Interconnections Between the Oral and Gut Microbiomes: Reversal of Microbial Dysbiosis and the Balance Between Systemic Health and Disease.

The human microbiota represents a complex array of microbial species that influence the balance between the health and pathology of their surrounding environment. These microorganisms impart important biological benefits to their host, such as immune regulation and resistance to pathogen colonization. Dysbiosis of microbial communities in the gut and mouth precede many oral and systemic diseases such as cancer, autoimmune-related conditions, and inflammatory states, and can involve the breakdown of innate barriers, immune dysregulation, pro-inflammatory signaling, and molecular mimicry. Emerging evidence suggests that periodontitis-associated pathogens can translocate to distant sites to elicit severe local and systemic pathologies, which necessitates research into future therapies. Fecal microbiota transplantation, probiotics, prebiotics, and synbiotics represent current modes of treatment to reverse microbial dysbiosis through the introduction of health-related bacterial species and substrates. Furthermore, the emerging field of precision medicine has been shown to be an effective method in modulating host immune response through targeting molecular biomarkers and inflammatory mediators. Although connections between the human microbiome, immune system, and systemic disease are becoming more apparent, the complex interplay and future innovations in treatment modalities will become elucidated through continued research and cross-disciplinary collaboration.

Adjunctive dental therapies in caries-active children: Shifting the cariogenic salivary microbiome from dysbiosis towards non-cariogenic health.

BACKGROUND: The oral microbiome is a complex assembly of microbial species, whose constituents can tilt the balance towards progression of oral disease or sustained health. Recently we identified sex-specific differences in the salivary microbiome contained within caries-active and caries-free children. In this study, we sought to ascertain if adjunctive dental therapies, including povidone iodine and chlorhexidine, were effective in shifting the cariogenic microbiome from dysbiosis to non-cariogenic health. DESIGN: We recruited young children (ages 2-12 years) to enter five enrollment groups, with each group (N = 9-30 participants/group) receiving caries restorative and/or adjunctive therapies, either singularly or in combination (OHSU IRB #6535). Saliva specimens were collected pre- and post-treatment (4-8 weeks) of caries preventive measures, and oral microbiota were identified using next generation sequencing (HOMINGS, Forsyth Institute, Cambridge, MA). RESULTS: With the use of multi-dimensional scaling plots, support vector machine learning, odds ratio analysis, and other statistical methods, we have determined that treatment with povidone iodine can shift the composition of the salivary cariogenic microbiome to include higher proportions of aerobic microorganisms, such as Stentrophomonas maltophila, as well as non-cariogenic, anaerobic microorganisms including Poryphyromonas and Fusobacterium species. CONCLUSION: We have identified microorganisms that are associated with caries-active children and have determined that povidone iodine is an effective adjunctive therapy that has the potential to shift the composition of the cariogenic microbiome to one more closely aligned with non-cariogenic health.

Sex-specific differences in the salivary microbiome of caries-active children.

Background and Objectives: Dental caries is a chronic disease affecting young children and has multi-factorial risk factors. The purpose of this work was to identify sex-specific differences in the salivary microbiota within caries-active children. Design: Saliva specimens were collected from 85 children (boys: 41; girls: 44) between the ages of 2-12 years. Salivary microbial DNA was subjected to PCR amplification using V3-V4 16S rDNA-specific primers and next-generation sequencing. Results: Significant sex differences in salivary microbiota were found between caries-active boys versus caries-active girls. Neisseria flavescens, Rothia aeria, and Haemophilus pittmaniae were found at significantly higher levels in caries-active boys. In contrast, Lactococcus lactis, Selenomonas species HOT 126, Actinobaculum species HOT 183, Veillonella parvula, and Alloprevotella species HOT 473 were found at significantly higher levels in caries-active girls. Conclusion: We have found the acid-generating, cariogenic Lactococcus lactis to be much more abundant in caries-active girls than caries-active boys, indicating that this microorganism may play a more significant role in shaping the cariogenic microbiome in girls. In addition, in caries-active girls, Alloprevotella species HOT 473 was the only species that exhibited both significant sex differences (4.4-fold difference; p=0.0003) as well as high abundance in numbers (1.85% of the total microbial population).

Oral microbiota species in acute apical endodontic abscesses.

BACKGROUND AND OBJECTIVES: Acute apical abscesses are serious endodontic diseases resulting from pulpal infection with opportunistic oral microorganisms. The objective of this study was to identify and compare the oral microbiota in patients (N=18) exhibiting acute apical abscesses, originating from the demographic region in Portland, Oregon. The study hypothesis is that abscesses obtained from this demographic region may contain unique microorganisms not identified in specimens from other regions. DESIGN: Endodontic abscesses were sampled from patients at the Oregon Health & Science University (OHSU) School of Dentistry. DNA from abscess specimens was subjected to polymerase chain reaction amplification using 16S rRNA gene-specific primers and Cy3-dCTP labeling. Labeled DNA was then applied to microbial microarrays (280 species) generated by the Human Oral Microbial Identification Microarray Laboratory (Forsyth Institute, Cambridge, MA). RESULTS: The most prevalent microorganisms, found across multiple abscess specimens, include Fusobacterium nucleatum, Parvimonas micra, Megasphaera species clone CS025, Prevotella multisaccharivorax, Atopobium rimae, and Porphyromonas endodontalis. The most abundant microorganisms, found in highest numbers within individual abscesses, include F. nucleatum, P. micra, Streptococcus Cluster III, Solobacterium moorei, Streptococcus constellatus, and Porphyromonas endodontalis. Strong bacterial associations were identified between Prevotella multisaccharivorax, Acidaminococcaceae species clone DM071, Megasphaera species clone CS025, Actinomyces species clone EP053, and Streptococcus cristatus (all with Spearman coefficients >0.9). CONCLUSIONS: Cultivable and uncultivable bacterial species have been identified in endodontic abscesses obtained from the Portland, Oregon demographic region, and taxa identifications correlated well with other published studies, with the exception of Treponema and Streptococcus cristae, which were not commonly identified in endodontic abscesses between the demographic region in Portland, Oregon and other regions.

Children with severe early childhood caries: streptococci genetic strains within carious and white spot lesions.

BACKGROUND AND OBJECTIVES: Mutans streptococci (MS) are one of the major microbiological determinants of dental caries. The objectives of this study are to identify distinct MS and non-MS streptococci strains that are located at carious sites and non-carious enamel surfaces in children with severe early childhood caries (S-ECC), and assess if cariogenic MS and non-cariogenic streptococci might independently exist as primary bacterial strains on distinct sites within the dentition of individual children. DESIGN: Dental plaque from children (N=20; aged 3-6) with S-ECC was collected from carious lesions (CLs), white spot lesions (WSLs) and non-carious enamel surfaces. Streptococcal isolates (N=10-20) from each site were subjected to polymerase chain reaction (PCR) to identify MS, and arbitrarily primed-PCR for assignment of genetic strains. Primary strains were identified as ≥50% of the total isolates surveyed at any site. In several cases, strains were characterized for acidurity using ATP-driven bioluminescence and subjected to PCR-determination of potential MS virulence products. Identification of non-MS was determined by 16S rRNA gene sequencing. RESULTS: Sixty-four independent MS or non-MS streptococcal strains were identified. All children contained 1-6 strains. In many patients (N=11), single primary MS strains were identified throughout the dentition. In other patients (N=4), primary MS strains were identified within CLs that were distinct from primary strains found on enamel. Streptococcus gordonii strains were identified as primary strains on enamel or WSLs in four children, and in general were less aciduric than MS strains. CONCLUSIONS: Many children with S-ECC contained only a single primary MS strain that was present in both carious and non-carious sites. In some cases, MS and non-cariogenic S. gordonii strains were found to independently exist as dominant strains at different locations within the dentition of individual children, and the aciduric potential of these strains may influence susceptibility in the development of CLs.

Mutans streptococci genetic strains in children with severe early childhood caries: follow-up study at one-year post-dental rehabilitation therapy.

BACKGROUND: Genotypic strains of cariogenic mutans streptococci (MS) may vary in important virulence properties. In previous published studies, we identified 39 MS strains from pediatric patients undergoing full-mouth dental rehabilitation, including the removal and/or repair of carious lesions and application of antimicrobial rinse and fluoride varnish. OBJECTIVES: The objectives of this current 1-year follow-up study are to assess the variability of MS strains that occur at 1-year post-rehabilitation and characterize the xylitol-resistance properties of MS strains that predominate. METHODS: Plaque from five children with severe early childhood caries was collected 1-year post-rehabilitation. MS isolates were subjected to arbitrarily primed-polymerase chain reaction (AP-PCR) for identification of genetic strains and in vitro xylitol-inhibition experiments. To more precisely define strain distributions within each patient, we isolated large numbers of isolates per patient. RESULTS: MS strains diminished from several strains pre-rehabilitation, to one dominant strain at 1-year post-rehabilitation, with several new emergent strains. The majority of the clinical MS strains, as well as the Streptococcus mutans laboratory strains ATCC 25175 and 35668, were predicted to undergo 50% inhibition with 2.48-5.58% xylitol, with some clinical MS strains being significantly more resistant in vitro. CONCLUSIONS: Our follow-up study using patients from the original cohort demonstrates that specific MS strains are dominant at 1-year post-dental rehabilitation. Most of the clinical MS strains are similar in xylitol resistance to the attenuated S. mutans ATCC control strains, with some strains being more resistant to xylitol in vitro.

Children with severe early childhood caries: pilot study examining mutans streptococci genotypic strains after full-mouth caries restorative therapy.

PURPOSE: Genotypic strains of mutans streptococci (MS) may vary in important virulence properties and be differentially affected by specific components of full-mouth caries restorative therapy. The purpose of this pilot study was to identify mutans streptococci strains that predominate following caries restorative therapy. METHODS: Plaque from 7 children with severe early childhood caries was collected before and following therapy. MS isolates (N=828) were subjected to polymerase chain reaction (PCR) and arbitrarily primed-PCR (AP-PCR) for assignment within MS strains. Determining the longitudinal changes in MS strain distribution over time within each patient required the isolation of larger numbers of isolates per patient, but from fewer patients. RESULTS: Up to 39 genotypic strains of Streptococcus mutans and Streptococcus sobrinus, and 7 genotypic strains of non-MS streptococci were identified by AP-PCR and 16S ribosomal rRNA gene sequencing. The number of MS strains isolated from each patient were 3 to 7 prior to treatment, diminishing to 1 to 2 dominant MS strains in most patients 6 months following therapy. CONCLUSIONS: Caries restorative therapy resulted in shifts of specific mutans streptococcus and non-mutans streptococcus strains. The implications are that caries restorative therapy affects the distribution of MS strains, and that well-accepted practices for caries prevention should be more closely examined for efficacy.

Elastomeric-ligated vs self-ligating appliances: a pilot study examining microbial colonization and white spot lesion formation after 1 year of orthodontic treatment.

AIM: To (1) evaluate the use of adenosine triphosphate (ATP)-driven bioluminescence for quantification of total plaque bacteria in orthodontic patients, (2) compare plaque bacteria amounts at the bracket-tooth interface with use of elastomeric-ligated and self-ligating brackets after 1 year of orthodontic treatment, and (3) analyze formation of white spot lesions by photographic evaluation and laser-light fluorescence (DIAGNOdent). METHODS: Thirteen subjects had fixed orthodontic appliances placed where lateral incisors were bonded with either elastomeric-ligated or self-ligating brackets. Plaque bacteria were collected from incisor surfaces after 1 year and quantified using plating methods and ATP-driven bioluminescence. White spot lesions were evaluated by photographic and DIAGNOdent determinations. A 2 x 2 x 2 mixed-design ANOVA was conducted to determine differences in plaque retention between elastomeric-ligated and self-ligating brackets. RESULTS: ATP-driven bioluminescence values correlated to numbers of total plaque bacteria (r = 0.80). However, unlike findings published in the original pilot study, which described increased plaque retention with elastomeric-ligated brackets at 5 weeks postbonding, there were no significant differences in bacterial numbers or ATP-driven bioluminescence values surrounding the elastomeric-ligated vs self-ligating brackets after 1 year of orthodontic treatment. Based on photographic and DIAGNOdent determinations, white spot lesions were found relatively equally on teeth bonded with either bracket type. DIAGNOdent measurements were found to have moderate sensitivity (0.71) and good specificity (0.88) when compared to white spot lesions determined using photographic evaluation. CONCLUSION: ATP-driven bioluminescence can be used as an accurate assessment of total plaque bacteria in orthodontic patients. After 1 year of orthodontic treatment for patients in this pilot study, there appeared to be no differences in retention of plaque bacteria or white spot lesions comparing the bracket types. The use of DIAGNOdent has some limitations, but may prove to be useful to monitor white spot lesions longitudinally.

Application of adenosine triphosphate-driven bioluminescence for quantification of plaque bacteria and assessment of oral hygiene in children.

PURPOSE: Dentistry has undergone a shift in caries management toward prevention and improved oral hygiene and diagnosis. Caries prevention now represents one of the most important aspects of modern dental practice. The purpose of this cross-sectional study was to demonstrate the use of adenosine triphosphate- (ATP-) driven bioluminescence as an innovative tool for the rapid chairside enumeration of oral bacteria (including plague streptococci) and assessment of oral hygiene and caries risk. METHODS: Thirty-three pediatric patients (7- to 12-year-old males and females) were examined, and plague specimens, in addition to stimulated saliva, were collected from representative teeth within each quadrant. Oral specimens (n=150 specimens) were assessed by plating on enriched and selective agars, to enumerate total bacteria and streptococci, and subjected to adenosine triphosphate- (ATP-) driven bioluminescence determinations using a luciferase-based assay system. RESULTS: Statistical correlations, linking ATP values to numbers of total bacteria, oral streptococci and mutans streptococci, yielded highly significant r values of 0.854, 0.840, and 0.796, respectively CONCLUSIONS: Our clinical data is consistent with the hypothesis that ATP measurements have a strong statistical association with bacterial number in plague and saliva specimens, including numbers for oral streptococci, and may be used as a potential assessment tool for oral hygiene and caries risk in children.

Plaque retention by self-ligating vs elastomeric orthodontic brackets: quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence.

INTRODUCTION: Enamel decalcification is a common problem in orthodontics. The objectives of this randomized clinical study were to enumerate and compare plaque bacteria surrounding 2 bracket types, self-ligating (SL) vs elastomeric ligating (E), and to determine whether adenosine triphosphate (ATP)-driven bioluminescence could be used for rapid assessment of bacterial load in plaque. METHODS: Patients (ages, 11-17 years) were bonded with SL and E brackets in 14 maxillary and 12 mandibular arches by using a split-mouth design. Recall visits were at 1 and 5 weeks after bonding. Plaque specimens were assayed for oral bacteria and subjected to ATP-driven bioluminescence determinations with a luciferin-based assay. RESULTS: In most patients, teeth bonded with SL attachments had fewer bacteria in plaque than did teeth bonded with E brackets. At 1 and 5 weeks after bonding, the means for SL vs E brackets were statistically lower for total bacteria and oral streptococci (P <0.05). ATP bioluminescence values were statistically correlated to the total oral bacteria and oral streptococci, with correlation coefficients of 0.895 and 0.843, respectively. CONCLUSIONS: SL appliances promote reduced retention of oral bacteria, and ATP bioluminescence might be a useful tool in the rapid quantification of bacterial load and the assessment of oral hygiene during orthodontic treatment.

Herpesviruses in abscesses and cellulitis of endodontic origin.

Acute apical abscesses and cellulitis are severe endodontic diseases caused by opportunistic bacteria with possible coinfection with latent herpesviruses. The objectives of this study are to identify herpesviruses, including human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), herpes simplex virus-1 (HSV-1), and Varicella zoster virus (VZV) in patients (n = 31) presenting with acute apical abscesses and cellulitis of endodontic origin. Primary and nested polymerase chain reaction (PCR) was conducted using virus-specific primers and DNA isolated from cell-free abscess fluid. From patients exhibiting concurrent spontaneous pain (n = 28), nine abscesses contained HCMV, two abscesses contained EBV, one abscess contained HSV-1, and no abscesses contained VZV. Control PCR using genomic or recombinant templates showed detection limits to a single genomic copy of HCMV, 100 genomic copies for EBV, and 1 to 10 copies for HSV-1 with no cross-amplification between herpesviral DNA targets. Nested PCR was required for detection of herpesviral DNA in the abscess specimens, indicating that these viruses were present in low copy number. Filtration of abscess specimens and virus transfer experiments using human fibroblastic MRC-5 cells confirmed the presence of HCMV particles in several abscess specimens. We conclude that herpesviruses are present but not required for the development of acute apical abscesses and cellulitis of endodontic origin.

Herpesviruses in endodontic pathoses: association of Epstein-Barr virus with irreversible pulpitis and apical periodontitis.

Irreversible pulpitis and apical periodontitis are inflammatory diseases caused by opportunistic bacteria with possible co-infection with latent herpesviruses. The objectives of this study are to identify herpesviruses, including human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV-1), and Varicella zoster virus (VZV) in patients with irreversible pulpitis (n = 29) or apical periodontitis, either primary (n = 30) or previously treated (n = 23). Using primary and nested polymerase chain reaction (PCR) and reverse transcription-PCR, EBV DNA and RNA were present in endodontic pathoses in significantly higher percentages (43.9% and 25.6%, respectively) compared with healthy pulp controls (0% and 0%, respectively). HCMV DNA and RNA were found in measurable numbers in both endodontic patients (15.9% and 29.3%, respectively) and in healthy pulp controls (42.1% and 10.5%, respectively). HSV-1 DNA was found in low percentages in endodontic patients (13.4%), and only one patient showed the presence of VZV. In conclusion, EBV may be associated with irreversible pulpitis and apical periodontitis.

CRM 1-mediated degradation and agonist-induced down-regulation of beta-adrenergic receptor mRNAs.

The beta1-adrenergic receptor (beta1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of its 3' untranslated region (UTR) with RNA binding proteins, including the HuR nuclear protein. In a previous report [Kirigiti et al. (2001). Mol. Pharmacol. 60:1308-1324], we examined the agonist-mediated down-regulation of the rat beta1-AR mRNAs, endogenously expressed in the rat C6 cell line and ectopically expressed in transfectant hamster DDT1MF2 and rat L6 cells. In this report, we determined that isoproterenol treatment of neonatal rat cortical neurons, an important cell type expressing beta1-ARs in the brain, results in significant decreases in beta1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in beta1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the beta1-AR 3' UTR recognize complexes composed of HuR and multiple proteins, including CRM 1. Cell-permeable peptides containing the leucine-rich nuclear export signal (NES) were used as inhibitors of CRM 1-mediated nuclear export. When DDT1MF2 transfectants were treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of beta1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the beta1-AR mRNAs.

Role of Tyr(356(7.43)) and Ser(190(4.57)) in antagonist binding in the rat beta1-adrenergic receptor.

Site-directed mutagenesis and photoaffinity labeling experiments suggest the existence of at least two distinct binding orientations for aryloxypropanolamine competitive antagonists in the beta-adrenergic receptor (beta-AR), one where the aryloxy moiety is located near transmembrane alpha-helix 7 (tm 7) and another where it is near tm 5. To explore a hydrophobic pocket involving tms 1, 2, 3, and 7 for potential aryloxy interaction sites, we selected Tyr(356(7.43)) and Trp(134(3.28)) in the rat beta(1)-AR for site-directed mutagenesis studies. Ser(190(4.57)) was also investigated, as the equivalent residues are known antagonist interaction sites in the muscarinic M(1) and the dopamine D(2) receptors. Binding affinities (pK(i)) of a series of structurally diverse aryloxypropanolamine competitive antagonists were determined for wild type and Y356A, Y356F, W134A, and S190A mutant rat beta(1)-ARs stably expressed in Chinese hamster ovary cells. To visualize possible antagonist/receptor interactions, the compounds were docked into a three-dimensional model of the wild-type rat beta(1)-AR. The results indicate that Tyr(356(7.43)) is an important aromatic interaction site for five of the eight competitive antagonists studied, whereas none of the compounds appeared to interact directly with Trp(134(3.28)). Only two of the competitive antagonists interacted with Ser(190(4.57)) on tm 4. Overall, the results extend our understanding of how beta(1)-AR competitive antagonists bind to the hydrophobic pocket involving tms 1, 2, 3, and 7; highlight the importance of Tyr(356(7.43)) in this binding pocket; and demonstrate the involvement of tm 4 in competitive antagonist binding.

Agonist binding and activation of the rat beta(1)-adrenergic receptor: role of Trp(134(3.28)), Ser(190(4.57)) and Tyr(356(7.43)).

We investigated the role of Trp(134(3.28)), Ser(190(4.57)) and Tyr(356(7.43)) in agonist binding to, and activation of, the rat beta(1)-adrenergic receptor by comparing pK(i)s and functional responses of W134A, S190A and Y356F mutant receptors to wild type, all stably expressed in CHO cells. All three mutations significantly (P < 0.05) reduced adenylyl cyclase intrinsic activity (IA) compared to wild type in response to stimulation with both (-)-isoprenaline (53-88%) and (-)-RO363 (46-61%), and there was no significant correlation either between IA or pD(2) and pK(i) (P > 0.4), suggesting that changes in pK(i) were not sufficient to explain the fall in adenylyl cyclase activity. The most pronounced reduction in affinity (126-fold, P < 0.01) was displayed by xamoterol for the Y356F mutation, suggesting that xamoterol is able to directly interact with Tyr(356(7.43)). For the other agonists, the change in pK(i) values for the mutant receptors ranged from a 20-fold decrease to a 2-fold increase compared to the wild type. In a three-dimensional model of the rat beta(1)-adrenergic receptor, Trp(134(3.28)) and Tyr(356(7.43)) form part of a hydrophobic binding pocket involving residues in transmembrane helices 1, 2, 3 and 7. Our results suggest that Trp(134(3.28)) and Tyr(356(7.43)), together with Trp(353(7.40)), are able to interact via pi-pi interactions to stabilize the extracellular ends of transmembrane helices 3 and 7. Ser(190(4.57)) appears to be involved in a hydrogen bonding network, which maintains the spatial relationship between transmembrane helices 3 and 4. These interhelical interactions suggest that the three mutated residues stabilize the active receptor state by maintaining the proper packing of their respective transmembrane helix within the helix bundle, facilitating the appropriate movement and rotation of the transmembrane regions during the activation process.

Site-directed mutagenesis of the rat beta1-adrenoceptor. Involvement of Tyr356 (7.43) in (+/-)cyanopindolol but not (+/-)[125Iodo]cyanopindolol binding.

To determine the role played by Tyr(356 (7.43)) in the rat beta(1)-adrenoceptor in binding the antagonists (+/-)cyanopindolol (4-[3-(t-butylamino]-3-(2'-cyano-indoloxy)-2-propanolol) and its iodinated analogue (+/-)[(125)Iodo]cyanopindolol (1-(t-butylamino]-3-(2'-cyano-3'-iodo-indoloxy)-2-propanolol), Tyr(356 (7.43)) was mutated to either Phe or Ala and binding affinities determined for wild type and mutant rat beta(1)-adrenoceptors. Our results indicate that Tyr(356 (7.43)) is important for (+/-)cyanopindolol, but not (+/-)[(125)Iodo]cyanopindolol, binding and that (+/-)cyanopindolol adopts a "reverse" binding orientation whereas (+/-)[(125)Iodo]cyanopindolol cannot be accommodated in this binding mode. We define a "reverse" antagonist binding mode as one where the aryloxy moiety interacts with residues on transmembrane helices 1, 2, 3 and 7. The beta(1)-adrenoceptor site-directed mutagenesis results are the first to support a "reverse" antagonist binding orientation and the involvement of Tyr(356 (7.43)) in this binding mode.

Simian retrovirus serogroup 2 constitutive transport element recognizes the ribosomal L10-like protein and translocon gamma subunit-like protein in a yeast three-hybrid assay.

The simian retrovirus (SRV) serogroup 2 genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. In a previous report [Virology 264 (1999) 37], CTE RNA-protein complexes were detected using UV-crosslinking/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To identify these CTE-interacting cellular proteins, we utilized yeast three-hybrid interaction approaches using the complete IR as bait, modified to eliminate transcriptional termination signals recognized by RNA polymerase III, and identified several interactive clones from a Hela cell cDNA activation domain (AD) library. UV-crosslinking of RNA-protein complexes, using Hela cell extracts and the modified IR bait, were conducted prior to library screening, to verify appropriate interaction of CTE RNA-protein complexes. Over one million recombinants were screened, and our yeast hybrid results indicate that the CTE interacts with several molecules involved in cellular translational and translocation machinery, including the ribosomal L10-like protein and the tranlocon protein gamma subunit-like protein. UV-crosslinking/immunoblot assays have verified the interaction of the CTE region with molecules immunologically reactive to antibodies recognizing the ribosomal L10-like protein.

UV cross-linking/immunoprecipitation assay: glucocorticoid receptor-adrenergic receptor gene sequence interaction.

The rat beta 1-adrenergic receptor (beta 1-AR) gene contains glucocorticoid response element (GRE) half-sites at positions -2767 and -945. In electrophoretic mobility shift assay (EMSA) experiments, neither beta 1-AR GRE half-site recognized glucocorticoid receptors (GRs) obtained from baculovirus high-level expression systems or from mammalian cells. We have developed a sensitive UV cross-linking/immunoprecipitation assay, using a 524-bp fragment containing the prototypical GRE obtained from the rat tyrosine aminotransferase promoter sequence and using antibodies recognizing mammalian GR. Using this assay, we provide evidence that rat beta 1-AR gene sequences recognize mammalian GRs expressed in mouse 3T3 cells and that the site of GR interaction does not appear to specifically contain the beta 1-AR GRE half-sites. This represents one of the first reports demonstrating the utility of a UV cross-linking/immunoprecipitation assay in the detection of mammalian GR interaction with beta 1-AR sequences, is consistent with the lack of specific DNA-GR protein complexes observed in EMSA experiments using oligonucleotide probes containing the beta 1-AR GRE half-sites, and provides evidence that mammalian GR interaction occurs at complex rate beta 1-AR gene sequences.