We report the development of an advanced system for transfer and expression of exogenous genes in mammalian cells based on Moloney murine leukemia virus (Mo MuLV). Extensive deletion/mutagenesis analysis to identify cis-acting signals involved in virus transmission has led to the design of a family of novel, highly efficient retroviral vectors and a partner helper-free packaging cell line. The pBabe retroviral vector constructs transmit inserted genes at high titres and express them from the Mo MuLV Long Terminal Repeat (LTR). Each of these vectors has been constructed with one of four different dominantly acting selectable markers, allowing the growth of infected mammalian cells in the presence of G418, hygromycin B, bleomycin/phleomycin or puromycin, respectively. The high titre ecotropic helper free packaging cell line, omega E, was designed in conjunction with the pBabe vectors to reduce the risk of generation of wild type Mo MuLV via homologous recombination events. The omega E cell line was generated with separate gagpol and ecotropic env expression constructs with minimal sequence overlap and decreased sequence homology achieved by 'codon wobbling'. Homologous env coding sequences were deleted from the pBabe vectors without diminishing recombinant vector titre. Together, the pBabe vectors and omega E cell line should prove useful in experiments where highest frequencies of gene transfer, or concomitant expression of several different genes within a single cell are required with minimal risk of helper virus contamination.
OB-R is a high affinity receptor for leptin, an important circulating signal for the regulation of body weight. We identified an alternatively spliced transcript that encodes a form of mouse OB-R with a long intracellular domain. db/db mice also produce this alternatively spliced transcript, but with a 106 nt insertion that prematurely terminates the intracellular domain. We further identified G --> T point mutation in the genomic OB-R sequence in db/db mice. This mutation generates a donor splice site that converts the 106 nt region to a novel exon retained in the OB-R transcript. We predict that the long intracellular domain form of OB-R is crucial for initiating intracellular signal transduction, and as a corollary, the inability to produce this form of OB-R leads to the severe obese phenotype found in db/db mice.
The crystal structure of the tandem SH2 domains of human ZAP-70 in complex with a peptide derived from the zeta-subunit of the T-cell receptor reveals an unanticipated interaction between the two domains. A coiled coil of alpha-helices connects the two SH2 domains, producing an interface that constitutes one of the two critical phosphotyrosine binding sites. These and other unique features provide the molecular basis for highly selective association of ZAP-70 with the T-cell receptor.
The pJQl vector series has been designed to allow easy insertion and subsequent expression of exogenous genes in a wide variety of mammalian cells. The vectors share a common structure of a mammalian transcription unit composed of a promoter flanked 3' by a polylinker, an intron, and a transcriptional termination signal which is linked to a pBR 322 derived backbone as shown. Each of the six vectors possesses a different promoter: pJ3Q, the SV40 early promoter (1); pJ4Q: the Mo MuLV LTR (2); pJ5Q: the MMTV LTR (3); pJ5EQ: an MSV enhancer linked 5' to the MMTV LTR (4); pJ6Q: the rat a actin promoter (5); and pJ7Q: the SCMV IE94 promoter (6).To characterise the expression capabilities of these vectors in mammalian cells, the cat gene was inserted into each of the vectors and the resultant constructs were introduced transiently and stably (with pSV2 neo as a selection marker) into NIH 3T3 and HeLa cells by calcium phosphate transfection.All vectors led to high levels of stable and transient cat activity as well as dexamethasone inducibility in the cases of pJ5S and pJ5QE. Moreover, a striking difference in transient versus stable expression by pJ3Qcat and pJ7Qcat was noted in NIH 3T3 but not in HeLa cells. The actin promoter in pJ6Q has also been shown to be expressed efficiently in undifferentiated teratocarcinoma cells in which transcription from all other promoters mentioned here is severly repressed. For example, in contrast to drug resistance markers under control of viral promoters pJ6Qhygro can confer hygromycin resistance to these cells.
The requirements for transformation of rat embryo fibroblasts (REFs) by transfected ras and myc oncogenes were explored. Under conditions of dense monolayer culture, neither oncogene was able to transform REFs on its own. However, the introduction of a ras oncogene together with a selectable neomycin resistance marker into REFs allowed killing of the normal nontransfected cells and the outgrowth of colonies of ras transformants, 10% of which survived crisis and became tumorigenic. These cells expressed >10-fold-higher levels of ras p21 than tumorigenic cells cotransfected with ras and myc oncogenes. The myc oncogene similarly was unable to induce tumorigenic conversion of REFs unless especially refractile colonies of oncogene-bearing cells, produced by use of a cotransfected selectable marker, were picked and subcultured. Tumorigenic conversion of REFs by single transfected oncogenes appears to require special culture conditions and high levels of gene expression.In previous reports, we and others have described experiments which suggested that multiple oncogenes are required to mediate the transformation of normal cells into tumorigenic cells (8,13,14,16,18,20,23,26,32,(35)(36)(37)50). The normal cells used in our own experiments were rat embryo fibroblasts (REFs), which were found to be incom-. pletely transformed to tumorigenicity by a ras or myc oncogene when introduced by transfection. These data suggested that a single oncogene could not impart the tumorigenic phenotype to REFs. However, when two oncogenes such as ras and myc were concomitantly introduced into REFs, these cells were able to yield fibrosarcomas after injection into young syngeneic rats or nude mice.Such results indicated that oncogenes like ras and myc act in distinct and complementary ways on cellular phenotype. Moreover, this cooperation suggested as assay in which yet other oncogenes could be tested for the ability to act in place of myc or ras in transforming cells. In this way, it was learned that the polyomavirus middle T oncogene can stand in place of ras in a cotransfection assay. Conversely, the large T oncogenes of polyomavirus and simian virus 40 (N-terminal fragment) as well as the Ela antigen of adenovirus, the N-myc oncogene, and the p53 tumor antigen gene could replace myc in the cotransfection test (8,13,14,16,26,35,37,50). These results made it possible to define two functional classes of oncogenes. The first class includes the three ras oncogenes as well as the polyomavirus middle T oncogene. The second encompasses the myc, N-myc, p53, polyomavirus large T, SV40 large T (N-terminal-proximal), and adenovirus Ela oncogenes.In the present report, we further explore the consequences of these functional assignments. We describe several properties that distinguish these two oncogene types from one another and the conditions under which the requirement for MATERIALS AND METHODS Plasmids. pEJ6.6 is a plasmid carrying the Ha-ras oncogene from the EJIT24 human bladder carcinoma cell line (39). Plasmid pHO6T1 carries the same Ha-ras ...
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