Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco

Nagaki, Kiyotaka; Shibata, Fumiaki; Kanatani, Asaka; Kashihara, Kazunari; Murata, Minoru

doi:10.1007/s00299-011-1198-4

Cited by 14 publications

(11 citation statements)

References 41 publications

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“…By contrast, the monomeric lengths of newly emerged centromeric repeats vary widely, ranging from a few hundred base pairs to several kilobases (Gong et al ., ; Zhang et al ., ). Centromeric satellite repeats with ‘odd’ monomeric lengths, which are significantly deviated from the mono‐nucleosomal length, were also reported in several plant species (Lee et al ., ; Nagaki et al ., ; Neumann et al ., ; Iwata et al ., ; Melters et al ., ; Kowar et al ., ). Interestingly, these repeats were often restricted to a few centromeres and have not been spread to all centromeres (Lee et al ., ; Nagaki et al ., ; Neumann et al ., ; Iwata et al ., ).…”

Section: Discussionmentioning

confidence: 98%

Amplification and adaptation of centromeric repeats in polyploid switchgrass species

et al. 2018

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Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats from a single satellite repeat family. Why centromeres are dominated by a single satellite repeat and how the satellite repeats originate and evolve are among the most intriguing and long-standing questions in centromere biology. We identified eight satellite repeats in the centromeres of tetraploid switchgrass (Panicum virgatum). Seven repeats showed characteristics associated with classical centromeric repeats with monomeric lengths ranging from 166 to 187 bp. Interestingly, these repeats share an 80-bp DNA motif. We demonstrate that this 80-bp motif may dictate translational and rotational phasing of the centromeric repeats with the cenH3 nucleosomes. The sequence of the last centromeric repeat, Pv156, is identical to the 5S ribosomal RNA genes. We demonstrate that a 5S ribosomal RNA gene array was recruited to be the functional centromere for one of the switchgrass chromosomes. Our findings reveal that certain types of satellite repeats, which are associated with unique sequence features and are composed of monomers in mono-nucleosomal length, are favorable for centromeres. Centromeric repeats may undergo dynamic amplification and adaptation before the centromeres in the same species become dominated by the best adapted satellite repeat.

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Section: Discussionmentioning

confidence: 98%

Amplification and adaptation of centromeric repeats in polyploid switchgrass species

et al. 2018

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“…Given the fact that there are other examples of centromeric satellite monomers with non‐nucleosome unit sizes [e.g. 91‐, 92‐ and 411–bp CentGm families in soybean; 126–bp CentO‐C1 and 366–bp CentO‐C2 in Oryza rhizomatis ; 48‐, 68‐, 91‐ and 92–bp in Nicotiana tabacum (tobacco)], the conservation of centromeric satellite monomer length around 150–180 bp is not universal across species (Lee et al ., ; Gill et al ., ; Tek et al ., ; Nagaki et al ., ; Melters et al ., ).…”

Section: Discussionmentioning

confidence: 99%

“…As the antibody showed basal non‐specific binding to DNA (Tek et al ., ), all the sequences were increased in these pellet fractions (Pel). Such non‐specific binding has been more conspicuous in ChIP using magnetic beads than that using sepharose beads, as magnetic beads have lower non‐specific binding than sepharose (Nagaki et al ., , ,b; Tek et al ., ); however, the non‐centromeric controls and candidate centromeric repeats were significantly differently enriched. Khipu was not enriched in Pel compared with rDNA [ P = 0.48, Tukey's honestly significant difference (Tukey's HSD) test].…”

Section: Centpv1 and Centpv2 Are Co‐localized With The Anti‐gmcenh3 Amentioning

confidence: 99%

“…CENH3 homologs have been identified in all eukaryotic genomes studied so far (Jiang et al ., ). Recently, immunofluorescence and chromatin immunoprecipiation (ChIP) assays using anti‐CENH3 antibodies have successfully identified satellite repeats at functional centromeres from several plant species, confirming their functional significance at these centromeres (Zhong et al ., ; Nagaki et al ., , , ; Nagaki and Murata, ; Houben et al ., ; Tek et al ., , ).…”

Section: Introductionmentioning

confidence: 99%

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Identification and characterization of functional centromeres of the common bean

et al. 2013

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SUMMARYIn higher eukaryotes, centromeres are typically composed of megabase-sized arrays of satellite repeats that evolve rapidly and homogenize within a species' genome. Despite the importance of centromeres, our knowledge is limited to a few model species. We conducted a comprehensive analysis of common bean (Phaseolus vulgaris) centromeric satellite DNA using genomic data, fluorescence in situ hybridization (FISH), immunofluorescence and chromatin immunoprecipitation (ChIP). Two unrelated centromere-specific satellite repeats, CentPv1 and CentPv2, and the common bean centromere-specific histone H3 (PvCENH3) were identified. FISH showed that CentPv1 and CentPv2 are predominantly located at subsets of eight and three centromeres, respectively. Immunofluorescence-and ChIP-based assays demonstrated the functional significance of CentPv1 and CentPv2 at centromeres. Genomic analysis revealed several interesting features of CentPv1 and CentPv2: (i) CentPv1 is organized into an higher-order repeat structure, named Nazca, of 528 bp, whereas CentPv2 is composed of tandemly organized monomers; (ii) CentPv1 and CentPv2 have undergone chromosome-specific homogenization; and (iii) CentPv1 and CentPv2 are not likely to be commingled in the genome. These findings suggest that two distinct sets of centromere sequences have evolved independently within the common bean genome, and provide insight into centromere satellite evolution.

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“…Since its introduction, HaloTag technology has been used predominantly for applications in imaging [8, 31-35] and for applications that require protein immobilization on a solid support [36-38]. To a lesser extent, HaloTag has been used for protein purification [9, 28, 29].…”

Section: Discussionmentioning

confidence: 99%

Application of HaloTag technology to expression and purification of cannabinoid receptor CB2

Locatelli-Hoops

Sheen

Zoubak

et al. 2013

Protein Expression and Purification

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Expression of milligram quantities of functional, stable G protein-coupled receptors (GPCR) for high-resolution structural studies remains a challenging task. The goal of this work was to evaluate the usefulness of the HaloTag system (Promega) for expression and purification of the human cannabinoid receptor CB2, an important target for development of drugs for treatment of immune disorders, inflammation, and pain. Here we investigated expression in Escherichia coli cells of the integral membrane receptor CB2 as a fusion with the 34 kDa HaloTag at N- or C-terminal location, either in the presence or in the absence of the N-terminal maltose-binding protein (MBP). The CB2 was flanked at both ends by the tobacco etch virus (TEV) protease cleavage sites to allow for subsequent removal of expression partners. Expression by induction with either IPTG (in E. coli BL21(DE3) cell cultures) or by auto-induction (in E. coli KRX cells) were compared. While the N-terminal location of the HaloTag resulted in high levels of expression of the fusion CB2, the recombinant receptor was not functional. However, when the HaloTag was placed in the C-terminal location, a fully active receptor was produced irrespective of induction method or bacterial strain used. For purification, the fusion protein was captured onto HaloLink resin in the presence of detergents. Treatment with specific TEV protease released the CB2 upon washing. To our knowledge, this study represents the first example of expression, surface immobilization and purification of a functional GPCR using HaloTag technology.

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Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco

Cited by 14 publications

References 41 publications

Amplification and adaptation of centromeric repeats in polyploid switchgrass species

Amplification and adaptation of centromeric repeats in polyploid switchgrass species

Identification and characterization of functional centromeres of the common bean

Application of HaloTag technology to expression and purification of cannabinoid receptor CB2

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