Restriction Endonuclease Mapping Research Articles

Characterization of the Type 2 L-Asparaginase Gene in Thermohalophilic Bacterial from Wawolesea Hot Springs, Southeast Sulawesi, Indonesia.

<b>Background and Objective:</b> Type 2 L-asparaginase enzyme can be used as a cancer therapy agent and prevent acrylamide formation in food products. Enzymes produced by thermohalophilic bacteria can provide high activity at high temperatures so they are needed on an industrial scale. Hence, this study aims to determine the characteristics of the gene encoding type 2 L-asparaginase enzyme in the thermohalophilic bacterial isolate CAT3.4. <b>Materials and Methods:</b> This research is a type of exploratory research. The characteristics of the gene encoding type 2 L-asparaginase were determined using the PCR technique using the primer pairs AsnBac2-F2 (5'-CTCACGGGAATCTCCATAACTC-3') and AsnBac2-R2 (5'CAGCGATGTAACAGACAGCATC-3'). The characterization process was carried out in stages: Isolation of genomic DNA using a modified alkali-lysis method, nucleotide and protein similarity analysis using BLASTn analysis on the NCBI website, construction of a phylogenetic tree using the MEGAX program, restriction enzyme mapping and amino acid analysis using the Bioedit program. <b>Results:</b> The characterization results showed that the PCR product has a size of 1594 bp with a CDS of 1128 bp, has a similarity value of 100% with <i>Bacillus subtilis</i>, has seven restriction enzymes as molecular markers for the type 2 L-asparaginase gene at the species level: <i>Bsr</i>GI, <i>Dra</i>I, <i>Eco</i>RV, <i>Hind</i>III, <i>Hpy</i>CH4IV , <i>Ssp</i>I and <i>Tai</i>I, have dominant hydrophilic regions and are in the same subclass as <i>Bacillus subtilis</i> strain GOT9. <b>Conclusion:</b> The target gene was similar to the gene encoding type 2 L-asparaginase from <i>Bacillus subtilis</i> with a max identity of 98.85%, query coverage value of 100% and E-value of 0.

Development of PCR-RFLP for the detection of benzimidazole resistance polymorphisms in isotype 1 β-tubulin gene of Trichostrongylus colubriformis

Trichostrongylus spp. is a predominant gastrointestinal strongyle in small ruminants and the development of benzimidazole resistance in this species can significantly contribute to the overall resistance status in farms. The objective of the present study was to develop PCR-RFLP for genotyping Trichostrongylus colubriformis larvae for benzimidazole resistance polymorphisms at codons 167, 198 and 200 in the isotype 1 β-tubulin gene. A 246 bp region of the isotype 1 β-tubulin gene containing the three codons was amplified by semi-nested PCR. A modified reverse primer was used in the semi-nested PCR for substituting the third nucleotide downstream of codon 200 (C) with nucleotide T to create a restriction site for identifying the polymorphism at codon 200. Restriction endonuclease mapping of the 246 bp product was done and the restriction enzymes HpyCH4III, HpyCH4V and RsaI were selected for the identification of SNPs at codons 200, 198 and 167, respectively. RFLP with HpyCH4III yielded 147 and 99 bp fragments in the susceptible genotype (SS) and 147, 67 and 32 bp fragments in the resistant genotype (rr) at codon 200. HpyCH4V was expected to cut the 246 bp semi-nested PCR product into 205 and 41 bp fragments in the resistant genotype at codon 198, while the susceptible genotype yielded an uncut 246 bp product. RFLP with RsaI was expected to give two fragments of sizes 199 and 47 bp in the susceptible genotype and three fragments of sizes 199, 25 and 22 bp in the resistant genotype. A total of 150 T. colubriformis larvae were genotyped at the three codons. Benzimidazole resistance-associated SNPs were found at codon 200 while no polymorphism was identified at codons 167 and 198. Twenty eight per cent of the larvae genotyped were found to be homozygous resistant (rr, TAC/TAC), 60.67 per cent homozygous susceptible (SS, TTC/TTC) and 11.33 per cent heterozygous (rS, TTC/TAC) at codon 200 of isotype 1 β-tubulin gene. The overall frequency of the resistant allele (r, TAC) was 0.337 and that of the susceptible allele (S, TTC) was 0.663. The present study forms the first report of PCR-RFLP for genotyping T. colubriformis in goats at the three benzimidazole resistance-associated codons.

Restriction Enzyme Maps of Circular Sequences of BBTV-DNA Components and Bioinformatics of Their Amino Acids

Restriction Enzyme Maps of Circular Sequences of BBTV-DNA Components and Bioinformatics of Their Amino Acids

Pathogenic detection and molecular tracing of a bacillary dysentery outbreak by Shigella sonnei in Huainan city

Objective: To understand the virulence gene and drug resistance profile of Shigella sonnei outbreak in Huainan city, and conduct pathogenic traceability analysis. Methods: Water samples and feces related to an infectious diarrhea outbreak in Huainan city in August 2020 were collected for multiple pathogen detection. Virulence gene, drug sensitivity, pulse-field gel electrophoresis and whole genome sequencing of Shigella isolates were analyzed respectively. Results: 38 strains of Shigella sonnei were detected in 56 samples of mucilage feces with a positive rate 67.86%, and all serotypes were Shigella sonnei Phase I. Three strains of Shigella sonnei were detected by fluorescence PCR in the Gram-negative (GN) bacterial enrichment solution of terminal water and well water. Virulence genes were ipaH positive (38), ipaH/ial (31) and ipaH/ial/sen positive (1), respectively. The drug resistance spectrum showed that 9 of 14 antibiotics were 100% resistant, and only imipenem, chloramphenicol, ceftazidime and ciprofloxacin were effective drugs. XbaⅠ restriction enzyme map type of 36 isolates was completely consistent, and the ST type analysis of 3 strains was ST152. Whole genome sequencing and analysis verified that the outbreak was caused by a single clonal group of strains, and revealed that the isolates of the outbreak were clustered into a large cluster with 3 Chinese strains and 1 Korean strain in the database, far away from the strains of other countries. Conclusion: The outbreak is caused by a single clone of Shigella sonnei, which are low virulence strains and have multiple drug resistance.

Identification of restriction enzyme in the FSHR gene of indonesian local cattle

The restriction enzyme is important for genotyping using the PCR-RFLP technique. Therefore, this study aims to identify the restriction enzyme mapping in the partial sequence of the follicle-stimulating hormone receptor (FSHR) gene in Indonesian local cattle. A total of 29 samples sized 306 bp, were aligned with Genbank sequence acc no. NC_032660, resulting three polymorphic sites, namely g.193G>C, g.227T>C, and g.275A>C. Furthermore, the restriction mapping analysis using the NEBcutter program V2.0 showed that no enzyme recognized the SNP g.275A>C, while the SNP g.193G>C and g.227T>C were identified by the AluI and MscI enzymes, respectively. The AluI enzyme cuts at two positions (193 bp and 243 bp) in the G allele sample producing three fragments namely 50 bp, 63 bp, and 193 bp, meanwhile, in the C allele, the AluI cuts only in position 243 bp, hence, the fragment products are 63 bp and 243 bp. In contrast, the MscI enzyme was only recognized in the T allele, producing fragments sized 77 bp and 229 bp but failed to identify the restriction site along with the PCR products in the C allele. Based on the results, the SNPs (g.193G>C and g.227T>C) and restriction enzymes (AluI and MscI) are applicable for genotyping local Indonesian cattle using the PCR-RFLP technique in future studies.

Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs

The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed. One such method is Restriction Enzyme (RE) mapping. Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously. In this study, the sgRNA templates were initially cloned into the above PX459 plasmids. Subsequently, the accuracy of the constructs was determined by RE mapping. This method was established to screen for sgRNA-bearing PX459 plasmids. However, numerous anomalies were detected after ligation of sgRNA templates into RE digested PX459 plasmids. Our data suggest that RE mapping is only appropriate as an initial screen and that the identity of all plasmids with the correctly identified RE maps should be confirmed by Sanger sequencing.

Optimization of human interferon beta protein expression in Chinese hamster ovary cells

Background and aims: Human interferon beta-1a (hIFNβ-1a) is a 22.5-kDa glycoprotein used to treat diseases such as multiple sclerosis (MS). Because of appropriate post-translation modifications, protein isolation, and lack of toxicity in Chinese hamster ovary (CHO) cells, we cloned hIFNβ-1a encoding sequence into these cells by recombinant DNA technology to achieve stable expression of this recombinant protein. Methods: The hIFNβ-1a encoding sequence was designed based on the CHO cells’ codon usage and the Gene Bank data, and then syntactically constructed in the pUC57 vector. After confirmation, the synthesized sequence was cloned into the pcDNA3.1 expression vector by using EcoRI and XhoI sites via Escherichia coli DH5α competent cells. Then, the recombinant vector pcDNA-hHIFNβ1a was linearized by BglII and transfected into the CHO cells using lipofectamine. The transfected cells were proliferated and screened by gentamicin. Certain concentrations of zinc sulfate, DMSO, and glycerol were used to enhance protein expression. Finally, the recombinant protein expression was qualitatively evaluated using different techniques. Results: The hIFNβ1a integrity was confirmed by DNA sequencing and specific software. The construction and sub-cloning of hIFNβ1a-pcDNA3.1 in E. coli were confirmed by colony-PCR with specific primers and restriction enzyme mapping. The screening of transfected CHO cells was performed using gentamicin. The protein expression was confirmed by RT-PCR, MTT assay, SDS-PAGE, and Western blot. Comparison of the optimized and control samples demonstrated that chemical treatment enhanced the protein expression. Conclusion: We achieved the stable clones of CHO cells expressing the active form of human interferon beta.

A Molecular Basis for Hemoglobin-H Disease in American Blacks

We have applied gene counting and restriction endonuclease mapping techniques to the study of two American black families in which there were one or more cases of HbH disease. We found deletions of three of the four normal alpha-globin genes in individuals with HbH disease. In two of these individuals, the chromosome containing the single alpha gene could have originated by crossing over between mispaired alpha genes, resulting in a deletion of about 4.2 kilobases (kb).

Cloning in Plasmid Vectors: Blunt-End Cloning.

This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To obtain the maximum number of "correct" ligation products when cloning blunt-ended target fragments, the two components of DNA in the ligation reaction must be present at an appropriate ratio. If the molar ratio of plasmid vector to target DNA is too high, then the ligation reaction may generate an undesirable number of circular empty plasmids, both monomeric and polymeric; if too low, the ligation reaction may generate an excess of linear and circular homopolymers and heteropolymers of varying sizes, orientations, and compositions. For this reason, the orientation of the foreign DNA and the number of inserts in each recombinant clone must always be validated by restriction endonuclease mapping or some other means.

Translational Fusion of a G‐protein Coupled‐Receptor from the Hookworm Ancylostoma ceylanicum Expressed in Caenorhabditis elegans

Hookworms are infectious parasites that affect more than 500 million people worldwide, causing extensive morbidity and economic burden in developing countries. Mass drug administration is a current control method, but resistance is of concern. Many hookworm species, including those in the genus Ancylostoma, are specialists and require specific host signals to continue development upon infection. Little is known about the signaling mechanisms, although evolutionary conservation between parasitic nematodes and the model organism C. elegans suggests that G‐protein coupled receptors (GPCRs) expressed in amphid neurons are chemosensory detection candidates. The amphid neurons of C. elegans are exposed to the environment, even during the dauer stage, an alternate phase of larval development that is similar to the parasitic infective juvenile stage. Previous experiments involving A. ceylanicum transcriptional GFP fusions in C. elegans identified putative GPCR genes expressed in neurons during dauer stage. To confirm that hookworm proteins are neuronally localized under dauer conditions, a translational GFP fusion of a putative hookworm GPCR was generated. The entire coding sequence, plus introns, was cloned using homologous recombination‐based, yeast assembly methods to create a C‐terminal GFP fusion. The putative A. ceylanicum promoter consisted of the upstream intergenic region, which is approximately 5000 base pairs, and the transcriptional terminator from C. elegans unc‐54 was used. To generate the expression plasmid, amplicons containing homology to the adjacent segment in the assembly were amplified using polymerase chain reaction (PCR) and transformed into yeast. The plasmid was verified by junction‐confirming PCR analysis, restriction enzyme mapping, and sequencing. After microinjection, GFP expression in amphid neurons of stably transformed C. elegans during the dauer stage would support the hypothesis that this putative GPCR plays a role in reception of host signaling molecules and may provide a possible therapeutic target.Support or Funding InformationThis work was supported by the Salisbury University Faculty Mini‐Grant Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression.

The Orf virus (ORFV; Parapoxvirus) strain D1701 with an attenuated phenotype and excellent immunogenic capacity is successfully used for the generation of recombinant vaccines against different viral infections. Adaption for growth in Vero cells was accompanied by additional major genomic changes resulting in ORFV strain variant D1701-V. In this study, restriction enzyme mapping, blot hybridization and DNA sequencing of the deleted region s (A, AT and D) in comparison to the predecessor strain D1701-B revealed the loss of 7 open reading frames (ORF008, ORF101, ORF102, ORF114, ORF115, ORF116, ORF117). The suitability of deletion site D for expression of foreign genes is demonstrated using novel synthetic early promoter eP1 and eP2. Comparison of promoter strength showed that the original vegf-e promoter Pv as well as promoter eP2 display an up to 11-fold stronger expression than promoter eP1, irrespective of the insertion site. Successful integration and expression of the fluorescent marker genes is demonstrated by gene- and insertion-site specific PCR assays, fluorescence microscopy and flow cytometry. For the first time ORFV recombinants are generated simultaneously expressing transgenes in two different insertion loci. That allows production of polyvalent vaccines containing several antigens against one or different pathogens in a single vectored ORFV vaccine.

Frequency of Canine Parvovirus Type-2 Variants by PCR in Enteric and Healthy Dogs and Genotyping of Variants by Restriction Enzyme (RE) Mapping by DdeI Endonuclease Based on Partial VP-2 Gene Sequence

Canine parvovirus 2 (CPV-2) is the causative agent of myocarditis and acute hemorrhagic enteritis in dog populations. In this study, vaccinal strains and sixty wild strains (CPV-2a, CPV-2b) of CPV-2 from fecal samples of enteric (30 strains) and healthy (30 strains) dogs detected by Duplex-PCR, were compared together by application of RE mapping based on a partial sequence of VP2 gene (681 bp) by DdeI endonuclease. Our results showed that both CPV-2a and CPV-2b are pathogenic for dogs, but CPV-2a are more prevalent in healthy dogs while CPV-2b strains are more prevalent in enteric cases, significantly (p < 0.05). RE mapping based on DdeI restriction endonuclease demonstrated homology in the nucleotide sequence in amplified part of VP2 gene region in the genome of vaccinal and wild strains of CPV-2 detected in fecal samples of tested animals.

Genetic Analysis Using Partial Sequencing of Melanocortin 4 Receptor (MC4R) Gene in Bligon Goat

Melanocortin 4 Receptor gene is involved in sympathetic nerve activity, adrenal and thyroid functions, and media for leptin in regulating energy balance and homeostasis. The aim of this research was to perform genetic analysis of MC4R gene sequences from Bligon goats. Fourty blood samples of Bligon does were used for DNA extraction. The primers were designed after alignment of 12 DNA sequences of MC4R gene from goat, sheep, and cattle. The primers were constructed on the Capra hircus MC4R gene sequence from GenBank (accession No. NM_001285591). Two DNA polymorphisms of MC4R were revealed in exon region (g.998 A/G and g.1079 C/T). The SNP g.998 A/G was a non-synonymous polymorphism i.e., changing of amino acid from methionine (Met) to isoleucine (Ile). The SNP g.1079 C/T was a synonymous polymorphism. Restriction enzyme mapping on Bligon goat MC4R gene revealed three restriction enzymes (RsaI (GT’AC), Acc651 (G’GTAC_C), and KpnI (G_GTAC’C), which can recognize the SNP at g.1079 C/T. The restriction enzymes may be used for genotyping of the gene target using PCR-RFLP method in the future research.

English

Enterohemorrhagic Escherichia coli are important human food-borne pathogens. Recently, Shiga toxin-producing E. coli (STEC) causes life-threatening hemolytic-uremic syndrome (HUS). In this study, Stx2B gene, a subunit of Shiga toxin, was amplified via polymerase chain reaction (PCR) from the chromosomal DNA of clinical fecal sample using appropriate primers. The PCR product was cloned to commercially available plasmid pH6HTN His6HaloTag® T7 containing two purification tags, namely, six histadine tag and Halo tag. The integrity of the constructed plasmid was confirmed using restriction enzyme mapping and sequencing. Then, Stx2B protein expressed after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) in E. coli JM109 (DE3) under the control of the T7 promotor. The two step purification trains were used to purify native Stx2B. First step purification was Ni-immobilized metal ion affinity chromatography (IMAC) column, followed by second step using HaloLink resin. The native Stx2B was obtained after column cleavage of halo-tag using HaloTEV protease. Maximum protein expression of Stx2B economically was obtained using 1 mM IPTG for 4 h at 37°C. Protein identity was confirmed by a band at ~11.4 kDa using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and StxB2 yield was 450 µg ml-1 confirmed by Bradford assay. Recombinant Stx2B protein was produced in highly pure yield using HaloTag technology. Key words: Escherichia coli O157:H7, StxB gene, expression, HaloTag technology, purification.

Application of in-house virtual protein database performed in genomic-proteomic combined research on heavy-metal stressed onion roots.

To establish an in-house virtual protein database that can be employed in proteomic research on non-model plants. A total of 87,430 unigenes were obtained through transcriptome sequencing from onion roots. Of these, 24,305 unigenes were annotated and their nucleotide sequences of coding regions were translated into amino acid sequences. The corresponding 24,305 amino acid sequences were considered as an in-house virtual protein database. Thirty-two protein spots with significant differential abundance were selected. Their MS data were submitted to a restriction enzyme map which was converted from the in-house virtual protein database. A total of 27 proteins were finally matched. The in-house protein database is a feasible and innovative strategy for proteomic research on non-model plants.

Engineering and characterization of a symbiotic selection-marker-free vector-host system for therapeutic plasmid production.

The present study aimed to develop a symbiotic selection-marker-free plasmid and host system that would allow successful plasmid maintenance and amplification for use in gene therapy. Initially, the chromosomal aspartate‑semialdehyde dehydrogenase (asd) gene was disrupted in DH10B Escherichia coli using Red recombinase‑mediated homologous recombination. This method required the use of linear DNA fragments carrying kan‑kil genes, and/or homologous extensions to the targeted locus. The resultant auxotrophic cell wall‑deficient strain (DH10BΔasd) was evaluated as a symbiotic host for amplification of the marker‑free plasmid, allowing it to supply ASD activity. In order to construct the plasmid, an asd expression cassette was inserted, under the control of the nirB promoter, into a eukaryotic expression vector, and its kanamycin resistance gene was subsequently removed. The symbiotic plasmid and host system was assessed for numerous plasmid production and stability parameters, including structure, yield, plasmid‑retention rate, and bacterial storability, under various conditions. The presence of the plasmid was subsequently confirmed by growth test, restriction enzyme mapping, and sequencing. The plasmid yield and copy number produced in the symbiotic cells, in the absence of antibiotic selection, were shown to be similar to those produced under kanamycin selection, in the cells containing the precursor plasmid and kanamycin resistance gene. Furthermore, the results of the present study demonstrated that when inoculated with <1% inoculant volume, >98% of the cells in the culture retained the plasmid regardless of the number of passages. The strain was stable when stored at ‑70˚C, with negligible viability loss over 12 months. The constructed plasmid is stable and has potential in future gene therapy, while much work is still required.

Improving the ostrich genome assembly using optical mapping data.

BackgroundThe ostrich (Struthio camelus) is the tallest and heaviest living bird. Ostrich meat is considered a healthy red meat, with an annual worldwide production ranging from 12,000 to 15,000 tons. As part of the avian phylogenomics project, we sequenced the ostrich genome for phylogenetic and comparative genomics analyses. The initial Illumina-based assembly of this genome had a scaffold N50 of 3.59 Mb and a total size of 1.23 Gb. Since longer scaffolds are critical for many genomic analyses, particularly for chromosome-level comparative analysis, we generated optical mapping (OM) data to obtain an improved assembly. The OM technique is a non-PCR-based method to generate genome-wide restriction enzyme maps, which improves the quality of de novo genome assembly.FindingsIn order to generate OM data, we digested the ostrich genome with KpnI, which yielded 1.99 million DNA molecules (>250 kb) and covered the genome at least 500×. The pattern of molecules was subsequently assembled to align with the Illumina-based assembly to achieve sequence extension. This resulted in an OM assembly with a scaffold N50 of 17.71 Mb, which is 5 times as large as that of the initial assembly. The number of scaffolds covering 90% of the genome was reduced from 414 to 75, which means an average of ~3 super-scaffolds for each chromosome. Upon integrating the OM data with previously published FISH (fluorescence in situ hybridization) markers, we recovered the full PAR (pseudoatosomal region) on the ostrich Z chromosome with 4 super-scaffolds, as well as most of the degenerated regions.ConclusionsThe OM data significantly improved the assembled scaffolds of the ostrich genome and facilitated chromosome evolution studies in birds. Similar strategies can be applied to other genome sequencing projects to obtain better assemblies.

The promise of genomics for a “next generation” of advances in higher-level legume molecular systematics

The years since the first International Legume Conference in 1978 have seen a veritable revolution in molecular phylogenetics. The first two volumes of Advances in Legume Systematics series, which were based on that conference, contained no information on DNA-level variation. The next volume in the series, in 1987, had a chapter on the potential of DNA approaches and results of some early studies that used restriction enzyme mapping. The 1990s saw the application of chloroplast gene sequencing to family-level phylogenetic problems that culminated in the studies from the first decade of the present century that have provided the working phylogenetic hypotheses for the family. The first full legume nuclear genome sequences have appeared more recently, and, fueled by the advent of “next-generation” sequencing in the late 2000s, there is now a flood of genomic and transcriptomic data that is again revolutionizing biology in the way that the molecular revolution did previously. How to take advantage of this opportunity is a key question for legume systematists, particularly its own “next generation”. The mere availability of massive amounts of data does not guarantee that all phylogenetic problems in legumes will be resolved once and for all; “megadata” is not a panacea, and the apparent rapid radiation of the family and its constituent clades represents a serious technical challenge. Moreover, major analytical controversies are simmering, notably between proponents of concatenation – the conventional way to analyze genome-scale data – and those who favor a coalescent-based species tree approach. Assuming that a stable, well-resolved phylogeny based on the nuclear genome is eventually produced, it will be useful not only for classification, but also for addressing questions involving homoplasy, such as the potential multiple origins of nodulation in the family. The rich legacy of the International Legume Conferences, embodied in the Advances in Legume Systematics, is that the increasing refinement of phylogenetic hypotheses serves the full range of comparative studies on the myriad facets of this huge, diverse, and fascinating family.

BioMatriX: Sequence analysis, structure visualization, phylogenetics and linkage analysis workbench

The BioMatriX (Build Mine Xplore) is a bioinformatics work bench (http://www.bmx-biomatrix.blogspot.com) developed for biological science community to augment scientific research regarding genomics, proteomics, phylogenetics and linkage analysis in one platform. BioMatriX offers multi-functional services to perform specific tasks like DNA/RNA/Protein sequence analysis with graphical representations, sequence editing, sequence alignment, restriction enzyme mapping, protein structure visualization, mutation and structure superimposition programs along with phylogenetics tree construction supporting dendrograms, neighbor joining and unweighted pair group method with arithmetic mean (UPGMA) programs. Genomic studies like linkage programs are also implemented. Special emphasis has been paid to integrate all the resources in one software so that the researcher does not have to install numerous pieces of software to analyze his data. Keywords: Bioinformatics, linkage, visualizer, alignment, superimposition, phylogenetics

Centromere-associated repeat arrays on Trypanosoma brucei chromosomes are much more extensive than predicted

BackgroundAfrican trypanosomes belong to a eukaryotic lineage which displays many unusual genetic features. The mechanisms of chromosome segregation in these diploid protozoan parasites are poorly understood. Centromeres in Trypanosoma brucei have been localised to chromosomal regions that contain an array of ~147 bp AT-rich tandem repeats. Initial estimates from the genome sequencing project suggested that these arrays ranged from 2 - 8 kb. In this paper, we show that the centromeric repeat regions are much more extensive.ResultsWe used a long-range restriction endonuclease mapping approach to more accurately define the sizes of the centromeric repeat arrays on the 8 T. brucei chromosomes where unambiguous assembly data were available. The results indicate that the sizes of the arrays on different chromosomes vary from 20 to 120 kb. In addition, we found instances of length heterogeneity between chromosome homologues. For example, values of 20 and 65 kb were obtained for the arrays on chromosome 1, and 50 and 75 kb for chromosome 5.ConclusionsOur results show that centromeric repeat arrays on T. brucei chromosomes are more similar in size to those of higher eukaryotes than previously suspected. This information provides a firmer framework for investigating aspects of chromosome segregation and will allow epigenetic features associated with the process to be more accurately mapped.