A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and drought-tolerance mechanisms.

Plants that grow in extreme environments represent unique sources of stress-resistance genes and mechanisms. Ammopiptanthus mongolicus (Leguminosae) is a xerophytic evergreen broadleaf shrub native to semi-arid and desert regions; however, its drought-tolerance mechanisms remain poorly understood. Here, we report the assembly of a reference-grade genome for A. mongolicus, describe its evolutionary history within the legume family, and examine its drought-tolerance mechanisms. The assembled genome is 843.07 Mb in length, with 98.7% of the sequences successfully anchored to the nine chromosomes of A. mongolicus. The genome is predicted to contain 47 611 protein-coding genes, and 70.71% of the genome is composed of repetitive sequences; these are dominated by transposable elements, particularly long-terminal-repeat retrotransposons. Evolutionary analyses revealed two whole-genome duplication (WGD) events at 130 and 58 million years ago (mya) that are shared by the genus Ammopiptanthus and other legumes, but no species-specific WGDs were found within this genus. Ancestral genome reconstruction revealed that the A. mongolicus genome has undergone fewer rearrangements than other genomes in the legume family, confirming its status as a "relict plant". Transcriptomic analyses demonstrated that genes involved in cuticular wax biosynthesis and transport are highly expressed, both under normal conditions and in response to polyethylene glycol-induced dehydration. Significant induction of genes related to ethylene biosynthesis and signaling was also observed in leaves under dehydration stress, suggesting that enhanced ethylene response and formation of thick waxy cuticles are two major mechanisms of drought tolerance in A. mongolicus. Ectopic expression of AmERF2, an ethylene response factor unique to A. mongolicus, can markedly increase the drought tolerance of transgenic Arabidopsis thaliana plants, demonstrating the potential for application of A. mongolicus genes in crop improvement.

Mindsets and self-efficacy beliefs among individuals with type 2 diabetes.

Growth mindsets and self-efficacy beliefs have been known to predict and promote resilience, challenge seeking, and improved outcomes in areas such as education and intelligence. However, little is known about the role of these two potentially influential beliefs in the context of type 2 diabetes (T2D), specifically in terms of whether and in which domains (i.e., beliefs toward general life, general health, or condition-specific domains) these beliefs-or lack thereof-is prevalent among individuals with T2D. Given the lifelong challenges that individuals with diabetes often encounter with managing their disease, many may slip into a conceding negative belief that their diabetes is "too difficult to control" or simply "out of their hands," inhibiting proactive self-management efforts. Results from our study (n = 893) revealed that individuals with T2D had a significantly lower growth mindset towards their blood glucose level and lower self-efficacy towards their general health, blood glucose, and cholesterol levels compared to those without T2D. Among participants with T2D, further analyses showed a pattern of higher HbA1c among those with lower growth mindsets and self-efficacy toward their general health or blood glucose level. These findings identify the belief-domains that may pose barriers to necessary self-care behaviors, informing future interventions to promote improved diabetes care and management.

Advances in omics research on peanut response to biotic stresses.

Peanut growth, development, and eventual production are constrained by biotic and abiotic stresses resulting in serious economic losses. To understand the response and tolerance mechanism of peanut to biotic and abiotic stresses, high-throughput Omics approaches have been applied in peanut research. Integrated Omics approaches are essential for elucidating the temporal and spatial changes that occur in peanut facing different stresses. The integration of functional genomics with other Omics highlights the relationships between peanut genomes and phenotypes under specific stress conditions. In this review, we focus on research on peanut biotic stresses. Here we review the primary types of biotic stresses that threaten sustainable peanut production, the multi-Omics technologies for peanut research and breeding, and the recent advances in various peanut Omics under biotic stresses, including genomics, transcriptomics, proteomics, metabolomics, miRNAomics, epigenomics and phenomics, for identification of biotic stress-related genes, proteins, metabolites and their networks as well as the development of potential traits. We also discuss the challenges, opportunities, and future directions for peanut Omics under biotic stresses, aiming sustainable food production. The Omics knowledge is instrumental for improving peanut tolerance to cope with various biotic stresses and for meeting the food demands of the exponentially growing global population.

Identification of short open reading frames in plant genomes.

The roles of short/small open reading frames (sORFs) have been increasingly recognized in recent years due to the rapidly growing number of sORFs identified in various organisms due to the development and application of the Ribo-Seq technique, which sequences the ribosome-protected footprints (RPFs) of the translating mRNAs. However, special attention should be paid to RPFs used to identify sORFs in plants due to their small size (~30 nt) and the high complexity and repetitiveness of the plant genome, particularly for polyploidy species. In this work, we compare different approaches to the identification of plant sORFs, discuss the advantages and disadvantages of each method, and provide a guide for choosing different methods in plant sORF studies.

Long non-coding RNA PRNCR1 promotes ovarian cancer cell proliferation, migration and invasion by targeting the miR-653-5p/ELF2 axis.

Recent studies have shown that prostate cancer-associated long non-coding RNA, PRNCR1, plays crucial roles in the development of multiple human cancers. However, its role in ovarian cancer is barely known. This study was carried out to investigate the role of PRNCR1 and the underlying mechanisms in OC. The expression of PRNCR1 and miR-653-5p in OC cell lines and tissues were detected by qRT-PCR. The expression of ELF2 protein was evaluated by Western blot analysis. Cell proliferation was measured by colony formation and MTT assay. Cell invasion and migration were evaluated by Transwell and wound healing assay. Luciferase reporter assay and RNA-binding protein immunoprecipitation assay were performed to determine the interaction between miR-653-5p and PRNCR1, as well as between miR-653-5p and ELF2. In vivo tumor xenograft model was established to evaluate the role of PRNCR1 in tumor growth. Our results demonstrated that PRNCR1 was significantly upregulated in both OC cell lines and tissues, and high expression of PRNCR1 was correlated with poor survival of OC patients. Overexpression of PRNCR1 accelerated OC cell invasion, migration and proliferation. Besides, the expression of PRNCR1 was negatively correlated with the expression of miR-653-5p, while positively correlated with the expression of E74-like factor 2 in OC tissues. Importantly, ELF2 could target miR-653-5p, and PRNCR1 increased the expression levels of ELF2 by sponging miR-653-5p in OC cells. Furthermore, the miR-145-5p/ELF2 axis was involved in the regulation of PRNCR1 in OC progression in vivo. PRNCR1 promotes OC tumor progress via the miR-653-5p/ELF2 axis and might be a potential therapeutic target for OC.

The novel circ_0084904/miR-802/MAL2 axis promotes the development of cervical cancer.

Circular RNAs (circRNAs) have been identified as critical regulators in human cancers, including cervical cancer (CC). However, the precise action of circ_0084904 in cervical carcinogenesis remains to be elucidated. The levels of circ_0084904, microRNA (miR)-802, and Mal, T cell differentiation protein 2 (MAL2) were checked by quantitative real-time PCR (qRT-PCR) or western blot. Ribonuclease R (RNase R) and subcellular localization assays were used to detect the stability and localization of circ_0084904, respectively. Cell colony formation ability was assessed by colony formation assay. Cell cycle and apoptosis were detected by flow cytometry. Cell migration and invasion abilities were gauged by transwell assay. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were applied to determine the direct relationship between miR-802 and circ_0084904 or MAL2. The xenograft experiments were performed to evaluate the role of circ_0084904 in tumor growth in vivo. Circ_0084904 was markedly up-regulated in CC tissues and cell lines. Silencing endogenous circ_0084904 impeded cell colony formation, cell cycle progression, migration, invasion, epithelial-mesenchymal transition (EMT), and promoted apoptosis in vitro, as well as diminished tumor growth in vivo. Mechanistically, circ_0084904 targeted miR-802, and the effects of circ_0084904 silencing were mediated by miR-802. MAL2 was directly targeted and inhibited by miR-802, and MAL2 was a functional target of miR-802. Moreover, circ_0084904 modulated MAL2 expression via miR-802. Our study identified circ_0084904 as a novel oncogenic driver in CC depending on the modulation of the miR-802/MAL2 axis, establishing the notion that silencing of circ_0084904 might represent a promising targeted therapy for CC.

The R2R3-type MYB transcription factor MdMYB90-like is responsible for the enhanced skin color of an apple bud sport mutant.

The anthocyanin content in apple skin determines its red coloration, as seen in a Fuji apple mutant. Comparative RNA-seq analysis was performed to determine differentially expressed genes at different fruit development stages between the wild-type and the skin color mutant. A novel R2R3-MYB transcription factor, MdMYB90-like, was uncovered as the key regulatory gene for enhanced coloration in the mutant. The expression of MdMYB90-like was 21.3 times higher in the mutant. MdMYB90-like regulates anthocyanin biosynthesis directly through the activation of anthocyanin biosynthesis genes and indirectly through the activation of other transcription factors that activate anthocyanin biosynthesis. MdMYB90-like bound to the promoters of both structural genes (MdCHS and MdUFGT) and other transcription factor genes (MdMYB1 and MdbHLH3) in the yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay. Transgenic analysis showed that MdMYB90-like was localized in the nucleus, and its overexpression induced the expression of other anthocyanin-related genes, including MdCHS, MdCHI, MdANS, MdUFGT, MdbHLH3, and MdMYB1. The mutant had reduced levels of DNA methylation in two regions (-1183 to -988 and -2018 to -1778) of the MdMYB90-like gene promoter, which might explain the enhanced expression of the gene and the increased anthocyanin content in the mutant apple skin.

The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication.

High oil and protein content make tetraploid peanut a leading oil and food legume. Here we report a high-quality peanut genome sequence, comprising 2.54 Gb with 20 pseudomolecules and 83,709 protein-coding gene models. We characterize gene functional groups implicated in seed size evolution, seed oil content, disease resistance and symbiotic nitrogen fixation. The peanut B subgenome has more genes and general expression dominance, temporally associated with long-terminal-repeat expansion in the A subgenome that also raises questions about the A-genome progenitor. The polyploid genome provided insights into the evolution of Arachis hypogaea and other legume chromosomes. Resequencing of 52 accessions suggests that independent domestications formed peanut ecotypes. Whereas 0.42-0.47 million years ago (Ma) polyploidy constrained genetic variation, the peanut genome sequence aids mapping and candidate-gene discovery for traits such as seed size and color, foliar disease resistance and others, also providing a cornerstone for functional genomics and peanut improvement.

Identification of miRNAs and their targets in maize in response to Sugarcane mosaic virus infection.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play essential regulatory roles in plant development and environmental stress responses. Maize (Zea mays L.) is a global economically important food and forage crop. To date, a number of maize miRNAs have been identified as being involved in plant development and stress responses. However, the miRNA-mediated gene regulatory networks responsive to virus infections in maize remain largely unknown. In this study, the profiles of small RNAs in buffer- and Sugarcane mosaic virus (SCMV)-inoculated maize plants were obtained by high-throughput sequencing, respectively. A total of 154 known miRNAs and 213 novel miRNAs were profiled and most of the miRNAs identified were differentially expressed after SCMV infection. In addition, 70 targets of 13 known miRNAs and 3 targets of a novel miRNA were identified by degradome analysis. The results of Northern blotting and quantitative real-time PCR showed that the expression levels of the selected miRNAs and their targets were mostly influenced by SCMV infection at 12 days post inoculation, including up-regulation of miR168 and miR528, and down-regulation of miR159, miR397 and miR827. These results provide new insights into the regulatory networks of miRNAs and their targets in maize plants responsive to SCMV infection.

Knocking out of carotenoid catabolic genes in rice fails to boost carotenoid accumulation, but reveals a mutation in strigolactone biosynthesis.

KEY MESSAGE: Targeted mutations in five carotenoid catabolism genes failed to boost carotenoid accumulation in rice seeds, but produced dwarf and high tillering mutants when OsCCD7 gene was knocked out. Carotenoids play an important role in human diet as a source of vitamin A. Rice is a major staple food in Asia, but does not accumulate carotenoids in the endosperm because of the low carotenoid biosynthesis or the degradation in metabolism. In this study, the CRISPR/Cas9 system was investigated in the targeted knockout of five rice carotenoid catabolic genes (OsCYP97A4, OsDSM2, OsCCD4a, OsCCD4b and OsCCD7) and in an effort to increase ß-carotene accumulation in rice endosperm. Transgenic plants that expressed OsNLSCas9 and sgRNAs were generated by Agrobacterium-mediated transformation. Various knockout mutations were identified at the T0 generation of the transgenic rice by TILLING and direct sequencing of the PCR products amplified from the target sites. Carotenoids were not accumulated in both mono-allelic and bi-allelic knockout mutations of the five genes. However, transgenic plants with homozygous or bi-allelic mutations to the OsCCD7 gene were extremely dwarfish with more tillers and lower seed setting than other transgenic or nontransgenic plants. This phenotype was similar to the previously reported ccd7 mutants, which are defective in the biosynthesis of strigolactone, a plant hormone that regulates branching in plants and tiller formation in rice.

Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus.

Bevacizumab, a humanized monoclonal antibody (mAb) targeting to the vascular endothelial growth factor (VEGF), has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC) and Bevacizumab heavy chain (BHC) genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme-linked immunosorbent assay (ELISA) analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7-242.8 mg/Kg) in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target human VEGF (hVEGF) antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin) were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment.

Rapid proliferation and nucleolar organizer targeting centromeric retrotransposons in cotton.

Centromeric chromatin in most eukaryotes is composed of highly repetitive centromeric retrotransposons and satellite repeats that are highly variable even among closely related species. The evolutionary mechanisms that underlie the rapid evolution of centromeric repeats remain unknown. To obtain insight into the evolution of centromeric repeats following polyploidy, we studied a model diploid progenitor (Gossypium raimondii, D-genome) of the allopolyploid (AD-genome) cottons, G. hirsutum and G. barbadense. Sequence analysis of chromatin-immunoprecipitated DNA showed that the G. raimondii centromeric repeats originated from retrotransposon-related sequences. Comparative analysis showed that nine of the 10 analyzed centromeric repeats were absent from the centromeres in the A-genome and related diploid species (B-, F- and G-genomes), indicating that they colonized the centromeres of D-genome lineage after the divergence of the A- and D- ancestral species or that they were ancestrally retained prior to the origin of Gossypium. Notably, six of the nine repeats were present in both the A- and D-subgenomes in tetraploid G. hirsutum, and increased in abundance in both subgenomes. This finding suggests that centromeric repeats may spread and proliferate between genomes subsequent to polyploidization. Two repeats, Gr334 and Gr359 occurred in both the centromeres and nucleolar organizer regions (NORs) in D- and AD-genome species, yet localized to just the NORs in A-, B-, F-, and G-genome species. Contained within is a story of an established centromeric repeat that is eliminated and allopolyploidization provides an opportunity for reinvasion and reestablishment, which broadens our evolutionary understanding behind the cycles of centromeric repeat establishment and targeting.

Identification of peanut (Arachis hypogaea) chromosomes using a fluorescence in situ hybridization system reveals multiple hybridization events during tetraploid peanut formation.

The cultivated peanut Arachis hypogaea (AABB) is thought to have originated from the hybridization of Arachis duranensis (AA) and Arachis ipaënsis (BB) followed by spontaneous chromosome doubling. In this study, we cloned and analyzed chromosome markers from cultivated peanut and its wild relatives. A fluorescence in situ hybridization (FISH)-based karyotyping cocktail was developed with which to study the karyotypes and chromosome evolution of peanut and its wild relatives. Karyotypes were constructed in cultivated peanut and its two putative progenitors using our FISH-based karyotyping system. Comparative karyotyping analysis revealed that chromosome organization was highly conserved in cultivated peanut and its two putative progenitors, especially in the B genome chromosomes. However, variations existed between A. duranensis and the A genome chromosomes in cultivated peanut, especially for the distribution of the interstitial telomere repeats (ITRs). A search of additional A. duranensis varieties from different geographic regions revealed both numeric and positional variations of ITRs, which were similar to the variations in tetraploid peanut varieties. The results provide evidence for the origin of cultivated peanut from the two diploid ancestors, and also suggest that multiple hybridization events of A. ipaënsis with different varieties of A. duranensis may have occurred during the origination of peanut.

Artificial Chromosomes in Rice (Oryza sativa).

Chromosomes are the carriers of genetic material in biological organisms. Each chromosome has three essential components: a centromere, telomeres, and origins of replication. The understanding of the essential structural and functional organization of chromosomes has made it possible to produce artificial chromosomes (ACs), which are human-engineered minichromosomes. There are two approaches to make ACs: de novo assembly (bottom-up) and truncation of existing chromosomes (top-down). Rice (Oryza sativa) ACs are produced by telomere-mediated chromosome truncation, and may have many applications, such as genetic engineering to stack and express multiple genes in rice to combat diseases caused by bacteria, fungi, and viruses, to enhance tolerance of rice to environmental stresses such as drought, heat, and salinity, and to improve yield and quality. © 2016 by John Wiley & Sons, Inc.

In Situ Hybridization in Rice (Oryza sativa).

Fluorescence in situ hybridization (FISH) is widely used in cytogenetics to determine the localization of DNA sequences on target chromosomes, to provide visible information regarding the physical position of DNA sequences, to determine the abundance and distribution of repetitive sequences that comprise a large proportion of genomes, and to determine the relative chromosome positions of multiple sequences in physical mapping. By mapping on extended chromatin fibers, fiber-FISH can be used to determine the structure and organization of genes or DNA sequences with a high resolution (to a few kilobases). The protocols described here will provide procedures of FISH on metaphase chromosomes and extended chromatin fibers of rice (Oryza sativa). © 2016 by John Wiley & Sons, Inc.

Chromosome Preparation in Rice (Oryza sativa).

Chromosomes are the carriers of genetic material in biological organisms. Each chromosome has three essential components: a centromere, telomeres, and chromosome arms. Chromosome preparation is the basic technique for studies in cytogenetics, including the analysis of chromosomal behavior, chromosomal variation, karyotyping analysis, fluorescence in situ hybridization, immunostaining, and artificial chromosome technology. In this unit, we describe the basic protocol for rice (Oryza sativa) metaphase chromosome preparation from rice root tips, alternative chromosome preparation protocols using cultured cells, and different chromosome spreading techniques. © 2016 by John Wiley & Sons, Inc.

Plant artificial chromosome technology and its potential application in genetic engineering.

Genetic engineering with just a few genes has changed agriculture in the last 20 years. The most frequently used transgenes are the herbicide resistance genes for efficient weed control and the Bt toxin genes for insect resistance. The adoption of the first-generation genetically engineered crops has been very successful in improving farming practices, reducing the application of pesticides that are harmful to both human health and the environment, and producing more profit for farmers. However, there is more potential for genetic engineering to be realized by technical advances. The recent development of plant artificial chromosome technology provides a super vector platform, which allows the management of a large number of genes for the next generation of genetic engineering. With the development of other tools such as gene assembly, genome editing, gene targeting and chromosome delivery systems, it should become possible to engineer crops with multiple genes to produce more agricultural products with less input of natural resources to meet future demands.

Stable mitotic inheritance of rice minichromosomes in cell suspension cultures.

KEY MESSAGE: Suspension cell cultures of rice minichromosomes were established. The minichromosomes in suspension cultured cells were mitotically stable and had active gene expression, thus have the potential to be used as gene expression vectors to produce valuable bioactive products. The plant artificial chromosome (PAC) is a novel vector for plant genetic engineering to produce genetically modified crops with multiple transgenes, or to produce valuable bioactive products through the expression of multiple genes or biochemical pathways as a bioreactor. PAC is mainly constructed by engineered minichromosomes through telomere-mediated chromosome truncations. We have constructed rice minichromosomes in a previous study. Thus, the understanding of rice minichromosome inheritance under different culture conditions has potential importance for their utility in future studies and applications. In this study, we performed suspension cultures of three rice minichromosome-containing cell lines, 1004-111, 1008-100 and 1004-011. Two cell lines, 1004-111 and 1008-100, showed typical S growth pattern consisting of a lag phase, an active growing exponential phase and a stationary phase, whereas cell line 1004-011 grew very slowly and eventually died. Both 1004-111 and 1008-100 minichromosomes were stably transmitted in cell suspension cultures without any abnormality. Foreign gene expression was verified from 1004-111 and 1008-100 minichromosomes in suspension cultures. The stable mitotic inheritance of minichromosomes and gene expression from them indicated that rice minichromosomes could be maintained and propagated in cell suspension cultures. This study tested key parameters for suspension cultures of rice cell lines with minichromosomes, and proved in concept the potential for industrial use of PAC vectors as bioreactors.

A matched-group study protocol to evaluate the implementation of an Integrated Care Pathway programme for chronic obstructive pulmonary disease in Singapore.

INTRODUCTION: The treatment of chronic obstructive pulmonary disease (COPD) involves different care providers across care sites. This fragmentation of care increases the morbidity and mortality burden, as well as acute health services use. The COPD-Integrated Care Pathway (ICP) was designed and implemented to integrate the care across different sites from primary care to acute hospital and home. It aims to reduce the prevalence of COPD among the population in the catchment, reduce risk of hospital admissions, delay or prevent the progression of the disease and reduce mortality rate by adopting a coordinated and multidisciplinary approach to the management of the patients' medical conditions. This study on the COPD-ICP programme is undertaken to determine the impact on processes of care, clinical outcomes and acute care utilisation. METHODS AND ANALYSIS: This will be a retrospective, pre-post, matched-groups study to evaluate the effectiveness of the COPD-ICP programme in improving clinical outcomes and reducing healthcare costs. Programme enrolees (intervention group) and non-enrolees (comparator group) will be matched using propensity scores. Administratively, we set 30% as our target for proportion admission difference between programme and non-programme patients. A sample size of 62 patients in each group will be needed for statistical comparisons to be made at 90% power. Adherence with recommended care elements will be measured at baseline and quarterly during 1-year follow-up. Risk of COPD-related hospitalisations as primary outcome, healthcare costs, disease progression and 1-year mortality during 1-year follow-up will be compared between the groups using generalised linear regression models. ETHICS AND DISSEMINATION: This protocol describes the implementation and proposed evaluation of the COPD-ICP programme. The described study has received ethical approval from the NHG Domain Specific Review Board (DSRB Ref: 2013/01200). Results of the study will be reported through peer-review publications and presentations at healthcare conferences.

Identification of centromeric and telomeric DNA-binding proteins in rice.

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G-rich telomere repeats, respectively. However, the protein components are not fully understood. DNA-binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA-specific binding proteins, affinity pull-down technique was applied in this study to isolate rice centromeric and telomeric DNA-binding proteins. Fifty-five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO-binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA-binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull-down technique is effective in the isolation of sequence-specific binding proteins and will be applicable in future studies of centromere and telomere proteins.