Construction of a chromosome specific library of human MARs and mapping of matrix attachment regions on human chromosome 19.
ABSTRACT: Using a novel procedure a representative human chromosome 19-specific library was constructed of short sequences, which bind preferentially to the nuclear matrix (matrix attachment regions, or MARs). Judging by 20 clones sequenced so far, the library contains > 50% of human inserts, about 90% of which are matrix-binding by the in vitro test. Computer analysis of sequences of eight human MARs did not reveal any significant homologies with the EMBL Nucleotide Data Base entries as well as between MARs themselves. Eight MARs were assigned to individual positions on the chromosome 19 physical map. The library constructed can serve as a good source of MAR sequences for comparative analysis and classification and for further chromosome mapping of MARs as well.
Project description:Scaffold/matrix attachment regions (S/MARs) are essential regulatory DNA elements of eukaryotic cells. They are major determinants of locus control of gene expression and can shield gene expression from position effects. Experimental detection of S/MARs requires substantial effort and is not suitable for large-scale screening of genomic sequences. In silico prediction of S/MARs can provide a crucial first selection step to reduce the number of candidates. We used experimentally defined S/MAR sequences as the training set and generated a library of new S/MAR-associated, AT-rich patterns described as weight matrices. A new tool called SMARTest was developed that identifies potential S/MARs by performing a density analysis based on the S/MAR matrix library (http://www.genomatix.de/cgi-bin/smartest_pd/smartest.pl). S/MAR predictions were evaluated by using six genomic sequences from animal and plant for which S/MARs and non-S/MARs were experimentally mapped. SMARTest reached a sensitivity of 38% and a specificity of 68%. In contrast to previous algorithms, the SMARTest approach does not depend on the sequence context and is suitable to analyze long genomic sequences up to the size of whole chromosomes. To demonstrate the feasibility of large-scale S/MAR prediction, we analyzed the recently published chromosome 22 sequence and found 1198 S/MAR candidates.
Project description:In this study, we systemically investigated the positions and orientations of matrix attachment regions (MARs) in expression vectors to fully explore the mechanism for improving transgene expression. We constructed 14 vectors that incorporated human ?-globin MARs into pIRES-eGFP backbone vectors. The MARs flanked the eGFP expression cassette or promoter in a forward/reverse orientation. After stable transfection into CHO-K1 cells with these vectors, eGFP expression levels were increased significantly relative to that of the control vector (MAR-devoid) when two MARs flanking the expression cassette were incorporated, followed by those at the 5' site (upstream of the promoter). Simultaneously, the percentage of the eGFP-expressing cells was elevated to some extent. The vector with both MARs in forward orientation flanking the expression cassette yielded the highest transgene expression levels (2.5-fold). The orientation (forward or reverse) of the MARs did not present a significant difference when added in the same site. In addition, transgene expression levels were not exclusively dependent on transgene copy numbers. Bioinformatic analysis indicated that some specific transcription factors may contribute to the transcriptional process. In conclusion, two MARs in a forward orientation and flanking the expression cassette comprised the optimal construct for improving the stable transgene expression in the CHO-K1 cells. The effects may be related to specific transcription factors, such as PRDM1 and REL.
Project description:Microtubule-associated proteins (MAPs) ensure the fidelity of chromosome segregation by controlling microtubule (MT) dynamics and mitotic spindle stability. However, many aspects of MAP function and regulation are poorly understood in a developmental context. We show that mars, which encodes a Drosophila melanogaster member of the hepatoma up-regulated protein family of MAPs, is essential for MT stabilization during early embryogenesis. As well as associating with spindle MTs in vivo, Mars binds directly to protein phosphatase 1 (PP1) and coimmunoprecipitates from embryo extracts with minispindles and Drosophila transforming acidic coiled-coil (dTACC), two MAPs that function as spindle assembly factors. Disruption of binding to PP1 or loss of mars function results in elevated levels of phosphorylated dTACC on spindles. A nonphosphorylatable form of dTACC is capable of rescuing the lethality of mars mutants. We propose that Mars mediates spatially controlled dephosphorylation of dTACC, which is critical for spindle stabilization.
Project description:Nuclear matrix binding assays (NMBAs) define certain DNA sequences as matrix attachment regions (MARs), which often have cis-acting epigenetic regulatory functions. We used NMBAs to analyze the functionally important 15q11-q13 imprinting center (IC). We find that the IC is composed of an unusually high density of MARs, located in close proximity to the germ line elements that are proposed to direct imprint switching in this region. Moreover, we find that the organization of MARs is the same at the homologous mouse locus, despite extensive divergence of DNA sequence. MARs of this size are not usually associated with genes but rather with heterochromatin-forming areas of the genome. In contrast, the 15q11-q13 region contains multiple transcribed genes and is unusual for being subject to genomic imprinting, causing the maternal chromosome to be more transcriptionally silent, methylated, and late replicating than the paternal chromosome. We suggest that the extensive MAR sequences at the IC are organized as heterochromatin during oogenesis, an organization disrupted during spermatogenesis. Consistent with this model, multicolor fluorescence in situ hybridization to halo nuclei demonstrates a strong matrix association of the maternal IC, whereas the paternal IC is more decondensed, extending into the nuclear halo. This model also provides a mechanism for spreading of the imprinting signal, because heterochromatin at the IC on the maternal chromosome may exert a suppressive position effect in cis. We propose that the germ line elements at the 15q11-q13 IC mediate their effects through the candidate heterochromatin-forming DNA identified in this study.
Project description:BACKGROUND:The number of mobile health apps (MHAs), which are developed to promote healthy behaviors, prevent disease onset, manage and cure diseases, or assist with rehabilitation measures, has exploded. App store star ratings and descriptions usually provide insufficient or even false information about app quality, although they are popular among end users. A rigorous systematic approach to establish and evaluate the quality of MHAs is urgently needed. The Mobile App Rating Scale (MARS) is an assessment tool that facilitates the objective and systematic evaluation of the quality of MHAs. However, a German MARS is currently not available. OBJECTIVE:The aim of this study was to translate and validate a German version of the MARS (MARS-G). METHODS:The original 19-item MARS was forward and backward translated twice, and the MARS-G was created. App description items were extended, and 104 MHAs were rated twice by eight independent bilingual researchers, using the MARS-G and MARS. The internal consistency, validity, and reliability of both scales were assessed. Mokken scale analysis was used to investigate the scalability of the overall scores. RESULTS:The retranslated scale showed excellent alignment with the original MARS. Additionally, the properties of the MARS-G were comparable to those of the original MARS. The internal consistency was good for all subscales (ie, omega ranged from 0.72 to 0.91). The correlation coefficients (r) between the dimensions of the MARS-G and MARS ranged from 0.93 to 0.98. The scalability of the MARS (H=0.50) and MARS-G (H=0.48) were good. CONCLUSIONS:The MARS-G is a reliable and valid tool for experts and stakeholders to assess the quality of health apps in German-speaking populations. The overall score is a reliable quality indicator. However, further studies are needed to assess the factorial structure of the MARS and MARS-G.
Project description:The short arm of human chromosome 21 (21p) contains many different types of repetitive sequences and is highly homologous to the short arms of other acrocentric chromosomes. Owing to its repetitive nature and the lack of chromosome 21p-specific molecular markers, most physical maps of chromosome 21 exclude this region. We constructed a physical map of chromosome 21p using sequence tagged site (STS) content mapping of yeast artificial chromosomes (YACs). To this end, 39 STSs located on the short arm or near the centromere of chromosome 21 were constructed, including four polymorphic simple tandem repeats (STRs) and two expressed sequence tags (ESTs). Thirty YACs were selected from the St. Louis YAC library, the chromosome 21-enriched ICRF YAC library, and the CEPH YAC and megaYAC libraries. These were assembled in a YAC contig map ranging from the centromere to the rDNA gene cluster at 21p12. The total size of the region covered by YACs is estimated between 2.9 and 5 Mb. The integrity of the YAC contig was confirmed by restriction enzyme fingerprinting and fluorescence in situ hybridization (FISH). One gap with an estimated size of 400 kb remained near the telomeric end of the contig. This YAC contig map of the short arm of human chromosome 21 constitutes a basic framework for further structural and functional studies of chromosome 21p.
Project description:Existing non-verbal ability tests are typically protected by copyright, preventing them from being freely adapted or computerized. Working towards an open science framework, we provide 80 novel, open-access abstract reasoning items, an online implementation and item-level data from 659 participants aged between 11 and 33 years: the matrix reasoning item bank (MaRs-IB). Each MaRs-IB item consists of an incomplete matrix containing abstract shapes. Participants complete the matrices by identifying relationships between the shapes. Our data demonstrate age differences in non-verbal reasoning accuracy, which increased during adolescence and stabilized in early adulthood. There was a slight linear increase in response times with age, resulting in a peak in efficiency (i.e. a measure combining speed and accuracy) in late adolescence. Overall, the data suggest that the MaRs-IB is sensitive to developmental differences in reasoning accuracy. Further psychometric validation is recommended.
Project description:Scaffold or matrix attachment regions (S/MARs) are found in all eukaryotes. The pattern of distribution and genomic context of S/MARs is thought to be important for processes such as chromatin organization and modulation of gene expression. Despite the importance of such processes, much is unknown about the large-scale distribution and sequence content of S/ MARs in vivo. Here, we report the use of tiling microarrays to map 1358 S/MARs on Arabidopsis thaliana chromosome 4 (chr4). S/MARs occur throughout chr4, spaced much more closely than in the large plant and animal genomes that have been studied to date. Arabidopsis S/MARs can be divided into five clusters based on their association with other genomic features, suggesting a diversity of functions. While some Arabidopsis S/MARs may define structural domains, most occur near the transcription start sites of genes. Genes associated with these S/MARs have an increased probability of expression, which is particularly pronounced in the case of transcription factor genes. Analysis of sequence motifs and 6-mer enrichment patterns show that S/MARs are preferentially enriched in poly(dA:dT) tracts, sequences that resist nucleosome formation, and the majority of S/MARs contain at least one nucleosome-depleted region. This global view of S/MARs provides a framework to begin evaluating genome-scale models for S/MAR function. Contrast between DNA bound to nuclear scaffold/matrix and total genomic DNA in Arabidopsis Chr4 excluding the constitutive heterochromatin. Total of three biological replicates with two independent hybridizations on custom-designed NimbleGen high-density microarrays that include duplicate spots for each probe.
Project description:Bread wheat (Triticum aestivum) has a large and highly repetitive genome which poses major technical challenges for its study. To aid map-based cloning and future genome sequencing projects, we constructed a BAC-based physical map of the short arm of wheat chromosome 1A (1AS). From the assembly of 25,918 high information content (HICF) fingerprints from a 1AS-specific BAC library, 715 physical contigs were produced that cover almost 99% of the estimated size of the chromosome arm. The 3,414 BAC clones constituting the minimum tiling path were end-sequenced. Using a gene microarray containing ?40 K NCBI UniGene EST clusters, PCR marker screening and BAC end sequences, we arranged 160 physical contigs (97 Mb or 35.3% of the chromosome arm) in a virtual order based on synteny with Brachypodium, rice and sorghum. BAC end sequences and information from microarray hybridisation was used to anchor 3.8 Mbp of Illumina sequences from flow-sorted chromosome 1AS to BAC contigs. Comparison of genetic and synteny-based physical maps indicated that ?50% of all genetic recombination is confined to 14% of the physical length of the chromosome arm in the distal region. The 1AS physical map provides a framework for future genetic mapping projects as well as the basis for complete sequencing of chromosome arm 1AS.
Project description:BACKGROUND: Japanese amberjack/yellowtail (Seriola quinqueradiata) is a commonly cultured marine fish in Japan. For cost effective fish production, a breeding program that increases commercially important traits is one of the major solutions. In selective breeding, information of genetic markers is useful and sufficient to identify individuals carrying advantageous traits but if the aim is to determine the genetic basis of the trait, large insert genomic DNA libraries are essential. In this study, toward prospective understanding of genetic basis of several economically important traits, we constructed a high-coverage bacterial artificial chromosome (BAC) library, obtained sequences from the BAC-end, and constructed comprehensive female and male linkage maps of yellowtail using Simple Sequence Repeat (SSR) markers developed from the BAC-end sequences and a yellowtail genomic library. RESULTS: The total insert length of the BAC library we constructed here was estimated to be approximately 11 Gb and hence 16-times larger than the yellowtail genome. Sequencing of the BAC-ends showed a low fraction of repetitive sequences comparable to that in Tetraodon and fugu. A total of 837 SSR markers developed here were distributed among 24 linkage groups spanning 1,026.70 and 1,057.83 cM with an average interval of 4.96 and 4.32 cM in female and male map respectively without any segregation distortion. Oxford grids suggested conserved synteny between yellowtail and stickleback. CONCLUSIONS: In addition to characteristics of yellowtail genome such as low repetitive sequences and conserved synteny with stickleback, our genomic and genetic resources constructed and revealed here will be powerful tools for the yellowtail breeding program and also for studies regarding the genetic basis of traits.