Biomedical subjects

N Parkin

Publications and source records attributed to N Parkin.

6 recordsLinked to original sources

PubMed2001

A multiple-site-specific heteroduplex tracking assay as a tool for the study of viral population dynamics.

Rapidly evolving entities, such as viruses, can undergo complex genetic changes in the face of strong selective pressure. We have developed a modified heteroduplex tracking assay (HTA) capable of detecting the presence of single, specific mutations or sets of linked mutations. The initial application of this approach, termed multiple-site-specific (MSS) HTA, was directed toward the detection of mutations in the HIV-1 pro gene at positions 46, 48, 54, 82, 84, and 90, which are associated with resistance to multiple protease inhibitors. We demonstrate that MSS HTA is sensitive and largely specific to all targeted mutations. The assay allows the accurate and reproducible quantitation of viral subpopulations comprising 3% or more of the total population. Furthermore, we used MSS HTA in longitudinal studies of pro gene evolution in vitro and in vivo. In the examples shown here, populations turned over rapidly and more than one population was present frequently. To demonstrate the versatility of MSS HTA, we also constructed a probe sensitive to changes at positions 181 and 184 of the RT coding domain. Changes at these positions are involved in resistance to nevirapine and 2',3'-dideoxy-3'-thiacytidine (3TC), respectively. This assay easily detected the evolution of resistance to 3TC. MSS HTA provides a rapid and sensitive approach for detecting the presence of and quantifying complex mixtures of distinct genotypes, including genetically linked mutations, and, as one example, represents a useful tool for following the evolution of drug resistance during failure of HIV-1 antiviral therapy.

Antiviral Agents↗

PubMed2000

Reply

Explore the source record for details and available documents.

Journal Article↗

PubMed1992

An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA.

Synthesis of the pol gene products of most retroviruses requires ribosomes to shift frame once or twice in the -1 direction while translating gag-pol mRNA. The viral signals for frameshifting include a heptanucleotide sequence on which the shift occurs and higher-order RNA structure just downstream of the shift site. We have made site-directed mutations in two stems (S1 and S2) of a putative RNA pseudoknot that begins 7 nucleotides 3' of the previously identified shift site (A AAA AAC) in the gag-pro region of mouse mammary tumor virus (MMTV) RNA. The mutants confirm the predicted structure, show that loss of either S1 or S2 impairs frameshifting, and exclude alternative RNA structures as significant for frameshifting. The importance of the MMTV pseudoknot has been further demonstrated by showing that shift sites from two other retroviruses function more efficiently in the position of the MMTV site than in their native contexts. However, the MMTV pseudoknot cannot promote detectable frameshifting in the absence of a recognizable upstream shift site. In addition, the species of tRNA that reads the second codon in the shift site appears to be a critical determinant, since changing the 7th nucleotide in the MMTV gag-pro shift site from C to A, U, or G severely impairs frameshifting.

Base Sequence↗

PubMed1988

cis-acting translational effects of the 5' noncoding region of c-myc mRNA.

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.

Animals↗

PubMed1988

Developmental regulation of translation by the 5' noncoding region of murine c-myc mRNA in Xenopus laevis.

The translational efficiency of chloramphenicol acetyltransferase (CAT) mRNA containing a 5' noncoding sequence derived from exon 1 of the murine c-myc gene (360CAT) has been examined at different stages of Xenopus egg development. In contrast to its reduced translation in the Xenopus oocyte, 360CAT mRNA is translated as efficiently as CAT mRNA when injected into either mature Xenopus eggs or Xenopus embryos. No significant alteration of 360CAT mRNA stability was observed up to 10 h post-fertilization in Xenopus embryos as compared to that of CAT mRNA. The increase in 360CAT mRNA translational efficiency in Xenopus embryos was not observed with CAT mRNAs possessing other inhibitory 5' noncoding sequences. The increase in 360CAT mRNA translational efficiency is attributed to a trans-acting factor synthesized or activated following Xenopus oocyte maturation. The possible significance of the 5' noncoding region of c-myc mRNA in developmental expression of c-myc is discussed.

Animals↗

PubMedDate not supplied

HIV-1 drug resistance in newly infected individuals.

CONTEXT: There is concern that the widespread use of antiretroviral drugs to treat human immunodeficiency virus 1 (HIV-1) infection may result in the increased transmission of drug-resistant virus. OBJECTIVE: To determine the prevalence of drug resistance-conferring mutations and phenotypic resistance to antiretroviral agents in a cohort of individuals newly infected with HIV-1. DESIGN: Case series with genetic analyses of the HIV-1 plasma-derived pol gene using reverse transcriptase polymerase chain reaction followed by direct sequencing of polymerase chain reaction products. Phenotypic analysis was performed with a recombinant virus assay. SETTING AND PATIENTS: Eighty individuals referred, on average, 1.7 months after infection with HIV-1 to the Aaron Diamond AIDS Research Center between July 1995 and April 1999. Subjects were from large urban areas (65 from New York, NY; 11 from Los Angeles, Calif); 60 (75%) were white, and 75 (93.8%) were homosexual men. MAIN OUTCOME MEASURES: Prevalence of known resistance-conferring genotypes and reduced susceptibility to individual antiviral agents by phenotype. RESULTS: Thirteen individuals (16.3%) had genotypes associated with drug resistance to any antiretroviral agent. Virus with known resistance-conferring mutations to any nucleoside reverse transcriptase inhibitors was found in 10 individuals, to any nonnucleoside reverse transcriptase inhibitors in 6 subjects, and to any protease inhibitors in 2 cases. Multidrug-resistant virus was identified in 3 individuals (3.8%). Extensive polymorphism in the protease gene was identified. Interpretation of genotypes and phenotypes was concordant in 57 (85%) of the 67 cases in which both studies were performed. CONCLUSION: The prevalence of HIV-1 variants with known resistance-conferring genotypes to any antiretroviral agent in this cohort of 80 newly infected individuals is 16.3%. These data support expanded use of resistance testing in the setting of primary HIV-1 infection. Clinical trials should be initiated to establish whether therapy guided by resistance testing, compared with the use of empirical triple combination antiretroviral therapy, provides additional virological and immunological benefit when treating primary HIV-1 infection. Further efforts to expand the study of transmission of drug-resistant HIV-1 variants, particularly in cohorts with different epidemiological profiles, are indicated.

Adult↗