Generation of cell lines for drug discovery through random activation of gene expression: application to the human histamine H3 receptor.

Target-based high-throughput screening (HTS) plays an integral role in drug discovery. The implementation of HTS assays generally requires high expression levels of the target protein, and this is typically accomplished using recombinant cDNA methodologies. However, the isolated gene sequences to many drug targets have intellectual property claims that restrict the ability to implement drug discovery programs. The present study describes the pharmacological characterization of the human histamine H3 receptor that was expressed using random activation of gene expression (RAGE), a technology that over-expresses proteins by up-regulating endogenous genes rather than introducing cDNA expression vectors into the cell. Saturation binding analysis using [125I]iodoproxyfan and RAGE-H3 membranes revealed a single class of binding sites with a K(D) value of 0.77 nM and a B(max) equal to 756 fmol/mg of protein. Competition binding studies showed that the rank order of potency for H3 agonists was N(alpha)-methylhistamine approximately (R)-alpha- methylhistamine > histamine and that the rank order of potency for H3 antagonists was clobenpropit > iodophenpropit > thioperamide. The same rank order of potency for H3 agonists and antagonists was observed in the functional assays as in the binding assays. The Fluorometic Imaging Plate Reader assays in RAGE-H3 cells gave high Z' values for agonist and antagonist screening, respectively. These results reveal that the human H3 receptor expressed with the RAGE technology is pharmacologically comparable to that expressed through recombinant methods. Moreover, the level of expression of the H3 receptor in the RAGE-H3 cells is suitable for HTS and secondary assays.

Development of homogeneous high-affinity agonist binding assays for 5-HT2 receptor subtypes.

The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily consists of three members, 5-HT2A, 5-HT2B, and 5-HT2C. These receptors share high homology in their amino acid sequence, have similar signaling pathways, and have been indicated to play important roles in feeding, anxiety, aggression, sexual behavior, mood, and pain. Subtype-selective agonists and antagonists have been explored as drugs for hypertension, Parkinson's disease, sleep disorders, anxiety, depression, schizophrenia, and obesity. In this study, we report the development of homogeneous agonist binding assays in a scintillation proximity assay (SPA) format to determine the high-affinity binding state of agonist compounds for the human 5-HT2C, 5-HT2A, and 5-HT2B receptors. The 5-HT2 agonist 1-(4- [125I]iodo-2,5-dimethoxyphenyl)-2-aminopropane ([125I]DOI) was used to label the high-affinity sites for the 5-HT2A and 5-HT2C receptors. The high-affinity sites for the 5-HT2B receptor were labeled with [3H]lysergic acid diethylamide. Total receptor expression was determined with the 5-HT2 antagonist [3H]mesulergine for the 5-HT2B and 5-HT2C receptors, and [3H]ketanserin for the 5-HT2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT(2C) receptor), 4.0% (5-HT2A receptor), and 22% (5-HT2B receptor) of the total receptor population. Competition binding studies using known agonists indicated high Z' values of the agonist binding assays in SPA format (Z' > 0.70). The Ki values of 5-HT, (R)(-)DOI, and VER-3323 for the 5-HT2A, 5-HT2B, and 5-HT2C receptors by SPA format were equivalent to published data determined by filtration binding assays. These results indicate that agonist binding assays in SPA format can be easily adapted to a high throughput assay to screen for selective 5-HT2C receptor agonists, as well as for selectivity profiling of the compounds.

Creation of genome-wide protein expression libraries using random activation of gene expression.

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.

Abolition of an Inducible Highly Anionic Peroxidase Activity in Transgenic Tomato.

Locally induced expression of a highly anionic peroxidase has previously been correlated temporally and spatially with suberization of tissues responding to pathogen assault, wounding, or exogenously applied abscisic acid or fungal elicitors. DNA sequences corresponding to the 5[prime] regions of two tomato (Lycopersicon esculentum) genes encoding homologous anionic peroxidases were fused, inserted into a pTi-based plasmid designed to express a composite antisense transcript, and introduced into tomato via Agrobacterium-mediated transformation. RNA gel-blot analyses showed high expression of the antisense transcript in most transgenic plants and no detectable induction of native anionic peroxidase transcripts in wounded or abscisic acid or pathogen-treated tissues. Plants and fruits expressing the antisense transcript appeared normal in all respects. Electrophoretic analysis of anionic proteins from selected transgenic plants showed no detectable anionic peroxidase protein or activity. Depolymerization of polymeric material from the wound periderm of transgenic tomato fruits and analysis of the aliphatic products by gas-liquid chromatography/mass spectrometry showed that the content and composition of C16/C18 [omega]-hydroxy and dicarboxylic acids, characteristic of suberin, were not affected by the absence of the anionic peroxidase. Autofluorescence generated from cell wall phenolics at the wound lesion was also not affected by the absence of the highly anionic peroxidase.

Protein expression from an Escherichia coli/Bacillus subtilis multifunctional shuttle plasmid with synthetic promoter sequences.

A plasmid shuttle vector (pSP10) was designed and constructed to simplify screening of cloned DNA and to facilitate expression of the protein products. The plasmid contained the following features: (i) a selection gene, chloramphenicol acetyltransferase; (ii) an indicator gene encoding beta-galactosidase for visual identification of colonies containing DNA inserts; (iii) a cloning region immediately upstream from the indicator gene; (iv) origins of replication recognized by both Escherichia coli and Bacillus subtilis; and (v) a synthetic DNA expression control sequence, including -35 and -10 regions, ribosomal binding site, and transcriptional and translational start sites. The promoter region is a synthetic consensus sequence derived from published B. subtilis promoters. The plasmid has been shown to replicate actively in E. coli and B. subtilis and to confer chloramphenicol resistance to both hosts. DNA inserted at the cloning region inactivates the indicator gene, resulting in white colonies on 5'-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside plates. beta-Galactosidase has been expressed from pSP10 in both E. coli and B. subtilis. A comparison was made of the expression levels of beta-galactosidase from the same plasmid which had been modified to contain: (i) the synthetic control region, (ii) no promoter region, (iii) the synthetic control region cloned in the opposite orientation, or (iv) the tac promoter.

Identification of the mcrD gene product and its association with component C of methyl coenzyme M reductase in Methanococcus vannielii.

A mcrD-lacZ gene fusion has been constructed and expressed under lacP control in Escherichia coli. Antibodies raised against the product of this gene fusion have been used in Western blotting (immunoblotting) to demonstrate the gene product of mcrD (gpmcrD) in Methanococcus vannielii. The alpha, beta, and gamma subunit polypeptides of component C of methyl coenzyme M reductase (MR) were coprecipitated with gpmcrD when bound by antibodies raised either against MR or against gpmcrD-lacZ. This association of MR and gpmcrD did not withstand polyacrylamide gel electrophoresis under nondenaturing conditions.

An archaebacterial promoter sequence assigned by RNA polymerase binding experiments.

To identify an archaebacterial promoter sequence, nuclease protection studies with the purified RNA polymerase of Methanococcus vannielii were performed. The enzyme binds specifically both at protein-encoding (hisA and methyl CoM reductase, component C) and tRNA-rRNA genes. The binding region of the RNA polymerase extends from 30 base pairs (bp) upstream (-30) to 20 bp downstream (+20) from the in vivo transcription start site. This finding indicates that the archaebacterial enzyme recognizes promoters without transacting transcription factors. The DNA segment protected from nuclease digestion by bound RNA polymerase contains an octanucleotide sequence centered at -25, which is conserved between the protein-encoding and the stable RNA genes. According to the specific binding of the enzyme to only DNA-fragments harbouring this motif, we propose the sequence TTTATATA as the major recognition signal of the Methanococcus RNA polymerase. Comparison of this motif with published archaebacterial DNA sequences revealed the presence of homologous sequences at the same location upstream of 36 genes. We therefore consider the overall consensus TTTATAATA as a general element of promoters in archaebacteria. In spite of the specific binding of the enzyme, most preparations of the Methanococcus vannielii RNA polymerase are unable to initiate transcription at the correct sites in vitro. Here we present first evidence for the possible existence of a transcription factor conferring the ability to the enzyme to initiate and terminate transcription specifically in vitro.

A comparison of the methyl reductase genes and gene products.

The DNA sequences encoding component C of methyl coenzyme M reductase (mcr genes) in Methanothermus fervidus, Methanobacterium thermoautotrophicum, Methanococcus vannielii, and Methanosarcina barkeri have been published. Comparisons of transcription initiation and termination sites and of the amino acid sequences of the mcr gene products are presented. Structural features conserved within the amino acid sequences are identified and a comparison of methyl reductase with other disulfide bond synthesizing enzymes is presented.

Structure and comparative analysis of the genes encoding component C of methyl coenzyme M reductase in the extremely thermophilic archaebacterium Methanothermus fervidus.

A 6-kilobase-pair (kbp) region of the genome of the extremely thermophilic arachaebacterium Methanothermus fervidus which encodes the alpha, beta, and gamma subunit polypeptides of component C of methyl coenzyme M reductase was cloned and sequenced. Genes encoding the beta (mcrB) and gamma (mcrG) subunits were separated by two open reading frames (designated mcrC and mcrD) which encode unknown gene products. The M. fervidus genes were preceded by ribosome-binding sites, separated by short A + T-rich intergenic regions, contained unexpectedly few NNC codons, and exhibited inflexible codon usage at some locations. Sites of transcription initiation and termination flanking the mcrBDCGA cluster of genes in M. fervidus were identified. The sequences of the genes, the encoded polypeptides, and transcription regulatory signals in M. fervidus were compared with the functionally equivalent sequences from two mesophilic methanogens (Methanococcus vannielii and Methanosarcina barkeri) and from a moderate thermophile (Methanobacterium thermoautotrophicum Marburg). The amino acid sequences of the polypeptides encoded by the mcrBCGA genes in the two thermophiles were approximately 80% identical, whereas all other pairs of these gene products contained between 50 and 60% identical amino acid residues. The mcrD gene products have diverged more than the products of the other mcr genes. Identification of highly conserved regions within mcrA and mcrB suggested oligonucleotide sequences which might be developed as hybridization probes which could be used for identifying and quantifying all methanogens.

RNA polymerase-binding and transcription initiation sites upstream of the methyl reductase operon of Methanococcus vannielii.

RNA polymerase, purified from Methanococcus vannielii, was shown by exonuclease III footprinting to bind to a 49-base-pair (bp) region of DNA in the intergenic region upstream of mcrB. S1 nuclease protection experiments demonstrated that transcription initiation in vivo occurs within this region at 32 or 33 bp 5' to the ATG translation initiation codon of mcrB and 19 or 20 bp 3' to a TATA box.

Structure and expression of the genes, mcrBDCGA, which encode the subunits of component C of methyl coenzyme M reductase in Methanococcus vannielii.

The genes that encode the alpha, beta, and gamma subunits of component C of methyl coenzyme M reductase (mcrA, mcrB, and mcrG) in Methanococcus vannielii have been cloned and sequenced, and their expression in Escherichia coli has been demonstrated. These genes are organized into a five-gene cluster, mcrBDCGA, which contains two genes, designated mcrC and mcrD, with unknown functions. The mcr genes are separated by very short intergenic regions that contain multiple translation stop codons and strong ribosomebinding sequences. Although the genome of M. vannielii is 69 mol% A+T, there is a very strong preference in the mcrA, mcrB, and mcrG genes for the codon with a C in the wobble position in the codon pairs AA(U) (C) (asparagine), GA(U) (C) (aspartic acid), CA(U) (C) (histidine), AU(U) (C) (isoleucine), UU(U) (C) (phenylalanine), and UA(U) (C) (tyrosine). The mcrC and mcrD genes do not show this codon preference and frequently have U or A in the wobble position. As the codon pairs listed above are likely to be translated by the same tRNA with a G in the first anticodon position, the presence of C in the wobble position might ensure maximum efficiency of translation of transcripts of these very highly expressed genes.