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.

Agag is a partial retroviral core protein gene, mht and myc are cell-derived MH2-specific sequences, and c is the 3'-terminal retroviral vector sequence. Here we have determined the nucleotide sequence of 3.5 kb from the 3' end of Agag to the 3' end of molecularly cloned proviral MH2 DNA, in order to elucidate the genetic structure of the virus and to compare it with other mht-and myc-containing oncogenic viruses as well as with the chicken proto-myc gene. The following results were obtained: (i) Agag-mht forms a hybrid gene with a contiguous

Previously, we have reported two major alpha 2-adrenergic receptor transcripts in rat brain of 3.8 and 3.0 kb and the cloning and characterization of the rat brain complementary DNA (cDNA) (RB alpha 2C) specific for the 3.0-kb messenger RNA. In this report, we used rat brain cDNAs specific for the 3.0 and 3.8 kb transcripts, which encode the alpha 2C- and alpha 2A-adrenergic receptors, respectively, and the RNG alpha 2 cDNA, which encodes for the nonglycosylated alpha 2B-adrenergic receptor in rat, to study tissue-specific expression of the three alpha 2-adrenergic receptor genes in rat. To eliminate cross-hybridization of probes with transcripts from other alpha 2 genes, we subcloned fragments that encode for the highly divergent third cytoplasmic loop of each rat alpha 2-adrenergic receptor cDNA and used RNase protection analysis to detect specific transcripts. We show that the three rat alpha 2-adrenergic receptor genes have diverse patterns of tissue expression, and although transcripts specific for each alpha 2-adrenergic receptor gene are found in brain and kidney, the levels of expression of each subtype differ in these tissues. We speculate on the significance of tissue-specific expression of the alpha 2-adrenergic receptor genes.

Three subtypes of alpha 2-adrenergic receptors (alpha 2A, alpha 2B and alpha 2C) have been described that differ in their primary sequence and tissue-specific expression and are encoded by three distinct genes. Previous work has shown that the human alpha 2A-adrenergic receptor gene promoter consists of a TATA-box (TATAAA), palindromic sequence (CCCACGTGGG) and GC-box (GGGGCGG) motif. Sequence analysis of the putative promoter region of the rat alpha 2A-adrenergic receptor gene showed that these promoter regions are conserved in their sequence and relative location. We analysed the transcriptional activity of these regions using RINm5F, a rat insulinoma cell line that expresses the endogenous alpha 2A-adrenergic receptor gene. These results showed that the region from -484 to -92 has a negative effect on transcription, as deletion of this region in alpha 2A-adrenergic receptor gene-chloramphenicol acetyltransferase reporter constructs increased reporter gene activity. This region included the GC-box sequence which is a consensus binding site for the nuclear factor SP1, which is a positive activator of transcription. Gel-mobility-shift assays and supershift assays with an antibody that recognizes SP1 showed binding of the SP1 nuclear factor as well as other nuclear factors to this GC-box region. Additional nuclear factors bind to the downstream palindromic region. We suggest that positive- and negative-acting nuclear factors contribute to the activity of the alpha 2-adrenergic receptor promoter.

We have isolated a cDNA clone (RBa2B) and its homologous gene (GRa2B) encoding an a2B-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (a2-C4) and divergent from the rat kidney nonglycosylated a2B subtype (RNGa2). Transient expression of RBa2B in COS-7 cells resulted in high-affinity saturable binding (Kd = 0.25 nM) for [3H]rauwolscine and a high receptor number (B.n = 7.7 pmol/mg of protein) in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine > yohimbine > prazosin > oxymetazoline, with a pin-to-oxymetazoline K; ratio of0.34. This profile is characteristic of the a2B-adrenergic receptor subtype. Blotting analysis ofrat brain mRNA gave one major (3.0-kilobase) and two minor (4.6-and 2.3-kilobase) mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GRA2B may be transcriptionally active. These findings show that rat brain expresses an a2%-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated a2B subtype. Thus the rat expresses at least two divergent a2B-adrenergic receptors.Pharmacological studies have classified a2-adrenergic receptors as a2A and a2B on the basis of their different ligandbinding properties and, in particular, their relative affinities for oxymetazoline and prazosin (1, 2). The a2A subtype, which exhibits high affinity for oxymetazoline and low affinity for prazosin, is the sole a2 subtype found in human platelets, whereas the a2B subtype, which has high affinity for prazosin and low affinity for oxymetazoline, is the only a2 subtype found in neonatal rat lung (2). Biochemical analysis of partially purified a2-adrenergic receptors from human platelets and neonatal rat lung showed that the differences in their ligand-binding properties are due to differences in their primary structure (3). This has been substantiated by the molecular characterization of different DNA clones encoding different a2-adrenergic receptor subtypes (4-8).Recent molecular cloning data suggest that there are at least three distinct a2 receptor genes in humans: a2-C1O, encoding the human a2A receptor expressed in human platelets, maps to chromosome 10 (4, 7); a2-C4, encoding a human glycosylated a2B, maps to chromosome 4 (5); and a2-C2, encoding a nonglycosylated a2 that has some of the pharmacological characteristics of a2B (8), maps to chromosome 2.RNGa2, a cDNA isolated from a rat kidney library, is highly divergent from the human a2-C10 and a2-C4 subtypes and encodes, like the human a2-C2, a nonglycosylated receptor with some a2B subtype properties (6). This receptor may be the equivalent of the rat a2B adrenergic receptor previously studied in neonatal rat lung (3).The contribution of various a2-adrene...