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We report the analysis of the genome of a novel

The complete genome sequence was determined for an apparent alphabaculovirus isolated from larval cadavers of the brown tussock moth, Olene mendosa Hübner, collected during an epizootic in Coimbatore, India. The genome was determined to be a circular 142,291 bp molecule, and 147 ORFs and nine homologous regions were annotated for the sequence. Analysis of the sequence confirmed that this virus, Olene mendosa nucleopolyhedrovirus (OlmeNPV), was a member of genus Alphabaculovirus in family Baculoviridae. Phylogenies inferred from nucleotide and amino acid alignments indicated that OlmeNPV was part of a group of viruses that infect moths of genus Lymantria, suggesting that OlmeNPV may have shifted hosts from a Lymantria species to an ancestral Olene species at some point during its evolutionary history. OlmeNPV was most closely related to Lymantria xylina multiple nucleopolyhedrovirus isolate 5 (LyxyMNPV-5). The genomes of OlmeNPV and LyxyMNPV-5 were distinguished not only by differences in ORF content, but by a 27 kbp region of the genome that is inverted in LyxyMNPV-5 relative to OlmeNPV. Pairwise nucleotide distances between OlmeNPV and other Lymantria spp. alphabaculoviruses indicate that OlmeNPV represents a new baculovirus species.

A baculovirus isolate from a USDA Forest Service collection was characterized by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species Mythimna unipuncta (true armyworm) and was renamed Mythimna unipuncta nucleopolyhedrovirus #7 (MyunNPV#7). The occlusion bodies (OBs) and virions exhibited a size and morphology typical for OBs produced by the species of genus Alphabaculovirus, with occlusion-derived virions consisting of 2-5 nucleocapsids within a single envelope. The MyunNPV#7 genome was determined to be 148,482 bp with a 48.58% G+C nucleotide distribution. A total of 159 ORFs of 150 bp or larger were annotated in the genome sequence, including the 38 core genes of family Baculoviridae. The genome contained six homologous repeat regions (hrs) consisting of multiple copies of a 34-bp imperfect palindrome. Phylogenetic inference from concatenated baculovirus core gene amino acid sequence alignments placed MyunNPV#7 with group II alphabaculoviruses isolated from other armyworm and cutworm host species of lepidopteran family Noctuidae. MyunNPV#7 could be distinguished from other viruses in this group on the basis of differences in gene content and order. Pairwise nucleotide distances suggested that MyunNPV#7 represents a distinct species in Alphabaculovirus. The MyunNPV#7 genome was found to contain two copies of the late expression factor-7 (lef-7) gene, a feature not reported for any other baculovirus genome to date. Both copies of lef-7 encoded an F-box domain, which is required for the function of LEF-7 in baculovirus DNA replication.

The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a destructive crop pest native to North, Central, and South America that recently has spread to Africa and Asia. Isolates of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) have the potential to be developed as low-risk biopesticides for management of fall armyworm, and a commercially available formulation has been developed for control of fall armyworm in North and South America. In this study, the virulence (LC50 and LT50) of several SfMNPV isolates towards larvae of both corn-strain and rice-strain fall armyworm was assessed. Bioassays with corn-strain larvae revealed that the isolates could be organized into fast-killing (LT50 68 h post-infection) groups. Rice-strain larvae exhibited narrower ranges of susceptibility to baculovirus infection and of survival times in bioassays with different isolates. Two SfMNPV isolates with rapid speeds of kill (SfMNPV-459 from Colombia and SfMNPV-1197 from Georgia, USA) along with an isolate that killed corn-strain at relatively low concentrations (SfMNPV-281 from Georgia) were selected for the complete determination of their genome sequences. The SfMNPV-1197 genome sequence shared high sequence identity with genomes of a Nicaraguan isolate, while SfMNPV-281 formed a separate clade with a USA and a Brazilian isolate in phylogenetic trees. The SfMNPV-459 sequence was more divergent with the lowest genome sequence identities in pairwise alignments with other sequenced SfMNPV genomes, and was not grouped reliably with either the 1197 clade or the 281 clade. SfMNPV-459 contained homologs of two ORFs that were unique to another Colombian isolate, but these isolates were not placed in the same clade in phylogenetic trees. This study identifies isolates with superior properties for control of fall armyworm and adds to our knowledge of the genetics of SfMNPV.

The pathogenicity and genome sequence of isolate LdMNPV-HrB of the gypsy moth alphabaculovirus, Lymantria dispar multiple nucleopolyhedrovirus from Harbin, Heilongjiang, China, were determined. A stock of this virus from one passage through the gypsy moth New Jersey Standard Strain (LdMNPV-HrB-NJSS) exhibited 6.2- to 11.9-fold greater pathogenicity against larvae from a Harbin colony of L. dispar asiatica than both Gypchek and a Massachusetts, USA LdMNPV isolate (LdMNPV-Ab-a624). Sequence determination and phylogenetic analysis of LdMNPV-HrB and LdMNPV-HrB-NJSS revealed that these isolates were most similar to other east Asian LdMNPV isolates with 98.8% genome sequence identity and formed a group with the east Asian LdMNPV isolates which was separate from groups of isolates from Russia, Europe, and USA.

Operophtera brumata nucleopolyhedrovirus (OpbuNPV) infects the larvae of the winter moth, Operophtera brumata. As part of an effort to explore the pesticidal potential of OpbuNPV, an isolate of this virus from Massachusetts (USA)—OpbuNPV-MA—was characterized by electron microscopy of OpbuNPV occlusion bodies (OBs) and by sequencing of the viral genome. The OBs of OpbuNPV-MA consisted of irregular polyhedra and contained virions consisting of a single rod-shaped nucleocapsid within each envelope. Presumptive cypovirus OBs were also detected in sections of the OB preparation. The OpbuNPV-MA genome assembly yielded a circular contig of 119,054 bp and was found to contain little genetic variation, with most polymorphisms occurring at a frequency of < 6%. A total of 130 open reading frames (ORFs) were annotated, including the 38 core genes of Baculoviridae, along with five homologous repeat (hr) regions. The results of BLASTp and phylogenetic analysis with selected ORFs indicated that OpbuNPV-MA is not closely related to other alphabaculoviruses. Phylogenies based on concatenated core gene amino acid sequence alignments placed OpbuNPV-MA on a basal branch lying outside other alphabaculovirus clades. These results indicate that OpbuNPV-MA represents a divergent baculovirus lineage that appeared early during the diversification of genus Alphabaculovirus.

Isolates of the alphabaculovirus species, Chrysodeixis includens nucleopolyhedrovirus, have been identified that produce polyhedral occlusion bodies and infect larvae of the soybean looper, Chrysodeixis includens. In this study, we report the discovery and characterization of a novel C. includens-infecting alphabaculovirus, Chrysodeixis includens nucleopolyhedrovirus #1 (ChinNPV#1), that produces tetrahedral occlusion bodies. In bioassays against C. includens larvae, ChinNPV #1 exhibited a degree of pathogenicity that was similar to that of other ChinNPV isolates, but killed larvae more slowly. The host range of ChinNPV#1 was found to be very narrow, with no indication of infection occurring in larvae of Trichoplusia ni and six other noctuid species. The ChinNPV#1 genome sequence was determined to be 130,540 bp, with 126 open reading frames (ORFs) annotated but containing no homologous repeat (hr) regions. Phylogenetic analysis placed ChinNPV#1 in a clade with other Group II alphabaculoviruses from hosts of lepidopteran subfamily Plusiinae, including Chrysodeixis chalcites nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus. A unique feature of the ChinNPV#1 genome was the presence of two full-length copies of the he65 ORF. The results indicate that ChinNPV#1 is related to, but distinct from, other ChinNPV isolates.

The Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV), a pathogen of the Egyptian cotton leaf worm S. littoralis, was subjected to sequencing of its entire DNA genome and bioassay analysis comparing its virulence to that of other baculoviruses. The annotated SpliMNPV genome of 137,998 bp was found to harbor 132 open reading frames and 15 homologous repeat regions. Four unique genes not present in SpltMNPV were identified, as were 14 genes that were absent or translocated by comparison. Bioassay analysis of experimentally infected Spodoptera frugiperda revealed an extended killing time for SpliMNPV as compared to S. frugiperda MNPV (SfMNPV), but a level of mortality similar to that caused by infection with SfMNPV and superior to that of Autographa californica MNPV (AcMNPV). Although extensive similarity was observed between the genome structure and predicted translation products of SpliMNPV and Spodoptera litura MNPV (SpltMNPV), genetic distances between isolates of SpliMNPV and SpltMNPV suggest that they are in fact different species of genus Alphabaculovirus.

Isolates of the baculovirus species Lymantria dispar multiple nucleopolyhedrovirus have been formulated and applied to suppress outbreaks of the gypsy moth, L. dispar. To evaluate the genetic diversity in this species at the genomic level, the genomes of three isolates from Massachusetts, USA (LdMNPV-Ab-a624), Spain (LdMNPV-3054), and Japan (LdMNPV-3041) were sequenced and compared with four previously determined LdMNPV genome sequences. The LdMNPV genome sequences were collinear and contained the same homologous repeats (hrs) and clusters of baculovirus repeat orf (bro) gene family members in the same relative positions in their genomes, although sequence identities in these regions were low. Of 146 non-bro ORFs annotated in the genome of the representative isolate LdMNPV 5-6, 135 ORFs were found in every other LdMNPV genome, including the 37 core genes of Baculoviridae and other genes conserved in genus Alphabaculovirus. Phylogenetic inference with an alignment of the core gene nucleotide sequences grouped isolates 3041 (Japan) and 2161 (Korea) separately from a cluster containing isolates from Europe, North America, and Russia. To examine phenotypic diversity, bioassays were carried out with a selection of isolates against neonate larvae from three European gypsy moth (Lymantria dispar dispar) and three Asian gypsy moth (Lymantria dispar asiatica and Lymantria dispar japonica) colonies. LdMNPV isolates 2161 (Korea), 3029 (Russia), and 3041 (Japan) exhibited a greater degree of pathogenicity against all L. dispar strains than LdMNPV from a sample of Gypchek. This study provides additional information on the genetic diversity of LdMNPV isolates and their activity against the Asian gypsy moth, a potential invasive pest of North American trees and forests.

Spatially and temporally regulated somatic mutations can be achieved by using the Cre/loxP recombination system of bacteriophage P1. To develop a cell type-specific system of gene targeting in the peripheral nervous system, we generated the transgenic mouse lines expressing Cre recombinase under the control of the mouse peripherin gene promoter. The activity of the Cre recombinase during embryonic development was examined by mating the peripherin-Cre transgenic mice to the knock-in Cre-mediated recombination reporter strain, R26R. Analysis of F1 embryos from this cross showed specific excision of loxP-flanked sequences in the dorsal root ganglia, trigeminal ganglia, and olfactory epithelium, in a pattern very similar to the expression of the endogenous mouse peripherin gene, and the previously reported peripherin-lacZ transgenic mice. Thus, the peripherin-Cre mouse described here will provide a valuable tool for Cre-loxP-mediated conditional expression in the peripheral nervous system.

Previous work suggested that the cocaine analog [125I]RTI-55 labels a novel binding site in rat brain membranes, which is not associated with the dopamine (DA), serotonin (5-HT), or norepinephrine (NE) transporters [Rothman et al. 1995 J Pharmacol Exp Ther 274:385–395]. Here, we tested whether this site is a product of the DA transporter (DAT) gene. We used a T-antigen knock-in at the DAT gene that results in an effective DAT knock-out (KO) confirmed by Southern blot, DAT immunohistochemistry, and [125I]RTI-55 ligand binding. Brain membranes were prepared from frozen whole brain minus caudate of wild-type (WT) B6/Sv129, +/+ and −/− (KO) mice. KO mice were used at approximately 23 days of age. Binding surface analysis of [125I]RTI-55 binding to membranes prepared from the brains of WT mice, with 100 nM citalopram to block binding to the 5-HT transporter (SERT), revealed two binding sites: the DAT and a second site, replicating previous studies conducted with rat brains. In the absence of the DAT (−/− mice), binding surface analysis demonstrated that [125I]RTI-55 labeled two sites: the NET and a second site called site “X.” Structure–activity studies of site “X” demonstrated that high-affinity ligands for the DAT, NET, and SERT have low or negligible affinity for site “X.” The relatively high density of site “X” in brain membranes and the fact that the Ki values of cocaine and cocaethylene for site “X” are in the range achieved in the brain following cocaine administration suggests that site “X” could contribute to the pharmacological or toxicological effects of cocaine. Further progress in delineating the function of site “X” will depend on developing potent and selective agents for this site. Synapse 44:94–105, 2002. Published 2002 Wiley-Liss, Inc.

Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) has been formulated and applied to control outbreaks of the gypsy moth, L. dispar . To classify and determine the degree of genetic variation among isolates of L. dispar NPVs from different parts of the range of the gypsy moth, partial sequences of the lef-8 , lef-9 , and polh genes were determined for Lymantria spp. virus samples from host populations throughout the world. Sequence analysis confirmed that all L. dispar virus samples tested contained isolates of the species Lymantria dispar multiple nucleopolyhedrovirus ( Baculoviridae: Alphabaculovirus ). Phylogenetic inference based on the lef-8 sequences indicated that the LdMNPV isolates formed two groups, one consisting primarily of isolates from Asia, and one consisting primarily of isolates from Europe and North America. The complete genome sequence was determined for an isolate from the Asian group, LdMNPV-2161 (S. Korea). The LdMNPV-2161 genome was 163,138 bp in length, 2092 bp larger than the previously determined genome of LdMNPV isolate 5–6 (CT, USA). The two genome sequences were co-linear, with an overall nucleotide sequence identity of 97.5% and some differences in ORF content. In droplet-feeding bioassays against neonate L. dispar larvae, isolates LdMNPV-3029 (Virin-ENSh/Russia) and LdMNPV-Ab-a624 (MA, USA) killed neonate larvae with an LC 50 values that were 1.8- to 3.2-fold lower than a sample of Gypchek® (CT, USA) and isolates LdMNPV-3041 (Japan) and LdMNPV-2161. This study expands our knowledge about genetic variation among LdMNPV isolates and provides novel information on the distinct groups in which these NPVs occur.

To determine the genetic diversity within the baculovirus species Autographa calfornica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus), a PCR-based method was used to identify and classify baculoviruses found in virus samples from the lepidopteran host species A. californica, Autographa gamma, Trichoplusia ni, Rachiplusia ou, Anagrapha falcifera, Galleria mellonella, and Heliothis virescens. Alignment and phylogenetic inference from partial nucleotide sequences of three highly conserved genes (lef-8, lef-9, and polh) indicated that 45 of 74 samples contained isolates of AcMNPV, while six samples contained isolates of Rachiplusia ou multiple nucleopolyhedrovirus strain R1 (RoMNPV-R1) and 25 samples contained isolates of the species Trichoplusia ni single nucleopolyhedrovirus (TnSNPV; Alphabaculovirus). One sample from A. californica contained a previously undescribed NPV related to alphabaculoviruses of the armyworm genus Spodoptera. Data from PCR and sequence analysis of the ie-2 gene and a region containing ORF ac86 in samples from the AcMNPV and RoMNPV clades indicated a distinct group of viruses, mostly from G. mellonella, that are characterized by an unusual ie-2 gene previously found in the strain Plutella xylostella multiple nucleopolyhedrovirus CL3 (PlxyMNPV-CL3) and a large deletion within ac86 previously described in the AcMNPV isolate 1.2 and PlxyMNPV-CL3. PCR and sequence analysis of baculovirus repeated ORF (bro) genes revealed that the bro gene ac2 was split into two separate bro genes in some samples from the AcMNPV clade. Comparison of sequences in this region suggests that ac2 was formed by a deletion that fused the two novel bro genes together. In bioassays of a selection of isolates against T. ni, significant differences were observed in the insecticidal properties of individual isolates, but no trends were observed among the AcMNPV, TnSNPV, or RoMNPV groups of isolates. This study expands on what we know about the variation of AcMNPV, AcMNPV-like and TnSNPV viruses, provides novel information on the distinct groups in which AcMNPV isolates occur, and contributes to data useful for the registration, evaluation, and improvement of AcMNPV, AcMNPV-like, and TnSNPV isolates as biological control agents.

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus ) obtained worldwide from larvae of Heliothis virescens , Helicoverpa zea , and Helicoverpa armigera . Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes ( lef-8 , lef-9 , and polh ) indicated that 67 of these samples contained isolates of the H. zea – H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera , HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci ( orf5/orf5b , hr3-orf62 , orf26 , orf79 , orf124/orf117a , orf42 , and a part of the region between hr2 and hr3 ) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea , the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC 50 s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.

A PCR-based method was used to identify and distinguish among 40 uncharacterized nucleopolyhedrovirus (NPV) isolates from larvae of the moth Spodoptera frugiperda that were part of an insect virus collection. Phylogenetic analysis was carried out with sequences amplified from two strongly conserved loci (polh and lef-8) from the 40 isolates in the collection and from eight previously studied S. frugiperda NPV (SfMNPV) isolates. To further distinguish these isolates, analysis was also carried out with sequences from two less-conserved loci, hr4 and hr5. Phylogenetic inference from the sequence data could distinguish among several of the individual isolates and between different groups of isolates from Georgia (USA) and Colombia, South America. A stronger degree of bootstrap support for the phylogenetic trees was obtained with the hr4 and hr5 homologous repeat sequences. Sequencing and phylogenetic analysis detected a relatively high degree of larva-to-larva sequence divergence occurring among isolates of SfMNPV collected from the same field in Missouri, USA. Restriction endonuclease analysis of viral DNA from larvae infected with five isolates from Georgia, Missouri, Louisiana, Florida (USA), and Colombia allowed for further comparison with other previously reported isolates of SfMNPV. Bioassays with these five geographically distinct isolates detected minor differences in virulence. This study highlights the use of PCR to rapidly distinguish and characterize large numbers of historical baculovirus isolates from the same host using minimal quantities of material, and the use of sequences from homologous repeat regions to distinguish closely related isolates of the same NPV species.

Lebia grandis (Coleoptera: Carabidae), recorded as a parasitoid only on Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), is capable of parasitizing the false potato beetle, L. juncta, and also L. haldemani. Historical records show that L. decemlineata, while the only recorded host, was not present in much of the original range of L. grandis, and may not have been its host prior to its expansion into eastern North America, where L. juncta is endemic. Our laboratory comparisons suggest that L. juncta, the presumptive original host, best supports the development of the parasitoid larval L. grandis, based on 43.6% successful emergence of the adult carabid parasitoid, compared to 11.5% from the two other Leptinotarsa species. L. grandis adults accept eggs and larvae of all 3 Leptinotarsa species as adult food. Naive, newly-emerged adults show no preference when presented the 3 species of third-instar larvae, which they consume at a mean rate of 3.3 per day, a rate which does not differ significantly by sex, larval host, or weight at emergence. When presented with equal amounts by weight of the 3 species of Leptinotarsa eggs, such adults consume the equivalent of 23.0 L. decemlineata eggs per day, with consumption of L. juncta eggs 67% higher by weight than L. decemlineata consumption. Insight into the biotic and abiotic limitations on L. grandis should aid in determining its potential for suppression of Colorado potato beetle by biological control in diverse agroecosystems.

The soybean aphid, Aphis glycines (Hemiptera: Aphididae), is a pest of soybeans in Asia, and in recent years has caused extensive damage to soybeans in North America. Within these agroecosystems, generalist predators form an important component of the assemblage of natural enemies, and can exert significant pressure on prey populations. These food webs are complex and molecular gut-content analyses offer nondisruptive approaches for examining trophic linkages in the field. We describe the development of a molecular detection system to examine the feeding behaviour of Orius insidiosus (Hemiptera: Anthocoridae) upon soybean aphids, an alternative prey item, Neohydatothrips variabilis (Thysanoptera: Thripidae), and an intraguild prey species, Harmonia axyridis (Coleoptera: Coccinellidae). Specific primer pairs were designed to target prey and were used to examine key trophic connections within this soybean food web. In total, 32% of O. insidiosus were found to have preyed upon A. glycines, but disproportionately high consumption occurred early in the season, when aphid densities were low. The intensity of early season predation indicates that O. insidiosus are important biological control agents of A. glycines, although data suggest that N. variabilis constitute a significant proportion of the diet of these generalist predators. No Orius were found to contain DNA of H. axyridis, suggesting intraguild predation upon these important late-season predators during 2005 was low. In their entirety, these results implicate O. insidiosus as a valuable natural enemy of A. glycines in this soybean agroecosystem.

The time during which prey remains are detectable in the gut of a predator is an important consideration in the interpretation of molecular gut-content data, because predators with longer detectability times may appear on the basis of unweighted data to be disproportionately important agents of prey population suppression. The rate of decay in detectability, typically expressed as the half-life, depends on many variables; one that has not been explicitly examined is the manner in which the predator processes prey items. The influence of differences in feeding mode and digestive physiology on the half-life of DNA for a single prey species, the Colorado potato beetle Leptinotarsa decemlineata (Say), is examined in two predators that differ dramatically in these attributes: the pink ladybeetle, Coleomegilla maculata (DeGeer), which feeds by chewing and then ingesting the macerated material into the gut for digestion; and the spined soldier bug, Podisus maculiventris (Say), which physically and enzymatically processes the prey extra-orally before ingestion and further digestion in the gut. In order to standardize the amount of DNA consumed per predator, a single L. decemlineata egg was used as the prey item; all predators were third instars. The PCR assay yields estimated prey DNA half-lives, for animals maintained under field temperatures, of 7.0 h in C. maculata and 50.9 h in P. maculiventris. The difference in the prey DNA half-lives from these two predators underscores the need to determine detectabilities from assemblages of predators differing in feeding mode and digestive physiology, in order to weight positives properly, and hence determine the predators' relative impacts on prey population suppression.

Identification of arthropod predators is challenging when closely related species are found at a given locality. Identification of the immature stages is especially problematic, because distinguishing morphological features are difficult to use or have not been described. We used polymerase chain reaction (PCR) to distinguish closely related carabids and spiders, and to match eggs and larvae (or nymphs) with identified adult parents. Within the Carabidae, we amplified species-specific mitochondrial cytochrome oxidase I (COI) fragments for three species each in the genera Poecilus and Harpalus , and two each in Chlaenius and Bembidion. Within the Araneae, we amplified species-specific COI fragments for two Hibana species (Anyphaenidae), Pardosa milvina and Rabidosa rabida (Lycosidae), Frontinella communis and Grammonota texana (Linyphiidae), and Cheiracanthium inclusum (Miturgidae). We are able to correctly identify all immature stages tested — eggs, larvae (or nymphs) and pupae — by comparison of the amplified fragments with those of the adults. Using COI markers as species identifiers is a tenet of the Barcode of Life initiative, an international consortium to provide a molecular identifier for every animal species.

Chromosomal regions near the mu opioid receptor gene are implicated in morphine preference by quantitative trait loci studies. Differences in expression of the mu opioid receptor are expected to contribute to differences in inter-individual (humans) or strain-specific (mice) responses to painful stimuli, opiate drugs, and addictive behaviors. The search for relevant genetic elements is hindered by a lack of inter-strain (or inter-individual) genomic sequence information. This work describes 9.3 kb of DNA sequence surrounding exons 2 and 3 of the murine mu opioid receptor gene from both 129/Sv and C57BL/6 strains. While the exons are perfectly conserved, intronic sequences demonstrate approximately a 2.5% divergence between the strains. Polymorphism within these intronic regions may effect either primary transcript stability or C-terminal splicing. Homologous recombination frequencies of targeting vectors harboring mu opioid receptor gene sequences have also been compared in embryonic stem cells derived from these strains. Non-isogenic targeting reduces homologous recombination in both 129/Sv and C57BL/6 embryonic stem cells by greater than 15-fold. These findings are the first to examine C57BL/6 embryonic stem cells for non-isogenic targeting frequencies and to define polymorphisms that exist between these mouse strains which might contribute to opioid behaviors.