RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.Hepatitis delta virus (HDV) is a subviral human pathogen that increases the risk of severe liver disease in those infected with its helper, hepatitis B virus (34). The HDV genome is an ϳ1,680-nucleotide (nt) circular RNA that replicates through a circular RNA intermediate, the antigenome (21). Both the genome and antigenome possess extensive intramolecular complementarity and are predicted to form rod-shaped structures in which about 70% of the nucleotides are base paired (39).HDV produces two forms of the sole viral protein, hepatitis delta antigen (HDAg) (4), and both are translated from a single mRNA that is transcribed from the genomic RNA (16,18,40). The shorter form, HDAg-S, is required for RNA replication, while the longer form, HDAg-L, inhibits replication but is required for packaging the viral RNA with the envelope of hepatitis B surface antigen (19,35,42). The virus uses adenosine-to-inosine RNA editing activity of the host cell to produce HDAg-S and HDAg-L from the same open reading frame (6, 25). The editing does not occur on the mRNA directly but on adenosine 1012 of the antigenome (9, 32). The nucleotide change is subsequently passed to the genome during replication and to the mRNA during transcription. The ultimate effect is the conversion of the UAG amber termination codon of HDAg-S to a UGG tryptophan codon required to synthesize the slightly longer HDAg-L; because of the codon change produced by this editing event, the editing site is called the amber/W site (32).We previously sh...

We have constructed physical and genetic maps of the chromosomes of 21 Lyme disease agent spirochetes from geographically diverse locations. All have linear chromosomes whose lengths range from 935 to 955 kbp, and all contain multiple linear plasmids in the 16-to 175-kbp size range. The locations of 11 gene clusters on the chromosomes of these different isolates are indistinguishable at the resolution achieved in this study, indicating that the members of this related group of species have highly conserved chromosomal gene orders. However, chromosomal restriction endonuclease cleavage site maps are unique for nearly all isolates. The 22 chromosomal maps currently available define eight classes of Lyme disease agents. Four of these correspond to the previously proposed species Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, and Borrelia japonica. In addition, the North American isolates 21038, DN127 cl9-2, 25015, and CA55 typify four additional chromosomal types that are as phylogenetically distinct as the species listed above. These findings support the idea that comparison of restriction maps is currently the most robust and definitive method for determining overall chromosomal relationships among closely related bacteria. In the course of this work, we located on the chromosome the previously unmapped outer surface protein-encoding LA7 gene and genes homologous to the Escherichia coli priA, plsC, parE, and parC genes, and we have substantially refined the locations of the recA, fla, p22A, and flgE genes.

Papillary transitional cell tumors were found in the urinary bladders in 8 rats out of 80 that received 2600 milligrams per kilogram of body weight per day of a mixture of sodium cyclamate and sodium saccharin (10:1) for up to 105 weeks. From week 79 on, several of these rats received cyclohexylamine hydrochloride (125 milligrams per kilogram per day, the molecular equivalent of the conversion of about 10 percent of the cyclamate dosage to cyclohexylamine) in addition to the sodium cyclamate and sodium saccharin. In another study in which 50 rats were fed daily 15 milligrams of cyclohexylamine sulfate per kilogram of body weight for 2 years, eight males and nine females survived. One of the eight males had a tumor of the urinary bladder. In neither study were bladder tumors found in the control rats or in rats treated with lower doses of the compounds.

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