Cloning of the human thyrotropin beta-subunit gene and transient expression of biologically active human thyrotropin after gene transfection

A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human alpha- and TSH beta-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunoradiometric and in vitro iodide trapping assay, respectively.     

Expression of human erythropoietin cDNA in human lymphoblastoid Namalwa cells: the inconsistency of a stable expression level with transient expression efficiency

Recombinant plasmids for the expression of human erythropoietin (EPO) cDNA in Namalwa cells were constructed. From the results of the EPO expression efficiency in transiently transfected cells, it was found that the simian virus 40 (SV40) early promoter directs EPO synthesis more efficiently in Namalwa cells than does the long terminal repeat promoter of Rous sarcoma virus and that the 3'-noncoding sequence including splice junction and polyadenylation site derived from the rabbit beta-globin gene are more effective than those of the SV40 early gene. However, in stable transformants, no simple relationship was found between the expression level of EPO cDNA and the structure of the introduced expression vectors.     

Construction of a replication-competent murine retrovirus vector expressing the human immunodeficiency virus type 1 tat transactivator protein.

A replication-competent Akv murine leukemia virus-based vector encoding the human immunodeficiency virus tat cDNA under control of the simian virus 40 early promoter sequences was constructed. The simian virus 40 tat sequences were placed within the U3 region of the 3' long terminal repeat. The resulting virus, derived by transfection, replicated efficiently in mouse NIH 3T3 cells and maintained the tat cDNA insert. It has been suggested that Tat function requires the presence of a human-specific cofactor, which is absent in murine cells. However, infection of murine cells with the Akv virus encoding tat resulted in significant transactivation of a human immunodeficiency virus long terminal repeat-driven reporter gene, indicating that human cofactors are not always required for Tat function. The vector system described may be useful for introduction of foreign genes in vivo and in whole animals when virus spread is required for efficient infection and levels of gene expression.     

Expression and amplification of cloned rat liver tyrosine aminotransferase in nonhepatic cells

A full-length cDNA for the rat liver enzyme tyrosine aminotransferase has been used to construct mammalian expression vectors by recombinant DNA techniques. These vectors, which have employed either a simian virus 40 or a Rous sarcoma virus promoter, were transfected into a variety of nonhepatic mammalian cell lines in culture. Transient expression of tyrosine aminotransferase was readily observed after transfection into monkey COS cells and mouse L cells. Stable clones that express cloned tyrosine aminotransferase have been isolated from mouse L cells, hamster Wg1a fibroblasts, and Chinese hamster ovary (CHO) cells. A vector capable of expressing both tyrosine aminotransferase and dihydrofolate reductase was stimulated to undergo amplification by treatment with methotrexate in a CHO cell line deficient in the latter enzyme. Levels of tyrosine aminotransferase as much as 50-fold higher than typically seen in glucocorticoid-induced hepatoma cells were achieved in some CHO clones by this technique. The tyrosine aminotransferase produced at these highly amplified levels appeared structurally normal and had no major harmful effects on the cells.     

High level expression of human tissue-type plasminogen activator gene in mouse C127 cell.

We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.     

Cloning and expression of a novel variant of human interferon-gamma cDNA

A cDNA library was prepared from the poly(A) mRNA isolated from human peripheral blood lymphocytes which were induced by combined treatment with phytohemagglutinin and a phorbol ester. Recombinant plasmids containing human interferon-gamma (HuIFN-gamma) cDNAs were identified by the oligonucleotide-hybridization method. Nucleotide sequence analysis showed that the nucleotide and amino-acid sequences of HuIFN-gamma cDNA in plasmid pIFN gamma-G4 differed from the published data at amino acid position 9 (CAA for glutamine versus AAA for lysine). The cDNA in plasmid pIFN gamma-G4 was expressed under control of the simian virus 40 early promoter in monkey COS cells and a biologically active HuIFN-gamma was secreted from the cells. The cDNA was also inserted into an expression vector carrying an E. coli tryptophan promoter and was expressed in E. coli. The results suggest that the conversion from lysine to glutamine at amino acid position 9 might not affect the specific activity of HuIFN-gamma.     

Role of the promoter in the regulation of the thymidine kinase gene.

To identify the regulatory elements of the human thymidine kinase (TK) gene, we have established stable cell lines carrying different chimeric constructs of the TK gene. Our results can be summarized as follows. (i) When the TK coding sequence is under the control of the calcyclin promoter (a promoter that is activated when G0 cells are stimulated by growth factors), TK mRNA levels are higher in G1-arrested cells than in proliferating cells; (ii) when the TK coding sequence is under the control of the promoter of heat shock protein HSP70, steady-state levels of TK mRNA are highest after heat shock, regardless of the position of the cells in the cell cycle; (iii) the bacterial CAT gene under the control of the human TK promoter is maximally expressed in the S phase; (iv) the TK cDNA driven by the simian virus 40 promoter is also maximally expressed in the S phase; and (v) TK enzyme activity is always at a maximum in the S phase, even when the levels of TK mRNA are highest in nonproliferating cells. We conclude that although the TK coding sequence may also play some role, the TK promoter has an important role in the cell cycle regulation of TK mRNA levels.     

Establishment of stable mouse myeloma cells constitutively secreting human tissue-type plasminogen activator

Expression plasmids for human tissue-type plasminogen activator (t-pA) were introduced into mouse myeloma cells and stable cell lines constitutively secreting t-pA established by selection with mycophenolic acid. Expression of t-pA is driven either by the simian virus 40 early promoter or by immunoglobulin regulatory elements of either light or heavy chains of the mouse. The availability of myeloma cells secreting a heterologous protein is of importance for biotechnological applications, because large-scale fermentation of myeloma cells is well established.     

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.     

Establishment of human interleukin-4 producing Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were transfected with a human interleukin 4 (IL-4) expression plasmid in which human IL-4 cDNA is linked downstream of the human cytomegalovirus/human immunodeficiency virus chimeric promoter. The plasmid also contained a mouse dihydrofolate reductase (dhfr) gene, expression of which is directed by the SV40 early promoter. The resulting methotrexate-resistant, transformed cells constitutively secreted a high level of human IL-4. CHO cells producing human IL-4 were cultured on microcarriers in a perfusion cell culture system containing 1 l of culture medium, and a high level of human IL-4 (5 × 10⁴U ml⁻¹) was produced at a high cell density (1 × 10⁷cells per ml). Serum-free culture was also examined.     

Bacteriophage lambda vector for transducing a cDNA clone library into mammalian cells.

We have developed a bacteriophage lambda vector (lambda NMT) that permits efficient transduction of mammalian cells with a cDNA clone library constructed with the pcD expression vector (H. Okayama and P. Berg, Mol. Cell. Biol. 3:280-289, 1983). The phage vector contains a bacterial gene (neo) fused to the simian virus 40 early-region promoter and RNA processing signals, providing a dominant-acting selectable marker for mammalian transformation. The phage DNA can accommodate pcD-cDNA recombinants with cDNA of up to about 9 kilobases without impairing the ability of the phage DNA to be packaged in vitro and propagated in vivo. Transfecting cells with the lambda NMT-pcD-cDNA recombinant phage yielded G418-resistant clones at high frequency (approximately 10(-2]. Cells that also acquired a particular cDNA segment could be detected among the G418-resistant transformants by a second selection or by a variety of screening protocols. Reconstitution experiments indicated that the vector could transduce 1 in 10(6) cells for a particular phenotype if the corresponding cDNA was present as 1 functional cDNA clone per 10(5) clones in the cDNA library. This expectation was confirmed by obtaining two hypoxanthine-guanine phosphoribosyltransferase (HPRT)-positive transductants after transfecting 10(7) HPRT-deficient mouse L cells with a simian virus 40-transformed human fibroblast cDNA library incorporated into the lambda NMT phage vector. These transductants contained the human HPRT cDNA sequences and expressed active human HPRT.