Uninduced State Research Articles

EFFECTS OF VARIOUS CYTOCHROME <italic>P</italic>-450 INDUCERS ON TESTOSTERONE METABOLISM AND SEVERAL MONOOXYGENASE ACTIVITIES IN SPRAGUEDAWLEY (SpD), HIGH ALCOHOL SENSITIVITY (HAS) AND LOW ALCOHOL SENSITIVITY (LAS) RATS

Hydroxylation of testosterone (TST) has been shown to be regio- and stereo-specific for a number of cytochrome P-450 isoenzymes. Three rat lines [Sprague-Dawley (SpD), high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS)] were tested for this enzymatic specificity after treatment with phenobarbital, clofibrate, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile. These compounds are known to induce cytochrome P-450 2B, 4A, 1A and 3A1, respectively, in the rat. Induction efficiency was established by using the usual enzyme activities specific for these P-450s (pentoxyresorufin, lauric acid, ethoxyresorufin and nifedipine oxidase). Five mono hydroxylated TST metabolites were separated using a sensitive HPLC procedure. The hydroxylation of TST was found to be significantly different between the lines even in the uninduced state. The formation of the metabolites of TST, hydroxylated on 2 alpha or 7 alpha or 16 alpha positions and oxidated on carbon 17 (delta 4), was found to be significantly increased in SpD rats when compared with the HAS-LAS lines (P < 0.0001 in each case). When the HAS-LAS lines were compared, the quantity of 2 alpha and 16 alpha hydroxylated metabolites was found to be significantly lower in LAS rats (P < 0.05). These differences persisted, although in the opposite direction, after 3-methylcholanthrene (P < 0.01 for both 2 alpha and 16 alpha) and phenobarbital induction (P < 0.01 for 2 alpha).(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional attenuation control of the tylosin‐resistance gene tlrA in Streptomyces fradiae

The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase-coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome-mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD.

The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction

Expression of the beta-casein milk protein gene in the mammary epithelial cell line HC11 is primarily regulated at the transcriptional level. A 338-bp segment of promoter sequence 5' of the transcription start site is sufficient to confer inducibility by the lactogenic hormones insulin, glucocorticoid hormone, and prolactin. Positively and negatively acting promoter elements and specific DNA binding proteins have been identified. The binding of the mammary gland factor MGF to a site between -80 and -100 is indispensable for hormonal induction of transcription. Binding of MGF activity to DNA is greatly enhanced by the action of the lactogenic hormones. Repression of transcription in the uninduced state is mediated by a promoter element located adjacent to the MGF binding site at positions -110 to -150. This repressor element consists of two interacting protein binding sites. A nuclear factor that binds specifically to the proximal site between positions -110 and -120 has been characterized and found to be identical with the nuclear factor YY1 (delta, NF-E1). YY1 does not bind to the distal site. The simultaneous mutation in the proximal and the distal sites results in high, hormone-independent transcription. This finding suggests that YY1 plays a functional role in the repression and acts in conjunction with a second DNA binding protein. Comparison of YY1 DNA binding activity in uninduced and hormone-induced cells showed that relief of repression is not mediated by changes in the concentration or binding affinity of YY1. Infection of HC11 cells with a YY1-expressing recombinant retrovirus resulted in overexpression of YY1 but did not suppress hormonal induction. The addition of purified MGF decreased YY1 binding to its DNA recognition site in vitro. This finding indicates that MGF regulates the DNA binding activity of YY1 and thereby may cause the relief of transcriptional repression.

The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction.

Expression of the beta-casein milk protein gene in the mammary epithelial cell line HC11 is primarily regulated at the transcriptional level. A 338-bp segment of promoter sequence 5' of the transcription start site is sufficient to confer inducibility by the lactogenic hormones insulin, glucocorticoid hormone, and prolactin. Positively and negatively acting promoter elements and specific DNA binding proteins have been identified. The binding of the mammary gland factor MGF to a site between -80 and -100 is indispensable for hormonal induction of transcription. Binding of MGF activity to DNA is greatly enhanced by the action of the lactogenic hormones. Repression of transcription in the uninduced state is mediated by a promoter element located adjacent to the MGF binding site at positions -110 to -150. This repressor element consists of two interacting protein binding sites. A nuclear factor that binds specifically to the proximal site between positions -110 and -120 has been characterized and found to be identical with the nuclear factor YY1 (delta, NF-E1). YY1 does not bind to the distal site. The simultaneous mutation in the proximal and the distal sites results in high, hormone-independent transcription. This finding suggests that YY1 plays a functional role in the repression and acts in conjunction with a second DNA binding protein. Comparison of YY1 DNA binding activity in uninduced and hormone-induced cells showed that relief of repression is not mediated by changes in the concentration or binding affinity of YY1. Infection of HC11 cells with a YY1-expressing recombinant retrovirus resulted in overexpression of YY1 but did not suppress hormonal induction. The addition of purified MGF decreased YY1 binding to its DNA recognition site in vitro. This finding indicates that MGF regulates the DNA binding activity of YY1 and thereby may cause the relief of transcriptional repression.

Molecular design of expression systems: Comparison of different control configurations using molecular mechanism models

Molecular-level mathematical models have been used to evaluate the effectiveness of eight different configurations of repressor synthesis control on the regulation of cloned gene expression initiated from a promoter-operator sequence. Both single and dual-repressor situations were considered, employing genetically structured models for the lac and lambdaP(R) promoter-operators in example calculations. Simulation results suggest that the most effective mode of cloned gene expression control is a cross-regulation configuration carried on a multicopy plasmid. This system was able to control cloned gene transcription in the uninduced state over a broad range of plasmid copy number and also provided the highest overall transcription rate in the induced state. The general strategies suggested by these simulations should be applicable for other repressor-operator-promoter systems in diverse hosts.

Inhibition of chromatin assembly in Xenopus oocytes correlates with derepression of the mouse mammary tumor virus promoter.

The mouse mammary tumor virus (MMTV) promoter is positively regulated by glucocorticoid hormone via binding of glucocorticoid receptor to a specific response element. Upon addition of hormone, a nucleosome containing the glucocorticoid response element is removed or structurally altered, suggesting that the nucleosome interferes with transcription. Accordingly, inhibition of chromatin assembly should relieve the repression and result in an increased constitutive activity. We have tested this hypothesis by injecting nonspecific competitor DNA into Xenopus laevis oocytes to titrate endogenous histones. The coinjection of competitor DNA altered chromatin structure: nucleosomal ladders produced by micrococcal nuclease were disrupted, and the unique helical setting of the MMTV promoter in a nucleosome was lost, as shown by in situ DNase I footprinting. Basal MMTV transcription was drastically increased by competitor DNA, whereas a coinjected, constitutively active adenovirus 2 major late promoter was not stimulated. These results show that the uninduced MMTV promoter is under negative control and provide direct support for the theory that the nucleosomal organization maintains the repression of this promoter in its uninduced state.

Inhibition of chromatin assembly in Xenopus oocytes correlates with derepression of the mouse mammary tumor virus promoter.

The mouse mammary tumor virus (MMTV) promoter is positively regulated by glucocorticoid hormone via binding of glucocorticoid receptor to a specific response element. Upon addition of hormone, a nucleosome containing the glucocorticoid response element is removed or structurally altered, suggesting that the nucleosome interferes with transcription. Accordingly, inhibition of chromatin assembly should relieve the repression and result in an increased constitutive activity. We have tested this hypothesis by injecting nonspecific competitor DNA into Xenopus laevis oocytes to titrate endogenous histones. The coinjection of competitor DNA altered chromatin structure: nucleosomal ladders produced by micrococcal nuclease were disrupted, and the unique helical setting of the MMTV promoter in a nucleosome was lost, as shown by in situ DNase I footprinting. Basal MMTV transcription was drastically increased by competitor DNA, whereas a coinjected, constitutively active adenovirus 2 major late promoter was not stimulated. These results show that the uninduced MMTV promoter is under negative control and provide direct support for the theory that the nucleosomal organization maintains the repression of this promoter in its uninduced state.

Repression and induction of the nag regulon of Escherichia coll K‐12: the roles of nagC and nagA in maintenance of the uninduced state

The nag regulon located at 15.5 min on the Escherichia coli chromosome consists of two divergent operons, nagE and nagBACD, encoding genes involved in the uptake and metabolism of N-acetylglucosamine. Null mutations have been created in each of the genes by insertion of antibiotic resistance cartridges. The phenotypes of the strains carrying the insertions in nagE, B and A were consistent with the previous identification of gene products: nagE, EII(Nag), the N-acetylglucosamine specific transporter of the phosphotransferase system and nagB and nagA, the two enzymes necessary for the degradation of N-acetylglucosamine. Insertions in the nagC result in derepression of the nag genes, which is consistent with earlier observations that the nagC gene encodes the repressor of the regulon. Insertions in nagA also provoke a derepression, implying that nagA has a role in the regulation of the expression of the nag regulon as well as in the degradation of the amino-sugars. N-acetylglucosamine-6-phosphate, the intracellular product of N-acetylglucosamine transport and the substrate of the nagA gene product, is shown to be an inducer of the regulon and this suggests how nagA mutations result in derepression: the absence of N-acetylglucosamine-6-phosphate deacetylase allows N-acetylglucosamine-6-phosphate to accumulate and induce the regulon.

Nicotine metabolism in isolated perfused lung and liver of phenobarbital- and benzoflavone-treated rats

The kinetics of nicotine elimination was investigated in isolated perfused lung and liver of phenobarbital (PB)- and 5,6-benzoflavone (BF)-pretreated rats. The estimated kinetic parameters demonstrated a high nicotine elimination rate in rat lung approaching the capacity of liver when both organs were in an uninduced state. The concentration-time profiles of cotinine as the main metabolite were almost identical for isolated lung and liver. In both organs the cotinine plasma concentrations reached a plateau level after 60 min of perfusion. Pretreatment of rats with 5,6-benzoflavone did not affect the rate of nicotine elimination and cotinine formation either in the lung or in the liver. Phenobarbital treatment, however, induced nicotine clearance in lung approximately 2-fold. This effect is quantitatively lower than the PB-related 8-fold induction of hepatic nicotine elimination observed in a previous study. The present results also indicate that the turnover of cotinine is markedly enhanced after PB induction. The elimination half-lives and clearance values for cotinine as the substrate were approximately 10-fold increased in rat liver after PB pretreatment. Thus, an important contribution of extrahepatic tissues to nicotine metabolism in rats has to be assumed. Moreover, since cotinine elimination is significantly increased after PB induction it is questionable whether cotinine plasma concentrations can further be used as suitable parameter for nicotine consumption.

Regulation of 2‐acetylaminofluorene‐and 3‐methylcholanthrene‐mediated induction of multidrug resistance and cytochrome P450IA gene family expression in primary hepatocyte cultures and rat liver

Previous studies by this laboratory have indicated that expression of the multidrug resistance (mdr) gene can be increased in vivo by exposure to a variety of xenobiotics. Because of the nature of these compounds, it was proposed that mdr gene expression might, at least in part, be regulated by the arylhydrocarbon (Ah) receptor. In the present study, we used a primary hepatocyte culture model to examine the relationship between induction of cytochrome P450IA and mdr expression in vitro. Both 3-methylcholanthrene (MC) and 2-acetylaminofluorene (AAF) were efficient inducers of mdr expression in this model. Induction of mdr gene expression by both MC and AAF obeyed a log10 concentration/response relationship. In contrast, 2,3,7,8-tetrachlorodibenzo-P-dioxin did not induce mdr expression at concentrations that yielded maximum induction of cytochrome P450IA expression. These data suggest that mdr induction was not mediated via the Ah receptor. Nuclear run-off analysis indicated that both AAF and MC induced mdr expression by increasing transcription. Primer extension analysis indicated that mdr gene transcription was initiated at one major site 151 bp upstream of the ATG site in both the uninduced and induced state in vivo and in vitro. The sequence of the primer and the site of initiation of gene transcription indicate that the main gene induced was the mdr 1b gene.

Genetic evidence for similar negative regulatory domains in the yeast transcription activators GAL4 and LAC9

The GAL4 protein of Saccharomyces cerevisiae and the LAC9 protein of Kluyveromyces lactis are transcription activator proteins with similar structure and function. Greatest similarity occurs in the C region near the carboxy terminus, where 16 of 18 amino acids are identical. The function of the C region is unclear. Here we show that the structural similarity is reflected in functional similarity. Single amino acid changes in the C region of GAL4 and LAC9 create a similar phenotype: constitutive gene expression. In S. cerevisiae the constitutive phenotype caused by GAL4 mutants can be abolished by overproduction of GAL80. These results support a model in which the C region of GAL4 and LAC9 constitute similar negative regulatory domains that interact with GAL80 in S. cerevisiae and an unidentified GAL80 homolog in K. lactis. This protein-protein interaction prevents expression of the galactose operon in the uninduced state.

TNF stimulates expression of mouse MHC class I genes by inducing an NF kappa B-like enhancer binding activity which displaces constitutive factors.

We have dissected the mouse H-2Kb gene promoter in order to define the sequences responsible for induction by tumour necrosis factor (TNF-alpha). An enhancer element (-187 to -158) composed of two imperfect direct palindromic repeats has been shown to be necessary and sufficient for TNF-alpha induction of a heterologous promoter. A multimer of either repeat is also responsive, while a single copy is not: this is the situation in the beta 2-microglobulin (beta 2-m) promoter which contains a single palindrome and does not respond to TNF-alpha. We had previously found that the two repeats can bind a factor named KBF1. We show here that in the uninduced state the transcription factor AP2 binds to the interpalindromic region, while in TNF-treated cells an NF kappa B-like activity is induced which displaces both KBF1 and AP2 and binds to the two palindromes. This strongly suggests that induction of an NF kappa B-like activity is responsible for TNF-alpha stimulation of mouse MHC class I genes.

Comparison of Anti-Tac and Anti-Transferrin Receptor-Conjugated Liposomes for Specific Drug Delivery to Adult T-Cell Leukemia

Adult T-cell leukemia (ATL) is a rapidly progressive and usually fatal malignancy of mature T cells characterized by the expression of large numbers of interleukin-2 (IL-2) receptors on the cell surface. Anti-Tac, a monoclonal antibody directed against the IL-2 receptor, was conjugated to liposomes and compared with anti-transferrin receptor (anti-TFR) conjugates for specific binding, internalization, and intracellular drug delivery to ATL cells. Two independent assays were used: a fluorimetric assay with liposome encapsulated 1-hydroxypyrene-3,6,8-trisulfonic acid, a pH-sensitive fluorescent dye, and a growth inhibition assay using methotrexate-gamma-aspartate, a liposome-dependent cytotoxic drug. MT-1 and HUT-102 cell lines derived from patients with ATL were compared with Molt-4, a leukemia cell line that does not express IL-2 receptors in an uninduced state. Fluorimetric studies showed specific binding and internalization of anti-Tac-conjugated liposomes by HUT-102 and MT-1 but not by the Tac-negative cell line Molt-4, demonstrating the lack of nonspecific or Fc receptor-mediated uptake. Anti-TFR-conjugated liposomes were effectively bound and internalized by all three cell lines and consistently showed the highest degree of cellular liposome uptake. Drug-containing liposomes conjugated to anti-Tac were more than tenfold more effective in causing growth inhibition of ATL cells than the nonspecific control conjugates. Anti-Tac conjugates caused minimal growth inhibition of Molt-4 cells over the concentration range effective against the ATL cells. Anti-TFR-coupled liposomes gave better growth inhibition of HUT-102 and MT-1 cells (40- to 60-fold) than anti-Tac conjugates. Both anti-Tac-directed and anti-TFR-directed liposomes are effective for intracellular drug delivery to ATL cells and may represent a useful method of treatment in this disease.

Comparison of anti-Tac and anti-transferrin receptor-conjugated liposomes for specific drug delivery to adult T-cell leukemia

Adult T-cell leukemia (ATL) is a rapidly progressive and usually fatal malignancy of mature T cells characterized by the expression of large numbers of interleukin-2 (IL-2) receptors on the cell surface. Anti- Tac, a monoclonal antibody directed against the IL-2 receptor, was conjugated to liposomes and compared with anti-transferrin receptor (anti-TFR) conjugates for specific binding, internalization, and intracellular drug delivery to ATL cells. Two independent assays were used: a fluorimetric assay with liposome encapsulated 1-hydroxypyrene- 3,6,8-trisulfonic acid, a pH-sensitive fluorescent dye, and a growth inhibition assay using methotrexate-gamma-aspartate, a liposome- dependent cytotoxic drug. MT-1 and HUT-102 cell lines derived from patients with ATL were compared with Molt-4, a leukemia cell line that does not express IL-2 receptors in an uninduced state. Fluorimetric studies showed specific binding and internalization of anti-Tac- conjugated liposomes by HUT-102 and MT-1 but not by the Tac-negative cell line Molt-4, demonstrating the lack of nonspecific or Fc receptor- mediated uptake. Anti-TFR-conjugated liposomes were effectively bound and internalized by all three cell lines and consistently showed the highest degree of cellular liposome uptake. Drug-containing liposomes conjugated to anti-Tac were more than tenfold more effective in causing growth inhibition of ATL cells than the nonspecific control conjugates. Anti-Tac conjugates caused minimal growth inhibition of Molt-4 cells over the concentration range effective against the ATL cells. Anti-TFR- coupled liposomes gave better growth inhibition of HUT-102 and MT-1 cells (40- to 60-fold) than anti-Tac conjugates. Both anti-Tac-directed and anti-TFR-directed liposomes are effective for intracellular drug delivery to ATL cells and may represent a useful method of treatment in this disease.

Stable-light-emitting Escherichia coli as a biosensor.

We have studied possibilities for constructing Escherichia coli strains capable of producing stable light. Light production in E. coli is achieved by cloning the genes encoding bacterial luciferase from Vibrio harveyi. To gain the advantage of sensitive detection of light we transferred the genes under the control of a strong, regulatable promoter system. Stabilization of light produced by E. coli clones was accomplished by finding the optimal plasmid construction and growth conditions as well as suitable measuring buffers. The adjustment of the luciferase synthesis for bioluminescence measurements to a high but not harmful level gives healthy cells and stable luciferase. Cultivation at 30 degrees C in an uninduced state was found to be the most important factor in getting stable-light production. The overall cell metabolism being unstressed gives us the possibility of monitoring cell physiology and factors affecting it via bioluminescence reactions in vivo. To make the results easy to interpret the light emission has to be stable during a measurement period of one to several hours. In the case of the original light-producing bacteria, Vibrio and Photobacterium strains it has not thus far been possible to find conditions where light emission would be stable for several hours. Based on our findings an automated biosensor system can be developed to monitor the effects of biologically active compounds against stable-light-producing bacteria.

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene.

Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.

A Highly Inducible System of Gene Expression by Positive Feedback Production of Glucocorticoid Receptors

To investigate the function of a cloned gene, inducible enhancer/promoters are preferred for controlling the level of expression experimentally. Highly inducible gene expression systems with both low basal levels and enhanced maximal levels of expression are especially sought. We have developed a new expression system using a positive feedback mechanism. Glucocorticoid receptor cDNA was fused to the enhancer/promoter of mouse mammary tumor virus (MTV) to induce receptor production. Simultaneous introduction of this receptor plasmid into mouse Ltk- cells augmented the expression of the reporter gene linked with the MTV enhancer/promoter by 10- to 100-fold in an induced state, but did not significantly change the expression of that in an uninduced state. This novel system of gene expression is a good candidate for an efficient genetic switch.

Selection and properties of Pseudomonas aeruginosa variants resistant to beta-lactam antibiotics.

The relation between basal and inducible beta-lactamase production and resistance to beta-lactam compounds was studied in five clinical Pseudomonas aeruginosa isolates and their corresponding resistant variants selected in the presence of either piperacillin, ceftazidime or aztreonam. In all wild-type strains enzyme levels were barely detectable in the uninduced state and most beta-lactams, including sulbactam and clavulanic acid, exhibited poor induction potency. Imipenem proved to be the most potent inducer in both these strains and their resistant variants. In the variants selected by either piperacillin or ceftazidime enzyme production amounted to 1.28 units/mg protein of the cell-free supernatants following the addition of beta-lactams as inducers. Additionally, these variants exhibited the phenomenon of "non-specific" induction, i.e. the increase of enzyme production by either a complex nutrient medium or by addition of vitamins. Enzyme production in the aztreonam-resistant variants was identical to that in the wild-type strains with a single exception, where the entire derepression of beta-lactamase production in one of the variants took place. Derepression of the chromosomally mediated enzyme affects the susceptibility to ureidopenicillins more than that to carboxy-penicillins and cephalosporins, whereas the beta-lactamase-independent resistance results in increased resistance to all beta-lactams with the single exception of imipenem.

DNA repair in the metallothionein gene increases with transcriptional activation.

We have studied DNA repair in the Chinese Hamster Ovary (CHO) metallothionein (MT) gene after UV-light induced damage. The repair was examined comparatively with or without transcriptional activation of the gene by incubation in the presence of the heavy metal ZnCl2. Whereas the repair efficiency was very low in the uninduced state, it increased significantly after induction of the gene. The presence of ZnCl2 did not appear to change other repair parameters in the cells. The overall genome DNA repair efficiency after UV irradiation was similar whether or not the gene was induced and the preferential DNA repair pattern in the essential dihydrofolate reductase (DHFR) gene which we have previously described was unaffected by the presence of ZnCl2. Based upon repair analysis in two different restriction fragments containing the MT I gene, we conclude that the region of efficient repair after induction is considerably larger than the 1 kb size of the gene. The results suggest that the accessibility of a genomic region to DNA repair enzymes may be regulated by the local chromatin structure in a dynamic manner.

Different adenovirus E1A-controlled properties of transformed cells require different levels of E1A expression

We have transformed primary baby rat kidney cells with a plasmid containing the adenovirus(Ad) 12E1 region in which the E1A promoter was replaced by the dexamethasone inducible mouse mammary tumor virus promoter. In the uninduced state the level of E1A expression is less than 10% of that in the induced state. We have investigated the effects of decreasing the levels of E1A on a number of E1A-mediated processes. First, expression of the E1B region is reduced several-fold upon reducing E1A expression. Second, a radical change in cell morphology is observed. Third, despite the decrease in E1A expression, down-regulation of the class-I major histocompatibility complex genes by Ad12E1A is not affected. These results are discussed in terms of different threshold levels of E1A expression required for various E1A-mediated processes.