RFLPs revealed by cosmid 131 [HGM9 no. D17S78].
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A procedure for selection of specific cosmid clones by homologous recombination between cosmid clones from a library and sequences cloned into a plasmid has been developed. Cosmid libraries constructed in a rec- host strain are packaged in vivo into lambda particles. Appropriate aliquots are then introduced into a rec+ host containing the sequence used for selection cloned into a plasmid vector without sequence homology to the cosmid vector. After a short time for recombination, the cosmids are packaged in vivo. Cosmids that have taken up the plasmid by homologous recombination are isolated by plating under conditions selecting for the antibiotic resistance markers carried by both vectors. The recombined cosmids can lose the inserted sequence by another homologous recombination event and, after packaging in vivo, these revertants can be identified on appropriate indicator plates.
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Cosmid libraries are important tools for molecular analysis of plant genes because of their useful properties. Cosmids carry the cos site from the bacteriophage lambda (1–4); therefore, cosmid libraries have the advantage of being able to be packaged in vitro using commercially available highly efficient packaging extracts, just as lambda libraries can. The high efficiency of in vitro lambda packaging allows a cosmid library with large inserts to be more easily constructed than a simple plasmid library, which relies on transformation of competent Escherichia coli cells. In addition, cosmids contain an origin of replication for propagation in E. coli and possibly other bacteria. Since cosmids are propagated as plasmids, not as lambda phages, the genes for lambda reproduction are not needed; therefore, a cosmid vector can be much smaller than a lambda vector (Fig. 1). Since lambda phage packages about 37–53 kb of DNA (5), cosmids can have relatively large inserts of more than 40 kb, depending on vector sizes. Another advantage of cosmids is the ease with which to prepare the DNA because plasmid DNA is much more readily isolated than lambda phage DNA. Finally, the fact that cosmids are plasmids allows the recombinant molecules to be transferred from one bacterium to another through conjugation and from Agrobacterium to plant cells, as long as the necessary cis elements are present. Therefore, cosmid libraries offer a unique combination of very useful features, which make them important for many different molecular studies, including those of plant genes.
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Cosmids are plasmids that contain the phage lambda sequences (cos) required for packaging of the phage DNA into the virion. Induction of a lambda prophage in an Escherichia coli strain carrying a cosmid results in lysates containing phage particles that are filled with cosmid DNA. However, the lysates also contain a large excess of infectious phage particles which complicate use of the packaged cosmids. I report that cosmids packaged by induction of a strain carrying a prophage with an altered cos region results in lysates containing very high levels (>10(10)/ml) of particles that contain cosmid DNA together with very few infectious phage particles. These lysates can be used to transduce cosmid DNA into all of the cells of a growing culture with minimal physiological disturbance. When the cosmid carries a conditionally active origin of replication, transductional introduction of the cosmid under nonreplicative conditions provides a system of transient expression. Transient expression has been used to make a recA strain temporarily recombination proficient and to temporarily introduce a site-specific recombinase. Transductional introduction of a cosmid also allows absolute off-to-on transcriptional control of nonessential genes. Two examples are given showing that when a strain carrying a null mutation in the gene of interest is transduced with a packaged cosmid carrying a functional copy of that gene, the expression of the gene rapidly goes from absolutely off to high-level expression. Additional possible uses of in vivo-packaged cosmids are proposed.
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We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.
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We have constructed and characterised a series of approved, disabled cosmid vectors which we call Homer cosmids and have examined the optimal conditions for the construction of libraries of eukaryotic DNA segments using these vectors. Analysis of these libraries shows that most of the sequences we have tested for are present at the expected frequency and that the libraries can be stably propagated. We have also directly tested the stability of cosmid clones carrying tandemly repeated inserts. This work shows that it should be possible to clone most eukaryotic genes using cosmid vectors and that such cloning systems have considerable advantages over those more commonly used.
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