Pro-remodeling peptides modulate collagen α1(I) promoter activity in rat cardiac myofibroblasts.

Previous studies have extensively demonstrated the effect of endothelin-1 (ET-1), angiotensin II (Ang II), and TGF-ß1 on the stimulation of collagen type I expression in cardiac myofibroblasts. However, the role of pro-remodeling peptides in the transcriptional regulation of the collagen promoter remains unclear. Thus, the purpose of this study was to investigate the net regulatory effects of pro-remodeling peptides on collagen type I promoter activity. Constructs of various lengths (300 bp, 1.1 kbp, 1.7 kbp, 2.3 kbp and 3.5 kbp) of the rat collagen α1(I) promoter were transfected into cardiac myofibroblasts in vitro and promoter activity was measured using chloramphenicol acetyl transferase (CAT) assays. Reduced promoter activity occurred across all treatments in myofibroblasts transfected with the 1.7 kbp construct. ET-1 was unable to increase promoter activity with constructs 300, 1.1, and 1.7 kbp, but induced promoter activity in cells with the 2.3 kbp construct. Additionally, while a combination of pro-remodeling peptides induced promoter activity across constructs, the resultant increase in the 2.3 and 3.5 kbp constructs were comparable to that observed from ET-1 treatment alone. Lastly, cells transfected with the entire promoter sequence had the lowest promoter activity. This data suggests that the collagen promoter is tightly regulated and that pro-remodeling factors produce an overall net effect on collagen expression, rather than additive.

Product feedback regulation implicated in translational control of the Trypanosoma brucei S-adenosylmethionine decarboxylase regulatory subunit prozyme.

Human African sleeping sickness (HAT) is caused by the parasitic protozoan Trypanosoma brucei. Polyamine biosynthesis is an important drug target in the treatment of HAT. Previously we showed that trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme for biosynthesis of the polyamine spermidine, is activated by heterodimer formation with an inactive paralogue termed prozyme. Furthermore, prozyme protein levels were regulated in response to reduced AdoMetDC activity. Herein we show that T. brucei encodes three prozyme transcripts. The 3'UTRs of these transcripts were mapped and chloramphenicol acetyltransferase (CAT) reporter constructs were used to identify a 1.2 kb region that contained a 3'UTR prozyme regulatory element sufficient to upregulate CAT protein levels (but not RNA) upon AdoMetDC inhibition, supporting the hypothesis that prozyme expression is regulated translationally. To gain insight into trans-acting factors, genetic rescue of AdoMetDC RNAi knock-down lines with human AdoMetDC was performed leading to rescue of the cell growth block, and restoration of prozyme protein to wild-type levels. Metabolite analysis showed that prozyme protein levels were inversely proportional to intracellular levels of decarboxylated AdoMet (dcAdoMet). These data suggest that prozyme translation may be regulated by dcAdoMet, a metabolite not previously identified to play a regulatory role.

Synthetic promoters functional in Francisella novicida and Escherichia coli.

In this work, we describe the identification of synthetic, controllable promoters that function in the bacterial pathogen Francisella novicida, a model facultative intracellular pathogen. Synthetic DNA fragments consisting of the tetracycline operator (tetO) flanked by a random nucleotide sequence were inserted into a Francisella novicida shuttle plasmid upstream of a promoterless artificial operon containing the reporter genes cat and lacZ. Fragments able to promote transcription were selected for based on their ability to drive expression of the cat gene, conferring chloramphenicol resistance. Promoters of various strengths were found, many of which were repressed in the presence of the tetracycline repressor (TetR) and promoted transcription only in the presence of the TetR inducer anhydrotetracycline. A subset of both constitutive and inducible synthetic promoters were characterized to find their induction ratios and to identify their transcription start sites. In cases where tetO was located between or downstream of the -10 and -35 regions of the promoter, control by TetR was observed. If the tetO region was upstream of the -35 region by more than 9 bp, it did not confer TetR control. We found that three of three promoters isolated in F. novicida functioned at a comparable level in E. coli; however, none of the 10 promoters isolated in E. coli functioned at a significant level in F. novicida. Our results allowed us to isolate minimal F. novicida promoters of 47 and 48 bp in length.

Versatile synthesis of probes for high-throughput enzyme activity screening.

Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination.

Assessing the effect of RET transmembrane domain mutations in receptor self-association capability using the in vivo TOXCAT system.

Mutations of c-RET proto-oncogene with a unique localization within the human transmembrane receptor represent a challenge for contemporary molecular oncology techniques. RET transmembrane domain (TMD)-driven dimerization of the receptor leads to its permanent activation that eventually results in the development of medullary thyroid neoplasia. In this study, we describe the employment of the TOXCAT system which enables to investigate mutation-induced alterations in the strength of RET TMD dimerization in vivo. We suggest an improvement of the method by adding reporter gene quantification at the mRNA levels as a support to the commonly used reporter protein level. We have investigated possible changes in RET TMD dimerization in case of two germline RET TMD mutations found in in several individual cases and MEN2 families worldwide, p.Ala641Ser and p.Ser649Leu. According to our results, substitution of Ser-649 residue by leucine, found as a result of germline mutation, caused a significant decrease of RET TMD self-association in comparison to RET wild-type transmembrane domain. The impaired ability of self-association suggests a novel, yet unknown mechanism of tyrosine kinase domain activation, possibly independent of RET homodimerization.

Diversity and strength of internal outward-oriented promoters in group IIC-attC introns.

Integrons are genetic elements that incorporate mobile gene cassettes by site-specific recombination and express them as an operon from a promoter (Pc) located upstream of the cassette insertion site. Most gene cassettes found in integrons contain only one gene followed by an attC recombination site. We have recently shown that a specific lineage of group IIC introns, named group IIC-attC introns, inserts into the bottom strand sequence of attC sites. Here, we show that S.ma.I2, a group IIC-attC intron inserted in an integron cassette array of Serratia marcescens, impedes transcription from Pc while allowing expression of the following antibiotic resistance cassette using an internal outward-oriented promoter (P(out)). Bioinformatic analyses indicate that one or two putative P(out), which have sequence similarities with the Escherichia coli consensus promoters, are conserved in most group IIC-attC intron sequences. We show that P(out) with different versions of the -35 and -10 sequences are functionally active in expressing a promoterless chloramphenicol acetyltransferase (cat) reporter gene in E. coli. P(out) in group IIC-attC introns may therefore play a role in the expression of one or more gene cassettes whose transcription from Pc would otherwise be impeded by insertion of the intron.

Abrogation of contaminating RNA activity in HIV-1 Gag VLPs.

BACKGROUND: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. METHODS: HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. RESULTS: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/µg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. CONCLUSIONS: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice.

[Effects of space flight on protein content and electrophoresis in Glycyrrhiza uralensis].

OBJECTIVE: To investigate the space environment on the role of licorice mutagenesis analysis of proteins. METHOD: Licorice (Glycyrrhiza uralensis) seeds were carried by a recoverable satellite for 18 days (the average radiation dose in the flight recovery module was 0.102 m x d(-1), the orbit semidiameter 350 km, gravity 10(-6)). After return, The satellite-flown seeds and the unflown seeds (ground control) were planted in the fields of experimental farm. The leaves of each group were used for studying the effects of space flight on CAT, SOD activity, the protein content and electrophoresis. RESULT: After the space flight, CAT, SOD activity of licorice increased in varying degrees, the difference was significant (P<0.05), two types of enzyme activity of sample from Ordos were higher than that from Hangjinqi. The protein content of licorice increased in a certain extent, the difference was significant (P<0.05), while protein electrophoresis also showed differences, weak new bands appeared. CONCLUSION: These results indicated that spaceflight has effect on protein of licorice, these changes may be used as a tool for accelerating the progress in G. uralensis breeding.

Fiber type- and position-dependent expression of a myosin light chain-CAT transgene detected with a novel histochemical stain for CAT.

We recently generated and characterized transgenic mice in which regulatory sequences from a myosin light chain gene (MLC1f/3f) are linked to the chloramphenicol acetyltransferase (CAT) gene. Transgene expression in these mice is specific to skeletal muscle and graded along the rostrocaudal axis: adult muscles derived from successively more caudal somites express successively higher levels of CAT. To investigate the cellular basis of these patterns of expression, we developed and used a histochemical stain that allows detection of CAT in individual cells. Our main results are as follows: (a) Within muscles, CAT is detected only in muscle fibers and not in associated connective tissue, blood vessels, or nerves. Thus, the tissue specificity of transgene expression observed by biochemical assay reflects a cell-type specificity demonstrable histochemically. (b) Within individual muscles, CAT levels vary with fiber type. Like the endogenous MLC1f/3f gene, the transgene is expressed at higher levels in fast-twitch (type II) than in slow-twitch (type I) muscle fibers. In addition, CAT levels vary among type II fiber subtypes, in the order IIB greater than IIX greater than IIA. (c) Among muscles that are similar in fiber type composition, the average level of CAT per fiber varies with rostrocaudal position. This position-dependent variation in CAT level is apparent even when fibers of a single type are compared. From these results, we conclude that fiber type and position affect CAT expression independently. We therefore infer the existence of separate fiber type-specific and positionally graded transcriptional regulators that act together to determine levels of transgene expression.

Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells.

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.

Induction of MMP-1 (collagenase-1) by relaxin in fibrocartilaginous cells requires both the AP-1 and PEA-3 promoter sites.

OBJECTIVES - Relaxin induces the matrix metalloproteinase MMP-1 (collagenase-1) in TMJ fibrocartilaginous cells, and this response is potentiated by beta-estradiol. We identified the MMP-1 promoter sites and transcription factors that are induced by relaxin with or without beta-estradiol in fibrocartilaginous cells. MATERIAL AND METHODS - Early passage cells were transiently transfected with the pBLCAT2 plasmid containing specific segments of the human MMP-1 promoter regulating the chloramphenicol acyl transferase (CAT) gene and co-transfected with a plasmid containing the beta-galactosidase gene. The cells were cultured in serum-free medium alone or medium containing 0.1 ng/ml relaxin, or 20 ng/ml beta-estradiol or both hormones, and lysates assayed for CAT and beta-galactosidase activity. RESULTS - Cells transfected with the -1200/-42 or -139/-42 bp MMP-1 promoter-reporter constructs showed 1.5-fold and 3-fold induction of CAT by relaxin in the absence or presence of beta-estradiol, respectively. Relaxin failed to induce CAT in the absence of the -137/-69 region of the MMP-1 promoter, which contains the AP-1-and PEA3-binding sites. Using wild type or mutated minimal AP-1 and PEA-3 promoters we found that both these promoter sites are essential for the induction of MMP-1 by relaxin. The mRNAs for transcription factors c-fos and c-jun, which together form the AP-1 heterodimer, and Ets-1 that modulates the PEA-3 site, were upregulated by relaxin or beta-estradiol plus relaxin. CONCLUSION - These studies show that both the AP-1 and PEA-3 promoter sites are necessary for the induction of MMP-1 by relaxin in fibrocartilaginous cells.

Conditional MitoTimer reporter mice for assessment of mitochondrial structure, oxidative stress, and mitophagy.

Assessment of structural and functional changes of mitochondria is vital for biomedical research as mitochondria are the power plants essential for biological processes and tissue/organ functions. Others and we have developed a novel reporter gene, pMitoTimer, which codes for a redox sensitive mitochondrial targeted protein that switches from green fluorescence protein (GFP) to red fluorescent protein (DsRed) when oxidized. It has been shown in transfected cells, transgenic C. elegans and Drosophila m., as well as somatically transfected adult skeletal muscle that this reporter gene allows quantifiable assessment of mitochondrial structure, oxidative stress, and lysosomal targeting of mitochondria-containing autophagosomes. Here, we generated CAG-CAT-MitoTimer transgenic mice using a transgene containing MitoTimer downstream of LoxP-flanked bacterial chloramphenicol acetyltransferase (CAT) gene with stop codon under the control of the cytomegalovirus (CMV) enhancer fused to the chicken ß-actin promoter (CAG). When CAG-CAT-MitoTimer mice were crossbred with various tissue-specific (muscle, adipose tissue, kidney, and pancreatic tumor) or global Cre transgenic mice, the double transgenic offspring showed MitoTimer expression in tissue-specific or global manner. Lastly, we show that hindlimb ischemia-reperfusion caused early, transient increases of mitochondrial oxidative stress, mitochondrial fragmentation and lysosomal targeting of autophagosomes containing mitochondria as well as a later reduction of mitochondrial content in skeletal muscle along with mitochondrial oxidative stress in sciatic nerve. Thus, we have generated conditional MitoTimer mice and provided proof of principle evidence of their utility to simultaneously assess mitochondrial structure, oxidative stress, and mitophagy in vivo in a tissue-specific, controllable fashion.

An anti-apoptotic protein, Hax-1, inhibits the HIV-1 rev function by altering its sub-cellular localization.

The human immunodeficiency virus type 1 (HIV-1) Rev protein facilitates the nuclear export of viral mRNA containing the Rev response element (RRE). Although several host proteins co-operating with Rev in viral RNA export have been reported, little is known about the innate host defense factors that Rev overcomes to mediate the nuclear export of unspliced viral mRNAs. We report here that an anti-apoptotic protein, HS1-associated protein X-1 (Hax-1), a target of HIV-1 Vpr, interacts with Rev and inhibits its activity in RRE-mediated gene expression. Co-expression of Sam68 emancipates Rev activity from Hax-1-mediated inhibition. Hax-1 does not bind to RRE RNA by itself, but inhibits Rev from binding to RRE RNA in vitro. The impact of Hax-1 on Rev/RRE interactions in vitro correlates well with the reduced level of RRE-containing mRNA in vivo. Immunofluorescence studies further reveal that Hax-1 and Rev are cytoplasmic and nuclear proteins, respectively, when expressed independently. However, in Hax-1 co-expressing cells, Rev is translocated from the nucleus to the cytoplasm, where it is co-localized with Hax-1 in the cytoplasm. We propose that over-expression of Hax-1, possibly through binding to Rev, may interfere with the stability/export of RRE-containing mRNA and target the RNA for degradation.

GATA elements control repression of cardiac troponin I promoter activity in skeletal muscle cells.

BACKGROUND: We reported previously that the cardiac troponin I (cTnI) promoter drives cardiac-specific expression of reporter genes in cardiac muscle cells and in transgenic mice, and that disruption of GATA elements inactivates the cTnI promoter in cultured cardiomyocytes. We have now examined the role of cTnI promoter GATA elements in skeletal muscle cells. RESULTS: Mutation or deletion of GATA elements induces a strong transcriptional activation of the cTnI promoter in regenerating skeletal muscle and in cultured skeletal muscle cells. Electrophoretic mobility shift assays show that proteins present in nuclear extracts of C2C12 muscle cells bind the GATA motifs present in the cTnI promoter. However, GATA protein complex formation is neither reduced nor supershifted by antibodies specific for GATA-2, -3 and -4, the only GATA transcripts present in muscle cells. CONCLUSION: These findings indicate that the cTnI gene promoter is repressed in skeletal muscle cells by GATA-like factors and open the way to further studies aimed at identifying these factors.

The immediate early genes of human cytomegalovirus upregulate tumor necrosis factor-alpha gene expression.

Cytomegalovirus (CMV) is an important cause of disease in the immunocompromised patient and CMV infection is associated with predominantly mononuclear inflammatory response. Since products of the CMV immediate early (IE) gene region are potent trans-activators, we used the monocyte cell line THP-1 and a transient transfection assay to determine if these viral proteins upregulate expression of the TNF gene. The IE genes of CMV upregulated TNF gene activity as judged by increases in promoter activity, steady state mRNA, and protein production. The presence or absence of the 3' untranslated region of the TNF gene did not affect gene expression induced by the IE gene products. These studies suggest that activation of TNF gene expression by the CMV IE gene products may, in part, account for the inflammatory response associated with CMV infections.

Mycobacterium tuberculosis enhances human immunodeficiency virus-1 replication by transcriptional activation at the long terminal repeat.

Tuberculosis has emerged as an epidemic fueled by the large number of individuals infected with the human immunodeficiency virus, especially those who are injecting drug users. We found a striking increase from 4- to 208-fold in p24 levels in bronchoalveolar lavage fluid from involved sites of Mycobacterium tuberculosis infection vs uninvolved sites in three HIV+ patients. We used an in vitro cell culture model to determine if tuberculosis could activate replication of HIV-1. Mononuclear phagocyte cell lines U937 and THP-1 infected with HIV-1JR-CSF, in vitro and stimulated with live M. tuberculosis H37Ra, had a threefold increase in p24 in culture supernatants. Using the HIV-1 long terminal repeat with a chloramphenicol acetyltransferase (CAT) reporter construct, live M. tuberculosis increased transcription 20-fold in THP-1 cells, and cell wall components stimulated CAT expression to a lesser extent. The nuclear factor-kappa B enhancer element was responsible for the majority of the increased CAT activity although two upstream nuclear factor-IL6 sites may also contribute to enhanced transcription. Antibodies to TNF-alpha and IL-1 inhibited the increase in CAT activity of the HIV-1 long terminal repeat by M. tuberculosis from 21-fold to 8-fold. Stimulation of HIV-1 replication by M. tuberculosis may exacerbate dysfunction of the host immune response in dually infected individuals.

Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia.

Overexpression of plasma apolipoprotein CIII (apo CIII) causes hypertriglyceridemia in transgenic mice. A genetically variant form of the human apo CIII promoter, containing five single base pair changes, has been shown to be associated with severe hypertriglyceridemia in a patient population. In animals and in cultured cells the apo CIII gene is transcriptionally downregulated by insulin. In this study we demonstrate that, unlike the wild-type promoter, the variant promoter was defective in its response to insulin treatment, remaining constitutively active at all concentrations of insulin. The loss of insulin regulation was mapped to polymorphic sites at -482 and -455, which fall within a previously identified insulin response element. Loss of insulin regulation could result in overexpression of the apo CIII gene and contribute to the development of hypertriglyceridemia. The variant apo CIII promoter is common in the human population and may represent a major contributing factor to the development of hypertriglyceridemia.

The immunoglobulin heavy chain locus contains another B-cell-specific 3' enhancer close to the alpha constant region.

The transcription of immunoglobulin genes is controlled by variable region promoters and by enhancers, both of which are lymphoid specific. Because immunoglobulin genes are subject to an extremely complex regulation, we anticipated that there might be additional control elements for these genes. We therefore sought additional enhancers and demonstrate here that there is indeed another weak transcriptional enhancer just 3' to the mouse alpha constant region. This novel immunoglobulin enhancer is lymphoid specific and at two positions can bind members of the Oct family of transcription factors.

Gas1-induced growth suppression requires a transactivation-independent p53 function.

In normal cells, induction of quiescence is accompanied by the increased expression of growth arrest-specific genes (gas). One of them, gas1, is regulated at the transcriptional level and codes for a membrane-associated protein (Gas1) which is down regulated during the G0-to-S phase transition in serum-stimulated cells. Gas1 is not expressed in growing or transformed cells, and when overexpressed in normal fibroblasts, it blocks the G0-to-S phase transition. Moreover, Gas1 blocks cell proliferation in several transformed cells with the exception of simian virus 40- or adenovirus-transformed cell lines. In this paper, we demonstrate that overexpression of Gas1 blocks cell proliferation in a p53-dependent manner and that the N-terminal domain-dependent transactivating function of p53 is dispensable for Gas1-induced growth arrest. These data therefore indicate that the other intrinsic transactivation-independent functions of p53, possibly related to regulation of apoptosis, should be involved in mediating Gas1-induced growth arrest.

Do products of the myc proto-oncogene play a role in transcriptional regulation of the prothymosin alpha gene?

The Myc protein has been reported to activate transcription of the rat prothymosin alpha gene by binding to an enhancer element or E box (CACGTG) located in the first intron (S. Gaubatz et al., Mol. Cell. Biol. 14:3853-3862, 1994). The human prothymosin alpha gene contains two such motifs: in the promoter region at kb -1.2 and in intron 1, approximately 2 kb downstream of the transcriptional start site in a region which otherwise bears little homology to the rat gene. Using chloramphenicol acetyltransferase (CAT) reporter constructs driven either by the 5-kb human prothymosin alpha promoter or by a series of truncated promoters, we showed that removal of the E-box sequence had no effect on transient expression of CAT activity in mouse L cells. When intron 1 of the prothymosin alpha gene was inserted into the most extensive promoter construct downstream of the CAT coding region, a diminution in transcription, which remained virtually unchanged upon disruption of the E boxes, was observed. CAT constructs driven by the native prothymosin alpha promoter or the native promoter and intron were indifferent to Myc; equivalent CAT activity was observed in the presence of ectopic normal or mutant Myc genes. Similarly, expression of a transiently transfected wild-type prothymosin alpha gene as the reporter was not affected by a repertoire of myc-derived genes, including myc itself and dominant or recessive negative myc mutants. In COS-1 cells, equivalent amounts of the protein were produced from transfected prothymosin alpha genes regardless of whether genomic E boxes were disrupted, intron 1 was removed, or a repertoire of myc-derived genes was included in the transfection cocktail. More importantly, cotransfection of a dominant negative Max gene failed to reduce transcription of the endogenous prothymosin alpha gene in COS cells or the wild-type transfected gene in COS or L cells. Taken together, the data do not support the idea that Myc activates transcription of the intact human prothymosin alpha gene or reporter constructs that mimic its structure. Rather, they suggest that the human prothymosin alpha promoter and downstream elements are buffered so as to respond poorly, if at all, to transient fluctuations in transcription factors which regulate other genes.