Restriction insensitivity in bacteriophage T5. III. Characterization of EcoRI-sensitive mutants by restriction analysis.
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Chromosome Mapping↗
Biomedical subjects
F Brunel
Publications and source records attributed to F Brunel.
Cloning of bacteriophage T5 DNA fragments in plasmid pBR322 and bacteriophage lambda gtWES.
Bacteriophage T5 was digested with the restriction endonucleases HindIII and EcoRI and the resulting fragments were inserted into the plasmid pBR322 and the bacteriophage lambda gtWES as vectors. Approx. 15% of the phage genome was recovered in recombinant clones. The recombinants were characterized by restriction analysis, DNA/DNA hybridization employing Southern blots, and ability to complement or recombine with amber mutants of T5. The results obtained allow revisions of the physical map of the T5 genome and partial correlation of the physical map with the genetic map.
Chromosome Mapping↗
A new host-vector system allowing selection for foreign DNA inserts in bacteriophage lambda gtWES.
An improved vector (lambda gtWES.T5-622) for EcoRI fragments has been derived from EK2 vector lambda gtWES.lambdaB' by replacing the lambda B fragment with two identical 1.1 Md fragments from the pre-early region of bacteriophage T5. The new vector has two advantages which facilitate elimination of parental-type recombinants in an in vitro recombination experiment. Firstly, the 1.1 Md insert is too small to be re-inserted into lambda gtWES in a single copy. Secondly the 1.1 Md T5 fragment carries T5 gene A3 which prevents growth of phage retaining this fragment when the Excherichia coli host carries plasmid ColIb. Thus, essentially all plaques are due to phage with donor DNA inserts and are free of T5 DNA fragments. The size usually given as the theoretical minimum size for insertion into the lambda gt series of vectors is 0.66 Md. We have shown that this size is an underestimate and that the lower limit is about 1.6 Md. A precise estimate is difficult since there is strong selection, among phage having small inserts, for those which have acquired additional genetic material by duplication of the lambda DNA.
Bacteriophage lambda↗
Restriction insensitivity in bacteriophage T5 I. Genetic characterization of mutants sensitive to EcoRI restriction.
Unmodified bacteriophage T5 is able to grow normally on bacterial hosts carrying three different Escherichia coli restriction systems, EcoK, EcoPI, and EcoRI. Under the same conditions, the plating efficiency of bacteriophage gamma is less than 10(-9). At least in the case of EcoRI, this lack of in vivo restriction is not due to lack of restriction sites on the T5 DNA molecule. These observations suggest that bacteriophage T5 specifies one or more restriction protection systems. Mutants (ris) of T5 have been isolated which confer sensitivity to EcoRI restriction but not to EcoK or EcoPI. The mutations are located in the pre-early region of the genetic map but are too far apart to be alleles of a single gene. Complementation studies show that the ris mutants can be helped to grow on the EcoRI-restricting host by coinfection with T5+. This result provides evidence for a restriction protection function but does not necessarily show that the ris mutants are defective in such a system.
Coliphages↗
Restriction insensitivity in bacteriophage T5. II. Lack of EcoRI modification in T5+ and T5ris mutants.
Neither bacteriophage T5+ nor its EcoRI-sensitive ris mutants became modified during growth on an EcoRI-modifying host. For this reason, the rare ris plaques able to grow on the EcoRI-modifying host were always due to revertant phage rather than to modified ris mutants. The ris mutations resulted in the creation of new EcoRI cleavage sites in the terminally repetitious first-step transfer DNA, and analysis of T5 ris revertants showed loss of these sites and restoration of the wild-type restriction pattern. Natural EcoRI sites present in the second-step transfer DNA were never lost in T5ris revertants, indicating that these are irrelevant to in vivo restriction and are protected during growth on the restricting host.
Coliphages↗