PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5-CAATATCAACAA-3. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.

We have cloned and characterized an opioid receptor-like (ORL1; also referred to as NOP) receptor from a urodele amphibian, the rough-skinned newt Taricha granulosa The cDNA clone encodes a protein of 368 amino acids that contains the seven hydrophobic domains characteristic of G-protein-coupled receptors, and has the highest sequence identity to the frog (Rana pipiens) nociceptin-like and human ORL1 opioid receptors (79·6 and 68·4%, respectively). Saturation binding assays on membranes from COS-7 cells transiently expressing the newt ORL1 (nORL) receptor revealed a single, high-affinity (estimated K d , 0·1974 nM) binding site for the ORL1-specific agonistIn competition binding assays, the [ 3 H]oFQ-binding site, like the mammalian ORL1 receptor, had no affinity for the non-selective opioid receptor antagonist naloxone, the -selective agonists U69593 and U50488, or the µ-and -selective opioid receptor agonists DAMGO and DPDPE, respectively. However, the nORL receptor displayed higher affinities for the -selective agonists dynorphin A (1-13), dynorphin B, and dynorphin A (1-8) (K i values, 2·8, 151·8, and 183·0 nM, respectively) than its mammalian homologue. The tissue distribution of the nORL receptor, as determined by reverse transcriptase PCR, was also found to differ from reports on the mammalian ORL1 receptor, with mRNA detected in brain, spinal cord, and lung, but not detected in a number of other peripheral tissues reported to express the receptor in mammals. This is the first report describing the expression and characterization of an amphibian ORL1 receptor, and contributes to our understanding of the evolution of the opioid system.

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A full-length cDNA that encodes a kappa ( ) opioid receptor has been isolated from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa. The deduced protein contains 385 amino acids and possesses features commonly attributed to G protein-coupled receptors, such as seven putative transmembrane domains. The newt receptor has 75% sequence identity to opioid receptors cloned from mammals, and 66% sequence identity to the opioid receptor reported for the zebrafish, with the greatest divergence in the extracellular N-terminus, the second and third extracellular loops and the intracellular C-terminus. Membranes isolated from COS-7 cells expressing the newt receptor possessed a single, high-affinity (K d =1·5 nM) binding site for the -selective agonist U69593. In competition binding assays, the expressed newt receptor displayed high affinity for the -selective agonists GR89696, dynorphin A(1-13), U69593, U50488 and BRL52537, as well as the -selective antagonist nor-binaltorphimine and the non-selective antagonist naloxone. Rank order potencies and affinity constants were similar in competition binding studies that used either whole brain homogenates or membranes isolated from COS-7 cells expressing the newt receptor. The expressed receptor displayed essentially no affinity for the -selective agonist DPDPE ([d-penicillamine, d-penicillamine]enkephalin), but showed moderate affinity for the µ-selective agonist DAMGO ([d-Ala-MePhe, Gly-ol]enkephalin) and moderately high affinity for nociceptin (orphanin FQ), the endogenous ligand for the opioid receptor-like (ORL)1 receptor. These findings support the conclusions that a gene for the opioid receptor is expressed in amphibians and that the pharmacology of the newt receptor closely matches mammalian opioid receptors. However, the functional dichotomy between the classic opioid receptors ( , , µ) and ORL1 appears less strict in amphibians than in mammals.