Biomedical information @ the speed of light: implementing desktop access to publishers' resources at the Paterson Institute for Cancer Research.

Shortly after midnight every Thursday morning, a list server in Massachusetts delivers an electronic table of contents message to the Kostoris Medical Library at the Paterson Institute for Cancer Research in Manchester, UK. The messageins details of the latest edition of the New England Journal of Medicine, complete with hyperlinks to the full text of the content online. Publishers' electronic current awareness services have been integrated into the dissemination process of the Library service to enhance the speed of communication and access to full text content. As a means of promoting electronic journal use, a system of e-mail delivery coupled with fast Internet access has allowed a migration from paper-based current awareness alerting to a seamless online product.

Cloning, production and characterisation of wild type and mutant forms of the R.EcoK endonucleases.

The hsdR, hsdM and hsdS genes coding for R.EcoK restriction endonuclease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coli C3-6. The presence of the hsdSts-1 mutation has no effect on the R-M phenotype of this construct in bacteria grown at 42 degrees C. However, DNA sequencing indicates that the mutation is still present on the pBR322-hsdts-1 operon. The putative temperature-sensitive endonuclease was purified from bacteria carrying this plasmid and the ability to cleave and methylate plasmid DNA was investigated. The mutant endonuclease was found to show temperature-sensitivity for restriction. Modification was dramatically reduced at both the permissive and non-permissive temperatures. The wild type enzyme was found to cleave circular DNA in a manner which strongly suggests that only one endonuclease molecule is required per cleavage event. Circular and linear DNA appear to be cleaved using different mechanisms, and cleavage of linear DNA may require a second endonuclease molecule. The subunit composition of the purified endonucleases was investigated and compared to the level of subunit production in minicells. There is no evidence that HsdR is prevented from assembling with HsdM and HsdSts-1 to produce the mutant endonuclease. The data also suggests that the level of HsdR subunit may be limiting within the cell. We suggest that an excess of HsdM and HsdS may produce the methylase in vivo and that assembly of the endonuclease may be dependent upon the prior production of this methylase.

Mutation in the specificity polypeptide of the type I restriction endonuclease R.EcoK that affects subunit assembly.

We describe the isolation and characterization of a temperature-sensitive mutation within the hsdS gene of the type I restriction and modification system EcoK. This mutation appears to affect the ability of the HsdR subunit to interact with the HsdS subunit when forming an active endonuclease. We discuss the possibility that this mutant, together with another mutation described previously, may define a discontinuous domain, involved in protein-protein interactions, within the HsdS polypeptide.

Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes.

EcoR124 and EcoR124/3 are type I DNA restriction and modification systems. The EcoR124/3 system arose from the EcoR124 system some 15 years ago and at the electron microscopic DNA heteroduplex level the genes for both systems are still apparently identical. We have shown that the DNA sequences recognized by the two systems are GAA(N6)RTCG for EcoR124 and GAA(N7)RTCG for EcoR124/3. The sequences thus differ only in the length of the non-specific spacer. This difference nevertheless places the two specific domains of the EcoR124/3 recognition sequence 0.34 nm further apart and rotates them 36 degrees with respect to those of EcoR124, which implies major structural differences in the proteins recognizing these sequences. We have now determined the nucleotide sequences of the hsdS and hsdM genes of both systems and of the hsdR gene of EcoR124/3. The hsdS gene products provide DNA sequence specificity in both restriction and modification, the hsdM gene products are necessary for modification and all three hsd gene products are required for restriction. The only difference that we have detected between the two systems is that a 12 base-pair sequence towards the middle of the hsdS gene is repeated twice in the EcoR124 gene and three times in the EcoR124/3 gene. We have deleted one of the repeats in the EcoR124/3 gene and shown that this changes the specificity to that of EcoR124. Thus, the extra four amino acids in the middle of the EcoR124/3 hsdS gene product, which in an alpha-helical configuration would extend 0.6 nm, are sufficient to explain the differences in sequence recognition. We suggest that the EcoR124/3 system was generated by an unequal crossing over and argue that this kind of specificity change should not be rare in Nature.

The EcoR124 and EcoR124/3 restriction and modification systems: cloning the genes.

The Escherichia coli plasmid R124 codes for a type I restriction and modification system EcoR124 and carries genetic information, most probably in the form of a "silent copy," for the expression of a different R-M specificity R124/3. Characteristic DNA rearrangements have been shown to accompany the switch in specificity from R124 to R124/3 and vice versa. We have cloned a 14.2-kb HindIII fragment from R124 and shown that it contains the hsdR, hsdM, and hsdS genes which code for the EcoR124 R-M system. An equivalent fragment from the plasmid R124/3 following the switch in R-M specificity has also been cloned and shown to contain the genes coding for the EcoR124/3 R-M system. These fragments, however, lack a component present on the wild-type plasmid essential for the switch in specificity. Restriction fragment maps and preliminary heteroduplex analysis indicate the near identity of the genes that encode the two different DNA recognition specificities. Transposon mutagenesis was used to locate the positions of the hsdR, hsdM, and hsdS genes on the cloned fragments in conjunction with complementation tests for gene function. Indirect evidence indicates that hsdR is expressed from its own promoter and that hsdM and hsdS are expressed from a single promoter, unidirectionally.

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3.

R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac+ plasmid, is restriction-deficient (Res-). The Res- phenotype is not due to selection of pre-existing mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F+ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res+ revertants of strains carrying F'lac+ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.

The alternate expression of two restriction and modification systems.

Plasmids R124 and R124/3 carry genes coding for two different R-M systems and normally only one set of genes is expressed. These genes can be translocated to F plasmids that are compatible with the R factors and in strains carrying these F plasmids and an R factor a transacting regulatory mechanism switches off the expression of R-M genes on the introduced plasmid. Additionally the unexpressed genes on the introduced plasmid are expressed. The regulatory mechanism controlling the alternative expression of R124 and R124/3 R-M genes involves a physical rearrangement of DNA sequences.