Luciferase Sequences Research Articles

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site‐directed mutagenesis survey

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin.

The Influence of Insertion of a Critical Residue (Arg356) in Structure and Bioluminescence Spectra of Firefly Luciferase

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.

Effects of oxalate exposure on Madin-Darby canine kidney cells in culture: renal prothrombin fragment-1 mRNA expression

It has been suggested that renal tubular cell damage induced by oxalic acid, one of the components of urinary calculi, may be involved in a variety of ways in the development of urolithiasis. During our study on a calculus related protein, renal prothrombin fragment-1 (RPTF-1), we noted that this is an inflammation related substance that mediates an acute inflammatory reaction, one of the original roles of prothrombin. RPTF-1 is a part of prothrombin that is a coagulation factor known to be expressed in the renal tubule. We examined whether oxalic acid may cause cytotoxic effects on tubular epithelial cells and whether such chemical stimulation may promote the translation of RPTF-1 mRNA into RPTF-1 proteins. We used Madin-Darby canine kidney (MDCK) cells derived from the distal tubule of a dog kidney. In this study, the effects of oxalic acid in culture solution at different concentrations on cytotoxicity were assessed using a MTT assay. The location of active oxygen species was identified using dichlorofluorescein diacetate. After the prothrombin sequence of RPTF-1 was confirmed in MDCK cells, RPTF-1 mRNA expression was determined by RT-PCR. The gene sequence of the same promoter area was ligated, and a luciferase sequence was inserted downstream of the vector. The target sequence was transfected into MDCK cells and the relation between oxalic acid and prothrombin promoter was examined. In addition, the variable expression of RPTF-1 mRNA was quantitatively compared depending on oxalic acid concentrations using real-time PCR. When cytotoxicity was investigated, cells were not damaged but, by contrast, were stimulated and activated under oxalic acid below a certain concentration. The relation between cytotoxicity on the cultured MDCK cell membrane and active oxygen species was confirmed. Luminescence in MDCK cells containing the luciferase gene was detected by the addition of oxalic acid, which activated the prothrombin promoter. A part of the prothrombin gene sequence in the MDCK cells was detected and an increase in the expression of RPTF-1 mRNA in MDCK cells by the addition of oxalic acid was confirmed using real-time PCR. Increased expression of prothrombin by adding oxalic acid has already been demonstrated in previous studies. In this study, however, RPTF-1 mRNA was promoted by oxalic acid and a direct association between oxalic acid and RPTF-1 is indicated. This finding shows that increased oxalic acid in urine induces the expression of RPTF-1 in tubular epithelial cells and thereby causes the generation of active oxygen species.

HVps34 Is a Nutrient-regulated Lipid Kinase Required for Activation of p70 S6 Kinase

Mammalian cells respond to nutrient deprivation by inhibiting energy consuming processes, such as proliferation and protein synthesis, and by stimulating catabolic processes, such as autophagy. p70 S6 kinase (S6K1) plays a central role during nutritional regulation of translation. S6K1 is activated by growth factors such as insulin, and by mammalian target of rapamycin (mTOR), which is itself regulated by amino acids. The Class IA phosphatidylinositol (PI) 3-kinase plays a well recognized role in the regulation of S6K1. We now present evidence that the Class III PI 3-kinase, hVps34, also regulates S6K1, and is a critical component of the nutrient sensing apparatus. Overexpression of hVps34 or the associated hVps15 kinase activates S6K1, and insulin stimulation of S6K1 is blocked by microinjection of inhibitory anti-hVps34 antibodies, overexpression of a FYVE domain construct that sequesters the hVps34 product PI3P, or small interfering RNA-mediated knock-down of hVps34. hVps34 is not part of the insulin input to S6K1, as it is not stimulated by insulin, and inhibition of hVps34 has no effect on phosphorylation of Akt or TSC2 in insulin-stimulated cells. However, hVps34 is inhibited by amino acid or glucose starvation, suggesting that it lies on the nutrient-regulated pathway to S6K1. Consistent with this, hVps34 is also inhibited by activation of the AMP-activated kinase, which inhibits mTOR/S6K1 in glucose-starved cells. hVps34 appears to lie upstream of mTOR, as small interfering RNA knock-down of hVps34 inhibits the phosphorylation of another mTOR substrate, eIF4E-binding protein-1 (4EBP1). Our data suggest that hVps34 is a nutrient-regulated lipid kinase that integrates amino acid and glucose inputs to mTOR and S6K1.

Genomic structure and phylogenetic analysis of the luciferase gene of the firefly, Luciola lateralis (Coleoptera: Lampyridae)

The complete nucleotide sequence and the exon-intron structure of the luciferase gene of the firefly, Luciola lateralis (Coleoptera: Lampyridae) is described. The luciferase gene of the L. lateralis firefly spans 1,971 bp and consists of six introns and seven exons coding for 548 amino acid residues. From samples collected at Boun and Muju, Korea, three isoforms, with identical exon-intron structure, named BU, MJ1 and MJ2, respectively, were obtained. Although the amino acid sequences of MJ1 and MJ2 were identical to those of known luciferase genes of Korean origin, the BU type was novel, differing from each of the MJ1 and MJ2 types by one amino acid. The luciferase sequences of the Korean samples, including those previously revealed, differed only by one - two amino acid residues, but differed by five - six amino acid residues from that of the luciferase gene recorded from specimens from Japan, which suggest genetic divergence has occurred in this species. Phylogenetic analyses using both amino acid and nucleotide sequences further showed that the luciferase gene of the Japanese L. lateralis firefly is genetically distinguishable from that of Korean L. lateralis, suggesting the presence of a genetic subdivision between the L. lateralis dwelling in the Korean Peninsula and Japanese Islands.

Darwinian natural selection for orange bioluminescent color in a Jamaican click beetle.

The Jamaican click beetle Pyrophorus plagiophthalamus (Coleoptera: Elateridae) is unique among all bioluminescent organisms in displaying a striking light color polymorphism [Biggley, W. H., Lloyd, J. E. & Seliger, H. H. (1967) J. Gen. Physiol. 50, 1681-1692]. Beetles on the island vary in the color of their ventral light organs from yellow-green to orange and their dorsal organs from green to yellow-green. The genetic basis for the color variation involves specific amino acid substitutions in the enzyme luciferase. Here, we show that dorsal and ventral light color in P. plagiophthalamus are under separate genetic control, we resolve the allelic basis for color variation, and, through analyses of luciferase sequence variation, we demonstrate that natural selection has produced a long-term adaptive trend for longer wavelength (more orange) ventral light on Jamaica. Our results constitute a novel example connecting the selective fixation of specific nucleotides in nature to their precisely determined phenotypic effects. We also present evidence suggesting that a recently derived ventral orange luciferase allele on the island has deterministically increased in frequency. Thus, the current luciferase polymorphism for P. plagiophthalamus appears to be mirroring the long-term anagenic trend on Jamaica, revealing a possible ongoing adaptive color transition in progress.

Structure and evolution of the luciferin-regenerating enzyme (LRE) gene from the firefly Photinus pyralis.

To study the structural features of genes for the luciferin-regenerating enzyme (LRE), the entire gene along with 524 bp of upstream sequence was determined from Photinus pyralis (Coleoptera: Lampyridae). The LRE gene revealed an open reading frame composed of five exons divided by four introns ranging in size from 47 to 904 bp. The deduced LRE amino acid sequence showed identity to senescence marker protein-30 (SMP30) from a number of insects and mammals including four putative SMP30 sequences from Anopheles gambiae. Gene structure comparisons showed some intron/exon site conservation with A. gambiae and mammalian SMP30 proteins but not Drosophila. LRE and luciferase sequence comparisons revealed two conserved putative luciferin-binding sites. The evolution of LRE was discussed in relation to its function.

Determination of the half-life of the murine β4-galactosyltransferase-1 mRNA in somatic cells using the tetracycline-controlled transcriptional regulation system

The glycosyltransferases are recognized as a functional family of an estimated 300 distinct, intracellular, membrane-bound enzymes that are positioned along the secretory pathway and participate coordinately in the biosynthesis of the carbohydrate moieties on glycoconjugates. The full-length cDNA sequence for many of these proteins is now available yet little is known about the transcriptional or translational regulation of a given transcript or its decay rate in the cell. These issues are made more complex by the observations that transcription of a glycosyltransferase gene in different cells/tissues results in mRNAs with significantly different structures. For example, transcription of the murine β4-galactosyoltransferase-1 gene in somatic cells yields two transcripts of 3.9 and 4.1 kb. In contrast transcription of this gene in developing male germ cells results in transcripts of 2.9 and 3.1 kb which are distinguished from their somatic cell counterparts primarily by the deletion of ~1.7 kb of sequence in the 3'-untranslated region (UTR). With the long range goal of determining the role that the 3'-UTR serves in mRNA decay we have taken advantage of a recently developed methodology, the Tet-Off system, to determine the half-life of the mRNA encoding β4-galactosyltransferase-1 in the murine NIH 3T3 somatic cell line. We show that the β4-galactosyltransferase-1 mRNA has a half-life of ~84 min (range of 82-85 min) in 3T3 cells and that substitution of the galactosyltransferase coding sequence with the coding sequence of luciferase does not significantly alter the decay rate (~87 min; range of 84-91 min). This latter observation suggests that the β4-galactosyltransferase-1 coding sequence does not contain functional elements that affect the intrinsic stability of this mRNA.

Replication of a hepatitis A virus replicon detected by genetic recombination in vivo.

Unlike other picornaviruses, hepatitis A virus (HAV) replicates so inefficiently in cell culture that the study of its RNA biosynthesis presents a major experimental challenge. To assess viral RNA replication independent of particle formation, a subgenomic replicon representing a self-replicating RNA was constructed by replacing the P1 domain encoding the capsid proteins with the firefly luciferase sequence. Although translation of the HAV replicon was as efficient as a similar poliovirus replicon, the luciferase activity derived from replication of the HAV construct was more than 100-fold lower than that of poliovirus. The replication capacity of the HAV replicon was clearly demonstrated by its ability to recombine genetically with a non-viable, full-length HAV genome that served as capsid donor and thus to rescue a fully infectious virus. In contrast to a replication-deficient replicon, co-expression of the genetically marked and replication-competent HAV replicon with several lethally mutated HAV genomes resulted in the successful rescue of infectious HAV with a unique genetic marker. Our data suggest: (i) that autonomous HAV RNA replication does not require sequences for the HAV structural proteins; and (ii) that low-level genome replication can unequivocally be demonstrated by the rescue of infectious virus after co-expression with non-viable genomes.

Human Papillomavirus E2 Down-regulates the Human Telomerase Reverse Transcriptase Promoter

The transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene is a critical step in transformation and differentiation. Human papillomavirus E2 protein inhibits cell growth in HPV-infected cells and triggers apoptosis in HeLa cells. Because E2 induces cell growth suppression and senescence, we hypothesize that the protein may modulate cellular gene expression related to these processes. In this report, we demonstrate that E2 inhibits the hTERT promoter. The mapping of the E2-responsive region of hTERT reveals that Sp1 is important for E2-mediated repression of this promoter in 293T cells. Site-directed mutagenesis data on the hTERT promoter show that E2 does not abolish E-Box-mediated transcription and represses promoter activity via the Sp1 binding site. Furthermore, chromatin immunoprecipitation assays indicate that E2 is actively recruited to the hTERT promoter region. Our findings provide novel insights into the biological function of human papillomavirus E2.

Breast cancer: from estrogen to androgen receptor

To investigate the link existing between androgens and human breast cancer, the hormonal milieu present in pre- and post-menopausal women has been translated in an in vitro model utilizing a hormone dependent breast cancer cell line MCF-7 exposed to DHEA, DHEAS, androstenediol, T, DHT with or w/o E 2. DHEAS and androstenediol stimulate the growth of MCF-7 cell line but reduce cell proliferation induced by E 2 (1 nM). T and DHT (1–100 nM) instead inhibit MCF-7 cell proliferation independently on E 2 presence. When we focused our study on the most powerful androgen, DHT alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal growth rate and counteracts E 2 proliferative action by 68%. These data correlate well with cell cycle analysis showing an enhanced number of cells in G 0/G 1 phase after 6 days of DHT treatment. Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while immunohistochemistry indicates that it was mostly translocated into the nucleus. So we assumed that the enhanced activation of the AR might inhibit MCF-7 cells proliferation. This assumption is corroborated by the fact that the inhibitory effects induced by DHT on MCF-7 cell proliferation are abrogated in the presence of hydroxyflutamide. Therefore to better investigate the role of AR in inhibiting E 2 action at genomic level, MCF-7 cells were transiently cotransfected with the reporter plasmid XETL carrying firefly luciferase sequence under the control of an estrogen responsive element and the full length AR or with an AR carrying a mutation (Cis 574→Arg 574) which abolishes its binding to DNA. The over-expression of the AR markedly decreases E 2 signalling which furthermore appears inhibited by simultaneous exposure to DHT but reversed by addition of hydroxyflutamide. The inhibitory effect was no longer noticeable when MCF-7 cells were cotransfected with XETL and the mutant AR. Taken together these data demonstrate that gonadal androgens antagonize MCF-7 proliferation induced by E 2. This seems to be related to the inhibitory effects of the over-expressed AR on E 2 genomic action.

Mutational Spectrum in the PEX7 Gene and Functional Analysis of Mutant Alleles in 78 Patients with Rhizomelic Chondrodysplasia Punctata Type 1

Rhizomelic chondrodysplasia punctata (RCDP) is a genetically heterogeneous, autosomal recessive disorder of peroxisomal metabolism that is clinically characterized by symmetrical shortening of the proximal long bones, cataracts, periarticular calcifications, multiple joint contractures, and psychomotor retardation. Most patients with RCDP have mutations in the PEX7 gene encoding peroxin 7, the cytosolic PTS2-receptor protein required for targeting a subset of enzymes to peroxisomes. These enzymes are deficient in cells of patients with RCDP, because of their mislocalization to the cytoplasm. We report the mutational spectrum in the PEX7 gene of 78 patients (including five pairs of sibs) clinically and biochemically diagnosed with RCDP type I. We found 22 different mutations, including 18 novel ones. Furthermore, we show by functional analysis that disease severity correlates with PEX7 allele activity: expression of eight different alleles from patients with severe RCDP failed to restore the targeting defect in RCDP fibroblasts, whereas two alleles found only in patients with mild disease complemented the targeting defect upon overexpression. Surprisingly, one of the mild alleles comprises a duplication of nucleotides 45-52, which is predicted to lead to a frameshift at codon 17 and an absence of functional peroxin 7. The ability of this allele to complement the targeting defect in RCDP cells suggests that frame restoration occurs, resulting in full-length functional peroxin 7, which leads to amelioration of the predicted severe phenotype. This was confirmed in vitro by expression of the eight-nucleotide duplication-containing sequence fused in different reading frames to the coding sequence of firefly luciferase in COS cells.

Genetically Fused Protein A–Luciferase for Immunological Blotting Analyses

The gene expression plasmid pMALU5 for the fusion protein of protein A (SpA) with a complete sequence of firefly luciferase (Luc) was constructed. The fused gene was expressed in Escherichia coli, and the resulting SpA–Luc fusion protein was purified by one-step affinity chromatography on IgG–Sepharose. The protein retained both activities: IgG binding capability of protein A and enzymatic activity of luciferase. Blotting analyses were performed with the fusion protein to determine a tumor marker of α-fetoprotein (AFP). AFP was detected at the lowest detection limit of 5 pg by dot blotting and Western blotting. The SpA–Luc fusion protein provides a highly selective, sensitive, and versatile marker for blotting analyses.

Positive tetracycline control of expression of p15INK4B from an Epstein–Barr autonomous plasmid in a human melanoma cell line

Homozygous deletions in the region of chromosome 9p21 are frequent in human melanoma. Mutations in the p16INK4A cyclin-dependent kinase inhibitor (CDI) gene at this locus have implicated the product of this gene as a tumor suppressor. Less attention has been focused on the homologous, closely linked p15INK4B gene. To facilitate study of the phenotypic effects of restoring expression of the latter in aggressive melanoma cells lacking INK4 expression, we inserted the cDNA encoding p15INK4B into an autonomously maintained plasmid under positive tetracycline control (‘TET ON’ system). Similarly regulated luciferase and herpes thymidine kinase sequences were used as controls. We demonstrate that this system enabled efficient, and reasonably uniform, induction of p15INK4B expression in a human melanoma cell line exposed to the tetracycline derivative, doxycycline. Flow cytometry showed that this induction resulted in substantial accumulation of cells in the G0/G1 phase of the cell cycle. This system will facilitate detailed analysis of the cell cycle inhibitory mechanisms of this CDI in human melanoma cells.

A Highly Conserved Mechanism of Regulated Ribosome Stalling Mediated by Fungal Arginine Attenuator Peptides That Appears Independent of the Charging Status of Arginyl-tRNAs

The Arg attenuator peptide (AAP) is an evolutionarily conserved peptide involved in Arg-specific negative translational control. It is encoded as an upstream open reading frame (uORF) in fungal mRNAs specifying the small subunit of Arg-specific carbamoyl phosphate synthetase. We examined the functions of the Saccharomyces cerevisiae CPA1 and Neurospora crassa arg-2 AAPs using translation extracts from S. cerevisiae, N. crassa, and wheat germ. Synthetic RNA containing AAP and firefly luciferase (LUC) sequences were used to program translation; analyses of LUC activity indicated that the AAPs conferred Arg-specific negative regulation in each system. The AAPs functioned either as uORFs or fused in-frame at the N terminus of LUC. Mutant AAPs lacking function in vivo did not function in vitro. Therefore, trans-acting factors conferring AAP-mediated regulation are in both fungal and plant systems. Analyses of ribosome stalling in the fungal extracts by primer extension inhibition (toeprint) assays showed that these AAPs acted similarly to stall ribosomes in the region immediately distal to the AAP coding region in response to Arg. The regulatory effect increased as the Arg concentration increased; all of the arginyl-tRNAs examined appeared maximally charged at low Arg concentrations. Therefore, AAP-mediated Arg-specific regulation appeared independent of the charging status of arginyl-tRNA.

Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color.

Under physiological conditions firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen. In nature, bioluminescence emission by beetle luciferases is observed in colors ranging from green (approximately 530 nm) to red (approximately 635 nm), yet all known luciferases use the same luciferin substrate. In an earlier report [Branchini, B. R., Magyar, R. M., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319], we described the effects of mutations at His245 on luciferase activity. In the context of molecular modeling results, we proposed that His245 is located at the luciferase active site. We noted too that the H245 mutants displayed red-shifted bioluminescent emission spectra. We report here the construction and purification of additional His245 mutants, as well as mutants at residues Lys529 and Thr343, all of which are stringently conserved in the beetle luciferase sequences. Analysis of specific activity and steady-state kinetic constants suggested that these residues are involved in luciferase catalysis and the productive binding of substrates. Bioluminescence emission spectroscopy studies indicated that point mutations at His245 and Thr343 produced luciferases that emitted light over the color range from green to red. The results of mutational and biochemical studies with luciferase reported here have enabled us to propose speculative mechanisms for color determination in firefly bioluminescence. An essential role for Thr343, the participation of His245 and Arg218, and the involvement of bound AMP are indicated.

Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line

The growth of the adenocarcinoma cell line derived from human salivary gland (HSG) is regulated by all-trans-retinoic acid (t-RA), which binds to its specific receptor, retinoic acid receptors (RARs), located in the nucleus, and thereby transactivates target genes. In this study, we examined the binding characteristics of the nuclear extract of HSG cells to the retinoic acid response element (RARE) compared with those of in vitro translated RAR alpha and retinoid X receptor alpha (RXR alpha), a heterodimeric partner of RAR alpha. Gel shift analysis using anti-RAR alpha and anti-RXR alpha antibodies revealed that the translated RAR alpha bound to RARE as a heterodimer with RXR alpha. In contrast, the binding of the nuclear extract of HSG cells to RARE showed a heterogeneous pattern, suggesting the existence of several species of RXRs as well as RARs in the nuclei of HSG cells. We therefore tried to clone these putative RXRs by the polymerase chain reaction using degenerated oligonucleotide primers conserved across the RXR family. The DNA sequencing of the recombinant clones revealed the expression of RXR alpha and RXR beta. In addition, chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI), which is also an RXR family member, was cloned. To evaluate the transcriptional activity of RARs and RXRs endogenously expressed in HSG cells, we performed a transient transfection analysis. When HSG cells were transfected with a luciferase reporter plasmid containing two repeats of either the RARE of the RAR beta gene or that of cellular retinol-binding protein II gene, positioned upstream of a thymidine kinase promoter fused to the luciferase sequence, a 2-3-fold induction of luciferase activity was observed in both cases. These results suggest that RARs and RXRs endogenously expressed in HSG cells were transcriptionally active in vivo. Thus, our findings showed that RXR alpha, RXR beta, and COUP-TFI are expressed in HSG cells and suggest that these molecules function as heterodimeric partners of RARs and (or) competitive repressors for RAREs and are involved in cellular responses mediated by retinoids.

Cloning and sequencing of a cDNA for firefly luciferase from Photuris pennsylvanica

The first cDNA from the Photurinae subfamily of the Lampyridae encoding a firefly luciferase from lantern mRNA of Photuris pennsylvanica has been cloned, sequenced, the amino-acid sequence predicted and the sequence reported to GenBank. The cDNA was about 1.8 kb in length with the largest open reading frame coding for a 545-residue protein. The 5′ noncoding region is 61 bp long and the 3′ noncoding region is 135 bp in length. There is a 24-nucleotide poly(A) tail. When the amino-acid residues are aligned, P. pennsylvanica contains 154 (about 28% of the total residues) that are conserved in all 16 of the deduced luciferase sequences that are presently available. In this P. pennsylvanica luciferase, the amino acids at 276 of the positions are the same at corresponding positions of at least one of the other enzymes. There are two amino-acid differences between this luciferase and the unpublished sequence obtained by Dr. Keith Wood for a putative larval Photuris firefly luciferase cloned from a Maryland firefly. Signature amino-acid sequences and domains found in the deduced sequence are for adenylate kinase, the putative AMP-binding domain, luciferin 4-monooxygenase, 4-coumarate CoA ligase, long-chain fatty acid CoA ligase, 2-acylglycerophosphoethanolamine acyltransferase, the microbody-directing sequence, peptide-synthesizing complexes, and acyladenylate-synthesizing enzymes.

Evaluation of cationic liposomes for delivery of diphtheria toxin A-chain gene to cells infected with bovine leukemia virus.

We investigated whether cationic liposomes are efficient at delivering the gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (pLTR-DT) into BLV-infected cells and are also suitable for in vivo use. The transfection activity of the cationic liposomes composed of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dioleoyl phosphatidylethanolamine (DOPE) and dilauroyl phosphatidylcholine (DLPC) (1:2:2, molar ratio) (TMAG-liposome) and liposomes composed of phosphatidylserine (PS) (PS-liposome) was evaluated by the luciferase assay using a plasmid which contains the coding sequence of firefly luciferase under the control of the SR alpha promoter (pSR alpha/L-A delta 5). The TMAG-liposome gave highly efficient transfection in the presence of serum. On the other hand, PS-liposome showed inferior efficiency. When BLV-infected cells were co-transfected with a fixed amount of pSR alpha/L-A delta 5-entrapped TMAG-liposome and various amount of pLTR-DT-containing TMAG-liposome, the luciferase activity in the BLV-infected cells was inhibited by the addition of pLTR-DT-entrapped TMAG-liposome dose-dependently. The cationic TMAG-liposome containing pLTR-DT was successively added to BLV-infected cells in culture. The number of viable cells was markedly reduced by the cationic TMAG-liposome containing pLTR-DT. On the other hand, TMAG-liposome containing pSR alpha/L-A delta 5 showed no such effect. pLTR-DT entrapped by the cationic TMAG-liposome was not digested by the treatment with DNase I and with serum. These results suggest that the cationic liposomes, such as TMAG-liposome, may be efficient transfection reagent for the BLV-infected cells and can be utilized for DT-A gene delivery into the BLV-infected cells in vivo.

Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line

The growth of the adenocarcinoma cell line derived from human salivary gland (HSG) is regulated by all-trans-retinoic acid (t-RA), which binds to its specific receptor, retinoic acid receptors (RARs), located in the nucleus, and thereby transactivates target genes. In this study, we examined the binding characteristics of the nuclear extract of HSG cells to the retinoic acid response element (RARE) compared with those of in vitro translated RAR alpha and retinoid X receptor alpha (RXR alpha ), a heterodimeric partner of RAR alpha . Gel shift analysis using anti-RAR alpha and anti-RXR alpha antibodies revealed that the translated RAR alpha bound to RARE as a heterodimer with RXR alpha . In contrast, the binding of the nuclear extract of HSG cells to RARE showed a heterogenous pattern, suggesting the existence of several species of RXRs as well as RARs in the nuclei of HSG cells. We therefore tried to clone these putative RXRs by the polymerase chain reaction using degenerated oligonucleotide primers conserved across the RXR family. The DNA sequencing of the recombinant clones revealed the expression of RXR alpha and RXR beta . In addition, chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI), which is also an RXR family member, was cloned. To evaluate the transcriptional activity of RARs and RXRs endogenously expressed in HSG cells, we performed a transient transfection analysis. When HSG cells were transfected with a luciferase reporter plasmid containing two repeats of either the RARE of the RAR beta gene or that of cellular retinol-binding protein II gene, positioned upstream of a thymidine kinase promoter fused to the luciferase sequence, a 2-3-fold induction of luciferase activity was observed in both cases. These results suggest that RARs and RXRs endogenously expressed in HSG cells were transcriptionally active in vivo. Thus, our findings showed that RXR alpha , RXR beta , and COUP-TFI are expressed in HSG cells and suggest that these molecules function as heterodimeric partners of RARs and (or) competitive repressors for RAREs and are involved in cellular responses mediated by retinoids. Key words: retinoid X receptor, retinoic acid receptor, retinoic acid response element, COUP-transcription factor (COUP-TF).