Structure of the galactokinase gene of<i>Escherichia coli</i>, the last (?) gene of the<i>gal</i>operon

Debouck, Christine; Riccio, Andrea; Schümperli, Daniel; McKenney, Keith; Jeffers, Joseph; Rosenberg, Martin; Heusterspreute, Michel; Brunel, Françoise; Davison, John

doi:10.1093/nar/13.6.1841

Cited by 75 publications

(42 citation statements)

References 30 publications

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“…The galK and galT genes were translated into amino acid sequences and compared by dot plot analysis with the corresponding E. coli genes (11,26). The results are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Galactose utilization in Lactobacillus helveticus: isolation and characterization of the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes

Mollet

Pilloud

1991

J Bacteriol

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By complementing appropriate gal lesions in Escherichia coli K802, we were able to isolate the galactokinase (gaLK) and galactose-1-phosphate uridyl transferase (gall) genes of Lactobacillus helveticus. Tn1O transposon mutagenesis, together with in vivo complementation analysis and in vitro enzyme activity measurements, allowed us to map these two genes. The DNA sequences of the genes and the flanking regions were determined. These revealed that the two genes are organized in the order galK-galT in an operonlike structure. In an in vitro transcription-translation assay, the galK and galf gene products were identified as 44-and 53-kDa proteins, respectively, data which corresponded well with the DNA sequencing data. The deduced amino acid sequence of the galK gene product showed significant homologies to other prokaryotic and eukaryotic galactokinase sequences, whereas galactose-1-phosphate uridyl transferase did not show any sequence similarities to other known proteins. This observation, together with a comparison of known gal operon structures, suggested that the L. helveticus operon developed independently to a translational expression unit having a different gene order than that in E. coli, Streptococcus lividans, or Saccharomyces cerevisiae. DNA sequencing of the flanking regions revealed an open reading frame downstream of the galKT operon. It was tentatively identified as galM (mutarotase) on the basis of the significant amino acid sequence homology with the corresponding Streptococcus thermophilus gene.Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus are used together with Streptococcus thermophilus in the dairy industry as starter cultures for cheese, yogurt, and other dairy product fermentations. As a primary carbon and energy source, they utilize lactose, the only sugar present in milk, and ferment it to lactate. The uptake of lactose by these organisms occurs via a permeasetype system and is followed by a ,3-galactosidase-catalyzed cleavage of the sugar into its monosaccharide glucose and galactose moieties (18,40).In L. delbrueckii subsp. bulgaricus and S. thermophilus, the glucose moiety is metabolized, while the galactose moiety is excreted stoichiometrically into the medium (18). In fact, it has been observed that galactose efflux from these cells is coupled to uptake of lactose or methyl-p-D-thiogalactopyranoside from the external medium and that the transport system involved acts as a lactose-galactose antiporter (21, 39, 39a). However, L. helveticus, which is related to L. delbrueckii subsp. bulgaricus and difficult to distinguish taxonomically from it (22, 38), is able to ferment the glucose and galactose moieties of lactose and does not accumulate free galactose in the external medium (47). It can utilize galactose as an energy source. Hickey et al. (18) observed that there is no lactose or galactose phosphotransferase system in L. helveticus and suggested that the galactose moiety of the lactose is metabolized via the Leloir rather than the tagatose 6-phosphate pat...

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“…The galK and galT genes were translated into amino acid sequences and compared by dot plot analysis with the corresponding E. coli genes (11,26). The results are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We assume that these five regions are important for enzyme function and substrate binding. In particular, the conserved region at L. helveticus protein positions 331 to 350 contains a sequence motif found within the ATP binding sites of numerous protein kinases (11).…”

Section: Mgmentioning

confidence: 99%

Galactose utilization in Lactobacillus helveticus: isolation and characterization of the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes

Mollet

Pilloud

1991

J Bacteriol

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“…A 1,661-bp PvuII (second site shown in contains a portion of the hPDE-1 sequence fused to the first 56 amino acids of E. coli galactokinase (GalK) (14). Expression is driven by the nalidixic acid-inducible X phage promoter (PL).…”

Section: Methodsmentioning

confidence: 99%

Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase.

Livi¹,

Kmetz²,

McHale³

et al. 1990

Mol. Cell. Biol.

129

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We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

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“…The sequences were: 5′-CCGAATTCTGGGGACCAAAGCAG on 1% agarose gel and staining with ethidium bromide, or by on-line monitoring of absorbance at 254 nm with a TTTC-3′ and 5′-CCAAGCTTCACTGTTCACGACGGG TGT-3′. 18 The reaction medium contained, in 25 l of PCR UV-1 monitor (Pharmacia). The yield of purification is expressed as the amount of plasmid DNA recovered in buffer (Promega), 1.…”

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confidence: 99%

Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography

et al. 1997

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Plasmid DNA used for nonviral therapeutic gene transfer After extensive washing of the column, purified plasmid or nucleic acid vaccination has to be highly purified, devoid DNA is eluted using an alkaline buffer. The binding conof contaminating components such as bacterial proteins, ditions of the plasmid DNA on to the column have been endotoxins, or bacterial chromosomal DNA. We have optimized, as well as the hybridization sequence and the developed a new affinity chromatography technique for linker group between the matrix and the third strand oligoplasmid DNA purification: triple-helix affinity chromatonucleotide. The THAC technique makes it possible to purify graphy (THAC). This technique is based on the sequencein one step supercoiled plasmid DNA, and to significantly specific interaction of an oligonucleotide forming a triplereduce the level of contaminating RNA, endotoxins and helix with plasmid DNA. The oligonucleotide was covalently chromosomal DNA. In particular, a 100-fold reduction of linked to a chromatographic matrix, thus providing a rechromosomal DNA contamination over that obtained with usable affinity support. By inserting a suitable homopurine conventional techniques can be achieved through a single sequence in the plasmid DNA, it is possible to obtain a additional THAC step. Further improvements of THAC triple-helix interaction that will only be stable at mild acidic technology are possible, and we anticipate that this techpH and that will dissociate in alkaline conditions. A crude nique can be scaled up for integration into a full commerlysate from a recombinant E. coli, or a pre-purified plasmid cial-scale DNA production process. DNA, is thus applied at acidic pH on to a THAC column.

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Structure of the galactokinase gene ofEscherichia coli, the last (?) gene of thegaloperon

Cited by 75 publications

References 30 publications

Galactose utilization in Lactobacillus helveticus: isolation and characterization of the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes

Galactose utilization in Lactobacillus helveticus: isolation and characterization of the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes

Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase.

Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography

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