We developed an expression cDNA cloning system capable of generating high-complexity libraries with unidirectionally inserted cDNA fragments and allowing efficient plasmid rescue. As an application of this system, a cDNA library was constructed from an NIH 3T3 transformant induced by mouse hepatocellular carcinoma DNA. Transfection of NIH 3T3 cells by the library DNA led to the detection of several transformed foci from which identical plasmids with transforming ability could be rescued. Structure and sequence analysis of the cDNA clones revealed that the oncogene was created by recombinational events involving an unknown gene and the mouse homologue of the B-raf protooncogene. Detection of the same genetic rearrangement in independent primary transformants implied that generation of the oncogene occurred within the tumor rather than during DNA transfection or cDNA library construction. The high frequency at which clones were identified and the large sizes of some of the transforming cDNA inserts isolated suggest wide applicability of this mammalian expression cloning system for isolating cDNAs of biologic interest.

Certain dideoxynucleosides have been shown to markedly inhibit the infectivity of human T-lymphotropic virus type m/lymphadenopathy-associated virus, the causative agent of acquired immunodeficiency syndrome (AIDS). Our present studies demonstrate that these drugs are broad spectrum antiretroviral agents capable of inhibiting the infectivity of evolutionarily divergent mammalian type C and animal lentiviruses. Under some conditions, virus infectivity could be inhibited by more than six orders of magnitude. However, the potency of these agents was shown to be greatly influenced by cell-specified determinants. Drug exposure during the initial 24 hr was almost as effective as prolonged treatment on the inhibition of a single cycle of virus infection and expression. Moreover, virus infection was shown directly to be inhibited at the level of proviral DNA synthesis. Thus the time period during which reverse transcription and provirus integration occur is the critical period required for drug action.Our findings have implications concerning strategies to be considered in attempts to utilize 2',3'-dideoxynucleosides in control and treatment of retrovirus-induced diseases ofanimals and humans.In the past few years, efforts aimed at prevention and control of retroviral-induced diseases have gained increasing importance with accumulating evidence that such viruses are responsible for human as well as animal diseases. A potentially useful strategy has arisen from the application of a group of nucleoside analogues first synthesized over 20 years ago (1-5). These dideoxynucleosides were shown to inhibit the retroviral reverse transcriptase (6-8) as well as cellular DNA polymerases f3 and y, while polymerase a was relatively resistant (8-12). Initial studies aimed at demonstrating selective inhibition of viral replication by these drugs showed relatively modest effects (7,8). Recently, however, 3'-azido-3'-deoxythymidine (N3dThd; sometimes referred to as AZT) (13) and virtually all of the 2',3'-dideoxynucleosides (14) analyzed were found to powerfully and selectively inhibit replication of human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), § the retrovirus causative of acquired immunodeficiency syndrome (AIDS).The AIDS virus has been shown to be closely related to animal lentiviruses (15-17), whereas early investigations of these antiviral drugs focused upon distantly related type C viruses. Thus, it is not known whether the reported differences in their antiviral effects relate to host cell or viral specificities. Recently Mitsuya and his colleagues have shown that these nucleoside analogues act as chain terminators of the reverse transcription products of the incoming HTLV-III/LAV (18), although this need not be the only mechanism for antiretroviral activity. Our present studies demonstrate a broad spectrum of antiretroviral activity by these agents, but show considerable cell variation in the potency of different agents. Under optimal conditions, the selective inhibition of even a ...

A recombinant plasmid vector, pSV2-neo, coding for resistance to neomycin and the related antibiotic G-418, was transfected into the mouse myeloma line X63-Ag8.653 by a modification of the protoplast fusion technique. The time interval required to obtain 10(6) G-418 resistant cells was 20 days and the efficiency was 10(-4)-10(-5), which represents a significant advantage over classical methods of selecting mutant cells bearing a dominant selection marker. To investigate the efficiency of this marker in somatic cell hybrid selection, these cells were fused to the human myeloma line U-266 and the hybrids were selected either in HAT + G-418 or HAT + ouabain. The pSV2-neo vector was as efficient as ouabain as a dominant marker with respect to the number of viable hybrid colonies selected and their levels of immunoglobulin secretion. The reciprocal experiment was also performed: HAT-sensitive, mutant U-266 cells were transfected with pSV2-neo, clones selected in G-418 and fused with X63-Ag8.653 cells, and hybrids selected in ouabain plus G-418, yielding HAT-sensitive hybrid "heteromyelomas" that were effective fusion partners with human B lymphocytes.