Transcriptional control of the uvrD gene of Escherichia coli.

Transcription of the uvrD gene of Escherichia coli was studied using the Mud(Aprlac) gene fusion technique of Casadaban and Cohen [Proc. Natl. Acad. Sci. USA 76 (1979) 4530-4533]. Strains were isolated with Mud(Aprlac) inserted in both orientations and chromosome mobilisation experiments showed that transcription of uvrD was from ilvD towards metE. Constitutive expression of uvrD was approximately equivalent to 3000 protein molecules per cell. This level increased 1.5-fold following treatment with DNA damaging agents, an increase which was regulated by the recA and lexA genes. In addition, the constitutive expression of uvrD was reduced in strains containing either the recA56 mutation or a multi-copy plasmid carrying lexA+. These results indicate that uvrD is an SOS-inducible gene.

Cloning of the uvrD gene of E. coli and identification of the product.

The uvrD gene has been cloned from Escherichia coli chromosomal DNA into phage lambda, cosmid, and low-copy-number plasmid vectors. Comparison of the proteins encoded by the cloned fragments with those encoded by fragments in which the uvrD gene is inactivated by transposon insertion or by deletion shows that the uvrD gene product is a protein of Mr = 73000.

A new restriction endonuclease, TspEI, from the genus Thermus that generates cohesive termini compatible with those of EcoRI.

The detection, isolation and properties of the restriction endonuclease TspEI are described. The canonical recognition sequence (AATT) is the same as the 4-bp core of the 6-bp sequence (GAATTC) of EcoRI. Hydrolysis occurs 5' to the palindromic tetramer so that TspEI produces the same cohesive termini as EcoRI. TspEI therefore has an obvious application in producing partial digests of DNA for ligation to EcoRI-digested cloning vectors.