Restriction-fragment map of the temperate Bacillus subtilis bacteriophage SPO2.

The endonucleases BglI, BglII, EcoRI, SalI, SmaI, and XbaI were used to fragment the phage SPO2 DNA. Electrophoretic analysis using ethidiumbromide agarose gels showed the phage to have nine BglI sites, one BglII site, four EcoRI sites, one SalI site, one SmaI site, and six XbaI sites. Using partial digestions, multiple endonuclease digestion, and autoradiography the fragments were sized and ordered into a circular map of 23 Md. Such an analysis locates the endonuclease sites, indicates which endonucleases are potentially useful in cloning with SPO2, and allows insertions and/or deletions in the SPO2 DNA to be characterized.     

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.     

Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease

The five EcoRI² restriction sites in bacteriophage lambda DNA have been mapped at 0.445, 0.543, 0.656, 0.810, and 0.931 fractional lengths from the left end of the DNA molecule. These positions were determined electron-microscopically by single-site cleavage of hydrogen-bonded circular λ DNA molecules and by cleavage of various DNA heteroduplexes between λ DNA and DNA from well defined λ mutants. The DNA lengths of the EcoRI fragments are in agreement with their electrophoretic mobility on agarose gels but are not in agreement with their mobilities on polyacrylamide gels. These positions are different from those previously published by Allet et al. (1973). Partial cleavage of pure λ DNA by addition of small amounts of EcoRI endonuclease does not lead to random cleavage between molecules. Also, the first site cleaved is not randomly distributed among the five sites within a molecule. The site nearest the right end is cleaved first about ten times more frequently than either of the two center sites.     

HindII, HindIII,and HpaI restriction fragment maps of the left arm of bacteriophage λ DNA

The sites on the left arm of bacteriophage λ DNA cleaved by the restriction endonucleases isolated from Hemophilus influenzae strain Rc (HincII) and Rd (HindII+III), and Hemophilus parainfluenzae (HpaI) were localized on the λ physical map, and the fragments resulting from these cleavages were identified by gel electrophoresis. The restriction sites within the b2 region of λ were mapped by analysis of the digestion profiles of deletion and substitution derivatives of λ, as well as by digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. The restriction sites on the λ genome between the left vegetative end and the b2 region were mapped entirely by successive digestion experiments. The restriction fragment map for the right arm of λ may be found in the accompanying paper (Robinson and Landy, 1977).     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Kinetic studies.

The kinetics of the reactions of the EcoRI restriction endonuclease at individual recognition sites on the DNA from bacteriophage lambda were found to differ markedly from site to site. Under certain conditions of pH and ionic strength, the rates for the cleavage of the DNA were the same at each recognition site. But under altered experimental conditions, different reaction rates were observed at each recognition site. These results are consistent with a mechanism in which the kinetic stability of the complex between the enzyme and the recognition site on the DNA differs among the sites, due to the effect of interactions between the enzyme and DNA sequences surrounding each recognition site upon the transition state of the reaction. Reactions at individual sites on a DNA molecule containing more than one recognition site were found to be independent of each other, thus excluding the possibility of a processive mechanism for the EcoRI enzyme. The consequences of these observations are discussed with regard to both DNA-protein interactions and to the application of restriction enzymes in the study of the structure of DNA molecules.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

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.     

Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5

The DNA of bacteriophage T5 has been treated with restriction endonucleases EcoRi, HindIII, BamI, SmaI, PstI, SalI, KpnI and the electrophoretic pattern obtained in agarose gel has been analyzed in order to localize the specific cleavage sites on the T5 DNA. The localization of cleavage sites has been deduced from the electrophoretic pattern of double and partial digests, the digests of isolated restriction fragments and the digests of deletion mutant T5st(o) DNA.Four BamI cleavage sites have been found and localized on the physical map of T5 DNA at 0.21, 0.225, 0.685 and 0.725 fractional length. Endonuclease SmaI cleaves at 0.39, 0.59 and 0.69 fractional length. Endonuclease PstI cuts T5 DNA at 11 sites: 0.090, 0.210, 0.320, 0.510, 0.635, 0.670, 0.705, 0.770, 0.815, 0.840, 0.875 fractional length. Six KpnI cleavage sites have been mapped at 0.170, 0.215, 0.525, 0.755, 0.830, 0.850 fractional length. A complete cleavage map of the phage genome is presented for seven restriction enzymes.     

Survey and mapping of restriction endonuclease cleavage sites in bacteriophage T7 DNA.

A survey of restriction endonucleases having different cleavage specificities has identified 10 that do not cut wild-type bacteriophage T7 DNA, 11 that cut at six or fewer sites, four that cut at 18 to 45 sites, and 12 that cut at more than 50 sites. All the cleavage sites for the 13 enzymes that cut at 26 or fewer sites have been mapped. Cleavage sites for each of the 10 enzymes that do not cut T7 DNA would be expected to occur an average of 9 to 10 times in a random nucleotide sequence the length of T7 DNA. A possible explanation for the lack of any cleavage sites for these enzymes might be that T7 encounters enzymes having these specificities in natural hosts, and that the sites have been eliminated from T7 DNA by natural selection. Five restriction endonucleases were found to cut within the terminal repetition of T7 DNA; one of these, KpnI, cuts at only three additional sites in the T7 DNA molecule. The length of the terminal repetition was estimated by two independent means to be approximately 155 to 160 base-pairs.     

Characterization of a site-specific restriction endonuclease SphI from Streptomyces phaeochromogenes

A new type-II restriction endonuclease SphI, has been partially purified from Streptomyces phaeochromogenes. SphI recognizes the hexanucleotide sequence 5′-GCATG↓C and cleaves it at the position marked by the arrow. This nucleotide sequence is present twice in SV40 DNA, four times in λ DNA and only once in the cloning vehicles pBR322, pBR325, pBR327 and pBR328.     

Physical mapping of the HindIII, EcoRI, Sal and Sma restriction endonuclease cleavage fragments from bacteriophage T5 DNA.

Summary The DNA of bacteriophage T5⁺ (molecular weight 76×10⁶ dalton) has been dissected by various specific endonucleases. The restriction enzymes HindIII and EcoRI produce 16 and 7 fragments respectively, whereas Sal and Sma produce 4 fragments each. Complete cleavage maps were established for the enzymes EcoRI, Sal and Sma and an almost complete map for HindIII. Furthermore the location and size of the deletions St 20, St 14, b3, St 0 and b1 were determined. The correlation of the genetic and functional map of the phage with the arrangement of fragments produced by the different enzymes has been established.This paper is the 4^rd publication in the series Structure and Function of the Genome of Coliphage T5.     

Isolation and characterization of viable deletion mutants of Bacillus subtilis bacteriophage SPO2.

Spontaneous deletion mutants of the bacteriophage SPO2, which are viable and retain their temperate character, were isolated using a heat-EDTA enrichment step. They were identified by endonuclease digestion and agarose-gel electrophoresis of phage DNA. Two of the nine mutants were characterized in detail. Both mutants have a 2.3 Md deletion removing the single BglII site and two of the XbaI fragments. The deletion extends 1.0 Md to one side of the former BglII site and 1.3 Md on the other side. This region of the SPO2 genome is non-essential for either lysogeny or viable phage production and thus is a suitable region for the insertion of exogenous DNA fragments.     

Rapid restriction mapping of DNA cloned in lambda phage vectors

A protocol for the rapid restriction mapping of phage λ clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive λ DNA termini by hybridisation with [³²P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the “ladder” of partial digestion products.     

A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes

Lambda derivatives are described that can be used for cloning DNA fragments of about 20 kilobase pairs (kb) generated by restriction enzymes EcoRi, HindIII, BamHI, MboI and BglII. Recombinants can be selected by their Spi- phenotype and their propagation is facilitated by the presence of a chi site.     

Physical mapping and characterization of bacteriophage 9NA genome

The DNA of bacteriophage 9NA, a virulent phage ofSalmonella typhimurium, is linear, double stranded, circularly permuted and is approximately 56 kilobase pairs long. The 9NA genome is partially methylated. A physical map of the DNA has been constructed using the restriction endonucleasesBamHI,BglII,SmaI andPvuII. The putative packaging end (‘pac’ end) and the direction of packaging of the concatemeric DNA has been postulated.