Statistical methodology for the identification and characterization of protein binding sites in a set of unaligned DNA fragments is presented. Each sequence must contain at least one common site. No alignment of the sites is required. Instead, the uncertainty in the location of the sites is handled by employing the missing information principle to develop an "expectation maximization" (EM) algorithm. This approach allows for the simultaneous identification of the sites and characterization of the binding motifs. The reliability of the algorithm increases with the number of fragments, but the computations increase only linearly. The method is illustrated with an example, using known cyclic adenosine monophosphate receptor protein (CRP) binding sites. The final motif is utilized in a search for undiscovered CRP binding sites.

It is generally accepted that cellular, but not humoral immunity, plays an important role in host defense against intracellular bacteria. However, studies of some of these pathogens have provided evidence that antibodies can provide immunity if present during the initiation of infection. Here, we examined immunity against infection by Ehrlichia chaffeensis, an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Studies with mice have demonstrated that immunocompetent strains are resistant to persistent infection but that SCID mice become persistently and fatally infected. Transfer of immune serum or antibodies obtained from immunocompetent C57BL/6 mice to C57BL/6 scid mice provided significant although transient protection from infection. Bacterial clearance was observed when administration occurred at the time of inoculation or well after infection was established. The effect was dose dependent, occurred within 2 days, and persisted for as long as 2 weeks. Weekly serum administration prolonged the survival of susceptible mice. Although cellular immunity is required for complete bacterial clearance, the data show that antibodies can play a significant role in the elimination of this obligate intracellular bacterium during active infection and thus challenge the paradigm that humoral responses are unimportant for immunity to such organisms.

NADPH-cytochrome P450 reductase (CPR) is an essential component for the function of many enzymes, including microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. In liver-Cpr-null (with liver-specific Cpr deletion) and Cpr-low (with reduced CPR expression in all organs examined) mouse models, a reduced serum cholesterol level and an induction of hepatic P450s were observed, whereas hepatomegaly and fatty liver were only observed in the liver-Cpr-null model. Our goal was to identify hepatic gene expression changes related to these phenotypes. Cpr-lox mice (with a floxed Cpr gene and normal CPR expression) were used as the control. Through microarray analysis, we identified many genes that were differentially expressed among the three groups of mice. We also recognized the 12 gene ontology terms that contained the most significantly changed gene expression in at least one of the two mouse models. We further uncovered potential mechanisms, such as an increased activation of constitutive androstane receptor and a decreased activation of peroxisomal proliferatoractivated receptor-␣ by precursors of cholesterol biosynthesis, that underlie common changes (e.g. induction of multiple P450s and suppression of genes for fatty acid metabolism) in response to CPR loss in the two mouse models. Additionally, we observed model-specific gene expression changes, such as the induction of a fatty-acid translocase (Cd36 antigen) and the suppression of carnitine O-palmitoyltransferase 1 (Cpt1a) and acyl-CoA synthetase long chain family member 1 (Acsl1), that are potentially responsible for the severe hepatic lipidosis and an altered fatty acid profile observed in liver-Cprnull mice.

c Escherichia coli AraC is a well-described transcription activator of genes involved in arabinose metabolism. Using complementary genomic approaches, chromatin immunoprecipitation (ChIP)-chip, and transcription profiling, we identify direct regulatory targets of AraC, including five novel target genes: ytfQ, ydeN, ydeM, ygeA, and polB. Strikingly, only ytfQ has an established connection to arabinose metabolism, suggesting that AraC has a broader function than previously described. We demonstrate arabinose-dependent repression of ydeNM by AraC, in contrast to the well-described arabinose-dependent activation of other target genes. We also demonstrate unexpected read-through of transcription at the Rho-independent terminators downstream of araD and araE, leading to significant increases in the expression of polB and ygeA, respectively. AraC is highly conserved in the related species Salmonella enterica. We use ChIP sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to map the AraC regulon in S. enterica. A comparison of the E. coli and S. enterica AraC regulons, coupled with a bioinformatic analysis of other related species, reveals a conserved regulatory network across the family Enterobacteriaceae comprised of 10 genes associated with arabinose transport and metabolism.

Although often considered to be ineffective against intracellular bacteria, Abs, in the absence of lymphocytes, have been shown previously to protect SCID mice from lethal infection by the obligate intracellular bacterium Ehrlichia chaffeensis, even when administered well after infection has been established. To identify characteristics of Abs that are critical for host defense during this intracellular infection, a panel of Ehrlichia-specific mAbs was generated and analyzed. Among 100 Abs recovered, 39 recognized an amino-terminal hypervariable region of an outer membrane protein (OMP), demonstrating that the OMPs are both antigenically variable and immunodominant. A subset of 16 representative OMP-specific Abs was further examined to identify characteristics that were essential for in vivo efficacy. The highly effective Abs recognized a linear epitope within the first hypervariable region of OMP-1g. Only IgG were found to be effective, and among the effective IgG, the following hierarchy was observed: IgG2a > IgG3 = IgG2b. The most striking characteristics of the highly effective Abs were their picomolar binding affinities and long binding t1/2. Thus, although epitope recognition and isotype use may contribute to efficacy, high affinity may be a critical characteristic of Abs that can act effectively during this intracellular bacterial infection.

As previously shown, 11 loci are required to complement human cytomegalovirus (HCMV) DNA replication in a transient-transfection assay (G. S. Pari and D. G. Anders, J. Virol. 67:6979-6988, 1993). Six of these loci encode known or candidate replication fork proteins, as judged by sequence and biochemical similarities to herpes simplex virus homologs of known function; three encode known immediate early regulatory proteins (UL36-38, IRS1/TRS1, and the major immediate early region spanning UL122-123); and two encode early, nucleus-localized proteins of unknown functions (UL84 and UL112-113). We speculated that proteins of the latter five loci might cooperate to promote and regulate expression of the six replication fork proteins. To test this hypothesis we made luciferase reporter plasmids for each of the replication fork gene promoters and measured their activation by the candidate effectors, expressed under the control of their respective native promoters, using a transient-cooperativity assay in which the candidate effectors were subtracted individually from a transfection mixture containing all five loci. The combination of UL36-38, UL112-113, IRS1, or TRS1 and the major immediate early region produced as much as 100-fold-higher expression than the major immediate early region alone; omitting any one of these four loci from complementing mixtures produced a significant reduction in expression. In contrast, omitting UL84 had insignificant (less than twofold), promoterdependent effects on reporter activity, and these data do not implicate UL84 in regulating HCMV early-gene expression. Most of the effector interactions showed significant positive cooperativity, producing synergistic enhancement of expression. Similar responses to these effectors were observed for the each of the promoters controlling expression of replication fork proteins. However, subtracting UL112-113 had little if any effect on expression by the UL112-113 promoter or by the simian virus 40 promoter-enhancer under the same conditions. Several lines of evidence argue that the cooperative interactions observed in our transient-transfection assays are important to viral replication in permissive cells. Therefore, the data suggest a model in which coordinate expression of multiple essential replication proteins during permissive infection is vitally dependent upon the cooperative regulatory interactions of proteins encoded by multiple loci and thus have broad implications for our understanding of HCMV biology.

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Glutamate-cysteine ligase (GCL) is the first and rate-limiting enzyme involved in the biosynthesis of glutathione (GSH). The GCL heterodimer is encoded by two genes: GLCLC, which directs synthesis of the catalytic subunit, and GLCLR, which encodes the regulatory subunit. We have previously identified a polymorphic GAG/CTC trinucleotide repeat within the 5' untranslated region of GLCLC. Here we report the further characterization of GLCLC polymorphism and the existence of five GLCLC alleles as defined by the trinucleotide repeat, which exhibits a range of 4 to 10 uninterrupted repeats. Significant variation in GLCLC allele frequencies was observed in four different ethnic populations examined. Interindividual variation in the capacity to produce GSH due to GLCLC polymorphism is hypothesized to influence the cellular response to environmental toxicants and chemotherapeutic agents. To test this hypothesis, the 60 tumor cell lines of the National Cancer Institute drug screening panel were genotyped for the GLCLC trinucleotide repeat, and the association of GLCLC genotype with GSH levels and drug sensitivity/resistance data was examined. Here we demonstrate an association between certain GLCLC alleles and GSH levels and/or drug sensitivity, providing evidence that suggests polymorphism of human GLCLC is functionally significant.