Positive regulation of the Vibrio cholerae porin OmpT by iron and fur.

The transcription factor Fur regulates the expression of a number of genes in Vibrio cholerae in response to changes in the level of available iron. Fur usually acts as a repressor, but here we show that Fur positively regulates the expression of ompT, which encodes a major outer membrane porin. OmpT levels increased when the bacteria were grown in medium containing relatively high levels of iron, and this effect required Fur. The level of ompT mRNA also is increased in the presence of iron and Fur. The effect of iron on OmpT levels was independent of the known ompT regulators ToxR and Crp, and it did not require RyhB, which has been shown to be responsible for positive regulation by iron of some V. cholerae genes. Electrophoretic mobility shift assays showed that Fur binds upstream of the ompT transcription start site in a region overlapping known binding sites for ToxR and Crp. These data suggest that Fur and iron positively regulate ompT expression through the direct binding of Fur to the ompT promoter.

Distribution of mRNAs encoding the arylhydrocarbon receptor, arylhydrocarbon receptor nuclear translocator, and arylhydrocarbon receptor nuclear translocator-2 in the rat brain and brainstem.

Dioxin exposure alters a variety of neural functions, most likely through activation of the arylhydrocarbon receptor (AhR) pathway. Many of the adverse effects, including disruption of circadian changes in hormone release and depressed appetite, seem to be mediated by hypothalamic and/or brainstem neurons. However, it is unclear whether these effects are direct or indirect, because there have been no comprehensive studies mapping the expression of components of the AhR pathway in the brain. Therefore, we used a sensitive in situ hybridization histochemical (ISHH) method to map the neural expression of AhR mRNA, as well as those of the mRNAs encoding the AhR dimerization partners, arylhydrocarbon receptor nuclear translocator (ARNT) and ARNT2. We found that AhR, ARNT, and ARNT2 mRNAs were widely distributed throughout the brain and brainstem. There was no neuroanatomic evidence that AhR is preferentially colocalized with ARNT or ARNT2. However, ARNT2, unlike ARNT expression, was relatively high in most regions. The most noteworthy regions in which we found AhR, ARNT, and ARNT2 mRNA were several hypothalamic and brainstem regions involved in the regulation of appetite and circadian rhythms, functions that are disrupted by dioxin exposure. These regions included the arcuate nucleus (Arc), ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), nucleus of the solitary tract (NTS), and the dorsal and median raphe nuclei. This neuroanatomic information provides important clues as to the sites and mechanisms underlying the previously unexplained effects of dioxins in the central nervous system.

Detection of estrogen receptor-beta messenger ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain.

Luteinizing hormone-releasing hormone (LHRH) neurons of the forebrain play a pivotal role in the neuroendocrine control of reproduction. Although serum estrogen levels influence many aspects of LHRH neuronal activity in the female, earlier studies were unable to detect estrogen receptors (ERs) within LHRH neurons, thus shaping a consensus view that the effects of estradiol on the LHRH neuronal system are mediated by interneurons and/or the glial matrix. The present studies used dual-label in situ hybridization histochemistry (ISHH) and combined LHRH-immunocytochemistry/125I-estrogen binding to readdress the estrogen-receptivity of LHRH neurons in the female rat. In ISHH experiments we found that the majority of LHRH neurons exhibited hybridization signal for the "beta" form of ER (ER-beta). The degree of colocalization was similar in topographically distinct populations of LHRH neurons and was not significantly altered by estradiol (67.2+/-1.8% in ovariectomized and 73.8+/-4.2% in ovariectomized and estradiol-treated rats). In contrast, the mRNA encoding the classical ER-alpha could not be detected within LHRH neurons. In addition, in vivo binding studies using 125I-estrogen revealed a subset of LHRH-immunoreactive neurons (8.8%) which accumulated the radioligand thus providing evidence for the translation of ER protein(s) within these cells. The findings that most LHRH neurons in the female rat express ER-beta mRNA and at least some are capable of binding 125I-estrogen challenge the current opinion that estrogen does not exert direct effects upon the LHRH neuronal system.

Analysis of cis-acting sequences involved in plus-strand synthesis of a turnip crinkle virus-associated satellite RNA identifies a new carmovirus replication element.

Satellite RNA C (satC) is a 356-base subviral RNA associated with turnip crinkle virus (TCV). A 3'-proximal element (3'-UCCCAAAGUAU) located 11 bases from the 3' terminus of satC minus strands can function as an independent promoter in an in vitro RNA-dependent RNA polymerase (RdRp) transcription system. Furthermore, in the absence of a 5'-proximal element, the 3'-proximal element is required for complementary strand synthesis in vitro. Site-directed mutagenesis was conducted to investigate the functional significance of this element and the 3' minus-strand terminal sequence "3'-OH-CCCUAU," which contains the minus-strand 3'-end sequence "3'-OH-CC(1-2)(A/U)(A/U)(A/U)" found in all carmovirus RNAs. Single mutations in the 3'-terminal sequence, which we have named the carmovirus consensus sequence (CCS), suppressed satC plus-strand synthesis to undetectable levels in protoplasts while still permitting some minus-strand synthesis. However, single and multiple mutations introduced into the 3'-proximal element had little or no effect on satC accumulation in protoplasts. In vivo genetic selection (SELEX) of the minus-strand 3'-terminal 21 bases revealed that all satC species accumulating in plants contained the 3' CCS. In addition, the 3'-proximal element preferentially contained a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to accumulation of satC in vivo.

Requirement of a 5'-proximal linear sequence on minus strands for plus-strand synthesis of a satellite RNA associated with turnip crinkle virus.

Viral RNA replication begins with specific recognition of cis-acting RNA elements by the viral RNA-dependent RNA polymerase (RdRp) and/or associated host factors. A short RNA element (3'-AACCCCUGGGAGGC) located 41 bases from the 5' end of minus strands of satellite RNA C (satC), a 356-base subviral RNA naturally associated with turnip crinkle virus (TCV), was previously identified as important for plus-strand synthesis using an in vitro RdRp assay (H. Guan, C. Song, A. E. Simon, 1997, RNA 3, 1401-1412). To examine the functional significance of this element in RNA replication, mutations were introduced into the consecutive C residues in the element. A single mutation of the 3'-most C residue resulted in undetectable levels of satC plus strands when transcripts were assayed in protoplasts and suppressed transcription directed by the element in vitro. However, satC minus strands were detectable at 6 h postinoculation (hpi) of protoplasts, accumulating to about 10% of wild-type levels at 24 hpi. This mutation, when in the plus-sense orientation, had little or no effect on minus-strand synthesis from full-length satC plus strands in vitro, suggesting that the 5'-proximal RNA element is required for satC plus-strand synthesis. In addition, in vivo genetic selection revealed a strict requirement for 10 of the 14 nucleotides of the element, indicating that the primary sequence is essential for RNA accumulation.

Minimal sequence and structural requirements of a subgenomic RNA promoter for turnip crinkle virus.

Infection of plants or protoplasts with turnip crinkle virus (TCV) results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs of 1.7 kb and 1.45 kb, respectively. Both of the sgRNA promoters were characterized previously and their secondary structures predicted by computer analysis [J. Wang and A. E. Simon (1997). Virology 232, 174-186]. Secondary structure-sensitive chemical and enzymatic probes have now been used to determine the structure of the promoter directing synthesis of the 1.45-kb sgRNA, namely the 1.45-kb sgRNA promoter, in solution. The newly obtained structure conforms with the previously predicted hairpin structure except for the hairpin base: four CG base pairs and a CA bulge are present instead of an A bulge. Studies of deletions within the 96-nucleotide (nt) 1.45-kb sgRNA promoter defined a minimal 30-nt core sequence as essential for promoter activity: a 21-nt hairpin and a 9-nt flanking single-stranded sequence. Mutational analysis in the stem section of the core promoter supported a role for the primary sequence and secondary structure in promoter activity. Sequence alterations in the flanking single-stranded region further suggest that the sequence CCCAUUA, encompassing the transcription start site, is required for efficient transcription of the 1.45-kb sgRNA by the TCV RNA-dependent RNA polymerase in vivo.

Analysis of sequences and predicted structures required for viral satellite RNA accumulation by in vivo genetic selection.

In vivo genetic selection was used to study the sequences and structures required for accumulation of subviral sat-RNA C associated with turnip crinkle virus (TCV). This technique is advantageous over site-specific mutagenesis by allowing side-by-side selection from numerous sequence possibilities as well as sequence evolution. A 22 base hairpin and 6 base single-stranded tail located at the 3'-terminus of sat-RNA C were previously identified as the promoter for minus strand synthesis. Approximately 50% of plants co-inoculated with TCV and sat-RNA C containing randomized sequence in place of the 22 base hairpin accumulated sat-RNA in uninoculated leaves. The 22 base region differed in sat-RNA accumulating in all infected plants, but nearly all were predicted to fold into a hairpin structure that maintained the 6 base tail as a single-stranded sequence. Two additional rounds of sat-RNA amplification led to four sequence family 'winners', with three families containing multiple variants, indicating that evolution of these sequences was occurring in plants. Three of the four sequence family winners had the same 3 bp at the base of the stem as wild-type sat-RNA C. Two of the winners shared 15 of 22 identical bases, including the entire stem region and extending two bases into the loop. These results demonstrate the utility of the in vivo selection approach by showing that both sequence and structure contribute to a more active 3'-end region for accumulation of sat-RNA C.

The coat protein of turnip crinkle virus is involved in subviral RNA-mediated symptom modulation and accumulation.

Some satellite (sat-) and defective interfering (DI) RNAs associated with plant viruses intensify or ameliorate the symptoms of the virus. We recently demonstrated that the TCV coat protein (CP) is involved in symptom modulation by sat-RNA C. Two additional subviral RNAs have now been tested for effect of the CP on symptom modulation. DI RNA G, which normally intensifies the symptoms of TCV, is able to attenuate symptoms if the TCV CP is replaced with the CP of cardamine chlorotic fleck virus. DI RNA G had no effect on the symptoms of TCV with a single base alteration in the CP open reading frame, unlike sat-RNA C, which was able to ameliorate the symptoms of the mutant TCV. Using a hybrid sat-RNA constructed from sat-RNA C and TCV (which shares a similar 3'-end region with DI RNA G), the 3'-terminal 53 bases of sat-RNA C were found to be involved in symptom attenuation, which was directly correlated with the lack of detectable viral genomic RNA in whole plants. Sat-RNA D had no effect on the symptoms of mutant or wild-type TCV. The accumulation of TCV subviral RNAs in plants and protoplasts was also found to be strongly influenced by the presence or absence of the wild-type TCV CP.

A novel 3'-end repair mechanism in an RNA virus.

Many positive-stranded RNA viruses contain short, single-stranded 3' ends that are vulnerable to degradation by host cellular RNases. Therefore, the existence of a 3'-end repair mechanism (analogous to cellular telomerases) must be required and/or advantageous for RNA viruses. Accordingly, we provide evidence suggesting that deletions of up to 6 nt from the 3' end of satellite (sat-) RNA C (a small parasitic RNA associated with turnip crinkle carmovirus) are repaired to the wild-type sequence in vivo and in vitro. The novel 3'-end repair mechanism involves the production of 4-8 nt oligoribonucleotides by abortive synthesis by the viral replicase using the 3' end of the viral genomic RNA as template. Based on our in vitro results, we postulate that the oligoribonucleotides are able to prime synthesis of wild-type negative-strand sat-RNA C in a reaction that does not require base pairing of the oligoribonucleotides to the mutant, positive-strand RNA template. The discovery of a 3'-end repair mechanism opens up new strategies for interfering with viral infections.

In vivo repair of 3'-end deletions in a TCV satellite RNA may involve two abortive synthesis and priming events.

RNA viruses that do not have the stabilizing features of poly(A) tails or amino acids covalently linked to their 3' ends must develop other means for protecting or repairing their genomes from damage caused by cellular RNases. We previously found that deletions in the single-stranded tails of a satellite RNA (sat-RNA D) associated with turnip crinkle virus are repaired in vivo (C. D. Carpenter and A. E. Simon, 1996, J. Virol. 70, 478-486). We now extend this analysis to show that sat-RNA D transcripts with 3'-end deletions of 5 bases give rise to wild-type sat-RNA, while deletions of 6 to 11 bases result in sat-RNA with additional deletions to the -14 position joined to internal TCV genomic RNA (or other) sequence followed by replacement of the terminal C1-2UGC1-3 motif. In addition, we have determined that the selection of internal TCV sequence used in the repair of sat-RNA D 3' ends is not random and generation of these short TCV segments likely involves primer-mediated synthesis of abortive products facilitated by base-pairing between internal regions of TCV genomic RNA and oligoribonucleotides generated by abortive cycling from the 3' end of the TCV genome.

Changes in locations of crossover sites over time in de novo generated RNA recombinants.

Recombinant RNAs generated in plants 3 weeks postinoculation with turnip crinkle virus (TCV) genomic RNA and an associated satellite RNA, sat-RNA D, have a majority of TCV crossover sites in a 24-nucleotide repeat (motif IIIA/IIIB) that forms part of a stable hairpin (Carpenter et al., 1995, J. Mol.Biol. 245, 608-622). To determine if parameters other than nucleotide sequence in the crossover region affect junction site selection, recombinants were assayed at various times postinoculation of plants and protoplasts. Populations of recombinants became progressively shorter in plants and larger in protoplasts. Levels of inoculated transcript and age of the plant were not substantial factors in the shifts in crossover site locations. The two most commonly cloned recombinant species were not amplified to detectable levels in protoplasts, suggesting that these molecules are not viable templates for replication. These results suggest that recombination between sat-RNA D and TCV is a very frequent event, and populations of recombinants are likely generated de novo in each infected cell and represent the original recombinant molecules rather than progeny of such molecules. Therefore, factors other than simple selection for recombinants that are more fit to replicate are probably responsible for the differences in junction sites in populations of sat-RNA D/TCV recombinants.

In vivo restoration of biologically active 3' ends of virus-associated RNAs by nonhomologous RNA recombination and replacement of a terminal motif.

Sequences at the 3' ends of plus-strand RNA viruses and their associated subviral RNAs are important cis elements for the synthesis of minus strands in vivo and in vitro. All RNAs associated with turnip crinkle virus (TCV), including the genomic RNA (4,054 bases) and satellite RNAs (sat-RNAs) such as sat-RNA D (194 bases), terminate with the motif CCUGCCC. While investigating the ability of in vivo-generated recombinants between sat-RNA D and TCV to be amplified in plants, we discovered that sat-RNA D, although truncated by as many as 15 bases in the chimeric molecules, was released from the chimeric transcripts and amplified to high levels. The "new" sat-RNA D molecules nearly all terminated with the motif (C1-2)UG(C1-3) (which may begin with 1 or 2 cytosines and end with 1, 2, or 3 cytosines), which was similar or identical to the natural sat-RNA D 3' end. The new sat-RNA D also contained between 1 and 22 bases of heterogeneous sequence upstream from the terminal motif, which, in some cases, was apparently derived from internal regions of either the plus or minus strand of the TCV genomic RNA. Since most of these internal genomic RNA sequences within TCV were not adjacent to (C1-2)UG(C1-3), at least two steps were required to produce new sat-RNA D 3' ends: nonhomologous recombination with the TCV genomic RNA followed by the addition or modification of the terminus to generate the (C1-2)UG(C1-3) motif.

Open reading frames of turnip crinkle virus involved in satellite symptom expression and incompatibility with Arabidopsis thaliana ecotype Dijon.

Carmoviruses are single-stranded, single component RNA viruses that include turnip crinkle virus (TCV) and the recently discovered cardamine chlorotic fleck virus (CCFV). Full-length, biologically active cDNAs were constructed for the TCV-M isolate and the Blue Lake isolate of CCFV. Using chimeric viruses constructed between isolates of TCV that produce mild or severe symptoms when coinoculated with a virulent satellite RNA, a Glu residue at position 1,144 in the polymerase open reading frame was identified as being involved in satellite-mediated symptom expression. To analyze viral determinants involved in resistance, chimeric viruses with precisely exchanged open reading frames were produced between TCV, which does not infect the Arabidopsis thaliana ecotype Dijon (Di-0), and CCFV, which can infect Di-0, TCV with the coat protein of CCFV was able to systemically infect Di-0 although whole plant hybridizations revealed that the hybrid virus spread more slowly than either of the two parental viruses. These results indicate that the two parental viruses. These results indicate that the coat protein is an important viral determinant in the resistance of Di-0 to TCV.

Involvement of a stem-loop structure in the location of junction sites in viral RNA recombination.

Recombination between RNAs associated with turnip crinkle virus is thought to occur during plus-strand synthesis at motifs resembling the 5'-ends of genomic, subgenomic and satellite RNAs. Common structural regions encompassing the motifs have been found for major crossover sites on two different minus-strand templates, with junctions preferentially located in a single-stranded region at the 3' base of a hairpin. Base changes, deletions and compensatory alteration constructed in and around the hairpin in the region of the turnip crinkle virus genomic RNA involved in recombination support the importance of the hairpin for normal crossover site selection. This region of the genomic RNA is also important for replication of the viral genomic RNA in plants and protoplasts, suggesting a common link between sequences required for recombination and viral replication.

Recombination between plus and minus strands of turnip crinkle virus.

Turnip crinkle virus (TCV), a small monopartite RNA virus, is associated with several subviral RNAs, some of which are the products of recombination between the genomic RNA and an unrelated satellite RNA (sat-RNA D). As part of a study on the nonrandom sequences found at the junctions of nearly all TCV recombinant molecules, we have characterized a novel recombinant (D/TCV950) that was formed from sat-RNA D and both the plus and minus strands of the genomic RNA. D/TCV950 (517 nt) is composed of sat-RNA D at the 5'-end, joined to four nontemplate derived uracil residues, and then three segments of TCV genomic RNA, one of which is from the complementary strand. Sequences at the right sides of the junctions were similar to sequences previously found at the right sides of junctions in other TCV-associated recombinant molecules. To our knowledge, this is the first report of a recombinant molecule formed from both coding and complementary strands of a single-stranded, positive-sense RNA virus.

Genes encoding glycine-rich Arabidopsis thaliana proteins with RNA-binding motifs are influenced by cold treatment and an endogenous circadian rhythm.

We have characterized the expression of two members of a class of Arabidopsis thaliana glycine-rich, putative RNA-binding proteins that we denote Ccr1 and Ccr2. Southern blot analysis indicates that Ccr1 and Ccr2 are members of a small gene family. Both Ccr1 and Ccr2 mRNA levels were influenced by a circadian rhythm that has an unusual phase for plants, with maximal accumulation at 6:00 PM and minimal accumulation at 10:00 AM. The level of CCR1 protein, however, remained relatively constant throughout the cycle. The transcript accumulation patterns of the Ccr1 and Ccr2 genes differed considerably from conditions that affect the expression of similar genes from maize, sorghum, and carrot. Levels of Ccr1 and Ccr2 mRNAs were unchanged in wounded plants, increased at least 4-fold in cold-stressed plants, and decreased 2- to 3-fold in abscisic acid-treated plants. Ccr1 transcript levels decreased in response to drought, whereas Ccr2 transcript levels increased under the same conditions. Based on the presence of additional Ccr transcripts in dark-grown plants, we propose that Ccr transcripts may be subjected to a light- or dark-mediated regulation.

Formation of multimers of linear satellite RNAs.

A 22-base region of turnip crinkle virus satellite-RNA C (sat-RNA C) is involved in the accumulation of monomeric and dimeric forms. Deletions within the region inhibited the accumulation of sat-RNA C monomers. However, normal ratios of dimers to monomers occurred if the 22 bases were replaced by 22 unrelated bases or if the location of this region was altered. Therefore, these specific 22 bases are not involved in the accumulation of sat-RNA C monomers. Examination of the sequences at the junctions of multimers of all three turnip crinkle virus sat-RNAs revealed the deletion of bases corresponding to the 3' and 5' ends of monomeric units as well as the addition of nucleotides not present in monomers. Based on these results, we present a model to explain the formation of multimers of linear subviral RNAs associated with turnip crinkle virus. Our model suggests that multimers are formed by the reinitiation of replication by the replicase before release of the nascent strand. We have previously proposed the same mechanism for the formation of defective interfering RNAs, chimeric sat-RNAs, and sat-RNA recombinants in the turnip crinkle virus system (Cascone, Carpenter, Li, and Simon. (1990). EMBO J. 9, 1709-1715).

Mutations in a satellite RNA of turnip crinkle virus result in addition of poly(U) in vivo.

Turnip crinkle virus (TCV) is associated with many subviral RNAs including satellite (sat-) RNAs which require a helper virus for infectivity. When plants were inoculated with TCV and transcripts of TCV sat-RNA C containing deletions of 3 to 8 nucleotides beginning at position 100 and extending toward the 5' end, some of the sat-RNA isolated from plants migrated more slowly than expected on denaturing polyacrylamide gels. Cleavage of the sat-RNA into two segments by digestion with RNase H following hybridization to an oligonucleotide complementary to internal sat-RNA sequence indicated that the 5' one-third of the molecule was involved in the abnormal gel migration. Sat-RNAs derived from transcripts with a deletion of bases in position 96-100 were cloned. Sequencing of the cDNAs revealed that the aberrant migration of the sat-RNAs was due to the presence of variable lengths of poly(U) 10 nucleotides downstream from the deletion at a position which already contained five U residues. Deletions extending toward the 3' end in the same region did not result in poly(U) additions. Mutations in the original five U residues along with the 5' deletions also did not lead to poly(U) additions. The insertion of poly(U) in TCV sat-RNA C may be a new example of replicase stuttering with the distinction that it only occurs following specific upstream mutations.

Structural analysis of the transmembrane domain of the epidermal growth factor receptor.

The ligand-binding domain of the epidermal growth factor (EGF) receptor is separated from the cytoplasmic protein tyrosine kinase domain by a predicted single transmembrane segment. Antipeptide antibodies prepared against the outer portion of the predicted transmembrane segment confirmed this area was exposed only when cells were treated with permeabilizing agents. To investigate structural requirements for signal transduction by the transmembrane domain, three types of mutant EGF receptor were prepared. The first type was designed to shorten the transmembrane domain, the second to place proline substitutions within this domain, and the third to make amino acid substitutions analogous to those present in the transforming c-erbB2/neu oncoprotein. Mutant human receptors were expressed in null recipient mouse B82L and Chinese hamster ovary cells. All receptors bound EGF and exhibited EGF-stimulated protein tyrosine kinase activity in vivo as assayed using a 125I-labeled monoclonal anti-phosphotyrosine antibody. EGF stimulated growth of cells expressing each mutant receptor with similar dose-response characteristics. In contrast to other growth factor receptors, the transmembrane domain of the EGF receptor is tolerant to a variety of changes which neither mimic EGF action by constitutive activation nor interfere with ligand-induced signal transduction.