Active Centromeres Research Articles

Peculiarities of karyotype of the Ukrainian aboriginal Hutsul breed of horse

Saving biodiversity and assessment of the genetic diversity of local breeds of domestic animals as priority subjects of protection in agrobiocenoses is one of the global challenges facing humanity in the 21st century. Hutsul horse is one of the oldest horse breeds in Ukraine, which according to the gene pool subject of horses in Ukraine belongs to the group “Local (mountain and ponies)” of the I category, which is already on the verge of extinction, and according to the FAO classification it is considered to be a subject of the gene pool threatened with extinction. Since the breeding chromosomal polymorphism of horses is insufficiently studied, it is timely to carry out a cytogenetic analysis of the characteristics of spontaneous mutagenesis in Hutsul horses. Karyotypic variability of Hutsul breed horses was determined using the methods of cytogenetic analysis and micronucleus test. The paper presents the results of cytogenetic analysis and micronucleus test of karyotypic variability of Hutsul breed horses. Asynchronous divergence of centromere regions of chromosomes occurs as a result of premature replication of centromere regions of heterochromatin associated with centromere activity. In the studied Hutsul horses, the magnitude of this variability corresponds to the spontaneous level for horses as a whole (2.2-9.1 %). No structural chromosomal abnormalities (chromosomal breaks) were revealed in Hutsul breed horses at Krai Neba LLC, and in the animals of the Hutsulshchina National Nature Park, the percentage of metaphase plates with chromosomal breaks was low (1.1 %), which indicates the stability of the karyotype of the studied animals. The results of the micronucleus test showed that the proportion of lymphocytes with micronuclei in the animals at the both farms was practically the same - 4.0-4.2. It can be concluded based on the data obtained that the animals under study were in ecologically clean conditions relative to the level of radionuclide contamination and were characterized by karyotype stability and reduced sensitivity to mutagenic factors of various nature.

Unequal contribution of two paralogous CENH3 variants in cowpea centromere function

In most diploids the centromere-specific histone H3 (CENH3), the assembly site of active centromeres, is encoded by a single copy gene. Persistance of two CENH3 paralogs in diploids species raises the possibility of subfunctionalization. Here we analysed both CENH3 genes of the diploid dryland crop cowpea. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of Vigna unguiculata. Both functional CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development. Hence, CENH3.2 of cowpea is likely at an early stage of pseudogenization and less likely undergoing subfunctionalization.

CenH3 distribution reveals extended centromeres in the model beetle Tribolium castaneum.

Centromeres are chromosomal domains essential for kinetochore assembly and correct chromosome segregation. Inconsistent in their underlying DNA sequences, centromeres are defined epigenetically by the presence of the centromere-specific histone H3 variant CenH3. Most of the analyzed eukaryotes have monocentric chromosomes in which CenH3 proteins deposit into a single, primary constriction visible at metaphase chromosomes. Contrary to monocentrics, evolutionary sporadic holocentric chromosomes lack a primary constriction and have kinetochore activity distributed along the entire chromosome length. In this work, we identified cCENH3 protein, the centromeric H3 histone of the coleopteran model beetle Tribolium castaneum. By ChIP-seq analysis we disclosed that cCENH3 chromatin assembles upon a repertoire of repetitive DNAs. cCENH3 in situ mapping revealed unusually elongated T. castaneum centromeres that comprise approximately 40% of the chromosome length. Being the longest insect regional centromeres evidenced so far, T. castaneum centromeres are characterized by metapolycentric structure composed of several individual cCENH3-containing domains. We suggest that the model beetle T. castaneum with its metapolycentromeres could represent an excellent model for further studies of non-canonical centromeres in insects.

Formation of the CenH3-Deficient Holocentromere in Lepidoptera Avoids Active Chromatin

Despite the essentiality for faithful chromosome segregation, centromere architectures are diverse among eukaryotes1,2 and embody two main configurations: mono- and holocentromeres, referring, respectively, to localized or unrestricted distribution of centromeric activity. Of the two, some holocentromeres offer the curious condition of having arisen independently in multiple insects, most of which have lost the otherwise essential centromere-specifying factor CenH33 (first described as CENP-A in humans).4-7 The loss of CenH3 raises intuitive questions about how holocentromeres are organized and regulated in CenH3-lacking insects. Here, we report the first chromatin-level description of CenH3-deficient holocentromeres by leveraging recently identified centromere components6,7 and genomics approaches to map and characterize the holocentromeres of the silk moth Bombyx mori, a representative lepidopteran insect lacking CenH3. This uncovered a robust correlation between the distribution of centromere sites and regions of low chromatin activity along B.mori chromosomes. Transcriptional perturbation experiments recapitulated the exclusion of B.mori centromeres from active chromatin. Based on reciprocal centromere occupancy patterns observed along differentially expressed orthologous genes of Lepidoptera, we further found that holocentromere formation in a manner that is recessive to chromatin dynamics is evolutionarily conserved. Our results help us discuss the plasticity of centromeres in the context of a role for the chromosome-wide chromatin landscape in conferring centromere identity rather than the presence of CenH3. Given the co-occurrence of CenH3 loss and holocentricity in insects,7 we further propose that the evolutionary establishment of holocentromeres in insects was facilitated through the loss of a CenH3-specified centromere.

High allelic diversity of the centromere-specific histone H3 (CENH3) in the legume sainfoin (Onobrychis viciifolia).

The centromere is a structurally and functionally specialized region on each eukaryotic chromosome and is essential for accurate and complete segregation of chromosomes during cell division. Centromeric nucleosomes differ from canonical nucleosomes by replacement of the histone H3 with its centromere-specific variant CENH3. CENH3 is essential for active centromeres in most eukaryotes. Homologs of CENH3 are identified in many organisms. Sainfoin (Onobrychis viciifolia) is an agriculturally important perennial forage and is a legume of the Fabaceae family. There is very limited information on the structure of the sainfoin genome and no data are available on its centromere structure. Here, we aim to characterize the sainfoin CENH3 homolog (OvCENH3). Using a sequence homology-based strategy with gene-specific primers, we were able to clone transcripts from sainfoin total RNA. The amplified clones were sequenced and compared by bioinformatics tools. Four distinct alleles of OvCENH3 were detected. Our study provides the first structural features on sainfoin centromeres with a possible allotetraploid origin for sainfoin. We discuss and compare our findings with that for other important legume species.

Chromosome number evolves at equal rates in holocentric and monocentric clades.

Despite the fundamental role of centromeres two different types are observed across plants and animals. Monocentric chromosomes possess a single region that function as the centromere while in holocentric chromosomes centromere activity is spread across the entire chromosome. Proper segregation may fail in species with monocentric chromosomes after a fusion or fission, which may lead to chromosomes with no centromere or multiple centromeres. In contrast, species with holocentric chromosomes should still be able to safely segregate chromosomes after fusion or fission. This along with the observation of high chromosome number in some holocentric clades has led to the hypothesis that holocentricity leads to higher rates of chromosome number evolution. To test for differences in rates of chromosome number evolution between these systems, we analyzed data from 4,393 species of insects in a phylogenetic framework. We found that insect orders exhibit striking differences in rates of fissions, fusions, and polyploidy. However, across all insects we found no evidence that holocentric clades have higher rates of fissions, fusions, or polyploidy than monocentric clades. Our results suggest that holocentricity alone does not lead to higher rates of chromosome number changes. Instead, we suggest that other co-evolving traits must explain striking differences between clades.

Rapid Birth or Death of Centromeres on Fragmented Chromosomes in Maize.

Comparative genomics has revealed common occurrences in karyotype evolution such as chromosomal end-to-end fusions and insertions of one chromosome into another near the centromere, as well as many cases of de novo centromeres that generate positional polymorphisms. However, how rearrangements such as dicentrics and acentrics persist without being destroyed or lost remains unclear. Here, we sought experimental evidence for the frequency and timeframe for inactivation and de novo formation of centromeres in maize (Zea mays). The pollen from plants with supernumerary B chromosomes was gamma-irradiated and then applied to normal maize silks of a line without B chromosomes. In ∼8,000 first-generation seedlings, we found many B-A translocations, centromere expansions, and ring chromosomes. We also found many dicentric chromosomes, but a fraction of these show only a single primary constriction, which suggests inactivation of one centromere. Chromosomal fragments were found without canonical centromere sequences, revealing de novo centromere formation over unique sequences; these were validated by immunolocalization with Thr133-phosphorylated histone H2A, a marker of active centromeres, and chromatin immunoprecipitation-sequencing with the CENH3 antibody. These results illustrate the regular occurrence of centromere birth and death after chromosomal rearrangement during a narrow window of one to potentially only a few cell cycles for the rearranged chromosomes to be recognized in this experimental regime.

Abstract 1203: Identification of 22 novel loci associated with susceptibility to testicular germ cell tumors

Abstract Background: Testicular germ cell tumors (TGCT) are the most common cancers in young men of European ancestry aged 20 to 39 years. The incidence of TGCT has doubled over the past 20 years, yet no robust environmental risk factors for disease have been identified. Although TGCTs are treatable by surgery, radiation, and platinum-based chemotherapy, multiple long-term toxicities of treatment often occur impacting morbidity and mortality. Due to their high heritability and homogenous cell of origin, TGCTs are well suited to genome-wide association methods. The Testicular Cancer Consortium (TECAC) has brought together the largest genome-wise association study (GWAS) study of TGCT to date. Methods: We conducted a GWAS of 5,602 cases and 5,006 controls aggregated from 12 locations in the US and Europe. Logistic regression models adjusted for study center and genomic ancestry. Genotypes were imputed against the Human Haplotype Reference Consortium. Meta-analysis was performed to combine GWAS results with summary statistics from five previously published TGCT studies, UK Biobank, deCODE Genetics, and an independent set of cases and controls, for a total of 10,156 cases and 179,683 controls. Biologic function of loci was explored using PAINTOR annotated with ATAC-seq data of four TGCT cell lines, SPATIAL-seq data of the NTERA2 TGCT cell-line, and publicly available data from ENCODE. Polygenic risk scores (PRS) were computed using subject-level data from the 5,602 cases and 5,006 controls, and effect sizes of the novel hits derived from the meta-analysis. Results: 22 novel and 45 previously reported loci associated with TGCT surpassed genome-wide significance (p < 5e-08). We discovered additional markers in known susceptibility loci and identified novel regions associated with germ cell development and sex determination (e.g., BCL11, AR), immune function (e.g., TNXB, ITIH5), and for the first time identified genes associated with kinetochore activity (e.g., PPP2R5A, ANAPC2). All identified risk SNPs to date account for 42.3% of heritability. Men in the highest 1% of PRS had over a 15-fold increased risk of TGCT compared to those at the median PRS, and PRS overall had an AUC of 74.29%. Conclusions: Results from our TGCT meta-analysis continue to provide insights into biological pathways affecting germ cell specification, expression, and epigenetic reprogramming, and sex determination. Our results also uniquely place TGCT as the only cancer type in which inherited variants implicating kinetochore activity, critical for chromosomal segregation, have been identified. Citation Format: John Pluta, Louisa Pyle, Timothy Bishop, Javier Benitez, Victoria Cortessis, Alberto Ferlin, Jourik Gietema, Mark Greene, Thomas Grotmol, Ramneek Gupta, Robert Hamilton, Michelle Hildebrandt, Lambertus Kiemeney, Davor Lessel, Thorunn Rafnar, Lorenzo Richiardi, Rolf Skotheim, Clare Turnbull, Fredrik Wiklund, Tongzhang Zheng, Ewa Rajpert- De Meyts, Stephen Schwartz, Katherine McGlynn, Peter Kanetsky, Katherine Nathanson. Identification of 22 novel loci associated with susceptibility to testicular germ cell tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1203.

Abstract 2391: Centromeric chromatin alteration is associated with increase in alphoid array replication fork speed in ovarian cancer cell lines

Abstract The centromere is well known as the center point of cell division, with aberrant centromeric function leading to mitotic dysfunction and chromosomal abnormalities. These abnormalities may in turn lead to diseases, including cancer and premature aging. Binding of CENPB to the CENPB box is orchestrated epigenetically, and has emerged as an important factor in the regulation of the centromeric-chromatin structural landscape. CENP-B depletion that resulted in euchromatization of the alphoid centromere array, also is associated with elevated centromeric DNA replication activity. Observation in two ovarian cancer cells from NCI-60 collection; OVCAR3 and SKOV3, identified as having low and high expression of CENPB RNA respectively, indicates the different level of centromeric DNA instability. Centromeric DNA damage fraction is more abundant and replication fork velocity is elevated in less CENPB expresser cells, indicates alphoid array alteration. We conclude that CENP-B is required for maintenance of centromeric chromatin structure and centromeric DNA integrity. Citation Format: Indri Erliandri, Christophe Redon, Jung-Hyun Kim, Haiqing Fu, Mirit Aladjem, William Reinhold, Vladimir Larionov. Centromeric chromatin alteration is associated with increase in alphoid array replication fork speed in ovarian cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2391.

A high-contiguity Brassica nigra genome localizes active centromeres and defines the ancestral Brassica genome

It is only recently, with the advent of long-read sequencing technologies, that we are beginning to uncover previously uncharted regions of complex and inherently recursive plant genomes. To comprehensively study and exploit the genome of the neglected oilseed Brassica nigra, we generated two high-quality nanopore de novo genome assemblies. The N50 contig lengths for the two assemblies were 17.1 Mb (12 contigs), one of the best among 324 sequenced plant genomes, and 0.29 Mb (424 contigs), respectively, reflecting recent improvements in the technology. Comparison with a de novo short-read assembly corroborated genome integrity and quantified sequence-related error rates (0.2%). The contiguity and coverage allowed unprecedented access to low-complexity regions of the genome. Pericentromeric regions and coincidence of hypomethylation enabled localization of active centromeres and identified centromere-associated ALE family retro-elements that appear to have proliferated through relatively recent nested transposition events (<1 Ma). Genomic distances calculated based on synteny relationships were used to define a post-triplication Brassica-specific ancestral genome, and to calculate the extensive rearrangements that define the evolutionary distance separating B. nigra from its diploid relatives.

Identification of Genes Encoding CENP-A and Heterochromatin Protein 1 of Lipomyces starkeyi and Functional Analysis Using Schizosaccharomyces pombe

Centromeres function as a platform for the assembly of multiple kinetochore proteins and are essential for chromosome segregation. An active centromere is characterized by the presence of a centromere-specific histone H3 variant, CENP-A. Faithful centromeric localization of CENP-A is supported by heterochromatin in almost all eukaryotes; however, heterochromatin proteins have been lost in most Saccharomycotina. Here, identification of CENP-A (CENP-AL.s.) and heterochromatin protein 1 (Lsw1) in a Saccharomycotina species, the oleaginous yeast Lipomyces starkeyi, is reported. To determine if these proteins are functional, the proteins in S. pombe, a species widely used to study centromeres, were ectopically expressed. CENP-AL.s. localizes to centromeres and can be replaced with S. pombe CENP-A, indicating that CENP-AL.s. is a functional centromere-specific protein. Lsw1 binds at heterochromatin regions, and chromatin binding is dependent on methylation of histone H3 at lysine 9. In other species, self-interaction of heterochromatin protein 1 is thought to cause folding of chromatin, triggering transcription repression and heterochromatin formation. Consistent with this, it was found that Lsw1 can self-interact. L. starkeyi chromatin contains the methylation of histone H3 at lysine 9. These results indicated that L. starkeyi has a primitive heterochromatin structure and is an attractive model for analysis of centromere heterochromatin evolution.

Sequence Composition Underlying Centromeric and Heterochromatic Genome Compartments of the Pacific Oyster Crassostrea gigas.

Segments of the genome enriched in repetitive sequences still present a challenge and are omitted in genome assemblies. For that reason, the exact composition of DNA sequences underlying the heterochromatic regions and the active centromeres are still unexplored for many organisms. The centromere is a crucial region of eukaryotic chromosomes responsible for the accurate segregation of genetic material. The typical landmark of centromere chromatin is the rapidly-evolving variant of the histone H3, CenH3, while DNA sequences packed in constitutive heterochromatin are associated with H3K9me3-modified histones. In the Pacific oyster Crassostrea gigas we identified its centromere histone variant, Cg-CenH3, that shows stage-specific distribution in gonadal cells. In order to investigate the DNA composition of genomic regions associated with the two specific chromatin types, we employed chromatin immunoprecipitation followed by high-throughput next-generation sequencing of the Cg-CenH3- and H3K9me3-associated sequences. CenH3-associated sequences were assigned to six groups of repetitive elements, while H3K9me3-associated-ones were assigned only to three. Those associated with CenH3 indicate the lack of uniformity in the chromosomal distribution of sequences building the centromeres, being also in the same time dispersed throughout the genome. The heterochromatin of C. gigas exhibited general paucity and limited chromosomal localization as predicted, with H3K9me3-associated sequences being predominantly constituted of DNA transposons.

Supernumerary B chromosomes of Aegilops speltoides undergo precise elimination in roots early in embryo development

Not necessarily all cells of an organism contain the same genome. Some eukaryotes exhibit dramatic differences between cells of different organs, resulting from programmed elimination of chromosomes or their fragments. Here, we present a detailed analysis of programmed B chromosome elimination in plants. Using goatgrass Aegilops speltoides as a model, we demonstrate that the elimination of B chromosomes is a strictly controlled and highly efficient root-specific process. At the onset of embryo differentiation B chromosomes undergo elimination in proto-root cells. Independent of centromere activity, B chromosomes demonstrate nondisjunction of chromatids and lagging in anaphase, leading to micronucleation. Chromatin structure and DNA replication differ between micronuclei and primary nuclei and degradation of micronucleated DNA is the final step of B chromosome elimination. This process might allow root tissues to survive the detrimental expression, or overexpression of B chromosome-located root-specific genes with paralogs located on standard chromosomes.

Centromere deletion in Cryptococcus deuterogattii leads to neocentromere formation and chromosome fusions.

The human fungal pathogen Cryptococcus deuterogattii is RNAi-deficient and lacks active transposons in its genome. C. deuterogattii has regional centromeres that contain only transposon relics. To investigate the impact of centromere loss on the C. deuterogattii genome, either centromere 9 or 10 was deleted. Deletion of either centromere resulted in neocentromere formation and interestingly, the genes covered by these neocentromeres maintained wild-type expression levels. In contrast to cen9∆ mutants, cen10∆ mutant strains exhibited growth defects and were aneuploid for chromosome 10. At an elevated growth temperature (37°C), the cen10∆ chromosome was found to have undergone fusion with another native chromosome in some isolates and this fusion restored wild-type growth. Following chromosomal fusion, the neocentromere was inactivated, and the native centromere of the fused chromosome served as the active centromere. The neocentromere formation and chromosomal fusion events observed in this study in C. deuterogattii may be similar to events that triggered genomic changes within the Cryptococcus/Kwoniella species complex and may contribute to speciation throughout the eukaryotic domain.

α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity.

Incorrect kinetochore-microtubule attachments during mitosis can lead to chromosomal instability, a hallmark of human cancers. Mitotic error correction relies on the kinesin-13 MCAK, a microtubule depolymerase whose activity in vitro is suppressed by α-tubulin detyrosination-a posttranslational modification enriched on long-lived microtubules. However, whether and how MCAK activity required for mitotic error correction is regulated by α-tubulin detyrosination remains unknown. Here we found that detyrosinated α-tubulin accumulates on correct, more stable, kinetochore-microtubule attachments. Experimental manipulation of tubulin tyrosine ligase (TTL) or carboxypeptidase (Vasohibins-SVBP) activities to constitutively increase α-tubulin detyrosination near kinetochores compromised efficient error correction, without affecting overall kinetochore microtubule stability. Rescue experiments indicate that MCAK centromeric activity was required and sufficient to correct the mitotic errors caused by excessive α-tubulin detyrosination independently of its global impact on microtubule dynamics. Thus, microtubules are not just passive elements during mitotic error correction, and the extent of α-tubulin detyrosination allows centromeric MCAK to discriminate correct vs. incorrect kinetochore-microtubule attachments, thereby promoting mitotic fidelity.

Epigenetic regulation of centromere function.

The centromere is a specialized region on the chromosome that directs equal chromosome segregation. Centromeres are usually not defined by DNA sequences alone. How centromere formation and function are determined by epigenetics is still not fully understood. Active centromeres are often marked by the presence of centromeric-specific histone H3 variant, centromere protein A (CENP-A). How CENP-A is assembled into the centromeric chromatin during the cell cycle and propagated to the next cell cycle or the next generation to maintain the centromere function has been intensively investigated. In this review, we summarize current understanding of how post-translational modifications of CENP-A and other centromere proteins, centromeric and pericentric histone modifications, non-coding transcription and transcripts contribute to centromere function, and discuss their intricate relationships and potential feedback mechanisms.

De Novo Centromere Formation: One's Company, Two's a Crowd.

Chromosomes containing two centromeres (dicentrics) trigger chromosome instability that is avoided by the enigmatic process of centromere inactivation. In this issue of Developmental Cell, Palladino etal. (2020) combine invivo chromosome engineering and Drosophila genetics to assess consequences of de novo centromere formation and clarify models of centromere inactivation.

Mitotic Spindle Attachment to the Holocentric Chromosomes of Cuscuta europaea Does Not Correlate With the Distribution of CENH3 Chromatin.

The centromere is the region on a chromosome where the kinetochore assembles and spindle microtubules attach during mitosis and meiosis. In the vast majority of eukaryotes, the centromere position is determined epigenetically by the presence of the centromere-specific histone H3 variant CENH3. In species with monocentric chromosomes, CENH3 is confined to a single chromosomal region corresponding to the primary constriction on metaphase chromosomes. By contrast, in holocentrics, CENH3 (and thus centromere activity) is distributed along the entire chromosome length. Here, we report a unique pattern of CENH3 distribution in the holocentric plant Cuscuta europaea. This species expressed two major variants of CENH3, both of which were deposited into one to three discrete regions per chromosome, whereas the rest of the chromatin appeared to be devoid of CENH3. The two CENH3 variants fully co-localized, and their immunodetection signals overlapped with the positions of DAPI-positive heterochromatic bands containing the highly amplified satellite repeat CUS-TR24. This CENH3 distribution pattern contrasted with the distribution of the mitotic spindle microtubules, which attached at uniform density along the entire chromosome length. This distribution of spindle attachment sites proves the holocentric nature of C. europaea chromosomes and also suggests that, in this species, CENH3 either lost its function or acts in parallel to an additional CENH3-free mechanism of kinetochore positioning.

Loss of centromere function drives karyotype evolution in closely related Malassezia species.

Genomic rearrangements associated with speciation often result in variation in chromosome number among closely related species. Malassezia species show variable karyotypes ranging between six and nine chromosomes. Here, we experimentally identified all eight centromeres in M. sympodialis as 3-5-kb long kinetochore-bound regions that span an AT-rich core and are depleted of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-A-rich regions in Malassezia furfur, which has seven chromosomes, and histone H3 depleted regions in Malassezia slooffiae and Malassezia globosa with nine chromosomes each. Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred nine-chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA and (b) centromere inactivation accompanied by changes in DNA sequence following chromosome-chromosome fusion. We propose that AT-rich centromeres drive karyotype diversity in the Malassezia species complex through breakage and inactivation.

Validation of Plasmodium vivax centromere and promoter activities using Plasmodium yoelii.

Plasmodium vivax is the leading cause of malaria outside Africa and represents a significant health and economic burden on affected countries. A major obstacle for P. vivax eradication is the dormant hypnozoite liver stage that causes relapse infections and the limited antimalarial drugs that clear this stage. Advances in studying the hypnozoite and other unique biological aspects of this parasite are hampered by the lack of a continuous in vitro laboratory culture system and poor availability of molecular tools for genetic manipulation. In this study, we aim to develop molecular tools that can be used for genetic manipulation of P. vivax. A putative P. vivax centromere sequence (PvCEN) was cloned and episomal centromere based plasmids expressing a GFP marker were constructed. Centromere activity was evaluated using a rodent malaria parasite Plasmodium yoelii. A plasmid carrying PvCEN was stably maintained in asexual-stage parasites in the absence of drug pressure, and approximately 45% of the parasites retained the plasmid four weeks later. The same retention rate was observed in parasites possessing a native P. yoelii centromere (PyCEN)-based control plasmid. The segregation efficiency of the plasmid per nuclear division was > 99% in PvCEN parasites, compared to ~90% in a control parasite harboring a plasmid without a centromere. In addition, we observed a clear GFP signal in both oocysts and salivary gland sporozoites isolated from mosquitoes. In blood-stage parasites after liver stage development, GFP positivity in PvCEN parasites was comparable to control PyCEN parasites. Thus, PvCEN plasmids were maintained throughout the parasite life cycle. We also validated several P. vivax promoter activities and showed that hsp70 promoter (~1 kb) was active throughout the parasite life cycle. This is the first data for the functional characterization of a P. vivax centromere that can be used in future P. vivax biological research.