The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation

Abstract

SUMMARYThe versatility of insertional inactivation of P-galactosidase activity for subcloning and sequencing has been enhanced by combining a chemically synthesized oligonucleotide which specifies nine 6-bp-cutter restriction sites including Bg/II, XhoI, NruI, &I, Sac1 and EcoRV in various configurations with existing polylinkers to create a set of highly versatile cloning sites. These improved polylinkers have been inserted into plasmids (the pICs) for routine cloning of double-stranded DNA, and into chimeric phage/plasmids (the pICEMs) for biological production of single stranded DNA. The most versatile polylinker specifies 17 restriction sites in the p-galactosidase a-complementing gene fragment. One of the new polylinkers was inserted into Ml3 DNA to produce a vector (M13mIC7) with nine cloning sites.

Replication promiscuity of DNA-β satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-β satellite localizes sequences involved in replication

“…The construction of infectious clones of AYVV and its associated satellite from A. conyzoides (pHNBin419 and pBinAYVVb) has been described by Tan et al (1995) and Saunders et al (2000). The BamHI insert of EpYVV clone pGEM-EYVVAM1 isolated from Eupatorium makinoi (Saunders et al, 2003) was cloned into BamHI/BglII-digested pIC19H (Marsh et al, 1984), and the insert was subsequently cloned into this BamHI-digested intermediate to produce pIC-EYVV2AM. The tandem repeat of the EpYVV genomic DNA was excised with HindIII and cloned into pBinPlus (van Engelen et al, 1995) to produce pBin-EYVVA.…”

DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum

“…Screening a D. discoideum AX3 ~,gtll cDNA library (Clonetech, Palo Alto, CA) with mAb 394 yielded two clones with large inserts. These inserts were excised with EcoRl, purified in agarose gels, and cloned into the EcoRI site of plC20R [3]. The inserts were sequenced on both strands using sequence-specific primers and a TT-polymerase sequencing kit (Pharmacia, Uppsala, Sweden).…”

Quiescence versus apoptosis: Myc abundance determines pathway of exit from the cell cycle

“…A 70 bp EcoRI/BglII multiple cloning site (MCS) from pIC-20R (Marsh et al, 1984) was inserted into the EcoRI/BamHI sites to generate seven unique restriction sites in place of the original two (yielding a plasmid designated as pTIC). This plasmid was further modi®ed by inserting a hygromycin resistance SalI/BamHI cassette (a gift of I Laird-O ringa) through blunt end ligation into the PvuII site of pTIC, yielding the hTIC plasmid.…”