Molecular cloning of the gene (ush) from Escherichia coli specifying periplasmic UDP-sugar hydrolase (5'-nucleotidase)

doi:10.1016/0378-1119(80)90111-0

Gene

Volume 12, Issues 3–4, December 1980, Pages 281-286

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Abstract

The gene (ush) specifying a periplasmic enzyme, UDP-sugar hydrolase (5'-nucleotidase), has been cloned using the restriction endonuclease HindIII and the relaxed plasmid pBR322. The source of DNA was a partially purified F-prime preparation and the hybrid plasmid was directly selected among the transformed cells. Cells carrying the hybrid plasmid synthesize about 20 times the usual amount of enzyme. The potential of the hybrid plasmid as a novel cloning vehicle, allowing direct selection of recombinant DNA and possibly secretion of cloned-gene products, is discussed.

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Cited by (14)

Identification and sequence analysis of a silent gene (ushA0) in Salmonella typhimurium
1986, Journal of Molecular Biology
The evolution of new or improved enzyme specificities in prokaryotes has been proposed to involve gene duplication, followed by silencing of one of the duplicates at the transcriptional or translational level. Such “silent gene intermediates” are distinct from “cryptic” genes, which are proposed to have a different role in evolution.
We describe the identification in Salmonella typhimurium of a silent gene (ush⁰) using the active (homologous) ushA gene (encoding UDP-sugar hydrolase) from Escherichia coli as a probe. The ushA⁰ gene has been cloned and, in the multicopy state, very weak expression can be detected; the gene product was shown to be immunologically and functionally related to the enzyme from E. coli.
The sequence of the ushA⁰ gene was found to be highly homologous to the previously determined sequence of the ushA⁰ gene, and the respective promoter and ribosomal-binding sites are also very similar. However, a presumed strong rho-independent terminator in the ushA gene is absent from ushA⁰; although a weak stem-and-loop structure is present in the 3′ region of ushA⁰, its structure is atypical of rho-independent terminators. The sequence analysis also revealed an insertion-sequence like sequence at the 3′ end of ushA⁰ with a convergent open reading frame terminating 116 base-pairs from the ushA⁰ stop codon. A deletion of the 5′ region of the open reading frame results in increased expression of ushA⁰, indicating that convergent transcription plays some role in the silencing of ushA⁰.
S. typhimurium contains a UDP-sugar hydrolase, biochemically and genetically distinct from that in E. coli, encoded by the ushB gene. Our results indicate that ushB is not strongly sequence-related to ushA⁰, and its gene product is not immunologically related to the ushA gene product. ushB is hence a functional duplicate of ushA and provides a rationale for the silencing of ushA⁰. This situation, and the DNA sequence comparison of ushA and ushA⁰, strongly suggests that rather than being a cryptic gene, ushA⁰ has been silenced recently during the evolution of S. typhimurium.
Positive selection vectors: a small plasmid vector useful for the direct selection of Sau3A-generated overlapping DNA fragments
1984, Gene
Characterisation of the ush gene of Escherichia coli and its protein products
1983, Gene
The Escherichia coli ush gene has been subcloned and the coding sequence delineated using BAL31 nuclease digestion. Synthesis of proteins encoded by the ush gene have been examined in “maxicells”; two proteins are made, one of which corresponds in M_r (61000) to purified uridine diphosphoglucose hydrolase and the other, less abundant, has an M_r of 43 000. A deletion at the 3′ end of the gene introduced by restriction endonuclease digestion, results in the synthesis of a truncated protein of the expected M_r of about 43 000. Precursors of all these proteins are observed in maxicells under conditions known to inhibit processing of secreted proteins. Whereas the precursor of the major ush-encoded protein is retained in the cytoplasm-plus-membrane fraction, unexpectedly the precursor of the truncated protein is secreted. The mature forms of both the normal and proncated proteins are secreted.
Studies on the UDP-sugar hydrolases from Escherichia coli and Salmonella typhimurium
1982, Archives of Biochemistry and Biophysics
A simple procedure for purification of the UDP-sugar hydrolase from Escherichia coli is described. The enzyme has an apparent molecular weight of 61,000. The UDP-sugar hydrolase from Salmonella typhimurium has been solubilized and partially purified from total cell membranes. According to several criteria, antibodies raised against the purified E. coli enzyme do not seem to react with partially purified Salmonella enzyme.
Substrate specificity of chimeric enzymes formed by interchange of the catalytic and specificity domains of the 5′-nucleotidase usha and the 3′-nucleotidase cpdb
2021, Molecules
Dynamic regulation of extracellular ATP in Escherichia coli
2017, Biochemical Journal

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Molecular cloning of the gene (ush) from Escherichia coli specifying periplasmic UDP-sugar hydrolase (5'-nucleotidase)

Abstract

Int. J. Biochem.

Gene

FEMS Microbiol. Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Gene

Pedigrees of some mutant strains of Escherichia coli K-12

Bacteriol. Revs.

Recalibrated linkage map of Escherichia coli K-12

Bacteriol. Rev.

Isolation of Escherichia coli mutants (cpdB) deficient in periplasmic 2'3'-cyclic phosphodiesterase and genetic mapping of the cpdB locus

J. Gen. Microbiol.

Genetic location of gene (ush) specifying periplasmic uridine diphosphate sugar hydrolase (5'-nucleotidase) in Escherichia coli K-12

J. Bacteriol.

Mutants of Escherichia coli K-12 “cryptic” or deficient in 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3'-nucleotidase (cyclic phosphodiesterase) activity

J. Bacteriol.