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C Hemenway

Publications and source records attributed to C Hemenway.

At least 19 recordsLinked to original sources
PubMed1999

Restoration of a stem-loop structure required for potato virus X RNA accumulation indicates selection for a mismatch and a GNRA tetraloop.

The 5' region of potato virus X (PVX) RNA contains a stem-loop structure, stem-loop 1 (SL1), that is required for efficient plus-strand RNA accumulation. To determine how changes to individual elements in SL1 are accommodated by the virus, we inoculated PVX transcripts containing modifications in the terminal tetraloop (TL), stem C (SC), and stem D (SD) regions onto Nicotiana benthamiana plants and analyzed progeny RNAs over a series of passages. Several progeny RNAs isolated from plants inoculated with the TL mutants containing changes to the first nucleotide of the GAAA motif or deletion of the entire TL sequence were found to contain multiple A insertions within the terminal loop region. The wild-type TL motif, GAAA, was recovered for all TL mutants by the second passage, suggesting that the sequence and potential structure of this element are crucial for PVX infection. Revertant RNAs isolated from plants inoculated with mutants in SD and the central region of SC indicated that increased stem length is tolerated. Restoration of SD length to the 4 bp typical of the wild-type PVX RNA was accompanied by A insertion into loop C. Mutants with a conversion of the C55-C78 mismatch to a G-C pair, relocation of this mismatch within the central region of SC, or deletion of C55-C78 were unable to infect protoplasts and plants. In contrast, the mutant with a conversion of the C55-C78 mismatch to an A-C mismatch, which exhibited low levels of PVX plus-strand RNA in protoplasts, was able to infect plants and quickly reverted to the wild-type C-C mismatch. These data indicate that important sequence and secondary structural elements within SL1 are required for efficient viral infection and that multiple A insertions within the TL and loop C regions, potentially by polymerase stuttering, accompany restoration of SL1 structure.

Adenosine↗
PubMed1998

Stem-loop structure in the 5' region of potato virus X genome required for plus-strand RNA accumulation.

Computer-generated thermodynamic predictions and solution structure probing indicated two stem-loop structures, stem-loop 1 (SL1; nt 32-106) and stem-loop 2 (SL2; nt 143-183), within the 5' 230 nt of potato virus X (PVX) RNA. Because the existence of SL1 was further supported by covariation analysis of several PVX strains, the functional significance of this structure was investigated by site-directed mutational analysis in a tobacco protoplast system. In general, mutations that reduced genomic plus-strand RNA accumulation similarly affected coat protein accumulation, indicating that subgenomic plus-strand RNA was also affected. In contrast, minus-strand RNA levels remained relatively unchanged. Mutational analysis of the stem C (SC) region of SL1 indicated that pairing was more important than sequence, which was consistent with the covariation analysis. Alterations that increased length and stability of either SC or stem D (SD) were deleterious to plus-strand RNA accumulation. The formation of internal loop C between SC and SD, as well as specific nucleotides within this loop, were also required. Several modifications were made to the terminal GAAA tetraloop, a motif known for enhanced RNA stability. Both GANA and GAAG motifs resulted in wild-type levels of RNA accumulation. However, a UUCG tetraloop was detrimental, indicating that the sequence of this element was important beyond just providing stabilization of the structure. These data indicate that multiple features of SL1 are critical for accumulation of PVX plus-strand RNA.

Base Sequence↗
PubMed1998

History of coat protein-mediated protection.

A decade of research has proven that plants can be genetically engineered to resist virus infection through expression of viral CP genes, as well as other viral genes and sequences. Additional opportunities for development of resistant plants will require research focused on mechanisms of protection, improvements in expression vector design, and transformation of new crop species. As each of these technologies is utilized singly or in combination to generate resistant crop varieties, the full impact of such engineered resistance will be realized.

Capsid Proteins↗
PubMed1997

Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation.

The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation of Nicotiana tabacum NT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.

Base Sequence↗
PubMed1996

Functional expression of the multidrug resistance-associated protein in the yeast Saccharomyces cerevisiae.

The multidrug resistance-associated protein (MRP) is a member of the ATP binding cassette superfamily of transporters which includes the mammalian P-glycoproteins (P-gp) family. In order to facilitate the biochemical and genetic analyses of MRP, we have expressed human MRP in the yeast Saccharomyces cerevisiae and have compared its functional properties to those of the mouse Mdr3 P-gp isoform. Expression of both MRP and Mdr3 in the anthracycline hypersensitive mutant VASY2563 restored cellular resistance to Adriamycin in this mutant. MRP and Mdr3 expression produced pleiotropic effects on drug resistance in this mutant, as corresponding VASY2563 transformants also acquired resistance to the anti-fungal agent FK506 and to the K+/H+ ionophore valinomycin. The appearance of increased cellular resistance to the toxic effect of Adriamycin (ADM) in MRP and Mdr3 transformants was concomitant with a reduced intracellular accumulation of [14C]ADM in spheroplasts prepared from these cells. Moreover, MRP and Mdr3, but not control spheroplasts, could mediate a time-dependent reduction in the overall cell-associated [14C]ADM from preloaded cells, suggesting the presence of an active ADM transport mechanism in MRP and Mdr3 transformants. Finally, human MRP was found to complement the biological activity of the yeast peptide pheromone transporter Ste6 and partially restored mating in a sterile ste6 null mutant. These findings suggest that despite their relatively low level of structural homology, MRP and P-gp share similar functional aspects, since both proteins can mediate transport of chemotherapeutic drugs and the a mating peptide pheromone in yeast.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1996

Synthesis of potato virus X RNAs by membrane-containing extracts.

Membrane-containing extracts isolated from tobacco plants infected with the plus-strand RNA virus, potato virus X (PVX), supported synthesis of four major, high-molecular-weight PVX RNA products (R1 to R4). Nuclease digestion and hybridization studies indicated that R1 and R2 are a mixture of partially single-stranded replicative intermediates and double-stranded replicative forms. R3 and R4 are double-stranded products containing sequences typical of the two major PVX subgenomic RNAs. The newly synthesized RNAs were demonstrated to have predominantly plus-strand polarity. Synthesis of these products was remarkably stable in the presence of ionic detergents.

Endonucleases↗
PubMed1996

The 5' nontranslated region of potato virus X RNA affects both genomic and subgenomic RNA synthesis.

A tobacco protoplast system was developed to analyze cis-acting sequences required for potato virus X (PVX) replication. Protoplasts inoculated with transcripts derived from a PVX cDNA clone or from clones containing mutations in their 5' nontranslated regions (NTRs) were assayed for RNA production by S1 nuclease protection assays. A time course of plus- and minus-strand-RNA accumulation indicated that both minus- and plus-strand PVX RNAs were detectable at 0.5 h postinoculation. Although minus-strand RNAs accumulated more rapidly than plus-strand RNAs, maximum levels of plus-strand RNAs were 40- to 80-fold higher. On the basis of these data, time points were chosen for determination of RNA levels in protoplasts inoculated with PVX clones containing deletions or an insertion in their 5' NTRs. Deletions of more than 12 nucleotides from the 5' end, internal deletions, and one insertion in the 5' NTR resulted in substantially decreased levels of plus-strand-RNA production. In contrast, all modified transcripts were functional for minus-strand-RNA synthesis, suggesting that elements in the 5' NTR were not essential for minus-strand-RNA synthesis. Further analysis of the 5' NTR deletion mutants indicated that all mutations that decreased genomic plus-strand-RNA synthesis also decreased synthesis of the two major subgenomic RNAs. These data indicate that cis-acting elements from different regions of the 5' NTR are required for plus-strand-RNA synthesis and that this process may be linked to synthesis of subgenomic RNAs.

Base Sequence↗
PubMed1994

Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands.

The peptidyl-prolyl isomerases FKBP12 and cyclophilin A (immunophilins) form complexes with the immunosuppressants FK506 and cyclosporin A that inhibit the phosphatase calcineurin. With the yeast two hybrid system, we detect complexes between FKBP12 and the calcineurin A catalytic subunit in both the presence and absence of FK506. Mutations in FKBP12 surface residues or the absence of the calcineurin B regulatory subunit perturb the FK506-dependent, but not the ligand-independent, FKBP12-calcineurin complex. By affinity chromatography, both FKBP12 and cyclophilin A bind calcineurin A in the absence of ligand, and FK506 and cyclosporin A respectively potentiate these interactions. Both in vivo and in vitro, the peptidyl-prolyl isomerase active sites are dispensable for ligand-independent immunophilin-calcineurin complexes. Lastly, by genetic analyses we demonstrate that FKBP12 modulates calcineurin functions in vivo. These findings reveal that immunophilins interact with calcineurin in the absence of exogenous ligands and suggest that immunosuppressants may take advantage of the inherent ability of immunophilins to interact with calcineurin.

Amino Acid Isomerases↗
PubMed1993

Corticosteroids improve survival of children with AIDS and Pneumocystis carinii pneumonia.

OBJECTIVE: To determine if corticosteroids administered in addition to antimicrobials improve survival in children with acquired immunodeficiency syndrome and severe Pneumocystis carinii pneumonia (PCP). DESIGN: Before-after, nonrandomized, case-comparison study. SETTING: Pediatric intensive care unit of a tertiary care teaching hospital in Florida. PARTICIPANTS: Eleven children infected with the human immunodeficiency virus (HIV) with confirmed PCP. SELECTION PROCEDURE: Infants with HIV infection and acute respiratory failure due to PCP were studied sequentially. INTERVENTION: The first seven infants were treated with antimicrobials alone while the next four received a 2-week course of methylprednisolone sodium succinate in addition to antimicrobials. MEASUREMENTS AND RESULTS: The two groups were similar with respect to age, route of HIV infection, stage of HIV disease, CD4 T-cell count, antiretroviral therapy, and respiratory parameters at intubation. All children treated with antimicrobials alone died while receiving mechanical ventilation. Survival of the children who received corticosteroid therapy was significantly improved (P < .05), and all were weaned from the ventilator. CONCLUSIONS: Corticosteroids administered in addition to antimicrobials and supportive care improve the short-term survival of HIV-infected children who have acute respiratory failure due to PCP.

AIDS-Related Opportunistic Infections↗
PubMed1990

Characterization of infectious transcripts from a potato virus X cDNA clone.

A full-length cDNA clone of potato virus X (PVX) has been constructed and fused to the bacteriophage T7 promoter in an in vitro transcription vector. Transcripts derived from this template (pMON 8660) were infectious when inoculated onto the local lesion host, Chenopodium amaranticolor. The infectivity of these transcripts was approximately 0.2% that of authentic PVX RNA. Lesions sampled from plants inoculated with these transcripts contained virus particles and virus aggregates typically observed in lesions from plants inoculated with authentic PVX RNA, as evidenced by electron microscopy. In addition, progeny virus isolated from these lesions was as infectious as progeny virus from an authentic PVX RNA infection when inoculated onto new local lesions plants. Infectious transcripts derived from PVX cDNA clones will facilitate analysis of the molecular aspects of PVX infection.

Antigens, Viral↗
PubMed1988

Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA.

Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP-antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. Plants expressing antisense RNA were protected only at low inoculum concentrations. The extent of this protection was even lower than that observed in plants expressing low levels of CP. In contrast to previous reports for plants expressing tobacco mosaic virus or alfalfa mosaic virus CP, inoculation of plants expressing high levels of PVX CP with PVX RNA did not overcome the protection. Specifically, lesion numbers on inoculated leaves and PVX levels on inoculated and systemtic leaves of the CP+ plants were reduced to a similar extent in both virus and RNA inoculated plants. Although these results do not rule out that CP-mediated protection involves inhibition of uncoating of the challenge virus, they suggest that PVX CP (or its RNA) can moderate early events in RNA infection by a different mechanism.

Journal Article↗
PubMed1987

DNase I-hypersensitive sites associated with expression and hormonal regulation of mouse C4 and Slp genes.

There are four major regions of DNase I hypersensitivity in the 5' regions of the genes for the murine fourth component of complement (C4) and its homologous neighbor, Slp (sex-limited protein). Hypersensitivity around the start site of transcription and approximately equal to 0.5 kilobases upstream correlates qualitatively with expression of these genes. Two hypersensitive sites, at -2.3 and -2.0 kilobases, map specifically to the Slp gene and correlate with its hormonal regulation. That is, these sites are more prominent in male liver chromatin and become more apparent in chromatin from females treated with testosterone. Further, these sites are established in males to a greater extent than in females prior to expression of Slp and may reflect gene-commitment events. Comparison of chromatin from mouse strains differing in C4 and Slp alleles indicates that the four regions of hypersensitivity may be necessary but are not sufficient for high levels of expression.

Animals↗
PubMed1987

Molecular genetics of androgen-dependent and -independent expression of mouse sex-limited protein.

Genes of the mouse S locus encoding C4 (the fourth component complement) and Slp (sex-limited protein) show extensive homology but are distinct in their function and regulation. In some mouse strains, such as B10.D2, Slp is androgen regulated, whereas in others, such as B10.W7R, expression of Slp is constitutive. We have previously shown that the B10.W7R strain has multiple Slp genes. In this report, we present the structure of the single C4 and four Slp genes of the B10.W7R S locus and compare the upstream flanking regions by partial sequence analysis and function in transfection assays. Of the four Slp genes, three (Slpw7.A, Slpw7.B, and Slpw7.C) have upstream and promoter regions very similar to those of C4. The fourth Slp gene (Slpw7.D) is instead virtually identical to the androgen-regulated allele (Slpd from the B10.D2 mouse) in upstream regions. In particular, far-upstream sequences from both Slpd and Slpw7.D render the bacterial chloramphenicol acetyltransferase gene hormonally responsive upon transfection into mammary carcinoma cell lines. The upstream sequences between 2 to 3 kilobases of the Slp promoter initiate transcription from multiple sites when fused proximal to the chloramphenicol acetyltransferase gene, and these transcripts are threefold more abundant in the presence of androgen. This behavior is similar for Slpd and Slpw7.D, which suggests that Slpw7.D may be androgen regulated but that this is masked in vivo by constitutive expression of the other Slp genes. Nonhomologous recombination is implicated not only in expanding the copy number of C4 and Slp genes in the B10.W7R mouse but also in creating hybrid genes with regulatory features of C4 and structural features of Slp.

Androgens↗
PubMed1986

Sequence comparison of alleles of the fourth component of complement (C4) and sex-limited protein (Slp).

cDNA clones specific for the fourth component of complement (C4) and its androgen-regulated isotype, sex-limited protein (Slp), have been isolated from two mouse haplotypes (H-2d and H-2w7) that show differential C4 activity and differential regulation of Slp. Clones were first isolated using a cDNA probe enriched by subtractive hybridization. Subsequent screening has resulted in cDNAs spanning the entire C4d mRNA, as well as much of C4w7, Slpw7 and a short region of Slpd. The cDNAs for C4 and Slp show extensive sequence homology, but can be distinguished using oligonucleotide probes synthesized to regions of greatest sequence divergence. Sequence differences between C4 and Slp indicate structurally important features of C4 that have been altered in Slp such that Slp is unable to participate in the complement pathway. Of the few nucleotide differences between C4d and C4w7, a single base change resulting in one less glycosylation site in the C4w7 alpha chain could account for its 4-fold reduced hemolytic efficiency. Sequence comparison of multiple alleles of C4 and Slp indicates that possible gene conversion events occurred in the H-2w7 strain that has multiple Slp genes.

Alleles↗