New Mutants (new + mutant)

Distribution by Scientific Domains

Selected Abstracts

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

FEBS JOURNAL, Issue 8 2008
Camila A. O. Dias
Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal ,-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins , eIF5AK56A and eIF5AQ22H,L93F, and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression. [source]

A simple in vivo assay for measuring the efficiency of gene length-dependent processes in yeast mRNA biogenesis

FEBS JOURNAL, Issue 4 2006
Macarena Morillo-Huesca
We have developed a simple reporter assay useful for detection and analysis of mutations and agents influencing mRNA biogenesis in a gene length-dependent manner. We have shown that two transcription units sharing the same promoter, terminator and open reading frame, but differing in the length of their 3,-untranslated regions, are differentially influenced by mutations affecting factors that play a role in transcription elongation or RNA processing all along the transcription units. In contrast, those mutations impairing the initial steps of transcription, but not affecting later steps of mRNA biogenesis, influence equally the expression of the reporters, independently of the length of their 3,-untranslated regions. The ratio between the product levels of the two transcription units is an optimal parameter with which to estimate the efficiency of gene length-dependent processes in mRNA biogenesis. The presence of a phosphatase-encoding open reading frame in the two transcription units makes it very easy to calculate this ratio in any mutant or physiological condition. Interestingly, using this assay, we have shown that mutations in components of the SAGA complex affect the level of mRNA in a transcript length-dependent fashion, suggesting a role for SAGA in transcription elongation. The use of this assay allows the identification and/or characterization of new mutants and drugs affecting transcription elongation and other related processes. [source]

Clinical and virological characteristics of lamivudine resistance in chronic hepatitis B patients: A single center experience

Jian Sun
Abstract We have investigated the characteristics of lamivudine-resistant strains in patients with chronic hepatitis B in Guangdong, China, where the predominant genotypes are B and C. Two hundred forty-seven patients treated with lamivudine in Nanfang Hospital were followed-up. Patients with hepatitis B e antigen (HBeAg) positive and hepatitis B virus (HBV)-DNA levels over 7.5,,106 copies/ml at baseline had a shorter time to the selection of YMDD mutant (P,=,0.02 and 0.00, respectively). The detection of YMDD mutant precedes HBV-DNA breakthrough and alanine transaminase (ALT) flare in about 2 and 3 months, respectively. The ALT flare after the appearance of YMDD mutants was more evident in HBeAg positive patients than HBeAg negative patients (P,=,0.02). After emergence of YMDD mutant, the HBV-DNA level was significantly higher in genotype C patients compared with genotype B patients (P,=,0.02). No significant difference of YMDD mutant pattern was found between patients with genotype B and C. Four kinds of new mutants were found in over two patients including rtL80I, rtG172E, rtG174C, and rtG172E/rtG174C. In vitro transfection and real-time analysis showed that rtG172E, rtG174C, and rtG172E/rtG174C mutants had a decreased replication competence compared with wild type (33%, 27%, and 15% of the wild type HBV, respectively). Our result suggest that genotypic monitoring of YMDD mutant is important for the management of patients treated with lamivudine. J. Med. Virol. 75:391,398, 2005. 2005 Wiley-Liss, Inc. [source]

Change of the unbinding mechanism upon a mutation: A molecular dynamics study of an antibody,hapten complex

PROTEIN SCIENCE, Issue 10 2005
Raffaele Curcio
Abstract We study forced unbinding of fluorescein from the wild type (WT) and a mutant [H(H58)A] of the single-chain variable-fragment (scFv) anti-fluorescein antibody FITC-E2 by molecular dynamics simulations using various pulling techniques. A large number of long simulations were needed to obtain statistically meaningful results as both the wild type and the H(H58)A mutant unbinding occurs through multiple pathways, often with metastable intermediates. For the wild type, the rate-limiting step in the unbinding process corresponds to the breaking of the non-native interactions characteristic of a specific intermediate. The H(H58)A mutation disfavors the occurrence of this intermediate. Two events where the hapten partially unbinds in the absence of pulling force are observed in extensive equilibrium simulations of the wild type, and their analysis indicates that forced unbinding and spontaneous unbinding proceed along similar pathways. The different unbinding mechanisms observed in the simulations suggest a possible reason for the difference in the experimental off-rate between the two antibodies. We predict mutations that are expected to modulate the occurrence of the unbinding intermediate. For two such new mutants [H(H58)A and S(H52)A], our predictions are validated in silico by additional simulations. The accompanying paper in this issue by Honegger et al. reports the X-ray structure of FITC-E2 with a derivative of fluorescein, which was used as the starting conformation for the work presented here. [source]

The BOS loci of Arabidopsis are required for resistance to Botrytis cinerea infection

Paola Veronese
Summary Three Botrytis -susceptible mutants bos2, bos3, and bos4 which define independent and novel genetic loci required for Arabidopsis resistance to Botrytis cinerea were isolated. The bos2 mutant is susceptible to B. cinerea but retains wild-type levels of resistance to other pathogens tested, indicative of a defect in a response pathway more specific to B. cinerea. The bos3 and bos4 mutants also show increased susceptibility to Alternaria brassicicola, another necrotrophic pathogen, suggesting a broader role for these loci in resistance. bos4 shows the broadest range of effects on resistance, being more susceptible to avirulent strain of Pseudomonas syringae pv. tomato. Interestingly, bos3 is more resistant than wild-type plants to virulent strains of the biotrophic pathogen Peronospora parasitica and the bacterial pathogen P. syringae pv. tomato. The Pathogenesis Related gene 1 (PR-1), a molecular marker of the salicylic acid (SA)-dependent resistance pathway, shows a wild-type pattern of expression in bos2, while in bos3 this gene was expressed at elevated levels, both constitutively and in response to pathogen challenge. In bos4 plants, PR-1 expression was reduced compared with wild type in response to B. cinerea and SA. In bos3, the mutant most susceptible to B. cinerea and with the highest expression of PR-1, removal of SA resulted in reduced PR-1 expression but no change to the B. cinerea response. Expression of the plant defensin gene PDF1-2 was generally lower in bos mutants compared with wild-type plants, with a particularly strong reduction in bos3. Production of the phytoalexin camalexin is another well-characterized plant defense response. The bos2 and bos4 mutants accumulate reduced levels of camalexin whereas bos3 accumulates significantly higher levels of camalexin than wild-type plants in response to B. cinerea. The BOS2, BOS3, and BOS4 loci may affect camalexin levels and responsiveness to ethylene and jasmonate. The three new mutants appear to mediate disease responses through mechanisms independent of the previously described BOS1 gene. Based on the differences in the phenotypes of the bos mutants, it appears that they affect different points in defense response pathways. [source]