Due to the high number of drug-resistant HIV-1 mutants generated by highly active antiretroviral therapy (HAART), there is continuing demand for new types of inhibitors. Both the assembly of the Gag polyprotein into immature and mature HIV-1 particles are attractive candidates for the blocking of the retroviral life cycle. Currently, no therapeutically-used assembly inhibitor is available. One possible explanation is the lack of a reliable and simple assembly inhibitor screening method. To identify compounds potentially inhibiting the formation of both types of HIV-1 particles, we developed a new fluorescent high-throughput screening assay. This assay is based on the quantification of the assembly efficiency in vitro in a 96-well plate format. The key components of the assay are HIV-1 Gag-derived proteins and a dual-labelled oligonucleotide, which emits fluorescence only when the assembly of retroviral particles is inhibited. The method was validated using three (CAI, BM2, PF74) reported assembly inhibitors.

• Klíčová slova
• Assay, Assembly, CA, Capsid, HIV, High-throughput screening, Inhibitor, Retrovirus,
• MeSH
• genové produkty gag metabolismus   MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 účinky léků   fyziologie   MeSH
• látky proti HIV farmakologie   MeSH
• lidé MeSH
• preklinické hodnocení léčiv metody   MeSH
• rychlé screeningové testy metody   MeSH
• sestavení viru účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty gag MeSH
• látky proti HIV MeSH

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

• Klíčová slova
• CAH *, IP6 *, M-PMV *, MLV *, RSV *, SP domain *, assembly *, hexamer *, immature *, polyanion *,
• MeSH
• Alpharetrovirus fyziologie   MeSH
• Betaretrovirus fyziologie   MeSH
• buněčná membrána chemie   metabolismus   MeSH
• Gammaretrovirus fyziologie   MeSH
• genové produkty gag chemie   metabolismus   MeSH
• interakce hostitele a patogenu * MeSH
• kultivované buňky MeSH
• polyelektrolyty chemie   metabolismus   MeSH
• Retroviridae fyziologie   ultrastruktura   MeSH
• sestavení viru * MeSH
• virion MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty gag MeSH
• polyanions MeSH Prohlížeč
• polyelektrolyty MeSH

Chromatin Assembly Factor 1 (CAF-1) is a major nucleosome assembly complex which functions particularly during DNA replication and repair. Here we studied how the nucleosome landscape changes in a CAF-1 mutant in the model plant Arabidopsis thaliana. Globally, most nucleosomes were not affected by loss of CAF-1, indicating the presence of efficient alternative nucleosome assemblers. Nucleosomes that we found depleted in the CAF-1 mutant were enriched in non-transcribed regions, consistent with the notion that CAF-1-independent nucleosome assembly can compensate for loss of CAF-1 mainly in transcribed regions. Depleted nucleosomes were particularly enriched in proximal promoters, suggesting that CAF-1-independent nucleosome assembly mechanisms are often not efficient upstream of transcription start sites. Genes related to plant defense were particularly prone to lose nucleosomes in their promoters upon CAF-1 depletion. Reduced nucleosome occupancy at promoters of many defense-related genes is associated with a primed gene expression state that may considerably increase plant fitness by facilitating plant defense. Together, our results establish that the nucleosome landscape in Arabidopsis is surprisingly robust even in the absence of the dedicated nucleosome assembly machinery CAF-1 and that CAF-1-independent nucleosome assembly mechanisms are less efficient in particular genome regions.

• Klíčová slova
• Arabidopsis thaliana, CAF-1, GSE87421, chromatin, plant defense,
• MeSH
• Arabidopsis genetika   imunologie   metabolismus   MeSH
• chromatin genetika   MeSH
• faktor 1 pro uspořádání chromatinu genetika   metabolismus   MeSH
• imunita rostlin genetika   MeSH
• mutace MeSH
• nukleozomy genetika   metabolismus   MeSH
• oprava DNA genetika   MeSH
• počátek transkripce MeSH
• promotorové oblasti (genetika) genetika   MeSH
• replikace DNA genetika   MeSH
• restrukturace chromatinu genetika   MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromatin MeSH
• faktor 1 pro uspořádání chromatinu MeSH
• nukleozomy MeSH

Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.

• Klíčová slova
• HIV-1, IP6, assembly, capsid, immature, mature, polyanion,
• MeSH
• genové produkty gag - virus lidské imunodeficience chemie   genetika   metabolismus   MeSH
• HIV-1 chemie   genetika   MeSH
• missense mutace MeSH
• polyelektrolyty MeSH
• polymery chemie   MeSH
• sestavení viru * MeSH
• substituce aminokyselin MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty gag - virus lidské imunodeficience MeSH
• polyanions MeSH Prohlížeč
• polyelektrolyty MeSH
• polymery MeSH

Chromatin Assembly Factor 1 (CAF1) is a three-subunit H3/H4 histone chaperone responsible for replication-dependent nucleosome assembly. It is composed of CAC 1-3 in yeast; p155, p60, and p48 in humans; and FASCIATA1 (FAS1), FAS2, and MULTICOPY SUPPRESSOR OF IRA1 in Arabidopsis thaliana. We report that disruption of CAF1 function by fas mutations in Arabidopsis results in telomere shortening and loss of 45S rDNA, while other repetitive sequences (5S rDNA, centromeric 180-bp repeat, CACTA, and Athila) are unaffected. Substantial telomere shortening occurs immediately after the loss of functional CAF1 and slows down at telomeres shortened to median lengths around 1 to 1.5 kb. The 45S rDNA loss is progressive, leaving 10 to 15% of the original number of repeats in the 5th generation of mutants affecting CAF1, but the level of the 45S rRNA transcripts is not altered in these mutants. Increasing severity of the fas phenotype is accompanied by accumulation of anaphase bridges, reduced viability, and plant sterility. Our results show that appropriate replication-dependent chromatin assembly is specifically required for stable maintenance of telomeres and 45S rDNA.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• DNA rostlinná genetika   metabolismus   MeSH
• faktor 1 pro uspořádání chromatinu genetika   metabolismus   MeSH
• inzerční mutageneze MeSH
• mutace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• restrukturace chromatinu MeSH
• ribozomální DNA genetika   metabolismus   MeSH
• RNA ribozomální genetika   metabolismus   MeSH
• telomery metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• faktor 1 pro uspořádání chromatinu MeSH
• FAS protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• ribozomální DNA MeSH
• RNA ribozomální MeSH
• RNA, ribosomal, 45S MeSH Prohlížeč

Dysfunction of chromatin assembly factor 1 in FASCIATA mutants (fas) of Arabidopsis thaliana results in progressive loss of telomeric DNA. Although replicative telomere shortening is typically associated with incomplete resynthesis of their ends by telomerase, no change in telomerase activity could be detected in vitro in extracts from fas mutants. Besides a possible telomerase malfunction, the telomere shortening in fas mutants could presumably be due to problems with conventional replication of telomeres. To distinguish between the possible contribution of suboptimal function of telomerase in fas mutants under in vivo conditions and problems in conventional telomere replication, we crossed fas and tert (telomerase reverse transcriptase) knockout mutants and analyzed telomere shortening in segregated fas mutants, tert mutants, and double fas tert mutants in parallel. We demonstrate that fas tert knockouts show greater replicative telomere shortening than that observed even in the complete absence of telomerase (tert mutants). While the effect of tert and fas mutations on telomere lengths in double mutants is additive, manifestations of telomere dysfunction in double fas tert mutants (frequency of anaphase bridges, onset of chromosome end fusions, and common involvement of 45S rDNA in chromosome fusion sites) are similar to those in tert mutants. We conclude that in addition to possible impairment of telomerase action, a further mechanism contributes to telomere shortening in fas mutants.

• MeSH
• Arabidopsis enzymologie   genetika   metabolismus   MeSH
• chromozomy rostlin genetika   metabolismus   MeSH
• faktor 1 pro uspořádání chromatinu genetika   metabolismus   MeSH
• mutace * MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• sestřihové faktory MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• At2g20020 protein, Arabidopsis MeSH Prohlížeč
• faktor 1 pro uspořádání chromatinu MeSH
• FAS protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• sestřihové faktory MeSH
• telomerasa MeSH
• TERT protein, Arabidopsis MeSH Prohlížeč

Tubulin self-assembly into microtubules is a fascinating natural phenomenon. Its importance is not just crucial for functional and structural biological processes, but it also serves as an inspiration for synthetic nanomaterial innovations. The modulation of the tubulin self-assembly process without introducing additional chemical inhibitors/promoters or stabilizers has remained an elusive process. This work reports a versatile and vigorous strategy for controlling tubulin self-assembly by nanosecond electropulses (nsEPs). The polymerization assessed by turbidimetry is dependent on nsEPs dosage. The kinetics of microtubules formation is tightly linked to the nsEPs effects on structural properties of tubulin, and tubulin-solvent interface, assessed by autofluorescence, and the zeta potential. Moreover, the overall size of tubulin assessed by dynamic light scattering is affected as well. Additionally, atomic force microscopy imaging reveals the formation of different assemblies reflecting applied nsEPs. It is suggested that changes in C-terminal modification states alter tubulin polymerization-competent conformations. Although the assembled tubulin preserve their integral structure, they might exhibit a broad range of new properties important for their functions. Thus, these transient conformation changes of tubulin and their collective properties can result in new applications.

• Klíčová slova
• microtubules, nanosecond pulsed electric field, self-assembly, tubulin,
• MeSH
• elektřina * MeSH
• hydrodynamika MeSH
• kinetika MeSH
• kvarterní struktura proteinů MeSH
• mikrotubuly metabolismus   MeSH
• molekulární modely MeSH
• multimerizace proteinu * MeSH
• tubulin chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• tubulin MeSH

BACKGROUND: There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. RESULTS: We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. CONCLUSIONS: We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

• Klíčová slova
• 7DS, Assembly, BAC, Next-generation sequencing, SASSY, Saccharum spp, Triticum aestivum,
• Publikační typ
• časopisecké články MeSH

UNLABELLED: The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-Å cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly. IMPORTANCE: Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses, and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroviruses, such as HIV, Rous sarcoma virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

• MeSH
• cirkulární dichroismus MeSH
• elektronová kryomikroskopie MeSH
• konformace proteinů MeSH
• Masonův-Pfizerův opičí virus fyziologie   MeSH
• molekulární modely MeSH
• nukleokapsida - proteiny chemie   genetika   metabolismus   ultrastruktura   MeSH
• sestavení viru * MeSH
• uvolnění viru z buňky MeSH
• virové plášťové proteiny chemie   genetika   metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nukleokapsida - proteiny MeSH
• virové plášťové proteiny MeSH

PREMISE OF THE STUDY: Red clover (Trifolium pratense) is an important forage plant from the legume family with great importance in agronomy and livestock nourishment. Nevertheless, assembling its medium-sized genome presents a challenge, given current hardware and software possibilities. Next-generation sequencing technologies enable us to generate large amounts of sequence data at low cost. In this study, the genome assembly and red clover genome features are presented. METHODS: First, assembly software was assessed using data sets from a closely related species to find the best possible combination of assembler plus error correction program to assemble the red clover genome. The newly sequenced genome was characterized by repetitive content, number of protein-coding and nonprotein-coding genes, and gene families and functions. Genome features were also compared with those of other sequenced plant species. KEY RESULTS: Abyss with Echo correction was used for de novo assembly of the red clover genome. The presented assembly comprises ∼314.6 Mbp. In contrast to leguminous species with comparable genome sizes, the genome of T. pratense contains a larger repetitive portion and more abundant retrotransposons and DNA transposons. Overall, 47 398 protein-coding genes were annotated from 64 761 predicted genes. Comparative analysis revealed several gene families that are characteristic for T. pratense. Resistance genes, leghemoglobins, and nodule-specific cystein-rich peptides were identified and compared with other sequenced species. CONCLUSIONS: The presented red clover genomic data constitute a resource for improvement through molecular breeding and for comparison to other sequenced plant species.

• Klíčová slova
• Fabaceae, Trifolium pratense, assessment of assembly software, de novo assembly, genome annotation, red clover,
• MeSH
• DNA rostlinná analýza   MeSH
• genom rostlinný * MeSH
• mapování chromozomů MeSH
• rostlinné geny * MeSH
• rostlinné proteiny genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• Trifolium genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• rostlinné proteiny MeSH