Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells.

The bioavailability of drugs administered topically or orally depends on their metabolism by epithelial enzymes such as the cytosolic sulfotransferases (SULT). Reverse transcriptase-polymerase chain reaction (RT-PCR) methods were established to detect expression of 8 SULT genes and 4 arylsulfatase (ARS) genes in human tissues of epithelial origin and in cultures of normal and transformed (cancer) cells. The results indicate: (i) SULT 1A1, 1A3, ARSC, and ARSD genes are ubiquitously expressed; (ii) expression is frequently similar between cell lines and corresponding tissues; (iii) SULT gene expression in normal cultured cells is generally comparable to the expression in associated transformed (cancer) cell lines; (iv) SULT 1A1 promoter usage is mainly tissue specific; however, both promoters are frequently used in SULT 1A3 expression; and (v) the expression profile of SULT 1A1, 1A3, 1E1, and 2B1a/b suggests that one or more of these isoforms may be involved in the cutaneous sulfoconjugation of minoxidil and cholesterol.

Secondary structures in the capsid protein coding sequence and 3' nontranslated region involved in amplification of the tobacco etch virus genome.

The 3'-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3' nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370-4379, 1996). To investigate the sequence and secondary structure requirements within the CP cis-active region and the 3' NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (beta-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-coding cis-active sequence and at several distinct locations within the 3' NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding cis-active sequence, a base-paired structure between two segments of the CP-coding cis-active sequence and a contiguous 14-nucleotide segment of the 3' NTR, and a base-paired structure near the 3' terminus of the 3' NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3' terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3' NTR are coadapted for genome amplification function through a requirement for base pair interactions.

Green-fluorescent protein fusions for efficient characterization of nuclear targeting.

The green-fluorescent protein (GFP) from Aequorea victoria has been shown to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly suited for applications in vivo, since the mechanism of fluorophore formation involves an intramolecular autoxidation and does not require exogenous co-factors. Unlike standard histochemical procedures of fixation and staining required for analysis of the cellular or tissue-specific expression of other popular reporter molecules, such as the beta-glucuronidase (GUS) marker, analysis of GFP can be done in living cells with no specific pretreatments. This implies that GFP might also be particularly suited for studies of intracellular protein targeting. In this paper, the use of GUS is compared with that of GFP for the analysis of nuclear targeting in tobacco. A novel oligopeptide motif from a tobacco protein is described which confers nuclear localization of GUS. The use of this oligopeptide and two from potyviral proteins to target GFP to the nucleus is examined. An essential modification of GFP is described, which specifically increases its molecular weight to eliminate its passive penetration into the nucleus. Three examples of the targeting of these enlarged GFP molecules to the nucleus are illustrated. GFP, in combination with confocal microscopy, offers significant advantages over traditional methods of studying nuclear targeting.

Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification.

A mutational analysis was conducted to investigate the functions of the tobacco etch potyvirus VPg-proteinase (NIa) protein in vivo. The NIa N-terminal domain contains the VPg attachment site, whereas the C-terminal domain contains a picornavirus 3C-like proteinase. Cleavage at an internal site separating the two domains occurs in a subset of NIa molecules. The majority of NIa molecules in TEV-infected cells accumulate within the nucleus. By using a reporter fusion strategy, the NIa nuclear localization signal was mapped to a sequence within amino acid residues 40 to 49 in the VPg domain. Mutations resulting in debilitation of NIa nuclear translocation also debilitated genome amplification, suggesting that the NLS overlaps a region critical for RNA replication. The internal cleavage site was shown to be a poor substrate for NIa proteolysis because of a suboptimal sequence context around the scissile bond. Mutants that encoded NIa variants with accelerated internal proteolysis exhibited genome amplification defects, supporting the hypothesis that slow internal processing provides a regulatory function. Mutations affecting the VPg attachment site and proteinase active-site residues resulted in amplification-defective viruses. A transgenic complementation assay was used to test whether NIa supplied in trans could rescue amplification-defective viral genomes encoding altered NIa proteins. Neither cells expressing NIa alone nor cells expressing a series of NIa-containing polyproteins supported increased levels of amplification of the mutants. The lack of complementation of NIa-defective mutants is in contrast to previous results obtained with RNA polymerase (NIb)-defective mutants, which were relatively efficiently rescued in the transgenic complementation assay. It is suggested that, unlike NIb polymerase, NIa provides replicative functions that are cis preferential.

Long-distance movement factor: a transport function of the potyvirus helper component proteinase.

Transport of viruses from cell to cell in plants typically involves one or more viral proteins that supply dedicated movement functions. Transport from leaf to leaf through phloem, or long-distance transport, is a poorly understood process with requirements differing from those of cell-to-cell movement. Through genetic analysis of tobacco etch virus (TEV; potyvirus group), a novel long-distance movement factor was identified that facilitates vascular-associated movement in tobacco. A mutation in the central region of the helper component proteinase (HC-Pro), a TEV-encoded protein with previously described activities in aphid-mediated transmission and polyprotein processing, inactivated long-distance movement. This mutant virus exhibited only minor defects in genome amplification and cell-to-cell movement functions. In situ histochemical analysis revealed that the mutant was capable of infecting mesophyll, bundle sheath, and phloem cells within inoculated leaves, suggesting that the long-distance movement block was associated with entry into or exit from sieve elements. The long-distance movement defect was specifically complemented by HC-Pro supplied in trans by a transgenic host. The data indicate that HC-Pro functions in one or more steps unique to long-distance transport.

Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus.

The tobacco etch potyvirus (TEV) capsid protein (CP) is necessary for cell-to-cell and long distance transport of the virus in plants. In this study, the transport phenotypes of TEV mutants containing CPs with a substitution of the highly conserved Ser122 (termed S122W) within the core domain, or with a deletion of sequences encoding 17 amino acid residues comprising most of the variable C-terminal domain (delta C), were analyzed. The S122W and delta C mutant genomes were amplified to levels comparable to parental virus in protoplasts. The S122W mutant was encapsidation-defective, although in transgenic plants expressing wild-type CP a small number of virions were observed after prolonged incubation. Cells infected by the delta C mutant produced virions, indicating that the C-terminal domain is not necessary for encapsidation. The mutants exhibited unique defects in cell-to-cell and long distance movement in plants. The S122W mutant was confined to single, primarily inoculated epidermal cells in nontransgenic plants, but the cell-to-cell movement defect was rescued efficiently by transgenic CP. Long distance movement of this mutant was also rescued in transgenic plants, but accumulation in systemically infected tissue was low compared to parental virus. The delta C mutant exhibited a slow cell-to-cell movement phenotype in inoculated leaves and a complete inability to move systemically in nontransgenic plants. Transgenic CP was able to rescue partially the slow cell-to-cell movement defect of the delta C mutant, but not the long distance transport defect. Taken together with previous results, these data suggest that the core domain of TEV CP provides a function essential during cell-to-cell movement and that the variable N- and C-terminal regions exposed on the virion surface are necessary for long distance transport. In addition, trans-inhibition models are presented to account for the widely differing transgenic complementation efficiencies of the various movement-defective mutants.