Cis-acting elements and developmental regulators govern toxin gene expression in Bacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals such as elevated CO2 , and the trans-acting positive regulator, AtxA. No specific binding of AtxA to the toxin gene promoters has been demonstrated and no sequence-based similarities are apparent in the promoter regions of toxin genes. We hypothesized that the toxin genes possess common structural features that are required for positive regulation. To test this hypothesis, I performed an extensive characterization of the toxin gene promoters. I determined the minimal sequences required for atxA-mediated toxin gene expression and compared these sequences for structural similarities. In silico modeling and in vitro experiments indicated significant curvature within these regions. Random mutagenesis revealed that point mutations associated with reduced transcriptional activity, mostly mapped to areas of high curvature. This work enabled the identification of two potential cis-acting elements implicated in AtxA-mediated regulation of the toxin genes. In addition to the growth condition requirements and AtxA, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. Here I report that toxin gene expression also requires sigH, a gene encoding the RNA polymerase sigma factor associated with development in B. subtilis. In the well-studied B. subtilis system, σH is part of a feedback control pathway that involves AbrB and the major response regulator of sporulation initiation, Spo0A. My data indicate that in B. anthracis, regulatory relationships exist between these developmental regulators and atxA . Interestingly, during growth in toxin-inducing conditions, sigH and abrB expression deviates from that described for B. subtilis, affecting expression of the atxA gene. These findings, combined with previous observations, suggest that the steady state level of atxA expression is critical for optimal toxin gene transcription. I propose a model whereby, under toxin-inducing conditions, control of toxin gene expression is fine-tuned by the independent effects of the developmental regulators on the expression of atxA . The growth condition-dependent changes in expression of these regulators may be crucial for the correct timing and uninterrupted expression of the toxin genes during infection. ^

{\it cis\/} -acting elements and {\it trans\/} -acting factors that control serum amyloid A gene expression during inflammation

To understand how the serum amyloid A (SAA) genes are regulated, the cis-acting elements and trans-acting factors involved in the regulation of mouse SAA3 and rat SAA1 genes expression during inflammation were analyzed.^ To identify DNA sequences involved in the liver-specific expression of the mouse SAA3 gene, the 5$\sp\prime$ flanking region of this gene was analyzed by transient transfection studies. Results suggest that C/EBP, a liver-enriched transcription factor, plays an important role for the enhanced expression of the mouse SAA3 gene in hepatocytes.^ Transfection studies of the regulation of the expression of rat SAA1 gene indicated that a 322 bp fragment ($-$304 to +18) of the gene contains sufficient information for cytokine-induced expression of the reporter gene in a liver cell-specific manner. Further functional analysis of the 5$\sp\prime$ flanking region of the rat SAA1 gene demonstrated that a 65 bp DNA fragment ($-$138/$-$73) can confer cytokine-inducibility onto a heterologous promoter both in liver and nonliver cells. DNase I footprint and gel retardation assays identified five putative cis-regulatory elements within the 5$\sp\prime$ flanking region of the gene: one inducible element, a NF$\kappa$B binding site and four constitutive elements. Two constitutive elements, footprint regions I and III, were identified as C/EBP binding sites with region III having over a 10-fold higher affinity for C/EBP binding than region I. Functional analysis of the cis-elements indicated that C/EBP(I) and C/EBP(III) confer liver cell-specific activation onto a heterologous promoter, while sequences corresponding to the NF$\kappa$B element and C/EBP(I) impart cytokine responsiveness onto the heterologous promoter. These results suggest that C/EBP(I) possesses two functions: liver-specific activation and cytokine responsiveness. The identification of two cytokine responsive elements (NF$\kappa$B and C/EBP(I)), and two liver-specific elements (C/EBP(I) and C/EBP(III)) implies that multiple cis-acting elements are involved in the regulation of the expression of the rat SAA1 gene. The tissue-specific and cytokine-induced expression of rat SAA1 gene is likely the result of the interactions of these cis-acting elements with their cognate trans-acting factors as well as the interplay between the different cis-acting elements and their binding factors. (Abstract shortened with permission of author.) ^

Characterization of the MLC box: A conserved {\it cis\/}-acting DNA element present in the chicken MLC1s/1c gene

The slow/cardiac alkali myosin light chain (MLC1s/1c) is a member of a multigene family whose protein products are essential for activation of the myosin ATPase. In the adult, the MLC1s/1c isoform is expressed in both cardiac and slow-twitch skeletal muscles, while it is expressed by all skeletal muscles during development.^ To elucidate the molecular mechanisms that underlie the transcriptional regulation of MLC1s/1c gene expression, the immediate 5$\sp\prime$ flanking region of the gene was isolated and shown to be capable of directing reporter gene expression. Analysis of this region revealed a 110 bp muscle-specific enhancer that includes a myocyte-specific enhancer-binding factor 2 (MEF-2) site, E-boxes, which are potential binding sites for the basic-helix-loop-helix proteins such as MyoD, and a MLC box. The focus of the thesis was to identify the role of the MLC box in expression of the MLC1s/1c gene.^ The MLC box is a member of the family of CArG box containing cis-acting DNA elements. Mutagenesis showed that the MLC box is necessary, but not sufficient, for the expression of a reporter gene linked to the 5$\sp\prime$ flanking region of the MLC1s/1c gene. Linker scanner and site-directed mutagenesis identified a number of potential sites within the 110 bp muscle-specific enhancer that may cooperate with the MLC box. These are the MEF-2 site, the E-box site, and a 10 bp element located upstream of the MEF-2 site that does not have sequence similarity with any known cis-acting element. The MLC box is capable of binding to factors present in muscle nuclear extracts, as well as to human recombinant serum response factor (SRF). Binding of SRF to the MLC box was correlated with the ability of the 5$\sp\prime$ flanking region of the MLC1s/1c gene to drive reporter gene expression. Results suggest a model in which binding of SRF to the MLC box activates expression of the MLC1s/1c gene while binding of the factors present in the nuclear extracts suppresses the expression of the gene. (Abstract shortened with permission of author.) ^

{\it cis\/} -acting determinants in the control of MuSVts110 RNA splicing

Like other simple retroviruses the murine sarcoma virus ts110 (MuSVts110) displays an inefficient mode of genome splicing. But, unlike the splicing phenotypic of other retroviruses, the splicing event effected upon the transcript of MuSVts110 is temperature sensitive. Previous work in this laboratory has established that the conditionally defective nature of MuSVts110 RNA splicing is mediated in cis by features in the viral transcript. Here we show that the 5$\sp\prime$ splice site of the MuSVts110 transcript acts as a point of control of the overall splicing efficiency at both permissive and nonpermissive temperatures for splicing. We strengthened and simultaneously weakened the nucleotide structure of the 5$\sp\prime$ splice site in an attempt to elucidate the differential effects each of the two known critical splicing components which interact with the 5$\sp\prime$ splice site have on the overall efficiency of intron excision. We found that a transversion of the sixth nucleotide, resulting in the formation of a near-consensus 5$\sp\prime$ splice site, dramatically increased the overall efficiency of MuSVts110 RNA splicing and abrogated the thermosensitive nature of this splicing event. Various secondary mutations within this original transversion mutant, designed to selectively decrease specific splicing component interactions, lead to recovery of inefficient and thermosensitive splicing. We have further shown that a sequence of 415 nucleotides lying in the downstream exon of the viral RNA and hypothesized to act as an element in the temperature-dependent inhibition of splicing displays a functional redundancy throughout its length; loss and/or replacement of any one sequence of 100 nucleotides within this sequence does not, with one exception detailed below, diminish the degree to which MuSVts110 RNA is inhibited to splice at the restrictive temperature. One specific deletion, though, fortuitously juxtaposed and activated cryptic consensus splicing signals for the excision of a cryptic intron within the downstream exon and markedly potentiated--across a newly defined cryptic exon--the splicing event effected upon the upstream, native intron. We have exploited this mutant of MuSVts110 to further an understanding of the process of exon definition and intron definition and show that the polypyrimidine tract and consensus 3$\sp\prime$ splice site, as well as the 5$\sp\prime$ splice site, within the intron at the 3$\sp\prime$ flank of the defined exon are required for the exon's definition; implying that definition of the downstream intron is required for the in vivo definition of the proximal, upstream exon. Finally; we have shown, through the construction of heterologous mutants of MuSVts110 employing a foreign 3$\sp\prime$ end-forming sequence, that efficiency of transcript splicing can be increased--to a degree which abrogates its thermosensitive nature--in direct proportion to increasing proximity of the 3$\sp\prime$ end-forming signal to the terminal 3$\sp\prime$ splice site. ^

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^

Evolution of cis-regulatory elements in a repetitive sequence adjacent to a sea urchin aboral ectoderm -specific gene

The creation, preservation, and degeneration of cis-regulatory elements controlling developmental gene expression are fundamental genome-level evolutionary processes about which little is known. In this study, critical differences in cis-regulatory elements controlling the expression of the sea urchin aboral ectoderm-specific spec genes were identified and explored. In genomes of species within the Strongylocentrotidae family, multiple copies of a repetitive sequence element termed RSR were present, but RSRs were not detected in genomes of species outside Strongylocentrotidae. RSRs are invariably associated with spec genes, and in Strongylocentrotus purpuratus, the spec2a RSR functioned as a transcriptional enhancer displaying greater activity than RSRs from the spec1 or spec2c paralogs. Single base-pair differences at two cis-regulatory elements within the spec2a RSR greatly increased the binding affinities of four transcription factors: SpCCAAT-binding factor at one element and SpOtx, SpGoosecoid, and SpGATA-E at another. The cis-regulatory elements to which SpCCAAT-binding factor, SpOtx, SpGoosecoid, and SpGATA-E bound were recent evolutionary acquisitions that could act either to activate or repress transcription, depending on the cell type. These elements were found in the spec2a RSR ortholog in Strongylocentrotus pallidus but not in the RSR orthologs of Strongylocentrotus droebachiensis or Hemicentrotus pulcherrimus. These results indicate that spec genes exhibit a dynamic pattern of cis-regulatory element evolution while stabilizing selection preserves their aboral ectoderm expression domain. ^

Concentrations of metals and POPs in liver tissue of wild sea bird chicks from the Norwegian arctic region

Lysosomal membrane stability, lipofuscin (LF), malondialdehyde (MDA), neutral lipid (NL) levels, as well as halogenated organic compounds (HOCs), Cr, Cd, Pb and Fe concentrations were analyzed in liver of black-legged kittiwake (BK), herring gull (HG), and northern fulmar (NF) chicks. There were significant species differences in the levels of NL, LF and lysosomal membrane stability. These parameters were not associated with the respective HOC concentrations. LF accumulation was associated with increasing Cr, Cd and Pb concentrations. HG presented the lowest lysosomal membrane stability and the highest. LF and NL levels, which indicated impaired lysosomes in HG compared to NF and BK. Lipid peroxidation was associated with HOC and Fe2+ levels. Specific HOCs showed positive and significant correlations with MDA levels in HG. The study indicates that contaminant exposure can affect lysosomal and lipid associated parameters in seabird chicks even at low exposure levels. These parameters may be suitable markers of contaminant induced stress in arctic seabirds.

C1-C8 hydrocarbons in DSDP Leg 64 Holes sediments

Sediments from the Baja California Continental Margin Transect - Sites 474 and 476 - showed small amounts of C2-C8 hydrocarbons and functionalized compounds (alkenes) typical of organic-rich, Recent, cold (<30°C) marine sediments. In contrast, some samples from Sites 477, 478, 479, and Hole 481A in the Guaymas Basin, an active spreading center, showed the characteristics of thermally generated hydrocarbons. These include an increase (sometimes exponential) in amount and diversity of C2-C8 hydrocarbons and a decrease in alkenes in more thermally mature sediments. The results indicate that the injection of basaltic sills has minimal effect on C2-C8 hydrocarbon generation except in the immediate vicinity of the sill. The absence of light hydrocarbons close to the hottest sills suggests that the compounds distill away as they are formed in these areas of very active hydrothermal circulation. A sample of young sediment exposed to very high temperatures (>300°C) from deeper thermal sources at the hottest site, 477, showed a very limited hydrocarbon distribution, including primarily ethane, benzene, and toluene, together with smaller amounts of propane and butane.

Migration of C1 to C8 volatile organic compounds in sediments at DSDP Hole 75-530A

The distribution of C1 to C8 hydrocarbons in sediment samples from DSDP Leg 75, Hole 530A, indicates that significant amounts of methane and ethane have migrated from organic-rich to organic-lean shales in close proximity. Most compounds larger than ethane are not migrating out of black shales, where they occur in high concentrations. These results lead to a general model for assessing migration. In addition, three shale types are identified on the basis of organic carbon and pyrolysis products and patterns.

Organic carbon, age, lithology, and CPI-alkenes at DSDP Sites 57-440 and 56-436

"Bound" and "free" solvent-extractable lipids have been examined from Sections 440A-7-6, 440B-3-5, 440B-8-4, 440B-68-2, and 436-11-4. The compound classes studied include aliphatic and aromatic hydrocarbons, ketones, alcohols, and carboxylic acids. Carotenoids and humic acids have also been examined. The quantitative results are considered in terms of input indicators, diagenesis parameters, and structural classes. A difference in input is deduced across the Japan Trench, with a higher proportion of autochthonous components on the western inner trench slope compared with the more easterly, outer trench, wall and greater input in the early Pleistocene than in the Miocene. A variety of diagenetic transformations is observed at Site 440 as sample depth increases. Results are compared with those of samples from Atlantic Cretaceous sediments and from the Walvis Bay high productivity area.

C1-C7 volatile organic compounds in sediments at DSDP Legs 56-57 Holes

Volatile C1-C7 components in sediments were examined for Japan Trench DSDP Sites 438, 439, 435, 440, 434 and 436, proceeding from west to east. Levels of all components are lowest in the highly fractured sediments of Sites 440 and 434. A number of alkenes, furans, and sulfur compounds were detected in concentrations higher than noted in any other DSDP sediments examined to date. The types, amounts, and specificity of occurrence are similar to those for 1-meter gravity cores we have examined which bear a significant biological imprint. Site 436 shows high levels of saturated and aromatic hydrocarbons, as well as olefins, including traces of dimethycyclopentanes and the highest level of cyclohexene detected in any DSDP sediment we have examined to date. The results from Site 436 were unexpected, considering the low organic-carbon content, absence of biogenic methane, and evidence of an aerobic depositional environment at this site.