Novel bacteriophage and method for breeding thereof

Claim 1

A method for breeding a novel bacteriophage having cohesive ends between the late promoter and the coat gene whose DNA comprises (1) in the upstream portion from the late promoter necessary for the transcription of genetic information for the biosynthesis of coat proteins to the cohesive end, no more than two cleavage sites for a restriction enzyme selected from the group consisting of EcoR I, HindIII and BamH I; (2) in the downstream portion from said late promoter to the cohesive end, one or more cleavage sites for said restriction enzyme, and; (3) the att gene which covalently bonds to the bacterial genome upon lysogenization, the method comprising: mating bacteriophage (a), said bacteriophage (a) being a lambdoid bacteriophage whose DNA has, in the upstream portion from the late promoter necessary for transciption of genetic information for the biosynthesis of coat proteins to the cohesive end, not more than two cleavage sites for said restriction enzyme, with bacteriophage (b), said bacteriophage (b) being a lambdoid bacteriophage whose DNA has one or more cleavage sites for said restriction enzyme in the downstream portion from said late promoter to the cohesive end, wherein at least one of the lambdoid bacteriophages in the mating contains the att gene, and wherein each of said bacteriophages (a) and (b) are labeled with selectable genetic markers, said bacteriophage (a) having a marker in the upstream portion from the late promoter and said bacteriophage (b) having a marker in the downstream portion from the late promoter; selecting recombinant bacteriophages under suitable conditions, said recombinant. bacteriophages containing both of the markers of said bacteriophages (a) and (b); purifying plaques formed by said recombinant bacteriophages; and screening said bacteriophages to determine that the proper number of restriction cleavage sites are in the selected bacteriophage in the proper places.

Claim 2

A method for breeding a novel bacteriophage having cohesive ends between the late promoter and the coat gene whose DNA comprises (1) in the upstream portion from the late promoter necessary for the transcription of genetic information for the biosynthesis of coat proteins to the cohesive end, no more than two cleavage sites for a restriction enzyme selected from the group consisting of EcoR I, HindIII and BamH I; (2) in the downstream portion from said late promoter to the cohesive end, one or more cleavage sites for said restriction enzyme, and; (3) the att gene which covalently bonds to the bacterial genome upon lysogenization, the method comprising: mating bacteriophage (a), said bacteriophage (a) being a lambdoid bacteriophage whose DNA has, in the upstream portion from the late promoter necessary for transciption of genetic information for the biosynthesis of coat proteins to the cohesive end, not more than two cleavage sites for said restriction enzyme, with bacteriophage (b), said bacteriophage (b) being a lambdoid bacteriophage whose DNA has one or more cleavage sites for said restriction enzyme in the downstream portion from said late promoter to the cohesive end, wherein at least one of the lambdoid bacteriophages in the mating contains the att gene, and wherein each of said bacteriophages (a) and (b) are labeled with selectable genetic markers, said bacteriophage (a) having a marker in the upstream portion from the late promoter and said bacteriophage (b) having a marker in the downstream portion from the late promoter; selecting recombinant bacteriophages under suitable conditions, said recombinant. bacteriophages containing both of the markers of said bacteriophages (a) and (b); purifying plaques formed by said recombinant bacteriophages; and screening said bacteriophages to determine that the proper number of restriction cleavage sites are in the selected bacteriophage in the proper places.

Claim 16

A method for constructing a recombinant lambdoid bacteriophage having cohesive ends between the late promoter and the coat gene, whose DNA in a region downstream from the late promoter to the cohesive end, comprises: a segment of DNA which encodes a non-lambdoid protein, said segment of DNA being inserted at a restriction enzyme site such that production of said protein is under the transcriptional control of the bacteriophage late promoter, comprising the steps of: (1) digesting with a restriction endonuclease a lambdoid bacteriophage DNA sample having a cleavage site for said enzyme in the region downstream from its later promoter to the cohesive end, and having not more than 2 cleavage sites for said enzyme in the region upstream from its late promoter; (2) providing a DNA sample containing a DNA segment having ends which can be joined by DNA ligase to the ends produced by said restriction endonuclease; (3) mixing and lubricating the two DNA samples of (1) and (2) together with DNA ligase under conditions where DNA ligase can convalently join the digested DNA samples; (4) transfecting bacterial recipients with the covalently joined DNA; (5) selecting plaque forming bacteriophages which contain in the region downstream from the late promoter the DNA segment which encodes the protein; and (6) growing said transfected bacteria under conditions for producing bacteriophages.

Claim 17

A method for constructing a recombinant lambdoid bacteriophage having cohesive ends between the late promoter and the coat gene, whose DNA in a region downstream from the late promoter to the cohesive end, comprises: a segment of DNA which encodes a non-lambdoid protein, said segment of DNA being inserted at a restriction enzyme site such that production of said protein is under the transcriptional control of the bacteriophage late promoter, comprising the steps of: (1) digesting with a restriction endonuclease a lambdoid bacteriophage DNA sample having a cleavage site for said enzyme in the region downstream from its later promoter to the cohesive end, and having not more than 2 cleavage sites for said enzyme in the region upstream from its late promoter; (2) providing a DNA sample containing a DNA segment having ends which can be joined by DNA ligase to the ends produced by said restriction endonuclease; (3) mixing and lubricating the two DNA samples of (1) and (2) together with DNA ligase under conditions where DNA ligase can convalently join the digested DNA samples; (4) transfecting bacterial recipients with the covalently joined DNA; (5) selecting plaque forming bacteriophages which contain in the region downstream from the late promoter the DNA segment which encodes the protein; and (6) growing said transfected bacteria under conditions for producing bacteriophages.