Biomaterial Database

Construction and use of SPP1v, a viral cloning vector for Bacillus subtilis. 1982

H Heilmann, and J N Reeve

A unique BamHI restriction site has been inserted into a nonessential region of the genome of a deletion mutant of phage SPP1. Construction of this phage, designated SPP1 v, required the in vitro conversion of a BclI site to a BamHI site. SPP1 v has been used as a vector phage to clone BamHI, BglII and BclI-generated restriction fragments of DNA. A direct selection for recombinants has been developed. Transfection with SPP1 v requires intact, genomic-length molecules, and cleavage with BamHI destroys the transfecting ability of this DNA. Recombinants in which the BamHI site has been destroyed by ligation to Bg/II or BclI-generated fragments of DNA become resistant to BamHI digestion after ligation and are active in transfection. Cloning of DNA containing BamHI sites has been accomplished by using the enzyme Bst1503I to methylate BamHI sites before insertion, and so to protect them during the BamHI digestion used to select against vector molecules. The in vitro construction of SPP1 v generated XmaIII sites directly adjacent to, and on both sides of the inserted BamHI site. This permits precise excision of cloned DNA even when cloning destroys the BamHI insertion site. Restriction-enzyme generated fragments of DNA in the size range of 0 to 4 Md have been cloned, including a full-length copy of plasmid pUB110, almost the complete sequence of plasmid pBR322, and a sequence of DNA that carried the lambda cos site.

UI	MeSH Term	Description	Entries
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D002874	Chromosome Mapping	Any method used for determining the location of and relative distances between genes on a chromosome.	Gene Mapping,Linkage Mapping,Genome Mapping,Chromosome Mappings,Gene Mappings,Genome Mappings,Linkage Mappings,Mapping, Chromosome,Mapping, Gene,Mapping, Genome,Mapping, Linkage,Mappings, Chromosome,Mappings, Gene,Mappings, Genome,Mappings, Linkage
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004262	DNA Restriction Enzymes	Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1.	Restriction Endonucleases,DNA Restriction Enzyme,Restriction Endonuclease,Endonuclease, Restriction,Endonucleases, Restriction,Enzymes, DNA Restriction,Restriction Enzyme, DNA,Restriction Enzymes, DNA
D004269	DNA, Bacterial	Deoxyribonucleic acid that makes up the genetic material of bacteria.	Bacterial DNA
D005822	Genetic Vectors	DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition.	Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D001412	Bacillus subtilis	A species of gram-positive bacteria that is a common soil and water saprophyte.	Natto Bacteria,Bacillus subtilis (natto),Bacillus subtilis subsp. natto,Bacillus subtilis var. natto
D001435	Bacteriophages	Viruses whose hosts are bacterial cells.	Phages,Bacteriophage,Phage