A small plasmid for recombination-based screening

We reported recently the construction of the 4.4-kb RoK-derived pMADI plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with Co1E1 and therefore permits recombination-based screening of 2 libraries that contain Co1E1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the [pi]-encoding pir gene required for R6K replication. To supply [pi] [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans we employed pPR1[Delta]22pir 116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger 2 inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1(1991) 120-123].Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29693/1/0000025.pd

Plasmids for recombination-based screening

To facilitate recombination-based screening, we constructed the ColE1-based plasmid, [pi]G4, that confers chloramphenicol resistance, contains a polylinker with multiple unique restriction enzyme recognition sequences, and contains the genetic marker, supF. To facilitate recombination-based screening followed by rapid DNA sequencing, we inserted the selectable marker, supF, into each of 20 high-copy-number (hcn) pUC-derived NoC plasmids that were designed for multiplex DNA sequencing. To facilitate recombination-based screening of common cDNA libraries that often contain ColE1 sequences, we constructed a supF-carrying plasmid whose replication was driven from an R6K replicon that does not share sequence homology with ColE1. Furthermore, we incorporated a useful polylinker and increased the copy number of this plasmid to create the 4.4-kb hcn plasmid, pMAD1. Thus, these plasmids allow: (1) background-free transformation of cells by a supF plasmid carrying an antibiotic-resistance marker; (2) simultaneous performance of the recombination-based assay and DNA sequencing; and (3) screening bacteriophage cDNA libraries that contain ColE1 sequences by recombination with a supF plasmid that is not homologous to ColE1 derivatives.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29122/1/0000161.pd

The murine situs inversus viscerum (iv) gene responsible for visceral asymmetry is linked tightly to the Igh-C cluster on chromosome 12

The iv gene controls left-right determination during murine organogenesis. To map this gene, we analyzed backcross progeny produced by mating (C57BL/6J x MEV/Ty)F1-iv/+ heterozygotes to C57BL/6J-iv homozygotes. Hybridization of a murine ecotropic virus probe and several homeotic box gene probes coupled with analysis of dominant visible markers enabled us to exclude the iv locus from much of the mouse genome. Spurred by a recent report that mapped the iv gene to mouse chromosome 12 which was not excluded by our previous work, we used the polymerase chain reaction on our larger cohort to determine that the iv gene is indeed linked tightly to the Igh-C locus on this chromosome: we observed 0/156 recombinants between the iv and Igh-C loci. Combining data from the two studies demonstrates that the murine iv gene is close (1/201 recombinants) to the Igh-C cluster on chromosome 12.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28508/1/0000305.pd