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The objective of this research was to clone and characterize the galactokinase gene (gal0 from Streptococcus therrnophilus F4 10. Partially digested genomic DNA was cloned into pBR322 and transformed into galK Escherichia coli, and a galactose-fermenting transformant was isolated. Restriction analysis revealed that the transformant resulted from a Sau3A-HindIIl 4.0-kb fragment. Galactokinase activity in the recombinant was 10 times that of the parent strain. Analysis of the DNA squence showed the presence of a 1.3-kb open reading frame that had high homology with the galK gene from other organisms. A putative ribosome-binding site, start and stop codons, and -10 and -35 sequences were identified. The predicted protein had a molecular mass of 49 kDa, which corresponded to the estimated size of a band apparent by SDS-PAGE. Amino acid sequence homologies with other galactokinases ranged from SO to 62% similarity. Northern blots were performed between the galK gene and mRNA from S. rhemphilus. No hybridization signals were observed for cells grown in glucose, but cells grown in lactose or galactose gave moderate and strong signals. The results suggest that repression of the galK gene by glucose may be responsible for the galactose-releasing phenotype in these strains.