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Plasmids comprising transgene insertions in four lines of transgenic mice have been retrieved by plasmid rescue into a set of Escherichia coli strains with mutations in different members of the methylation-dependent restriction system (MDRS). Statistical analysis of plasmid rescue frequencies has revealed that the MDRS loci detect differential modifications of the transgene insertions among mouse lines that show distinctive patterns of transgene expression. Plasmids in mice that express hybrid insulin transgenes during development can be readily cloned into E. coli strains carrying mutations in two of the MDRS loci, merA and mcrB. In mice in which transgene expression is inappropriately delayed into adulthood, plasmids can only be cloned into E. coli that carry mutations in all known MDRS activities. Differential cloning frequencies in the presence or absence of the various methylation-dependent restriction genes represent a further way to distinguish regions of mammalian chromosomes. These multiply deficient E. coli strains will also facilitate the molecular cloning of modified chromosomal DNA.

Sam68 is a multimeric 68-kDa RNA-binding nuclear protein of unknown function that interacts with, and is tyrosine-phosphorylated by, the oncogenic protein Src during mitosis. Random homozygous knock-out (RHKO) is a retroviral-based antisense RNA strategy that can identify chromosomal genes whose functional disablement leads to reversible tumorigenic capabilities. Here we report that RHKO-induced Sam68 deficiency results in neoplastic transformation of murine NIH3T3 fibroblasts. Whereas simple haploinsufficiency of Sam68 produced by insertion mutagenesis in a single chromosomal allele did not detectably affect cell growth, reduction of Sam68 protein to <25% of the wild type level was associated with anchorage-independent growth, defective contact inhibition, and the ability to form metastatic tumors in nude mice. These properties were reversed by cessation of RHKO inactivation. Our findings, which indicate that the Sam68 protein level can prominently affect cell proliferation, implicate Sam68 function in tumorigenesis. Consistent with these results is evidence that cells undergoing mitosis show a dramatic reduction in the level of Sam68 protein.

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Random homozygous knockout (RHKO) is an antisense RNA strategy capable of identifying genes whose homozygous functional inactivation yields a selectable phenotype in cells growing in culture. Using this approach, we isolated NIH 3T3 fibroblast clones that showed the ability to form colonies on 0.5% agar and tumors in nude mice. The gene inactivated in one of these clones was found to encode VASP (vasodilatorstimulated phosphoprotein), a previously identified protein that binds to components of the cadherin-catenin junctional complex and has been implicated in cell-cell interactions, the formation of actin filaments, and the transmission of signals at the cytoskeleton-membrane interface. Fibroblasts made deficient in VASP by RHKO showed loss of contact inhibition, and consequently, continued cell division past confluence. Restoration of VASP function by reversal of RHKO yielded cells that had lost the neoplastic capabilities acquired during RHKO. Overproduction of VASP mRNA in the sense or antisense orientation from expression constructs introduced by transfection into naive NIH 3T3 fibroblasts also resulted in neoplastic transformation, implying that normal cell growth may require the maintenance of VASP expression within a narrow range. Our results implicate VASP in tumorigenesis and/or cancer progression.Vasodilator-stimulated phosphoprotein (VASP), a prolinerich founding member of the Ena-VASP protein family, was discovered both as a substrate of cyclic AMP-and cyclic GMPdependent protein kinases and a component of the actin-based cytoskeleton (10,(25)(26)(27)65). VASP is associated with focal adhesions, actin filaments, and highly dynamic membrane regions (50) and has been shown to be a multiligand protein that binds to actin, profilin, ActA, zyxin, and vinculin (9, 12, 51, 52). Vinculin in turn associates with tensin, ␣-actinin, ␣-catenin, and talin (4,31,63,66,68), and zyxin and profilin are involved in actin filament assembly and organization (3,14,43,56,60). In vivo evidence indicates that VASP is a crucial factor in the formation of actin filaments and the integration of signals transmitted between the cytoskeleton, the cytoskeleton-membrane interface, and two cyclic nucleotide signal transduction pathways (2,25,65). Additionally, recent data indicate that VASP is functionally homologous to the Drosophila melanogaster Enabled (Ena) locus, which was identified initially as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase (1). However, the biological role of VASP has remained largely unknown.Recently, a novel gene discovery strategy termed random homozygous knockout (RHKO) was shown to be capable of identifying genes whose functional inactivation in murine fibroblasts leads to reversible cellular transformation (40). This approach uses a promoter within a randomly inserted, chromosomally integrated gene search vector (GSV) to produce antisense transcripts complementary to those originating in the chromosomal gene containing the GSV, and consequently, also complementary to transcri...