Complex chimeras to map ligand binding sites of GPCRs.

Family 1a GPCRs are thought to bind small molecule ligands in a pocket comprising sequences from non-contiguous transmembrane helices. In this study, receptor-ligand binding determinants were defined by building a series of complex chimeras where multiple sequences were exchanged between related G-protein coupled receptors. Regions of P2Y(1), P2Y(2) and BLT(1) predicted to interact with nucleotide and leukotriene ligands were identified and receptors were engineered within their transmembrane helices to transpose the ligand binding site of one receptor on to another receptor. Ligand-induced activation of chimeras was compared with wild-type receptor activation in a yeast reporter gene assay. Binding of ligand to a P2Y(2)/BLT(1) chimera confirmed that the ligand binding determinants of BLT(1) are located in the upper regions of the helices and extracellular loops of this receptor and that they had been successfully transferred to a receptor that normally binds unrelated ligands.

Fatty acid activation of the peroxisome proliferator activated receptor, a member of the nuclear receptor gene superfamily.

The peroxisome proliferator activated receptor is a member of the steroid receptor gene superfamily, sharing amino acid sequence homology with other receptors and also showing similarities at the level of gene structure. This receptor is activated both by xenobiotic compounds that induce peroxisome proliferation and by fatty acids at physiological concentrations. Upon activation the receptor mediates transcription of responsive genes through binding to peroxisome proliferator response elements. These genes include those encoding peroxisomal enzymes and members of the cytochrome P450 family of drug metabolizing enzymes. It is therefore possible that the peroxisome proliferator activated receptor may play a crucial role in regulating lipid homeostasis.

Structure of the mouse peroxisome proliferator activated receptor alpha gene.

We have isolated and characterised genomic clones spanning the mouse peroxisome proliferator activated receptor alpha gene (PPAR alpha). The gene contains eight exons spanning at least 30 kilobases. The DNA binding domain of PPARa consists of two exons, each encoding one of the zinc fingers. Three exons encode the ligand binding domain and 3' untranslated regions of PPAR alpha. There are two 5' untranslated exons and one exon encoding the N-terminal domain. The 5' flanking region is GC rich and contains several putative SP1 binding sites. The structure of the PPAR alpha gene is unique but shares similar features with related members of the nuclear hormone receptor family.

Heterogeneity in the 5' untranslated region of the rat glucocorticoid receptor mRNA.

We have cloned several novel sequences upstream from the first coding exon of the rat glucocorticoid receptor (GR) mRNA using PCR. Analysis of these sequences in RNase protection assays showed that one of the cloned sequences represents the major GR 5' non-coding exon which is expressed in all tissues studied, both at different stages of development and under different hormonal conditions. This major exon is homologous to the human GR 5' untranslated region (UTR). Three other sequences were cloned, but could not be detected in the RNase protection assay, suggesting that they are only minor transcripts, at least under the varying conditions of GR expression studied. One of these sequences is identical to a previously described rat GR cDNA sequence, while another was shown to be contiguous with the rat genomic DNA sequence.

Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor.

The rat peroxisome-proliferator-activated receptor (PPAR) was expressed in insect cells and was shown to bind to a cognate PPAR response element (PPRE) from the acyl-CoA oxidase gene. Upon purification, PPAR was no longer able to bind DNA, although binding could be restored by addition of insect cell extracts. We investigated whether the retinoid X receptor (RXR) could supplement for this accessory activity. The rat RXR alpha cDNA was cloned and it was found that addition of in vitro-translated RXR alpha to purified PPAR facilitated binding of PPAR to a PPRE. Furthermore, an additional activity, which appeared to be distinct from rRXR alpha, was found in COS cell nuclear extracts that enabled binding of PPAR to a PPRE. Transient expression of RXR alpha in CHO cells was found to be essential for the response of a chloramphenicol acetyltransferase reporter construct containing PPREs to activators of PPAR. These results raise the possibility of convergence of the PPAR and retinoid-dependent signaling pathways on promoters containing PPRE-like responsive elements.

Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and -R (polyhedrin gene) region.

The nucleotide sequence of a 9.4-kbp region including the polyhedrin gene of the C6 strain of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome was determined. These data provide a complete description of the EcoRI-I fragment, which is used to produce transfer vectors for inserting foreign genes into the AcMNPV. Ten potential open reading frames (ORFs) were identified in the complete sequence, on either strand of DNA. The largest of these was 1629 nucleotides in length and was located downstream from the polyhedrin coding sequences, but on the opposite strand of DNA. Northern blot hybridization analysis of ORF 8 (1629) identified an RNA of 2000 nucleotides which was produced in infected cells from 12 hr p.i. and remained until at least 48 hr p.i. S1 nuclease mapping and analysis of cDNA clones located the 3' end of the mRNA at a site 16 nucleotides downstream of the polyhedrin coding sequences. The 5' end of the mRNA was mapped using primer extension analysis of polyadenylated RNA. The mRNA start site was positioned within a late/very late consensus transcription initiation motif (ATAAG), 428 nucleotides upstream from the potential ATG translation initiation codon. The biological significance of the putative gene product was assessed by inserting a synthetic oligonucleotide in the carboxyl terminal coding sequences of ORF 8 (1629) to prematurely terminate translation. Recombinant viruses containing this mutation were not isolated, suggesting that the ORF 1629 gene product is essential for virus replication.

Functional analysis of a 603 nucleotide open reading frame upstream of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus.

The sequence of the 2000 nucleotides immediately upstream of the polyhedrin gene of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus has been determined. Comparative analysis of the data identified a 603 nucleotide open reading frame (ORF) separated from the polyhedrin gene coding sequences by 156 nucleotides and in the opposite strand of DNA. Northern hybridization analysis of polyadenylated RNA from infected cells highlighted a 3.7 kb species produced maximally at 12 h post-infection, but not in the presence of cycloheximide. Preliminary nuclease S1 analysis of the 5' end of this RNA suggested that it initiated at a position very close to that of the polyhedrin mRNA start site. Deletion of a portion of the ORF 603 from viruses containing the normal polyhedrin gene and the lacZ gene in lieu of polyhedrin did not affect replication in cell culture or the production of beta-galactosidase protein. A virus which lacked the ORF 603 gene but produced polyhedrin had similar infectivity in Trichoplusia ni larvae compared to the wild-type virus. The chloramphenicol acetyltransferase (CAT) gene was also inserted in lieu of the ORF 603 in a virus containing the lacZ gene instead of the polyhedrin (Ac.CAT.lacZ). Analysis of CAT expression revealed that a maximum level was reached at 16 h p.i. and that transcription was initiated in Ac.CAT.lacZ at the same site as for the normal gene.

Multiple cis-active elements in the long control region of bovine papillomavirus type 1 (BPV-1).

A 1.0 kb region of the BPV-1 genome (the long control region, LCR), contains controls for transcription and the origin of replication. Transcription directed by the LCR is activated by the viral encoded E2 protein. To define the essential cis acting elements that are required to control transcription we have constructed a series of deletions throughout the LCR. We have identified three important domains in the LCR, two of which respond to E2. We have analysed the ability of small subcloned regions of the E2 responsive domains to act as enhancers in a heterologous assay system. This has led to the identification of five independent E2 responsive elements. We have shown that a fragment of only 38 base pairs is sufficient to respond to activation by E2. We also present evidence to suggest two types of E2 responsiveness that result in strong or weak activation. Strong response is correlated with the presence of the sequence 5'-ACCG/TNNNC/TCGGTGC-3' whereas weak response is correlated with the presence of a related sequence 5'-ACC(N)6GGT-3'. The contribution of these multiple elements to viral transcription is discussed.