RESUMEN
The high variability of the influenza A virus poses a significant threat to public health, therefore monitoring viral strains and studying their genetic properties are important tasks. One part of this monitoring includes sequencing of influenza A viruses of any subtype and analysis of their whole genomes, which is especially important in cases of interspecies adaptation and reassortment. High-throughput sequencing technologies have significantly extended the capabilities of influenza virus epidemiological surveillance. The preparation stages for next generation sequencing (NGS) of influenza A virus include whole genome amplification using one-step RT-PCR, the results of which vary greatly depending on the sample type and quality, that, in turn, affects the coverage of virus fragments and the sequencing results in general. In this work, we propose to supplement the aforementioned technique of whole genome amplification of influenza A virus with sequential suppression PCRs to obtain an even coverage of viral segments of different lengths, which allows sequencing of samples with lower read coverage without decreasing the sequencing quality.
Asunto(s)
Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Virus de la Influenza A , Virus de la Influenza A/genética , Reacción en Cadena de la PolimerasaRESUMEN
The family of proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria exhibits striking diversity of features, including several different types of autocatalytically synthesized chromophores. Here we report 11 new members of the family, among which there are 3 red-emitters possessing unusual features, and discuss the similarity relationships within the family in structural, spectroscopic, and evolutionary terms. Phylogenetic analysis has shown that GFP-like proteins from representatives of subclass Zoantharia fall into at least four distinct clades, each clade containing proteins of more than one emission color. This topology suggests multiple recent events of color conversion. Combining this result with previous mutagenesis and structural data, we propose that (i) different chromophore structures are alternative products synthesized within a similar autocatalytic environment, and (ii) the phylogenetic pattern and color diversity in reef Anthozoa is a result of a balance between selection for GFP-like proteins of particular colors and mutation pressure driving the color conversions.
Asunto(s)
Proteínas Luminiscentes/química , Secuencia de Bases , Color , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/clasificación , Datos de Secuencia Molecular , Filogenia , Terminología como AsuntoRESUMEN
We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission lambda(max)=615-640 nm). Moreover, coupled site-directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.
Asunto(s)
Proteínas Luminiscentes/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Escherichia coli/genética , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , TransfecciónRESUMEN
BACKGROUND: Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (approximately 485 nm), green (approximately 505 nm), yellow (approximately 540 nm), and red (>580 nm) emitters. RESULTS: Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence. CONCLUSIONS: Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals.