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At least 919 records · Page 51Linked to original sources
PubMed2005

[The molecular mechanisms of the effects of murine interferon-gamma transgenic expression on allergen-induced allergic model via adenoviral vector].

OBJECTIVE: To investigate adenoviral vector mediated murine interferon-gamma (mIFN-gamma) transgene expression and its effect on allergen-induced airway inflammation and multiple interleukin (IL) cytokines (IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and IL-18) expression in a murine allergic model. METHODS: Forty-eight mice were divided into 7 groups by random digits table: a negative control group (A), allergic model groups I, II, III (B, D, F), gene therapy groups I, II, III (C, E, G). Except for group A, mice of the other groups were peritoneally sensitized with ovalbumin (OVA, 15 microg/mouse) twice on day 0 and day 5, and challenged by inhalation of 0.5% OVA (20 ml per time) twice per day from day 12 to 14. On day 15, AdCMVmIFN-gamma (5 x 10(9) PFU/mouse) solution 50 microl was administrated by nasal drip in groups C, E and G. For groups A, B, D and F, 0.9% NaCl 50 microl was administrated by nasal drip. Groups A, B and C were sacrificed on day 18. Groups D and E were sacrificed on day 21. Groups F and G were sacrificed on day 25. The concentration of mIFN-gamma in bronchioalveolar lavage (BALF) was measured by enzyme-linked immunosorbant assay. Accumulation of inflammatory cells and eosinophils (EOS) were quantified by cell count and histopathological analysis. Multi-cytokine expression was tested by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: (1) mIFN-gamma was efficiently expressed after gene therapy. The concentration of mIFN-gamma in BALF was (729.0 +/- 104.7) pg/ml 3 days after gene therapy (group C); it was (984.5 +/- 119.1) pg/ml after 6 days (group E); and (310.6 +/- 59.7) pg/ml after 10 days (group G). (2) The total cell number in BALF of group B and C was (318 +/- 41) x 10(3)/ml and (137 +/- 12) x 10(3)/ml, respectively (P < 0.01); the constituent ratio of EOS in BALF was 0.715 +/- 0.054 and 0.452 +/- 0.016, respectively (P < 0.01). The total cell number in BALF of group D and E was (183 +/- 23) x 10(3)/ml and (92 +/- 6) x 10(3)/ml, respectively (P < 0.01); the constituent ratio of EOS was 0.393 +/- 0.065 and 0.083 +/- 0.038, respectively (P < 0.01). The total cell number in BALF of groups F and G was (196 +/- 7) x 10(3)/ml and (98 +/- 15) x 10(3)/ml, respectively (P < 0.01); the constituent ratio of EOS was 0.253 +/- 0.035 and 0.068 +/- 0.025, respectively (P < 0.01). (3) The histopathological results showed that in the gene therapy groups, the infiltration of inflammatory cells was markedly reduced and the damage of airway epithelium was alleviated. (4) For group D and E, the ratio of IL-10 mRNA abundance to that of home gene was 0.14 +/- 0.10 and 0.49 +/- 0.27, respectively (P < 0.01); that of IL-12 was 0.15 +/- 0.05 and 0.63 +/- 0.17, respectively (P < 0.01); that of IL-13 was 0.76 +/- 0.17 and 0.37 +/- 0.10, respectively (P < 0.01). For group F and G, the ratio of IL-10 mRNA abundance to that of home gene was 0.13 +/- 0.04 and 0.27 +/- 0.17, respectively (P = 0.019); that of IL-12 was 0.14 +/- 0.05 and 0.35 +/- 0.21, respectively (P = 0.006); that of IL-13 was 0.57 +/- 0.24 and 0.30 +/- 0.09, respectively (P = 0.003). However, there were no statistically significant changes in IL-4, IL-5, IL-6, and IL-18 in lung tissue between allergic model groups (B, D, F) and gene therapy groups (C, E, G, P > 0.05). CONCLUSIONS: (1) mIFN-gamma gene transferred via adenoviral vector could abrogate the infiltration of EOS in OVA-induced allergic model. (2) The molecular mechanisms for effect by AdCMVmIFN-gamma transgenic therapy on murine allergic model involve the indirect action by the upregulation of IL-10 and IL-12 expression and the downregulation of IL-13 expression locally in the lungs, in addition to the direct effects of locally overexpressed mIFN-gamma.

Adenoviridae↗
PubMed1990

Cell surface expression of hybrid murine/human MHC class II beta alpha dimers. Key influence of residues in the amino-terminal portion of the beta 1 domain.

To aid in the identification of key residues responsible for the control of class II MHC beta-alpha dimer assembly and expression, a series of cotransfections of human plus mouse beta- and alpha-genes was performed. The resulting expression data were correlated with the sequences of the relevant proteins to identify residues that played critical roles in these processes. For the I-E/DR homologues good expression was seen for both E beta DR alpha and DR beta E alpha combinations involving several allelically variable beta-chains of each species. These results are consistent with the sequence conservation seen for I-E and DR gene products, and indicate that the species-specific differences that do exist play little role in controlling dimer formation or transport. For A beta chains, a more complex picture was seen. A beta d, but not A beta k or A beta b, was found to coexpress with human alpha-chains. Not only did A beta d show expression with the homologous DQ alpha-chain, but it also was expressed with DR alpha and DP alpha. These data indicate that species-specific residues do not control dimer expression under these conditions and confirm that allelically polymorphic residues have a crucial role in this process. Mapping studies using recombinant A beta genes established the importance of the residues in the amino-terminal half of the beta 1 domain in the differences observed among the A beta alleles. Sequence comparison of DR beta, DP beta, DQ beta, E beta, and A beta chains in this region revealed a single residue (position 12) conserved in most chains and differing in a nonconservative fashion between A beta d vs A beta b or k. A beta d has the conserved lysine at this position, whereas A beta b has methionine and A beta k has glutamine. To test whether this residue actually was important physiologically, a lysine codon was created in a recombinant A beta gene possessing the amino-terminal sequence of the kappa haplotype, and the ability of this mutant chain to be expressed with various mouse A alpha-chains was examined. This mutant chain was shown to gain the ability to be efficiently expressed with A alpha d without losing its ability to be expressed with A alpha k. These data reemphasize the special role played by allelically polymorphic residues in Ia expression and identify one such polymorphic site as position 12.(ABSTRACT TRUNCATED AT 400 WORDS)

Alleles↗
PubMed1989

Intronic positioning maximizes co-expression and co-amplification of nonselectable heterologous genes.

The overproduction of heterologous gene products in mammalian cells is often a prerequisite for studies of protein structure, function, and therapeutic efficacy. We report that recombinant fusion of a nonselectable reporter template into the intronic sequences of an amplifiable minigene gives dramatically enhanced co-expression efficiency in stable, primary transformants. Further incremental selective pressure for marker gene amplification results in the rapid acquisition of very high expression levels from the intronically positioned reporter. Nested within a constitutively expressing gene, these templates can be independently regulated at moderate gene dosage levels. Intronic positioning may therefore be of general utility for a variety of recombinant studies.

Animals↗
PubMed1999

Purification, cloning, and heterologous expression of a catalytically efficient flavonol 3-O-galactosyltransferase expressed in the male gametophyte of Petunia hybrida.

Flavonols are plant-specific molecules that are required for pollen germination in maize and petunia. They exist in planta as both the aglycone and glycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3GalTase) that is expressed exclusively in the male gametophyte and controls the formation of a pollen-specific class of glycosylated flavonols. Thus an essential step to understanding flavonol-induced germination is the characterization of F3GalTase. Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length cDNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTase cDNA in Escherichia coli yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferases from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis showed that the k(cat)/K(m) values of rF3GalTase, using kaempferol and quercetin as substrates, approaches that of a catalytically perfect enzyme. rF3GalTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. The biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.

Amino Acid Sequence↗
PubMed2003

The future of recombinant coagulation factors.

Hemophilias A and B are X chromosome-linked bleeding disorders, which are mainly treated by repeated infusions of factor (F)VIII or FIX, respectively. In the present review, we specify the limitations in expression of recombinant (r)FVIII and summarize the bioengineering strategies that are currently being explored for constructing novel rFVIII molecules characterized by high efficiency expression and improved functional properties. We present the strategy to prolong FVIII lifetime by disrupting FVIII interaction with its clearance receptors and demonstrate how construction of human-porcine FVIII hybrid molecules can reduce their reactivity towards inhibitory antibodies. While the progress in improving rFIX is impeded by low recovery rates, the authors are optimistic that the efforts of basic science may ultimately lead to higher efficiency of replacement therapy of both hemophilias A and B.

Factor IX↗
PubMed2006

Multiple co-transfection and co-expression of human beta-1,3-N-acetylglucosaminyltransferase with human calreticulin chaperone cDNA in a single step in insect cells.

Human beta-1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is indispensable for the conversion of lacto-N-triose II into lacto-N-tetraose and lacto-N-neotetraose. In this paper, we report multiple co-transfection in a single step using two different human cDNAs in an insect cell, beta3GnT2 and calreticulin chaperone respectively. This minimized the time required to isolate stably expressing cell line from 12 weeks to 4 weeks and simplified the isolation technique to a one-step process. We tried to insert as much cDNA as possible and used various concentrations of two antibiotics, Blasticidin and Geneticin, at 25-1500 microg/ml respectively during co-transfection for the selection of an efficiently expressing stable cell line with no adverse effects. A stably expressing cell line was isolated which expressed beta3GnT2 and chaperone simultaneously, which gave an activity of 10.1 m-units/ml compared with 6.7 m-units/ml by a cell only carrying beta3GnT2. In this study we correlated the activity of beta3GnT2 with the amount of beta3GnT2 and human calreticulin cDNA in a stably expressing insect cell line simultaneously expressing calreticulin chaperone. When the amounts of chaperone and beta3GnT2 cDNA were in a rough ratio of 1:1, the beta3GnT2 activity was at a high level. In order to achieve better expression levels of beta3GnT2 with less cost and time, efficient ways have to be devised.

Animals↗
PubMed1998

Incorporation of fowl plague virus hemagglutinin into murine leukemia virus particles and analysis of the infectivity of the pseudotyped retroviruses.

We describe retrovirus particles carrying the fowl plague virus (FPV) hemagglutinin (HA). When expressed in cells providing Moloney murine leukemia virus (MoMLV) Gag and Pol proteins and a lacZ retroviral vector, FPV HA was found to be efficiently expressed, correctly processed, and stably incorporated into retroviral particles. HA-bearing retroviruses were infectious with a wide host range and were only 10-fold less infectious than retroviruses carrying wild-type MLV retroviral envelopes. We also coexpressed HA proteins in retroviral particles with chimeric MoMLV-derived envelope glycoproteins that efficiently retarget virus attachment but are only weakly fusogenic. Our results suggest that HA can in some cases enhance the fusion ability of these retroviral particles, depending on the cell surface molecule that is used as a receptor.

Animals↗
PubMed1986

Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.

We have totally synthesized a gene that codes for rat hepatic cytochrome b5. The 5' flanking region was designed for efficient expression of this gene in Escherichia coli by incorporating an optimum ribosome binding site and spacer region. Both a soluble form, analogous to the protease-treated microsomal protein, as well as the complete cytochrome with hydrophobic membrane anchor, was constructed and expressed. Transformants with the gene for the soluble protein overproduce authentic cytochrome b5 to a level of 8% of the total cell protein. The complete cytochrome is expressed to a lesser extent with most of the protein found in the cell membrane fraction. This represents complete synthesis and bacterial expression of a mammalian metalloprotein gene. Cytochrome b5 is normally a six-coordinate low spin heme protein with histidine-39 and histidine-63 as axial ligands. We have replaced histidine-63 with a methionine residue by cassette mutagenesis, utilizing specific restriction enzyme sites engineered into the synthetic gene. The resultant protein has histidine-39 as sole axial ligand and is five-coordinate high spin in the ferric resting state, as indicated by optical and electron spin resonance spectroscopy. The ability to generate mutant cytochrome b5 in high yield is a crucial step in understanding heme protein folding, protein-protein recognition and binding, and biological electron transfer processes.

Animals↗
PubMed2002

Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system.

Targeted recombination was used to select mouse hepatitis virus isolates with stable and efficient expression of the gene encoding the enhanced green fluorescent protein (EGFP). The EGFP gene was inserted into the murine coronavirus genome in place of the nonessential gene 4. These viruses expressed the EGFP gene from an mRNA of slightly slower electrophoretic mobility than mRNA 4. EGFP protein was detected on a Western blot of infected cell lysates and EGFP activity (fluorescence) was visualized by microscopy in infected cells and in viral plaques. Expression of EGFP remained stable through at least six passages in tissue culture and during acute infection in the mouse central nervous system. These viruses replicated with similar kinetics and to similar final extents as wild-type virus both in tissue culture and in the mouse central nervous system (CNS). They caused encephalitis and demyelination in animals as wild-type virus; however, they were somewhat attenuated in virulence. Isogenic EGFP-expressing viruses that differ only in the spike gene and express either the spike gene of the highly neurovirulent MHV-4 strain or the more weakly neurovirulent MHV-A59 strain were compared; the difference in virulence and patterns of spread of viral antigen reflected the differences between parental viruses expressing each of these spike genes. Thus, EGFP-expressing viruses will be useful in the studies of murine coronavirus pathogenesis in mice.

Animals↗
PubMed2004

Expression of a modified Cry1Ie gene in E. coli and in transgenic tobacco confers resistance to corn borer.

The wild-type Cry1Ie gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Cry1Ie gene (designated as Cry1Iem) was cloned into prokaryotic expression vector pET28b and its expression in E. coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E. coli revealed that Cry1Iem protein had a similar toxicity to corn borer as wild-type Cry1Ie. Cry1Iem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. Transgenic tobacco plants carrying Cry1Iem showed insecticidal activity against corn borer.

Animals↗
PubMedDate not supplied

[Effect of lox-sites of the Cre lox recombination system on promoterless bar gene expression in transgenic plants].

We demonstrate that localization of lox site between the right border of T-DNA and promoterless bar gene (RB-lox-bar-) led to its highly efficient expression in transgenic plants of Nicotiana tabacum and N. africana. Plasmid vectors used in gene integration experiments contained neomycin phosphotransferase II (npt II) gene under nos promoter as well. Transgenic plants were selected according to their capacity to grow on the medium with kanamycin and then they were tested on the selective medium containing phosphinothricin. 80% of transgenic plants expressed bar gene at the level similar to that in plants transformed with the bar gene under widely used constitutive promoter. Transformation of plants with the plasmid vector containing only promoterless bar gene near T-DNA right border (RB-bar-) and with the vector containing lox site and promoterless bar gene in the middle of the construction (-lox-bar-) led to obtaining no more than 4.5% of transgenic plants resistant to phosphinothricin. PCR analyses confirmed both the absence of tandem repeats and of plasmid recombination resulting in transference of bar gene under promoter in plasmid vector. Nos-terminator situated between the lox site and the right border of T-DNA did not decrease bar gene expression.

DNA, Bacterial↗
PubMed1984

Sequences in the long terminal repeats of the Moloney murine sarcoma virus-124 genome which control transforming gene function.

The role of long terminal repeat (LTR) sequences in the efficient expression of Moloney murine sarcoma virus (MSV-124) transforming gene function was investigated. Recombinant plasmids containing a single LTR positioned 3' of v-mos were subjected to sequential deletions, and the relative transforming efficiency of these recombinants was analyzed in the NIH/3T3 transfection assay. Recombinants lacking CAAT, TATA, and poly(A) signals within the LTR were able to transform with an efficiency comparable to that of the wild-type MSV-124 genome. Deletion of one of the two 74-bp tandem repeat units within the LTR did not abolish v-mos gene function, whereas removal of both 74-bp repeat units completely eliminated transforming activity. The addition of a fragment containing only a single 74-bp unit and 29-bp downstream sequences derived from the LTR to a position 3' of v-mos led to efficient activation of v-mos transforming function. Residual potentiating activity for v-mos expression was retained even when the distance between v-mos and the 3' LTR was increased by several kilobase pairs. All these findings are consistent with the concept that the potentiating action of the LTR in its 3' position is due to activator/enhancer sequences localized to one of its 74-bp repeats. A permuted MSV-124 molecule, whose single LTR was localized 5' of v-mos, was very inefficient at transformation. However, its transforming activity could be increased by approximately 1000-fold by tandemization of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

RNA polymerase II-controlled expression of antigenomic RNA enhances the rescue efficacies of two different members of the Mononegavirales independently of the site of viral genome replication.

De novo generation of negative-strand RNA viruses depends on the efficient expression of antigenomic RNA (cRNA) from cDNA. To improve the rescue system of Borna disease virus (BDV), a member of the Mononegavirales with a nuclear replication phase, we evaluated different RNA polymerase (Pol) promoters for viral cRNA expression. Human and mouse Pol I promoters did not increase the recovery rate of infectious BDV from cDNA compared to the originally employed T7 RNA polymerase system. In contrast, expression of viral cRNA under the control of an RNA Pol II promoter increased the rescue efficacy by nearly 20-fold. Similarly, rescue of measles virus (MV), a member of the Mononegavirales with a cytoplasmic replication phase, was strongly improved by Pol II-controlled expression of viral cRNA. Analysis of transcription levels derived from different promoters suggested that the rescue-enhancing function of the Pol II promoter was due mainly to enhanced cRNA synthesis from the plasmid. Remarkably, correct 5'-terminal processing of Pol II-transcribed cRNA by a hammerhead ribozyme was not necessary for efficient rescue of BDV or MV. The correct 5' termini were reconstituted during replication of the artificially prolonged cRNA, indicating that the BDV and MV replicase complexes are able to recognize internal viral replication signals.

Borna disease virus↗
PubMed1994

Densovirus of Aedes aegypti as an expression vector in mosquito cells.

We have constructed an infectious DNA clone containing the genome of Aedes aegypti densovirus (AeDNV) in a bacterial plasmid. When this clone was transfected into Aedes albopictus C6/36 mosquito cells, the AeDNV genome rescued from the plasmid and replicated as the wild-type virus. To investigate the cloned virus as an expression vector, the reporter gene encoding beta-galactosidase (beta-gal) was inserted into four large open reading frames (ORF) observed in the AeDNV genome. When these recombinant constructs were transfected into Aedes albopictus C6/36 cells, the beta-gal was expressed efficiently from the right ORF (encoding capsid proteins, Vps) and the mid ORF (encoding putative nonstructural protein 2). A low level of expression was found from the left ORF (encoding nonstructural protein 1, NS1), and no expression was detected from the ORF observed on the minus strand of the AeDNV genome. The expression from the right, mid, and left ORFs can be trans-activated with NS1. A putative nuclear targeting sequence observed in the N-terminus of the AeDNV Vps is presumed to be responsible for transport of the chimeric beta-gal into nucleus. The recombinant AeDNV genomes (carrying the beta-gal gene) supplied with the AeDNV capsid proteins can be packaged into infectious transducing particles. Our results indicate that the genome of AeDNV can serve as a vector for delivery and expression of foreign genes in mosquito cells with subsequent targeting of the product to the desired cell compartment.

Aedes↗
PubMed2002

Purification and reactiongenicity of human immunodeficiency virus type 2 total external glycoprotein expressed in Pichia Pastoris.

The purification of human immunodeficiency virus type 2 total external glycoprotein gp105 expressed in Pichia pastoris was investigated. Expression conditions were optimized by an orthogonal test. The results from tests of variance analyses showed that the most important parameter for efficient expression of total gp105 in P. pastoris is adequate aeration during methanol induction. The optimum induction conditions for gp105 expression were: more than 85% aeration, induction for 3 days, the initial pH 6.0-7.0 and a final methanol concentration of 1.0%. Under these conditions, the expressed total gp105 was secreted into fermentation broth and reached a yield of 23%, approximately 141 mg/l. Expressed gp105 was isolated and purified by salting out and Sephadex G-100 chromatography and the yield of gp105 was 40%. gp105 was purified to electrophoretic purity and its isoelectric point (pI) was about 5.2 by SDS-PAGE and isoelectrofocusing. The purified gp105 contained approximately 35% carbohydrate, which proved that the expressed gp105 was a glycoprotein. Its N-terminal amino acid was arginine by Dansyl-Cl and the result indicated that expressed gp105 was secreted and cleaved correctly. The results from gp105 ELISA demonstrated that the purified total gp105 showed good reactiongenicity and antigenic specificity.

Cloning, Molecular↗
PubMed2001

Promoter analysis of the Drosophila melanogaster gene encoding transcription elongation factor TFIIS.

The promoter region of the Drosophila melanogaster TFIIS gene was characterized by transient expression assay. Serial deletion analysis of the promoter region showed that the promoter region between -112 and +113 is required for the efficient expression of the D. melanogaster TFIIS gene. The results also suggest that the DNA fragments between -112 and -54 and between +94 and +113 contain the vital elements for the expression. The importance of these fragments was further substantiated by the findings that the sequences in these fragments of the D. melanogaster TFIIS gene are conserved in the 5'-flanking regions of the Drosophila virilis TFIIS gene. The comparison of the nucleotide sequences in the 5'-flanking region of the D. melanogaster and D. virilis TFIIS genes revealed that the three regions, -85--59, +76-+126, and the vicinity of the transcription initiation site of the D. melanogaster TFIIS gene, are conserved. It is very interesting that the long downstream DNA between +76 and +126 is highly conserved with 90% identities between the two species. The downstream promoter region between +94 and +113 of the D. melanogaster TFIIS gene was further analyzed by transient expression and band mobility shift assays. The results obtained suggest that the region between +94 and +113 is probably recognized by nuclear factors and that the sequence (+98)AGTAAACAACAT(+109) seems to make a great contribution to promoter activity of the D. melanogaster TFIIS gene.

Animals↗
PubMed1992

High-level production and purification of Escherichia coli N-acetylneuraminic acid aldolase (EC 4.1.3.3).

The Escherichia coli gene which encodes N-acetylneuraminic acid aldolase was isolated by the polymerase chain reaction, cloned into the inducible expression vector pTTQ18, and overexpressed in E. coli. The high yield of aldolase was achieved through both optimum growth of cells and efficient expression of the aldolase gene (20-30% soluble cellular protein). The recombinant enzyme was purified to homogeneity with an activity of 1.2-2.2 U/mg, which compared favorably with that of commercial preparations of E. coli aldolase (1.1 U/mg) and Clostridium perfringens aldolase (0.4 U/mg). The cloning strategy, fermentation conditions, purification protocol, and activity assay are described.

Amino Acid Sequence↗
PubMed1998

Effects of ras transformation on the induction of the IL-1 receptor related T1 gene in response to mitogens, anisomycin, IL-1 and TNFalpha.

The T1 gene gives rise to two transcripts encoding a 62 kDa membrane-bound and a 37 kDa secreted protein with similarity to the type I IL-1 receptor. It is weakly expressed in proliferating but not in resting fibroblasts and is strongly induced during the entry of quiescent cells into the cell cycle. Here we show that the T1 gene is also transcriptionally activated in response to the treatment of fibroblasts with cycloheximide and anisomycin. These protein synthesis inhibitors are known to stimulate the JNK and p38/RK signal transduction pathways. We provide evidence that anisomycin triggers T1 gene induction through the stimulation of the p38/RK MAP kinase. This observation is in line with our finding that physiological activators of the p38/RK pathway, the proinflammatory cytokines IL-1 and TNFalpha, stimulate T1 gene expression efficiently. Growth factor mediated T1 gene induction is a delayed early event, requiring ongoing protein synthesis. In contrast, anisomycin induces T1 gene expression at concentrations which block translation completely. Thus, transcriptional induction of the T1 gene via the p38/RK pathway is an immediate early event not requiring de novo protein synthesis. The T1 gene is strongly induced by various mitogens in quiescent NIH3T3 fibroblasts but not in ras transformed NIH3T3 cells. In contrast, all of the three tested agent which activate the p38/RK pathway, IL-1, TNFalpha, and anisomycin led to strong T1 gene expression in normal and ras transformed NIH3T3 cells alike. Thus, the T1 gene can be induced through the activation of at least two MAP kinase pathways: signaling through the ERK pathway can occcur in normal but not in ras transformed NIH3T3 cells, whereas the signaling through the p38/RK pathway is not affected by ras transformation.

3T3 Cells↗