Nucleotide sequence of the gene encoding the RsrI methyltransferase.
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Base Sequence↗
Biomedical subjects
F H Stephenson
Publications and source records attributed to F H Stephenson.
Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases.
The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.
Amino Acid Sequence↗
Plasmid positive selection vectors.
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Bacteriophages↗
Positive-selection vectors utilizing lethality of the EcoRI endonuclease.
The construction and use of a series of positive-selection vectors are described. These plasmids encode EcoRI endonuclease, the synthesis of which is under the control of the lacUV5 promoter. The pKG2 plasmid encodes a wild-type EcoRI endonuclease. In the absence of EcoRI methylase, the endonuclease is lethal. Cloning into any of the unique restriction sites within the endonuclease-coding gene allows survival of the transformed EcoRI-methylase-less host. The pKGW and pKGS plasmids encode an altered EcoRI endonuclease which, when repressed in a lacIQ host, allows survival in the absence of the methylase. Induction with IPTG, however, results in cell death as a result of high-level EcoRI synthesis. Cloning into any of the unique restriction sites within the EcoRI gene of pKGW or pKGS allows survival of derepressed transformed cells. These vectors strongly select for cloning events which inactivate the endonuclease gene.
Chromosome Mapping↗
Rearrangements between the operators in the bacteriophage lambda.
The left operator mutant lambda v2s develops poorly during infection as a result of constitutive expression of the left operon. A revertant of lambda v2s, designated lambda iri, was found to contain an inversion of the cI region with the inversion endpoints to be within the lambda operators OL and OR. Formation of the inversion is facilitated by a translocation of right operator OcR mutant sequence to the left operator in lambda v2s. The inversion in lambda iri positions wild-type OR sequence at OL returning control of the left operon to repression by the lambda cro repressor.
Bacteriophage lambda↗
A CII-responsive promoter within the Q gene of bacteriophage lambda.
A site within the phage lambda Q gene shares homology with the CII-activated promoters, pE and pI, and is oriented in the direction opposite to that of Q gene transcription. A DNA fragment containing this site can serve as a template for CII-activated transcription in vitro. To ask if this presumptive CII control site functions as a CII-activated promoter in vivo, a restriction fragment containing this promoter has been cloned on a plasmid so that synthesis of beta-galactosidase will be under its control. When CII protein is supplied in trans from a compatible plasmid, this promoter, designated PaQ, is activated to produce beta-galactosidase. A promoter positioned within the Q gene which can be activated by CII protein to initiate transcription in an anti-sense direction should result in an interference with Q gene expression, enhancing CII regulation of late functions, and adding to the list of known CII controls on the lysogenic response.
Bacteriophage lambda↗