Bacterial Chloramphenicol Acetyltransferase Gene Research Articles

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 β-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.

Abstract C16: miR-148a regulates the expression of PTEN in hepatitis B associated hepatocellular carcinoma

Abstract Previous work has shown that hepatitis B virus encoded X antigen (HBx) is a trans-regulatory protein that alters the activity of selected transcription factors and cytoplasmic signal transduction pathways. The resulting changes in host gene expression are thought to contribute importantly to the development and progression of hepatocellular carcinoma (HCC). In this context, HBx up-regulates the expression of a unique gene, URG11, which in turn up-regulates β-catenin, thereby contributing importantly to hepatocarcinogenesis. Changes in the expression levels of microRNAs (miRNAs) are also characteristic of many tumors, including HCC. To determine whether altered miRNA expression contributes to the mechanism whereby HBx and URG11 contribute to HCC, recombinant retroviruses encoding HBx, URG11, or the bacterial chloramphenicol acetyltransferase (CAT) gene (the latter used as a negative control) were used to infect separate cultures of HepG2 and Hep3B cells. When these cells were compared by miRNA array analysis, both HBx and URG11 were shown to promote the expression of miR-148a. This was confirmed by quantitative (q)RT/PCR analysis. Elevated levels of miR-148a strongly correlated with HBx positive staining in liver samples from infected patients by qRT/PCR (P< 0.001). To study the function of miR-148a, anti-miR-148a was introduced into HepG2 and Hep3B cells stably expressing HBx or stably over-expressing URG11. Anti-miR-148a suppressed cell proliferation (P < 0.01), cell cycle progression (P < 0.01), cell migration (P < 0.01), anchorage independent growth in soft agar (P < 0.01) and subcutaneous tumor formation in SCID mice (P < 0.05). Introduction of anti-miR-148a also increased PTEN mRNA and protein expression, suggesting that PTEN was targeted by miR-148a. This was verified in experiments where anti-miR-148a failed to suppress PTEN expression when co-transfected with reporter gene mutants of the 3′UTR of PTEN mRNA. When HBx positive HepG2 and Hep3B cells were transiently transfected with URG11 specific siRNA, both URG11 and miR-148a levels were suppressed, suggesting that up-regulated expression of miR-148a in HBx positive cells was URG11 dependent. Finally, anti-miR-148a blocked the stimulation of Akt signaling by HBx and URG11, resulting in decreased expression of β-catenin (P < 0.05). Thus, miR-148a appears to contribute a central role to HBx/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C16.

Transgenic models for proliferative and hyperfunctional thyroid diseases.

Mouse transgenic models that develop thyroid diseases were generated. All transgenes were driven by the thyroid specific promoter of the thyroglobulin gene. The tissue specificity of the promoter was investigated by using the bacterial chloramphenicol acetyl transferase gene as reporter. The expression of an adenosine A2a receptor resulted in the permanent activation of the cAMP cascade. As a consequence, the transgenic mice developed severe hyperthyroidism and a large goiter, demonstrating in vivo the role of the cAMP cascade in the promotion of both function and proliferation of the thyroid cell. These mice constitute a model for autonomous hyperfunctional adenoma and non-autoimmune familial hyperthyroidism, where mutant thyrotropin receptors stimulate the cAMP cascade constitutively. In another transgenic model, the function of the retinoblastoma susceptibility gene product RB1 (and of related proteins) was inhibited by expression of the E7 oncoprotein of human papillomavirus type 16. The result was the development of a differentiated and normofunctional colloid goiter, with the progressive development of differentiated malignant lesions. This model suggests the essential role of RB1 and related proteins in the negative control of proliferation that characterizes thyroid cells in the adult. Other transgenic models of thyroid diseases are discussed.

The hepatitis B x antigen effector, URG7, blocks tumour necrosis factor α-mediated apoptosis by activation of phosphoinositol 3-kinase and β-catenin

Hepatitis B x antigen (HBxAg) contributes significantly to the pathogenesis of chronic infection and development of hepatocellular carcinoma. To discern some of its operative pathways, HepG2 cells were stably transduced with HBx or the bacterial chloramphenicol acetyltransferase (CAT) gene. Differential gene expression has previously revealed an upregulated gene, clone 7 (URG7), that conferred resistance to anti-Fas killing on HepG2X cells. Given that tumour necrosis factor alpha (TNFalpha) is also an important mediator of chronic hepatitis, and partially shares signalling with Fas, experiments were designed to test whether URG7 blocks TNFalpha killing of HepG2X cells. HepG2X cells expressing URG7 and HepG2 cells overexpressing URG7 in the absence of HBxAg were resistant to TNFalpha killing compared with HepG2CAT cells. URG7 small interfering RNA restored the sensitivity of HepG2X cells to TNFalpha killing. Killing was associated with the activation of caspases 3 and 8, suggesting that URG7 blocked these caspases. This resistance was also associated with activation of phosphoinositol 3-kinase/Akt. Given that Akt and HBxAg also activate beta-catenin, experiments were designed to determine whether URG7 blocked apoptosis via activation of beta-catenin. Both HBxAg and URG7 activated fragments of the beta-catenin promoter, and also promoted expression of beta-catenin target genes. Hence, URG7 inhibits TNFalpha-mediated killing by blocking one or more caspases in the apoptotic pathway and by activating phosphoinositol 3-kinase and beta-catenin, thereby overriding the apoptotic signalling of TNFalpha. This suggests that URG7 helps to protect virus-infected hepatocytes during chronic hepatitis B virus infection.

In vivo competition identifies positive cis-regulatory elements required for lineage-specific gene expression in the sea urchin embryo.

Several cis-regulatory elements within the 5' regulatory region of the lineage-specific CyIIIa actin gene have been identified by in vivo competition. Sea urchin eggs were coinjected with a fusion construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by the CyIIIa regulatory domain, together with molar excesses of various DNA subfragments that are derived from this region. Each subfragment studied includes one or several known sites where highly specific interactions occur in vitro with nuclear DNA-binding proteins. Coinjection of excess molecules of some of these subregions results in a decrease in the activity of the CyIIIa-CAT fusion gene, as a function of the molar subfragment: CyIIIa-CAT ratio. This result implies that these sites complete with cis sequences linked to the CAT reporter gene for limited factors that positively regulate CyIIIa transcription in the embryo, and demonstrates the functional importance of a number of the DNA-protein interactions that have been observed in vitro.

Imaging Update: New Windows, New Views

The recent progress in our understanding of the molecular-genetic mechanisms active in many diseases and the application of new biologically based approaches in therapy are exciting new developments that have emerged following mapping of the human genome. Novel “molecular therapies” have been

Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus

A mammalian expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used to demonstrate that CAT could be successfully used as a reporter system in fish cells growing at low temperatures. We then constructed a viral haemorrhagic septicaemia virus (VHSV) minigenome by cloning the CAT reporter gene between the viral leader and trailer sequences. This construct was used in transfection experiments with helper VHSV to demonstrate that the minigenome can be encapsidated and transcribed by helper virus proteins. In addition, passaging of viruses collected from cells expressing the minigenome showed that the minigenome was being packaged and replicated in the presence of helper virus. These experiments provide the initiating steps for a reverse genetics system for VHSV.

Transcriptional regulation of 2',3'-cyclic nucleotide 3'-phosphodiesterase gene expression by cyclic AMP in C6 cells.

It was recently shown that the two transcripts encoding the isoforms of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP1 and CNP2) are differentially regulated during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only CNP2 mRNA is present, whereas in differentiating oligodendrocytes, both CNP1 and CNP2 mRNAs are expressed. This pattern of CNP expression is likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation. Here, we report the influence of increased intracellular cyclic AMP (cAMP) levels on the transcription of both CNP1 and CNP2 mRNAs in rat C6 glioma cells. We found that the transcription of CNP1 mRNA was significantly increased in comparison with that of CNP2 mRNA in cells treated with cAMP analogues to elevate intracellular cAMP levels. This up-regulation of CNP1 expression (a) is due to an increase of transcription, (b) requires de novo protein synthesis, and (c) requires the activity of protein kinase A. These results are physiologically significant and support the idea that a cAMP-mediated pathway is part of the molecular mechanisms regulating the expression of CNP1 in oligodendrocytes. The regulation of CNP1 promoter activity by cAMP was then investigated in stably transfected C6 cell lines containing various deletions of the CNP promoter directing the bacterial chloramphenicol acetyltransferase gene. We showed that the sequence between nucleotides -126 and -102 was essential for the cAMP-dependent induction of CNP1 expression. Gel retardation analysis showed that two protein-DNA complexes are formed between this sequence and nuclear factors from C6 cells treated or not treated with cAMP. This suggests that the induction of CNP1 mRNA transcription is not mediated by changes in binding of nuclear factors that interact directly with the -126/-102 sequence. Sequence analysis of this region revealed the presence of a putative activator protein-2 (AP-2) binding site. It is interesting that mutagenesis of this region resulted in a significant reduction in transcriptional responses to cAMP, implying a possible role for the AP-2 factor in the expression of CNP1. In addition, we have shown that putative binding sites for activator protein-4 and nuclear factor-1 adjacent to the AP-2 site are required for efficient induction of CNP1 expression by cAMP. Taken together, our results show that the cAMP-dependent accumulation of CNP1 mRNA appears to depend on the synergistic interaction of several regulatory elements.

Transcription factor Sp3 activates the liver/bone/kidney-type alkaline phosphatase promoter in hematopoietic cells

The promoter region of the liver/bone/ kidney-type alkaline phosphatase gene was examined to define the cis-acting regulatory sequences and transcription factors responsible for its expression in hematopoietic cells. Transient transfection experiments revealed that regions deleted up to -154 base pairs upstream from the transcription initiation site had significant activities to induce bacterial chloramphenicol acetyltransferase gene. The shortest DNA fragment was found to contain three GC boxes in addition to a TATA box. Electrophoretic mobility shift assay and Southwestern analysis showed that Sp3 could bind to the fragment. Western blot analysis also detected Sp3 protein in eluate from the DNA probe mixed with the nuclear extracts. Through the use of Drosophila Schneider cells that lack the Sp1 family of transcription factors, Sp3 was shown to activate the basal promoter in a dose-dependent manner. When the amount of Sp3 was limited, the most proximal GC box was found to be critical for the basal promoter activity.

Sequence analysis of the human glycoprotein hormone α-subunit gene 5′-flanking DNA and identification of a potential regulatory element as an Alu repetitive sequence

The nucleotide sequence of the human glycoprotein hormone α-subunit (GPHα) gene 5′-flanking DNA was determined from −1637 to +49 relative to the cap site (+1). Comparison of the upstream sequence of the human gene with those of rhesus and mouse demonstrates regions with variable identity. When the 1.7 kb fragment was used to drive the expression of chloramphenicol acetyltransferase (CAT) in transiently transfected HeLa cells, it was found that CAT activity was elevated about 3-fold when the fragment was truncated from −1637 to −846, suggesting the presence of a negative regulatory element in the distal 5′-flanking DNA. This overlaps an Alu repetitive sequence (ARS) located between nucleotides −1330 and −1007. Gel mobility shift and DNase protection analyses identified a protein binding site centered around −1100 in the ARS second monomer. The GPHα upstream ARS was cloned in both orientations in positions upstream and downstream from the bacterial CAT gene under control of the herpes simplex virus thymidine kinase (tk) promoter. DNA-mediated transient transfection of these plasmids revealed a marked inhibition (79–82%) of CAT production by the ARS when it was cloned upstream from the tk promoter and in the same orientation as that found in the GPHα 5′-flanking DNA. Smaller decreases (29–57%) were produced by the ARS cloned upstream from the tk promoter in the reverse orientation. In marked contrast, the Alu repetitive element had little or no effect when cloned in either orientation downstream from the tk-CAT gene. Introduction of a second ARS downstream from the CAT reporter gene in vectors already containing an ARS upstream from the tk promoter significantly reduced the strong negative effect elicited by the upstream repetitive element. When compared to the Blur 8 Alu element, the GPHα upstream ARS differs markedly with respect to its effect on tk-CAT expression in transient assays and as a substrate for DNA binding proteins present in HeLa nuclear extracts. Together, the transient expression results demonstrate that ARS elements can influence expression of nearby class II promoters. The extent of this effect depends on element position and orientation, cell type, the particular ARS (e.g., GPHα or Blur 8), and whether copies were present both upstream and downstream from the transcription unit.

Rescue of synthetic salmonid rhabdovirus minigenomes.

Synthetic T7-driven cDNA minigenomes containing the bacterial chloramphenicol acetyltransferase gene as a reporter were derived from the genome of two salmonid novirhabdoviruses, infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). We showed that an exogenous IHNV RNA minigenome transfected into fish cells could be rescued following IHNV infection as it was replicated, encapsidated and transcribed. When cells were infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3), transfected with the plasmid carrying the IHNV minigenome (genomic- and antigenomic-sense) and superinfected with IHNV, rescue of the minigenome was more efficient. Heterologous VHSV/IHNV rescue experiments failed. Finally, when the IHNV N, P and L proteins were expressed from cDNAs in cells, the minigenome was also successfully rescued, indicating that the nucleocapsid proteins were biologically functional. These data represent the first example of rescue experiments for non-mammalian rhabdoviruses replicating at a low temperature.

TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p.

Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro.

Farnesoid X Receptor Responds to Bile Acids and Represses Cholesterol 7α-Hydroxylase Gene (CYP7A1) Transcription

Cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor alpha (RXRalpha)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 microM) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXRalpha/FXR. The concentration of CDCA required to inhibit 50% of reporter activity (IC(50)) was determined to be approximately 25 microM without FXR and 10 microM with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides -148 and -128 was the FXR response element, but RXRalpha/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells.

Increased expression after X-irradiation of MUC1 in cultured human colon carcinoma HT-29 cells.

The effect of X‐irradiation on production of MUC1 was studied with human colon carcinoma HT‐29 cells. As evaluated by immunocytochemical staining, the percentages of MUC1‐positive cells in cells at 4 days after 6 Gy irradiation and in unirradiated control cells were 52±3.5% (n=6) and 26±2.8% (n=6), respectively. Flow‐cytometric analysis of living cells showed that MUC1 began to rise from day 1, reaching a plateau by day 4 after 6 Gy irradiation. Western blot analysis with monoclonal antibody MY.1E12 against glycosylated MUC1 (mature form) showed dose‐dependent increases of two bands (500 and 390 kDa) corresponding to two polymorphic MUC1 alleles. Premature forms of MUC1 (350 and 240 kDa) were detectable with monoclonal antibody HMFG‐2 only in irradiated cells, suggesting that new core protein synthesis had been induced. The transcriptional activity of the MUC1 gene was analyzed in terms of transient expression of MUC1‐CAT reporter plasmids containing 5′‐flanking sequences of the MUC1 gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The results of CAT assay indicate that enhanced expression of MUC1 in irradiated HT‐29 cells was due to upregulation of MUC1 transcription, and required the upstream promoter.

Use of a cryptic splice donor site in the chloramphenicol acetyltransferase (CAT)-SV40 small-t antigen cassette generates alternative transcripts in transgenic rats.

The bacterial gene chloramphenicol acetyltransferase (CAT) is a widely used reporter in both in-vitro and in-vivo studies of genetic regulation. We have recently generated novel rat transgenic lines carrying an arylalkylamine N-acetyltransferase (AA-NAT) promoter-reporter construct in which CAT (with associated SV40 small-t antigen sequence) is the reporter. In addition to the predicted transgene transcript (1.9 kb), we identified an abundant 1.5 kb transcript which derives from an alternative splicing event that utilises a cryptic splice donor site located within the CAT gene. The native CAT open reading frame (ORF) is lost in the 1.5 kb transcript, and a western analysis has shown that protein deriving from an aberrant open reading frame is not expressed at detectable levels.

Rescue of a bovine respiratory syncytial virus genomic RNA analog by bovine, human and ovine respiratory syncytial viruses confirms the "functional integrity" and "cross-recognition" of BRSV cis-acting elements by HRSV and ORSV.

The nucleotide sequences of the 3' leader and 5' trailer regions were determined for genomic RNA of bovine respiratory syncytial virus (BRSV) strain A-51908. The leader and trailer sequences are '45' and '161' nucleotides in length, respectively. The functionality of BRSV leader and trailer sequences and their recognition by HRSV and ovine respiratory syncytial virus (ORSV) proteins were examined with a in vitro transcribed BRSV genomic RNA analog carrying the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of BRSV transcription signals. Upon transfection into BRSV, HRSV or ORSV infected cells, the BRSV minireplicons were 'rescued' such that the reporter gene was expressed, the minigenome was replicated and packaged into micrococcal nuclease resistant-infectious minireplicons. The passage of infectious minireplicons could be blocked by a polyclonal BRSV neutralizing antiserum. Bovine parainfluenza virus-3, a heterologous paramyxovirus was inactive in rescuing BRSV genomic RNA analog. Mutational substitution of the G residue at position 4 of leader sequence in the BRSV genomic RNA analog, with an A or U residue inhibited its transcription and replication, while replacement with a C residue had no significant effect on rescue. These results show that the cis-acting elements of BRSV are functional and are also recognized by the proteins of HRSV and ORSV. The helper virus complemented rescue system developed here will be useful for characterizing the cis-acting elements of BRSV.

Genetic targeting: the serotonin N-acetyltransferase promoter imparts circadian expression selectively in the pineal gland and retina of transgenic rats.

The arylalkylamine N-acetyltransferase (AA-NAT) gene is strongly expressed in the rat primarily in the pineal gland; low levels of expression are also found in the retina. AA-NAT catalyzes the key regulatory step controlling rhythmic melatonin output: the acetylation of serotonin. In the rat, the AA-NAT gene is expressed at night. This is controlled partly by cyclic AMP (cAMP) acting through a composite cAMP-responsive element-CCAAT site located upstream of the transcription start point. In the present study, we have extended our previous in vitro findings and found that additional elements in the 5' flanking region and first intron play an important role in the regulation of the AA-NAT gene. This led us to test the influence of an AA-NAT 5' flanking segment on the expression of the bacterial chloramphenicol acetyltransferase gene in a rat transgenic model. The results of this study clearly demonstrate that the segment of the AA-NAT gene that encompasses the minimal promoter and the first intron is able to confer the highly specific pineal/retinal and time-of-day patterns of AA-NAT gene expression. This advance also provides a tool that selectively targets genetic expression to pinealocytes and retinal photoreceptors, providing new experimental opportunities to probe gene expression in these tissues.

Heterologous Gene Expression in Avian Cells: Potential as a Producer of Recombinant Proteins

We have explored the possibility of using avian cells for the expression of human proteins. We found that various avian cells including quail fibrosarcoma cells (QF), duck embryo cells (DE) and primary chicken embryo fibroblasts (CE) could efficiently be transfected with DNA by calcium phosphate coprecipitation, and that promoters which are transcriptionally active in mammalian cells also functioned well in these avian cells. Among the promoters we tested, the major immediate early promoter of human cytomegalovirus drove the highest level of chloramphenicol acetyl transferase (CAT) expression, outperforming the SV40 early promoter and the RSV LTR. Using the bacterial CAT gene as a reporter, we found that levels of CAT activity were higher in QF and DE cells than in mammalian cells such as CHO, HeLa, Vero and 293T cells. We further cloned a sequence encoding human erythropoietin (EPO) and compared its expression in QF and mammalian cells. Consistent with the CAT data, in transient transfection assays, QF cells produced higher levels of EPO than the mammalian cell lines tested. QF cells which can be passaged permanently were stably transfected with an EPO expression vector. The subcloned QF line was able to produce up to 1,700 U/ml EPO from 3 × 10⁶ cells in 72 h. Purified QF-produced EPO showed a broad but discrete protein band, ranging from 33 to 41 kD and was as biologically active as CHO-produced EPO. Although a number of factors still remain to be optimized, our results demonstrate the potential of avian cells such as QF as producers of heterologous proteins.

Demonstration that orf2 encodes the feline immunodeficiency virus transactivating (Tat) protein and characterization of a unique gene product with partial rev activity.

The long PCR technique was used to amplify the three size classes of viral mRNAs produced in cells infected by feline immunodeficiency virus (FIV). We identified in the env region a new splice acceptor site that generated two transcripts, each coding for an 11-kDa protein, p11(rev), whose function is unknown. The small-size class of mRNAs included two bicistronic orf2/rev mRNAs and two rev-like mRNAs, consisting only of the second exon of rev and coding for a 15-kDa protein, p15(rev). p15(rev) contained the minimal effector domain of Rev and was sufficient to mediate partial Rev activity. The bicistronic mRNAs encoded two distinct proteins, one of 23 kDa corresponding to Rev and a 9-kDa protein encoded by the orf2 gene. The orf2 gene product is a protein of 79 amino acids with characteristics similar to those of the Tat (transactivator) proteins of the ungulate lentiviruses. Transient expression assays, using the FIV long terminal repeat (LTR) to drive transcription of the bacterial gene for chloramphenicol acetyltransferase demonstrated that the orf2 gene transactivates gene expression an average of 14- to 20-fold above the basal level. Deletion mutants of the FIV LTR were generated to locate sequences responsive to transactivation mediated by the orf2 gene. A 5' deletion mutant that removed the AP1 site resulted in residual low-level transactivation by orf2. Further experiments using LTR mutants with internal deletions identified three regions located between positions -126 and -47 relative to the cap site that were important for orf2-directed transactivation. These regions include the AP1 site, a C/EBP tandem repeat, and an ATF site.

Efficiency of expression of transfected genes depends on the cell cycle.

Lipofection, a lipid-mediated DNA transfection procedure, was used to transfect synchronized L929 mouse fibroblast cells with a reporter plasmid containing the bacterial chloramphenicol acetyltransferase gene. The efficiency of gene expression was investigated on transfection of cells at different stages of the cell cycle. Our data show that expression of the reporter gene was minimal when transfection was performed in G0-phase and parallel experimental data disproved the possibility that the reduced expression observed was due to differential uptake at different times in the cell cycle. Investigation into the condensation state of the plasmid has shown that the low chloramphenicol acetyltransferase gene expression could be a direct consequence of the packaging of the plasmid into condensed chromatin when transfection occurs in G0-phase. The inactivation of the reporter gene is not reversed by growth of the cells in high serum or by treatment with Trichostatin A, a specific inhibitor of histone deacetylase, suggesting that the inactive chromatin formed in G0-phase cells lacks associated histone acetylase activity. In contrast, the high activity seen when cells in S-phase are transfected is enhanced even further by treatment with Trichostatin A.