Avian carcinoma virus MH2 has been grouped together with MC29, CMII, and OK10, because all of these viruses share a transformation-specific sequence termed myc. A 5.2-kilobase (kb) DNA provirus of MH2 has been molecularly cloned. The complete genetic structure of MH2 is 5'-delta gag(1.9-kb)-mht(1.2-kb)-myc(1.3-kb)-delta env(?) and noncoding c-region (0.2-kb)-3'. delta gag, delta env, and c are genetic elements shared with nondefective retroviruses, whereas mht is a unique, possibly MH2 transformation-specific, sequence. Hybridizations with normal chicken DNA and cloned chicken c-myc DNA indicate that the mht sequence probably derives from a normal cellular gene that is distinct from the c-myc gene. The genetic structure of MH2 suggests that the delta gag and mht sequences function as a hybrid gene that encodes the p100 putative transforming protein. The myc sequence of MH2 appears to encode a second transforming function. Therefore, it seems that MH2 contains two genes with possible oncogenic function, whereas MC29, CMII, and OK10 each carries a single hybrid delta gag-myc transforming gene. It is remarkable that, despite these fundamental differences in their primary structures and mechanisms of gene expression, MH2 and MC29 have very similar oncogenic properties.

The clinical manifestations, epidemiology, and pathology of hepatitis B virus (HBV) infection are well established (1). However, due to the lack of an in vitro culture system in which the virus can be efficiently propagated, little is known about the replicative cycle of the virus or about its direct effect on the metabolism of infected cells. Although these difficulties have not yet been overcome, it is possible to study the expression of viral genes in mammalian cells transfected with cloned HBV DNA sequences (2-4).As we have reported (2), it is possible to obtain expression of viral proteins in HeLa cells after simple transfection with recircularized cloned HBV genomes. Further studies of the expression of HBV genes in transfected HeLa cells were severely limited by the absence of an efficient selection system for isolating the small number of cells producing viral proteins. Here, we describe the selection of 3T3 cells containing HBV DNA by use of a method developed by Wigler et aL (5) for introducing and amplifying nonselectable genetic elements in methotrexate-sensitive mouse cells. Although this method could not be applied to HeLa cells because ofthe high frequency with which they developed methotrexate resistance, it allowed the selection ofHBV-transfected 3T3 clones that efficiently synthesized and secreted HBV surface antigen (HBsAg). We further report that conditions that lead to amplification of methotrexate resistance result not only in amplification of HBV DNA sequences but also in increased HBsAg production.MATERUILS AND METHODS Cell Lines and Culture Conditions. NIH 3T3 cells (mouse fibroblasts) were maintained in Dulbecco's modified Eagle's medium (DME medium) supplemented with 10% fetal bovine serum, penicillin at 250 units/ml, and streptomycin at 0.2 tkg/ ml. A derivative of the hamster line A29 (6), containing at least 40 copies of a mutated gene for dihydrofolate reductase (generously provided by R. Axel) was grown in DME medium with 3x nonessential amino acids supplemented with methotrexate at 40 tkg/ml, 10% calf serum, and antibiotics as above. All cultures were maintained at 370C in a moist atmosphere containing 5% CO2.Construction and Cloning of a Plasmid Containing Tandem Copies of the HBV Genome in a Head-to-Tail Arrangement. HBV DNA sequences were excised from pHBV-1, a recombinant plasmid constructed by inserting EcoRI-cleaved DNA from Dane particles into the EcoRI site of plasmid pBR322 (2). These sequences were purified by electrophoresis on 1% agarose, collected by electroelution, and ligated (7) to pHBV-1 that had been partially digested with EcoRI. The ligated DNA was used to transfect Escherichia coli (strain 294). DNA was extracted from tetracycline-and ampicillin-resistant colonies after chloramphenicol amplification (8).Colonies containing plasmids with tandem head-to-tail HBV insertions were identified by restriction analysis of the isolated plasmid DNA. Fig. 1 shows the restriction analysis of one such plasmid, pTHBV-1, which was used for the transfection experiments descri...

Epinephrine stimulation of rat alpha 2D, alpha 2B, and alpha 2C adrenergic receptor subtypes, expressed stably in Chinese hamster ovary (CHO) cells, caused a rapid, transient activation of mitogen-activated protein kinase (MAPK), with subtype-specific different efficiencies. The order of activation was CHO-2B approximately CHO-2D much greater than CHO-2C. Pertussis toxin blocked the stimulation of MAPK enzymatic activity and the parallel MAPK phosphorylation, demonstrating that these responses are mediated by pertussis toxin-sensitive Gi proteins. Contrary to what has been reported for the alpha 2A subtype expressed in rat-1 fibroblasts, epinephrine did not cause any detectable activation of p21ras in the CHO transfectants. Furthermore, combined application of epinephrine and phorbol myristate acetate had a potent cooperative but not additive effect in clones CHO-2D and CHO-2B but not in CHO-2C, suggesting that protein kinase C is probably differently involved in the signaling by the three alpha 2 receptor subtypes. These results show that in CHO cells, the different alpha 2 adrenergic receptor subtypes utilize differential pathways to activate MAPK in a p21ras-independent way.

A common cellular sequence was independently transduced by avian carcinoma virus MH2 (v-mht) and murine sarcoma virus (MSV) 3611 (v-raf). Comparison of the nucleotide sequences of v-mht and v-raf revealed a region of homology that extends over 969 nucleotides. The homology between the corresponding amino acids was about 95 percent with only 19 of 323 amino acids being different. With this example, 5 of the 19 known different viral onc genes have been observed in viruses of different taxonomic groups. These data indicate that (i) the number of cellular proto-onc genes is limited because, like other viruses of different taxonomic groups, MH2 and MSV 3611 have transduced the same onc gene-specific sequences from different cell species and (ii) that specific deletion and linkage of the same proto-onc sequences to different viral vector elements affect the oncogenic potential of the resulting viruses. The difference in transformation capabilities of MH2 and MSV 3611 serves as an example.