Construction and expression of a hybrid plasmid containing the Escherichia coli thrA and thrB genes

The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense coon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.The thrA gene is the first structural gene of the threonine operon of Escherichia coil K-12 (1, 2). It is composed of two parts, thrAl and thrA2 and codes for a bifunctional enzyme, aspartokinase I-homoserine dehydrbgenase I (EC 2.7.2.4 and EC 1.1.1.3). The native enzyme (3) is a tetramer with each chain carrying, on discrete domains, the aspartokinase I and homoserine dehydrogenase I activities, which are regulated allosterically by L-threonine. Limited proteolysis of the native enzyme leads to a homodimeric fragment having the same COOHterminal sequence as the native enzyme having only the dehydrogenase activity and no longer inhibited by threonine (3). On the other hand, a polypeptide chain synthesized by an ochre mutant that has the same NH2 terminus as the native enzyme assembles as a tetramer having only the aspartokinase activity, still regulated by threonine (3). The determination of the primary structure of aspartokinase I homoserine-dehydrogenase I seemed warranted for a number of reasons. Sequence information was important to understand enzyme structure-function relationships and to elucidate the allosteric properties of the enzyme. It should permit the study of possible evolutionary relationships between the different proteins coded by the threonine operon and the homology with the isofunctional enzymes in E. coli, aspartokinase II-homoserine dehydrogenase II, coded by metL, and aspartokinase III coded by lysC.

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“…The pBR322 hybrid plasmid containing the thrA and thrB genes (pIPII) was constructed as described (12).…”

Section: Methodsmentioning

confidence: 99%

“…Analysis of the nucleotide sequences was done with the programs of Staden (19)(20)(21) (12). Genetic and biochemical analysis showed that this fragment contained the entire thrA gene (Fig.…”

Section: Methodsmentioning

confidence: 99%

Nucleotide sequence of the thrA gene of Escherichia coli.

Katinka¹,

Cossart²,

Sibilli³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…Incubation of the DNA with the different restriction endonucleases was performed according to standard procedures [l 51. The generated fragments were analysed on horizontal 0.7 "/, or 1 "/, agarose slab gels containing 1 yg/ml ethidium bromide, in Tris/borate/EDTA buffer (see [13]). The migration of the fragments obtained was compared to those of a set of internal and external size-markers (pBR322 or simian virus 40 DNA cleaved by different restriction enzymes), with lengths ranging from 5200 to 200 base pairs.…”

Section: Molecular Cliaracterization Of Cloned Hurnun Papillornu Virumentioning

confidence: 99%

Molecular Cloning, Refined Physical Map and Heterogeneity of Methylation Sites of Papilloma Virus Type 1a DNA

1980

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The entire genome of human papilloma virus type 1a was cloned in Escherichia coli using the plasmid pBR322 as vector. The integrity and the homogeneity of the viral DNA thus obtained was confirmed by restriction endonucleases analysis. Viral DNA isolated from a single wart was partially methylated at only one out of the four HpaII sites, d(C‐C‐G‐G). Recognition sites for BglI, BglII, PstI and PvuII restriction endonucleases were located on the cloned genome.

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“…Cossart et a1. 17 ) also discussed the possibility that promoter-down mutation occurred on· the threonine operon they cloned. We observed that No.…”

Section: Discussionmentioning

confidence: 99%