LX4211, a dual SGLT1/SGLT2 inhibitor, improved glycemic control in patients with type 2 diabetes in a randomized, placebo-controlled trial.

Thirty-six patients with type 2 diabetes mellitus (T2DM) were randomized 1:1:1 to receive a once-daily oral dose of placebo or 150 or 300 mg of the dual SGLT1/SGLT2 inhibitor LX4211 for 28 days. Relative to placebo, LX4211 enhanced urinary glucose excretion by inhibiting SGLT2-mediated renal glucose reabsorption; markedly and significantly improved multiple measures of glycemic control, including fasting plasma glucose, oral glucose tolerance, and HbA(1c); and significantly lowered serum triglycerides. LX4211 also mediated trends for lower weight, lower blood pressure, and higher glucagon-like peptide-1 levels. In a follow-up single-dose study in 12 patients with T2DM, LX4211 (300 mg) significantly increased glucagon-like peptide-1 and peptide YY levels relative to pretreatment values, probably by delaying SGLT1-mediated intestinal glucose absorption. In both studies, LX4211 was well tolerated without evidence of increased gastrointestinal side effects. These data support further study of LX4211-mediated dual SGLT1/SGLT2 inhibition as a novel mechanism of action in the treatment of T2DM.

Variation in plasma leptin levels in response to fasting in Antarctic fur seals (Arctocephalus gazella).

Plasma leptin levels were determined in 8 lactating female and 20 pup Antarctic fur seals (Arctocephalus gazella) during fasting periods of normal duration. Plasma leptin levels ranged from 1.35 3.19 ng x ml(-1) in lactating females and 1.79-4.80 ng x ml(-1) in pups and were not positively correlated with body mass or condition. A negative trend, however, was observed between plasma leptin levels and body condition in lactating females upon their arrival at the colony following a foraging trip (beginning of fast). In accordance with findings in other species, plasma leptin levels dropped significantly (P < 0.02) in response to the 17-19% drop in body mass experienced by pups during fasting. In contrast, plasma leptin levels in lactating females increased during the first 24 h of fasting before decreasing throughout the remaining 48 h of the fast. This unexpected result could be due to the high level of energy expenditure by seals as they swim back to the colony (i.e. post-exercise response) or may be influenced by the intense suckling activity experienced by females during the onshore fasting periods. The results of this study support recent findings in other carnivore species which suggest the primary physiological role of leptin in these species may not necessarily be as a signal of the magnitude of body energy reserves.

Ancient chronology. Astronomical orientation of the pyramids.

Spence speculates that Egypt's pyramid builders found true north by using a plumb line: when the stars Kochab and Mizar were seen on the same vertical, one was facing north. As evidence in support of this hypothesis, she points to the proposed interstar-line precession past the north celestial pole at a rate of 27' per century (cy). We argue that a mathematical error affects this result, which when corrected points more strongly to a different pair of stars. This suggests that the conventional ancient chronology, instead of being compressed, may actually have to be expanded slightly.

Fasting metabolism in Antarctic fur seal (Arctocephalus gazella) pups.

The metabolism of 52-73-day old Antarctic fur seal pups from Bird Island, South Georgia, was investigated during fasting periods of normal duration while their mothers were at sea foraging. Body mass decreased exponentially with pups losing 3.5-3.8% of body mass per day. Resting metabolic rate also decreased exponentially from 172-197 ml (O2) x min(-1) at the beginning of the fast and scaled to M(b)(0.74) at 2.3 times the level predicted for adult terrestrial mammals of similar size. While there was no significant sex difference in RMR, female pups had significantly higher (F(1,18)=6.614, P<0.019) mass-specific RMR than male pups throughout the fasting period. Fasting FMR was also significantly (t(15)=2.37, P<0.035) greater in females (823 kJ x kg(-1) x d(-1)) than males (686 kJ x kg(-1) x d(-1)). Average protein turnover during the study period was 19.3 g x d(-1) and contributed to 5.4% of total energy expenditure, indicating the adoption of a protein-sparing strategy with a reliance on primarily lipid catabolism for metabolic energy. This is supported by observed decreases in plasma BUN, U/C, glucose and triglyceride concentrations, and an increase in beta-HBA concentration, indicating that Antarctic fur seals pups adopt this strategy within 2-3 days of fasting. Mean RQ also decreased from 0.77 to 0.72 within 3 days of fasting, further supporting a rapid commencement of protein-sparing. However, RQ gradually increased thereafter to 0.77, suggesting a resumption of protein catabolism which was not substantiated by changes in plasma metabolites. Female pups had higher TBL (%) than males for any given mass, which is consistent with previous findings in this and other fur seal species, and suggests sex differences in metabolic fuel use. The observed changes in plasma metabolites and protein turnover, however, do not support this.

The effect of nasal patency on the clearance of radiolabeled saline in healthy volunteers.

PURPOSE: The objective of this study was to investigate the effect of nasal cavity patency on the penetration, deposition, and clearance of an aqueous isotonic saline solution. METHODS: The study was carried out as a single center, open, randomized, 2-way cross-over in healthy volunteers. Nasal patency was assessed using misting patterns on a cold metal surface at the beginning and end of study. 100 microl of technetium-99m radiolabeled saline solution was introduced into either the most or least patent nasal cavity using a purpose designed spray device. The distribution and residence time of the radiolabel was followed for 2 hours using gamma scintigraphy. RESULTS: The mean times to 50% clearance were 34+/-7 and 28+/-12 minutes (+/- s.d.) for the side view of the least and most patent nasal cavity respectively. Total clearance of the radiolabelled saline from the nose was not affected by patency. Between 7 and 35% of the radiolabelled saline solution remained in the nasal cavity at the end of imaging. Using endoscopy to track the clearance of an aqueous solution of food dye using the same delivery procedure, identified this region as hair in the nasal vestibule. The dye was seen to dry in this region along with the mucus. CONCLUSIONS: Nasal patency affects the initial, but not total clearance of solutions, however, the remaining solution may not be available for drug delivery.

Determination of baseline human nasal pH and the effect of intranasally administered buffers.

The nose is becoming a common route of drug administration, however, little is known about the pH of the human nasal cavity. Local pH may have a direct effect on the rate and extent of absorption of ionizable compounds and hence this study was performed to investigate normal pH values and whether pH could be manipulated by various buffers. Twelve healthy volunteers participated in a study to measure pH in the anterior and posterior sites of the nasal cavity. Miniature pH electrodes were placed 3 cm apart in the nasal cavity and a baseline was recorded for 30 min once the pH had stabilized. One hundred microlitres of isotonic solution was sprayed into the nostril and the pH was measured for 4 h post-dose. The following five formulations were tested: formulation A--sodium chloride (0.9%) at pH 7.2; formulation B--sodium chloride (0.9%) at pH 5.8; formulation C--Sorensens phosphate buffer (0.06 M) at pH 5. 8; formulation D--Sorensens phosphate buffer (0.13 M) at pH 5.8 and formulation E--formulation as (c) but adjusted to pH 5.0. Each formulation also contained saccharin sodium (0.5%) as a taste marker for nasal clearance. The time at which each subject detected the taste of saccharin was noted. The 30-minute baseline recording prior to administration of the nasal spray formulation demonstrates that there was both considerable intersubject and intrasubject variation in nasal pH. The average pH in the anterior of the nose was 6.40 (+0. 11, -0.15 S.D.) when calculated from H(+) values. The pH in the posterior of the nasal cavity was 6.27 (+0.13, -0.18 S.D.). The overall range in pH was 5.17-8.13 for anterior pH and 5.20-8.00 for posterior pH. Formulation A caused the pH in the anterior part of the nasal cavity to reach a maximum of 7.06 in 11.25 min from the baseline of pH 6.14 (P<0.05). The mean baseline pH was 6.5 for the posterior part of the nose which did not change over the recording period. Formulation B caused the anterior pH to increase from pH 6. 60 to 7.25 within the first minute. This fell back to a mean pH of 7.07 over the first hour which was still significantly above the baseline. It remained at this value for the remainder of the recording period. The initial average posterior pH was 6.32 and again this did not significantly change over the recording period. Formulation C produced a sustained increase in anterior nasal pH from a baseline pH of 6.57-7.12. A small transient decrease was observed in the pH in the posterior of the nose but baseline pH of 6. 6 was re-established within 15 min post dose. Formulation D significantly reduced anterior nasal pH from 6.30 to 5.87 by 30 min reaching a pH of 5.95 by 90 min where it remained for the remainder of the recording period. The posterior baseline pH was 6.3 and introduction of the pH 5.8 buffer caused a slow increase over 90 min to pH 6.6. Formulation E increased anterior pH from 6.1 to 6.7 for the remainder of the recording period. It had an insignificant effect on posterior nasal pH. The mean (+/-S.D.) time to taste saccharin for formulations A to E was 13.42+/-10.21, 14.67+/-8.37, 11.67+/-8.08, 10.08+/-7.6, 9.80+/-6.73 min, respectively. There was no significant difference between the clearance times for the different formulations. In conclusion, average baseline human nasal pH is approximately 6.3. Nasal anterior pH can be decreased when buffers of 0.13 M and above are used. Mildly acidic solutions produce an increase in pH presumably due to reflux bicarbonate secretion. Posterior nasal pH was not altered by administration of any buffer except the 0.13 M buffer at pH 5.8. This produced a rise in posterior pH.

Dissolution behaviour of sulphonamides into sodium dodecyl sulfate micelles: a thermodynamic approach.

The surface energies of four sulfonamides have been assessed from contact angle data, using the Lewis acid-base approach. From these data the free energy of adhesion between the drugs and sodium dodecyl sulfate (SDS) head groups and tails has been calculated. The most favored interaction was for adhesion to the SDS tails, rather than the head groups. The initial rotating disk dissolution rate (hereafter termed dissolution rate) of drug compacts has been measured in water and water with SDS micelles at a range of temperatures. The thermodynamic parameters of activation have been calculated from the rate data. Linear relationships exist between the enthalpy of transfer between water and SDS micelles and the free energy of adhesion between the drugs and both SDS head groups and SDS tails. The most nonpolar drugs had the most favored free energy of adhesion and the most favored enthalpy of transfer. The most polar drug had a disfavoured free energy of adhesion to the SDS head and a disfavoured enthalpy of transfer. This response demonstrates that the most important barrier to the passage from the aqueous fluid to the hydrophobic core of the micelle is the monopolar repulsion between the polar forces of the drug and head group surface energies. This provides a new insight into a possible mechanism of solubilization and offers the prospect of understanding even more complex partitioning behavior.

Identification and characterization of ZIIBC, a complex formed by cellular factors and the ZII site of the Epstein-Barr virus BZLF1 promoter.

The transition from latency to lytic Epstein-Barr virus replication is dependent on the Epstein-Barr virus BZLF1 gene product. Genetic and biochemical attempts to link cellular second-messenger signaling pathways that trigger this transition with the subsequent viral gene cascade have identified functional elements within the BZLF1 promoter (Zp) that appear to bind undefined cellular transcription factors. One of these previously identified sites, ZII, has homology to consensus AP-1 and CREB binding sites, implying a role for these factors in the inductive process. We have identified and characterized ZIIBC, a ZII site binding complex that is distinct from the factors previously proposed to bind this site. Active ZIIBC was found to be present in both uninduced and chemically induced cell extracts at approximately equivalent concentrations. Analysis of the DNA sequence requirements for the binding of ZIIBC to the ZII site shows that sequences homologous to AP-1 and CREB consensus sites are necessary but not sufficient for complex formation. Although the components of ZIIBC that directly contact DNA were found to be of the same molecular masses (26 and 36 kDa) in both uninduced and chemically induced cell extracts, a slight mobility difference between DNA-protein complexes formed by these two types of extracts is observable and indicates that ZIIBC is directly affected by chemical induction. The effects of ZIIBC binding to the ZII site on expression from Zp were evaluated, and they suggest that ZIIBC plays a critical role in the regulation of Zp expression.

EBNA-2 upregulation of Epstein-Barr virus latency promoters and the cellular CD23 promoter utilizes a common targeting intermediate, CBF1.

The EBNA-2 protein is essential for the establishment of a latent Epstein-Barr virus (EBV) infection and for B-cell immortalization. EBNA-2 functions as a transcriptional activator that modulates viral latency gene expression as well as the expression of cellular genes, including CD23. We recently demonstrated that EBNA-2 transactivation of the EBV latency C promoter (Cp) is dependent on an interaction with a cellular DNA-binding protein, CBF1, for promoter targeting. To determine whether targeting via CBF1 is a common mechanism for EBNA-2-mediated transactivation, we have examined the requirements for activation of the cellular CD23 promoter. Binding of CBF1 to a 192-bp mapped EBNA-2-responsive region located at position -85 bp to -277 bp upstream of the CD23 promoter was detected in electrophoretic mobility shift assays. The identity of the bound protein as CBF1 was established by showing that the bound complex was competed for by the CBF1 binding site from the EBV Cp, that the bound protein could be supershifted with a bacterially expressed fusion protein' containing amino acids 252 to 425 of EBNA-2 but was unable to interact with a non-CBF1-binding EBNA-2 mutant (WW323SR), and that in UV cross-linking experiments, the Cp CBF1 binding site and the CD23 probe bound proteins of the same size. The requirement for interaction with CBF1 was demonstrated in a transient cotransfection assay in which the multimerized 192-bp CD23 response region was transactivated by wild-type EBNA-2 but not by the WW323SR mutant. Reporter constructions carrying multimerized copies of the 192-bp CD23 response region or multimers of the CBF1 binding site from the CD23 promoter were significantly less responsive to EBNA-2 transactivation than equivalent constructions carrying a multimerized region from the Cp or multimers of the CBF1 binding site from the Cp. Direct binding and competition assays using 30-mer oligonucleotide probes representing the individual CBF1 binding sites indicated that CBF1 bound less efficiently to the CD23 promoter and the EBV LMP-1 promoter sites than to the Cp site. To investigate the basis for this difference, we synthesized a series of oligonucleotides carrying mutations across the CBF1 binding site and used these as competitors in electrophoretic mobility shift assays. The competition experiments indicated that a central core sequence, GTGGGAA, common to all known EBNA-2-responsive elements, is crucial for CBF1 binding. Flanking sequences on either side of this core influence the affinity for CBF1.(ABSTRACT TRUNCATED AT 400 WORDS)

The Epstein-Barr virus immortalizing protein EBNA-2 is targeted to DNA by a cellular enhancer-binding protein.

The Epstein-Barr virus nuclear antigen EBNA-2 is essential for Epstein-Barr virus-induced immortalization of B cells. EBNA-2 is a transcriptional activator capable of modifying the expression of specific viral and cellular genes. However, the mechanism of EBNA-2 transactivation has been an enigma. We used a fractionated extract of CA46 lymphoblastoid cells and bacterially expressed EBNA-2 polypeptides to demonstrate that EBNA-2 is targeted to the Epstein-Barr virus latency C promoter (Cp) through interaction with a cellular DNA binding protein designated Cp binding factor 1 (CBF1). A glutathione S-transferase-EBNA-2 fusion protein containing aa 252-425 of EBNA-2 interacted with CBF1 to yield a slowly migrating complex in an electrophoretic mobility shift assay. Mutation of EBNA-2 aa 323 and 324, which lie within a highly conserved amino acid motif, abolished the interaction with CBF1. This same mutation also abolished the ability of EBNA-2 to activate the Cp in a cotransfection assay. The binding site for CBF1 was localized to residues -359 to -388 of the Cp by using an electrophoretic mobility shift assay and DNase I footprinting. Introduction of multiple copies of the CBF1 binding site upstream of a minimal heterologous promoter conferred EBNA-2 responsiveness on that promoter. Mutation of a core sequence CNGTGGGAA abolished CBF1 binding, and the mutated sequence was unable to mediate EBNA-2 transactivation. The CBF1 core sequence also occurs in other EBNA-2-responsive promoters suggesting that CBF1 may mediate EBNA-2 transactivation of both cellular and viral targets.

Microtubules rich in post-translationally modified alpha-tubulin form distinct arrays in frog lens epithelial cells.

Isolated frog lens epithelia were stained with antibodies against tyrosinated, detyrosinated or acetylated alpha-tubulin and observed by several means including a scanning confocal microscope. The most prominent feature of Rana pipiens lens cells was a primary cilium close to the apical surface of the cells above the centrosome. This structure was associated with microtubules rich in modified alpha-tubulin. The cilium was less pronounced but still discernible in the cells of another species R. ridibunda. In both species, the modified (acetylated or detyrosinated) microtubules formed arrays spatially distinct from the unmodified (tyrosinated) microtubules. The modified microtubules formed a basket of microtubules with a curly distribution around the nucleus while the tyrosinated array consisted predominantly of rather straighter microtubules running from the apical centrosome to the cell periphery, down the lateral sides of the cells and across the basal surface adjacent to the lens capsule and basement membrane. It is concluded that the organization of modified microtubules previously described for several types of cultured cells may represent a remnant of the three-dimensional perinuclear array of such microtubules described here for the cells of an intact epithelium.

Definition of the sequence requirements for binding of the EBNA-1 protein to its palindromic target sites in Epstein-Barr virus DNA.

Interaction between the trans-acting DNA-binding protein EBNA-1 and cis-acting sequences in the ori-P region of Epstein-Barr virus DNA is required for maintenance of the viral plasmid state in latently infected B cells and is involved in the regulation of transcription during latency. In the Epstein-Barr virus genome, a total of 26 EBNA-1-binding sites occur within three clustered loci referred to as the family of repeats and dyad symmetry locus of ori-P and the separate BamHI-Q locus. Incubation of a bacterially expressed carboxy-terminal domain of EBNA-1 (28,000-molecular-weight EBNA-1 [28K EBNA-1]) with synthetic monomer and dimer consensus binding sites gave characteristic DNA-protein complexes in a mobility retardation assay. A similar approach with the naturally occurring Q locus confirmed that it contains two distinct but low-affinity binding sites. We then examined the precise sequence requirements for EBNA-1 binding, using a set of 30-base-pair oligonucleotides designed to contain symmetric point mutations within both halves of the palindromic target site. Analysis of all possible single substitutions between positions 1 and 10 in the consensus half-palindrome sequence revealed that positions 9 and 10 did not contribute to EBNA-1 binding and that considerable flexibility could be tolerated at positions 1 and 2. Positions 3 through 8 of the recognition site had the most stringent requirements, with transversions at these positions either reducing or eliminating binding. The relative spacing of the halves of the palindrome was also critical, since the addition or removal of 2 base pairs at the center of the sequence abolished binding. Similar results were obtained when a partially purified preparation of intact Raji EBNA-1 was substituted for the 28K EBNA-1, and the results were further supported by methylation interference studies which indicated contact points between EBNA-1 and the guanine residues at positions -8, -7, and +3 of the binding site. The three naturally occurring EBNA-1-binding loci have previously been shown to differ in their relative affinities for EBNA-1. The present study indicates that the sequence variations occurring within the family of repeats would not affect binding affinity, whereas certain base substitutions within the Q and dyad symmetry sites would be predicted to contribute to the observed lower affinities of these sites. An apparent Kd of 1.5 x 10(-11) M for binding of 28K EBNA-1 to a consensus recognition site was calculated from Scatchard analysis.

Nucleus-associated microtubules help determine the division plane of plant epidermal cells: avoidance of four-way junctions and the role of cell geometry.

To investigate the spatial relationship between the nucleus and the cortical division site, epidermal cells were selected in which the separation between these two areas is large. Avoiding enzyme treatment and air drying, Datura stramonium cells were labeled with antitubulin antibodies and the three-dimensional aspect of the cytoskeletons was reconstructed using computer-aided optical sectioning. In vacuolated cells preparing for division, the nucleus migrates into the center of the cell, suspended by transvacuolar strands. These strands are now shown to contain continuous bundles of microtubules which bridge the nucleus to the cortex. These nucleus-radiating microtubules adopt different configurations in cells of different shape. In elongated cells with more or less parallel side walls, oblique strands radiating from the nucleus to the long side walls are presumably unstable, for they are progressively realigned into a transverse disc (the phragmosome) as broad, cortical, preprophase bands (PPBs) become tighter. The phragmosome and the PPB are both known predictors of the division plane and our observations indicate that they align simultaneously in elongated epidermal cells. These observations suggest another hypothesis: that the PPB may contain microtubules polymerized from the nuclear surface. In elongated cells, the majority of the radiating microtubules, therefore, come to anchor the nucleus in the transverse plane, consistent with the observed tendency of such cells to divide perpendicular to the long axis. In nonrectangular isodiametric epidermal cells, which approximate regular hexagons in section, the radial microtubular strands emanating from the nucleus tend to remain associated with the middle of each subtending cell wall. The strands are not reorganized into a single dominant transverse bar, but remain as a starlike array until mitosis. PPBs in these cells are not as tight; they may only be a sparse accumulation of microtubules, even forming along non-diametrical radii. This arrangement is consistent with the irregular division patterns observed in epidermal mosaics of isodiametric D. stramonium cells. The various conformations of the radial strands can be modeled by springs held in two-dimensional hexagonal frames, and by soap bubbles in three-dimensional hexagonal frames, suggesting that the division plane may, by analogy, be selected by minimal path criteria. Such behavior offers a cytoplasmic explanation of long-standing empirically derived "rules" which state that the new cell wall tends to meet the maternal wall at right angles. The radial premitotic strands and their analogues avoid taking the longer path to the vertex of an angle where a cross wall is already present between neighboring cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Three-dimensional localisation of DNA in the nucleolus of Spirogyra by correlated optical tomography and serial ultra-thin sectioning.

Spirogyra nucleoli were shown by three-dimensional optical microscopy of DAPI fluorescence to contain DNA with a pattern and distribution matching those of the fibrillar centres. This was confirmed using different species with nucleoli showing different sizes of fibrillar centre. Much lower levels of fluorescence were seen corresponding to the dense fibrillar component. Nearly all the DAPI fluorescence arises from the fibrillar centres or from regions very close to their surface, indicating that this is the site of nucleolar transcription.

Localization of ribosomal and telomeric DNA sequences in intact plant nuclei by in-situ hybridization and three-dimensional optical microscopy.

We have combined the use of three-dimensional (3-D) fluorescence microscopy and computer image processing of images with in-situ hybridization to analyse the 3-D organization of interphase nuclei in plants. In sections of root tips of Pisum sativum, using cDNA probes, we have shown that telomeres are arranged around the nuclear periphery and that the ribosomal genes in this species appear to exist in discrete, 3-D domains.

The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements.

A 600-base-pair (bp) enhancer region upstream from the major IE94 gene of simian cytomegalovirus (SCMV) produces very strong basal expression of associated gene products. This domain consists of multiple sets of interspersed repetitive elements, including 11 copies of a conserved 16-bp palindromic sequence with the consensus CCATTGACGTCAATGG. These series I repeats contain an 8-bp core TGACGTCA that resembles the cyclic AMP (cAMP) response element (CRE) of cellular genes. In transient chloramphenicol acetyltransferase assays in K562 human erythroleukemia cells, a set of deleted variants of the IE94 promoter all responded up to 15-fold to induction by cAMP. However, successive removal of most of the SCMV 16-bp motifs reduced basal expression over 20-fold. The cAMP stimulation was also manifested at the steady-state RNA level after SCMV infection of K562 cells and was detectable within 1.5 h after treatment of DNA-transfected cells. Addition of a single 30-bp oligonucleotide encompassing the 16-bp palindrome conveyed up to 10-fold cAMP responsiveness onto a heterologous weak promoter but had no effect on basal expression. In contrast, two or more adjacent copies produced 20- to 40-fold increases in basal expression and provided greater than 200-fold activation in the presence of cAMP. Similar effects were obtained when the oligonucleotides were placed in a downstream location relative to the reporter gene. Studies with mutant oligonucleotides revealed that both the core CRE and the flanking sequence portions of the 16-bp elements were essential for enhancer function. Both components were also important for maximum cAMP responsiveness. Band shift assays with fractionated nuclear extracts from Raji lymphocytes revealed multiple competable complexes with cellular DNA-binding factors that recognized the series I elements. Three distinct CREB-like factors were detected that required only the core 8-bp elements for binding. We conclude that the 16-bp series I repeats provide a major contribution to the constitutive enhancer properties of the IE94 promoter and also act as functional CREs. The cAMP response properties appear likely to play a key role in reactivation of the virus from a latent state in appropriately differentiating cell types.

Interaction of the lymphocyte-derived Epstein-Barr virus nuclear antigen EBNA-1 with its DNA-binding sites.

The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 plays an integral role in the maintenance of latency in EBV-infected B lymphocytes. EBNA-1 binds to sequences within the plasmid origin of replication (oriP). It is essential for the replication of the latent episomal form of EBV DNA and may also regulate the expression of the EBNA group of latency gene products. We have used sequence-specific DNA-binding assays to purify EBNA-1 away from nonspecific DNA-binding proteins in a B-lymphocyte cell extract. The availability of this eucaryotic protein has allowed an examination of the interaction of EBNA-1 with its specific DNA-binding sites and an evaluation of possible roles for the different binding loci within the EBV genome. DNA filter binding assays and DNase I footprinting experiments showed that the intact Raji EBNA-1 protein recognized the two binding site loci in oriP and the BamHI-Q locus and no other sites in the EBV genome. Competition filter binding experiments with monomer and multimer region I consensus binding sites indicated that cooperative interactions between binding sites have relatively little impact on EBNA-1 binding to region I. An analysis of the binding parameters of the Raji EBNA-1 to the three naturally occurring binding loci revealed that the affinity of EBNA-1 for the three loci differed. The affinity for the sites in region I of oriP was greater than the affinity for the dyad symmetry sites (region II) of oriP, while the physically distant region III locus showed the lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can mediate differing regulatory functions through differential binding to its recognition sequence.