Control Of SV40 Early Promoter Research Articles

Inhibition of poly(ADP‐ribosyl)ation introduces an anomalous methylation pattern in transfected foreign DNA

The aim of this paper is to verify whether the control played by poly(ADP-ribosyl)ation on genomic DNA methylation, and in particular on CpG islands, can also be seen on foreign DNA transfected in cells where inhibition of the poly(ADP-ribosyl)ation process was obtained by treating them with 2 mM 3-aminobenzamide for 24 h. The CpG island-like pVHCk plasmid containing the bacterial chloramphenicol acyltransferase (CAT) gene under the control of SV40 early promoter was transfected in L929 mouse fibroblast cells. The bisulfite reaction, which is capable of immortalizing the methylation state of cytosine on DNA, was performed before amplification of the plasmid DNA fragment, then used for sequence analysis. Our results have shown that 1) when transfected in control cells, the plasmid maintains its characteristic unmethylated pattern, whereas this pattern is lost when the plasmid is transfected in cells treated with 3-aminobenzamide; and 2) the presence of new methyl groups on plasmid DNA is paralleled by a decrease of CAT reporter gene expression. These data confirm that poly(ADP-ribosyl)ation is a process tightly involved in protecting genomic DNA from full methylation and suggest the use of 3-aminobenzamide as a possible experimental strategy to mime other conditions of DNA hypermethylation in cells.

Engineering Chinese hamster ovary (CHO) cells to achieve an inverse growth - associated production of a foreign protein, beta-galactosidase.

Protein synthesis in mammalian cells can be observed in two strikingly different patterns: 1) production of monoclonal antibodies in hybridoma cultures is typically inverse growth associated and 2) production of most therapeutic glycoproteins in recombinant mammalian cell cultures is found to be growth associated. Production of monoclonal antibodies has been easily maximized by culturing hybridoma cells at very low growth rates in high cell density fed- batch or perfusion bioreactors. Applying the same bioreactor techniques to recombinant mammalian cell cultures results in drastically reduced production rates due to their growth associated production kinetics. Optimization of such growth associated production requires high cell growth conditions, such as in repeated batch cultures or chemostat cultures with attendant excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) cells is related to the S (DNA synthesis)-phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis is growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was found to be active primarily in the G1-phase and is expected to yield the desirable inverse growth associated production behavior. Our results in repeated batch cultures show that the protein synthesis kinetics in this resulting CHO cell line is indeed inverse growth associated. Results from continuous and high cell density perfusion culture experiments also indicate a strong inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated production.

Transfection of the ketogenic mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase cDNA into Mev-1 cells corrects their auxotrophy for mevalonate.

A somatic cell mutant of the Chinese hamster ovary (CHO)-K1 (called Mev-1), auxotrophic for mevalonate by virtue of a complete lack of detectable cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase activity, was transfected with a plasmid containing the cDNA for ketogenic mitochondrial HMG-CoA synthase under the control of SV40 early promoter. The resulting stable cell line (Mev-SM) was able to grow in the absence of mevalonate. Analysis of Western blot showed that the new cell line strongly expressed mitochondrial HMG-CoA synthase protein. Immunocytochemical studies using specific antibodies against mitochondrial HMG-CoA synthase showed that the protein was located exclusively inside the mitochondria. The prototroph cell line Mev-SM can incorporate labeled acetate into cholesterol in the absence of mevalonate. These results show that the new cell line may circumvent the lack of cytosolic HMG-CoA synthase activity by producing cholesterol-convertible HMG-CoA inside the mitochondria.

Ribozyme mediated destruction of influenza A virus in vitro and in vivo

Short catalytic RNAs with inherent, specific endoribonuclease activity, called ribozymes, have recently been shown to exist in nature. According to the structural models artificial ribozymes have been designed that can potentially hydrolyse any chosen target RNA sequence in trans at a specific site. We have constructed and characterized in vitro hammerhead and hairpin ribozymes designed to cleave viral RNA segment 5 of influenza A virus. Both ribozymes were functional under optimal in vitro conditions, but quantitative measurements indicate that the hammerhead ribozyme is considerably more efficient at this target site than the hairpin ribozyme. Mg2+ dependent hammerhead ribozyme-mediated cleavage reactions were enhanced at higher temperature and in presence of spermidine, but catalytic activities were retained also in cellular extract S-100 or nuclear extracts at physiological temperatures. Recombinant plasmids derived from transfection vector pSV2-neo were engineered to allow the expression of specific ribozymes under the control of SV40 early promoter or SV40 early+ late promoters. These plasmids were introduced by transfection into COS cells, and their expression and enzymatic activities were analyzed in stable cell lines after selection of neomycin-resistance. Several permanent ribozyme-expressing clones were established and characterized: ribozyme coding DNA sequences and synthesis of ribozyme RNA molecules in the transfected cells were determined and monitored by polymerase chain reactions. It was found that the highest levels (up to 70-80%) of resistance to influenza A virus strain X-31 super-infection was observed in COS cells transfected with plasmids containing SV40 early or SV40 early+late promoters coinciding with relatively high and constitutive rates of ribozyme expression. These results suggest the feasibility of developing ribozymes designed against influenza virus to achieve therapeutic value.

A high-level expression vector for human cells

We have developed a vector (pSupexp), for high-level expression of genes, that is dependent on transactivation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) by the HIV-1 transactivator protein. Tat. The foreign gene, expressed under transcriptional control of the HIV-1 LTR, and the tat gene, expressed under transcriptional control of SV40 early promoter, are expressed from the same plasmid. The vector also has the neomycin resistance-encoding gene ( neo), with G418 being used as a dominant selection marker for stable expression. We have cloned the bacterial cat gene into pSupexp and measured transient CAT production in human HeLa and A549 cells. Our results indicate that pSupexpCAT expresses about 25- to 68-fold higher levels of CAT activity as compared to other standard SV40- and Rous sarcoma virus-based vectors, and three- to fivefold more activity than the cytomegalovirus-based vector. Immunoprecipitation of the CAT protein also revealed a high level of production in human cells.

Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells

cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity.

Production of human thyroid-stimulating hormone in Chinese hamster ovary cells.

Human thyroid-stimulating hormone (hTSH) has been produced in Chinese hamster ovary (CHO) cells co-transformed with two plasmids: one carrying the alpha subunit cDNA with mouse dihydrofolate reductase gene and the other carrying hTSH beta subunit cDNA. Each cDNA was driven to expression under the control of SV40 early promoter. hTSH and its alpha subunit were secreted into culture media, and their secretion increased with exposure of the cells to increasing concentrations of methotrexate. Gel filtration analysis revealed that the molecular size of the hTSH was the same as that of natural hTSH. Furthermore, the CHO cell-produced hTSH elevated the cyclic AMP level in the rat thyroid cell line FRTL-5 in the same manner as natural hTSH does.

Molecular Cloning of Human and Murine Interleukin-2 Genes and Their Expression in Various Host Cells

Interleukin-2 (IL-2), originally defined as a lymphokine which permits clonal expansion of T-cell clones, appears to elicit multiple biological activities in immune regulation. IL-2 cDNA cloning was one possible means to elucidate the whole structure of the IL-2 molecule and to obtain the molecule as a pure lymphokine in large quantity. The cloning and expression of human IL-2 cDNA is previously reported by us. Recently, we have obtained mouse IL-2 cDNA clone. Sequence analysis of the cloned cDNAs revealed that the murine IL-2 consists of 169 amino acids including a signal peptide. Interestingly, murine IL-2 appears to contain 12 glutamine residues in a row at the N-terminal region. Expression of this cDNA under the control of SV40 early promoter resulted in a typical mouse IL-2 activity. To reveal the structure, particularly potential regulatory elements of the IL-2 gene, we have cloned human and mouse IL-2 genes. Both genes are contained in a 7 kb genomic DNA fragment and blocked by 3 introns. The sizes of corresponding introns and exons between human and mouse are very similar to each other. The homology upstream from the TATA box is significantly higher than exon homology, implying the presence of regulatory sequence in this region.