24 research outputs found
Capsid Assembly and Single Stranded DNA Genome Formation of Adeno-Associated Virus Type2 in Yeast Cells
No full textSaccharomyces cerevisiae has provided an array of genetic tools to study unknown aspects
of viral life cycles, supporting replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses). It also provides means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus) and as such represents a useful tool for vaccine development. To extend the utility of the S. cerevisiae expression system, we expressed AAV2 structural and nonstructural proteins in yeast cells, using both authentic AAV2 and heterologous yeast promoters. For the first time, we described the assembly of AAV2 virus-like particles from yeast-expressed AAV2 structural proteins. To do this we used AAV p40 promoter, whose activity in yeast cells resembled the one of yeast glycolytic promoters, resulting in the synthesis of the most abundant capsid protein VP3 when transformed yeast cells were
grown on glucose as a carbon source. The expression of other two VPs was induced from yeast, galactose inducible pGal1 promoter. Simultaneous production of all three VPs was achieved by growing the yeast cells in the medium containing both glucose and galactose, while their relative production levels were further optimized by varying amounts of each
carbon source in the induction medium, followed by the fine tuning of the induction time.
Moreover, we investigated the ability of the yeast Saccharomyces cerevisiae to carry out
the replication of a recombinant rAAV2. When a plasmid harboring the rAAV2 genome in
which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in
yeast with a plasmid expressing Rep68 from constitutive yeast promoter pADH, a
significant number of URA3+ clones were scored (more than 30-fold over controls).
Molecular analysis of low molecular weight DNA revealed that the single stranded DNA is
formed, in Rep68 and ITR dependent manner, and that the plasmid is entirely replicated.
The ss DNA contained the ITRs, URA3 gene and also vector sequences suggesting that ss
rAAV genomes were not obtained by the canonical AAV replication mechanism.
These results could open new prospects for using yeast cell in two ways: (i) as a model
system for studying viral and cellular factors involved in AAV2 capsid assembly and
packaging of rAAV ss genomes; and (ii) as a novel cell factory for developing superior
recombinant rAAV production technologies
Draft Genome Sequence of the Probiotic Yeast Kluyveromyces marxianus fragilis B0399
No full textHere, we report the draft genome sequence of Kluyveromyces marxianus fragilis B0399, the first yeast approved as a probiotic for human consumption not belonging to the genus Saccharomyces The genome is composed of 8 chromosomes, with a total size of 11.44 Mb, including mitochondrial DNA
Capsid protein expression and adeno-associated virus like particles assembly in <it>Saccharomyces cerevisiae</it>
No full textAbstract Background The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast. Results To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells. Conclusions Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.</p
Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae.
No full textThe budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast.To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5\% glucose and 5\% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells.Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology
Between two worlds : approaching Balkan oral music tradition through the use of technology as a compositional and performing medium
No full textThis text explores the problems of interpreting musical identity, meaning, and sociocultural value of a compositional work influenced by two traditions with different values: the modernist tradition based on Western European classical heritage, and the oral tradition of the Balkans. It also follows the process of transformation and recreation of the author's musical language: from classical, notation-oriented to a more intuitive, improvisational and live-performance based. Through detailing some of the
experiences of the author as a composer and a performer, it also discusses some observations on the ways in which this discrepancy between two traditions and practices has affected and still influences those creative practices in Serbia and the former Yugoslavia that relate to traditional music and its derivations. By identifying musical performance within certain socio-cultural contexts this dichotomy can be highlighted. As a result, a substantial part of this text focuses on investigating the
capacity of a technologically assisted composition and performance practice to overcome this issue. Technology is here perceived not only as an instrument for
recording, improvising, composing and performing but also as a medium which communicates musical value. In this study, the oral tradition from the Balkans was approached not only as a purely acoustic phenomenon, but it also included a raised awareness of the nature of the continuous fusion of various cultures in the region, as
well as existing cultural and religious antagonisms. This study investigates the problems of constructing musical identity as well as the meaning of an author’s creative practice in relation to the socio-cultural environment of its origin, whilst observing its reception by audiences outside the Balkan region. Socio-cultural environments are established through exploring the writings of the authors that depict the Balkans historical, cultural and musical spheres in relation to other cultural practices and influences
Genetic tracing and topography of spontaneous and stimulated cardiac regeneration in mice
No full textDespite recent efforts to stimulate endogenous cardiomyocyte proliferation for cardiac regeneration, the lack of reliable in vivo methods for monitoring cardiomyocyte replication has hindered our understanding of its mechanisms. Thymidine analogs, used to label proliferating cells, are unsuitable for long-term cardiac regeneration studies as their DNA incorporation elicits a damage response, leading to their elimination. Here we present CycleTrack, a genetic strategy based on the transcriptional activation of Cre recombinase from a temporally regulated cyclin B2 promoter segment, for permanent labeling of cardiomyocytes passing through the G2/M phase. Using CycleTrack, we visualized cardiomyocyte turnover in neonatal and adult mice under various conditions, including pregnancy, increased ventricular afterload, and myocardial infarction. CycleTrack also provided visual and quantitative evidence of ventricular remuscularization following treatment with pro-regenerative microRNAs. We identify the subendocardium as a key site of mitotic activity and provide a mode of cardiomyocyte division along their short axis. CycleTrack is a powerful tool to monitor cardiomyocyte renewal during regenerative interventions
Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.
No full textAdeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway
The frequency of colonies carrying the AAV genome increased when Rep68 is expressed.
No full text<p>The plasmid pAAVRepURA3, pAAVpokURA linearized with <i>Pvu</i>II and the control plasmid, pRepURA3, digested with <i>Xba</i>I, were co-transformed with the plasmid pGAD424 or pG.Rep68 containing the <i>LEU2</i> marker gene (A, B). (A) Representative plates comparing colonies obtained from the transformed yeast RSY12 strain with plasmid pRepURA and pGAD424 (i), pRepURA and pG.Rep68 (ii), pAAVRepURA and pGAD424 (iii); pAAVRepURA and pG.Rep68 (iv). (B) Transformed yeast cells were scored for their ability to form colonies on selective medium lacking leucine and uracil. The frequency was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023474#s2" target="_blank">material and methods</a>. Results are the mean of 4 independent experiments±standard deviation.</p
S1 and <i>Mung Bean</i> nuclease sensitivity.
No full text<p>(A, B) Low M<sub>r</sub> DNA from a URA3<sup>+</sup>LEU2<sup>+</sup> clone derived from co-transformation of pAAVRepURA with plasmid pG.Rep68, was digested with <i>Dpn</i>I (loaded in the lane 2), <i>Mbo</i>I (loaded in the lane 3) , S1 nuclease (in the lane 4) and was analyzed on Southern Blot using URA3 probe (A) or ITR probe (B). (C) Southern blot analysis of low M<sub>r</sub> of two URA3<sup>+</sup>LEU2<sup>+</sup> clone derived from co-transformation of pAAVRepURA with plasmid pG.Rep68 was digested with <i>Mung Bean</i> nuclease (in the lane lane 2 and 4) and compared with the not digested DNA (lane 1, 3). DNA was detected using the URA3 probe. The arrows indicate the ssDNA which disappeared after digestion with S1 and <i>Mung Bean</i> nuclease.</p
Diagram model showing ssDNA formation from a plasmid containing the AAV genome in yeast.
No full text<p>(A) For the sake of clarity, the plasmid carrying ITRs is depicted as linear molecule. Rep nicks at the <i>trs</i> (white star) (B). A replication complex is assembled and replication commences through the ITR towards the vector (C). The new synthesized ITR fold into a hairpin conformation displacing the replication strand. Such displacement determines a template switch so that the originally nicked strand is copied during replication. The replication passes through the other ITR and proceeds into the vector sequence (D). After replication fork has completed a full circle, Rep produces a second nick and the newly synthesized DNA is displaced as ssDNA (E). The new ssDNA is nicked again and two ssDNA containing only one complete ITR are formed (F). The missing ITR is repaired by gene correction mechanism (G).</p
