library included gene products required for maltose utilization (e.g., pyruvate dikinase), as well as growth-rate-related genes such as those encoding ribosomal proteins and RNA polymerase subunits. Our approach is applicable to studying gene regulation in organisms that are not amenable to classical genetic techniques.
Robinson
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Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202.
Robb
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Schreier
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D001105:Archaea / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics*; D008320:Maltose / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D012329:RNA, Bacterial; D012333:RNA, Messenger; D008320:Maltose