The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform

James A. Birchler - One of the best experts on this subject based on the ideXlab platform.

  • Production of Engineered Minichromosome Vectors via the Introduction of Telomere Sequences.
    Methods of Molecular Biology, 2016
    Co-Authors: Nathaniel D. Graham, Jon P. Cody, Nathan C. Swyers, Morgan E. Mccaw, Changzeng Zhao, James A. Birchler
    Abstract:

    Artificial Minichromosomes are non-integrating vectors capable of stably maintaining transgenes outside of the main chromosome set. The production of Minichromosomes relies on telomere-mediated chromosomal truncation, which involves introducing transgenes and telomere sequences concurrently to the cell to truncate an endogenous chromosomal target. Two methods can be utilized; either the telomere sequences can be incorporated into a binary vector for transformation with Agrobacterium tumefaciens, or the telomere sequences can be co-introduced with transgenes during particle bombardment. In this protocol, the methods required to isolate and introduce telomere sequences are presented. Following the methods presented, standard transformation procedures can be followed to produce Minichromosome containing plants.

  • Plant Minichromosomes.
    Current opinion in biotechnology, 2015
    Co-Authors: James A. Birchler, Nathaniel D. Graham, Jon P. Cody, Nathan C. Swyers, Morgan E. Mccaw
    Abstract:

    Plant Minichromosomes have the potential for stacking multiple traits on a separate entity from the remainder of the genome. Transgenes carried on an independent chromosome would facilitate conferring many new properties to plants and using Minichromosomes as genetic tools. The favored method for producing plant Minichromosomes is telomere-mediated chromosomal truncation because the epigenetic nature of centromere function prevents using centromere sequences to confer the ability to organize a kinetochore when reintroduced into plant cells. Because haploid induction procedures are not always complete in eliminating one parental genome, chromosomes from the inducer lines are often present in plants that are otherwise haploid. This fact suggests that Minichromosomes could be combined with doubled haploid breeding to transfer stacked traits more easily to multiple lines and to use Minichromosomes for massive scale genome editing.

  • Minichromosomes: Vectors for Crop Improvement
    Agronomy, 2015
    Co-Authors: Jon P. Cody, Nathaniel D. Graham, Nathan C. Swyers, Morgan E. Mccaw, Changzeng Zhao, James A. Birchler
    Abstract:

    Minichromosome technology has the potential to offer a number of possibilities for expanding current biofortification strategies. While conventional genome manipulations rely on random integration of one or a few genes, engineered Minichromosomes would enable researchers to concatenate several gene aggregates into a single independent chromosome. These engineered Minichromosomes can be rapidly transferred as a unit to other lines through the utilization of doubled haploid breeding. If used in conjunction with other biofortification methods, it may be possible to significantly increase the nutritional value of crops.

  • Engineered Minichromosomes in Plants: Structure, Function, and Applications
    International Review of Cell and Molecular Biology, 2015
    Co-Authors: Nathaniel D. Graham, Jon P. Cody, Nathan C. Swyers, Morgan E. Mccaw, Changzeng Zhao, James A. Birchler
    Abstract:

    Engineered Minichromosomes are small chromosomes that contain a transgene and selectable marker, as well as all of the necessary components required for maintenance in an organism separately from the standard chromosome set. The separation from endogenous chromosomes makes engineered Minichromosomes useful in the production of transgenic plants. Introducing transgenes to Minichromosomes does not have the risk of insertion within a native gene; additionally, transgenes on Minichromosomes can be transferred between lines without the movement of linked genes. Of the two methods proposed for creating engineered Minichromosomes, telomere-mediated truncation is more reliable in plant systems. Additionally, many plants contain a supernumerary, or B chromosome, which is an excellent starting material for Minichromosome creation. The use of site-specific recombination systems in Minichromosomes can increase their utility, allowing for the addition or subtraction of transgenes in vivo. The creation of Minichromosomes with binary bacterial artificial chromosome vectors provides the ability to introduce many transgenes at one time. Furthermore, coupling Minichromosomes with haploid induction systems can facilitate transfer between lines. Minichromosomes can be introduced to a haploid-inducing line and crossed to target lines. Haploids of the target line that then contain a Minichromosome can then be doubled. These homozygous lines will contain the transgene without the need for repeated introgressions.

  • Engineered Minichromosome Technology in Plants
    Recent Advancements in Gene Expression and Enabling Technologies in Crop Plants, 2015
    Co-Authors: James A. Birchler
    Abstract:

    Engineered Minichromosomes have been produced in plants using telomere-mediated truncation that cleaves chromosome arms and simultaneously places desired transgenes linked to an endogenous centromere. Proof-of-concept experiments have been successful, illustrating that site-specific recombination can occur at terminal sites on the chromosome and that in vivo modification of Minichromosomes is possible as demonstrated by selectable marker removal. Future developments are discussed that would amplify the utility of engineered Minichromosomes.

Ronald Hancock - One of the best experts on this subject based on the ideXlab platform.

  • repair of dna strand breaks in a Minichromosome in vivo kinetics modeling and effects of inhibitors
    PLOS ONE, 2013
    Co-Authors: Slawomir Kumala, Joanna Rzeszowskawolny, Krzysztof Fujarewicz, Dheekollu Jayaraju, Ronald Hancock
    Abstract:

    To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a 170 kb circular Minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin [1]. The rate of repair of single strand breaks in cells irradiated with photons was quantitated by determining the sensitivity of the Minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed fie ld electrophoresis. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in Minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minich romosome provides a useful system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair. Four compartments each containing one form of Minichromosome DNA were considered together with the four ordinary differential equations. Fitt ing to the experimental data depended on estimating parameters and initial conditions in normal conditions or when double strand break repair was inhibited. A number of conclusions which were not directly apparent from the experimental data illustrated the usefulness of modeling. First, when repair of double strand breaks was arrested, the single strand breaks in linear molecules were still repaired and circular molecules containing single strand breaks were converted to supercoiled molecules at close to the normal rate showing that the systems which repair single and double strand breaks operate independently, which has not been demonstrated previously as far as we are aware. Second, the calculated rate constants show that in an average linearised Minichromosome the double strand break was repaired three to four times faster than all the single strand b reaks, so that the rate limiting step for complete repair of Minichromosomes was the repair of single strand breaks.

  • dna of a circular Minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage an influence of chromatin topology on the accessibility of dna
    Nucleic Acids Research, 2012
    Co-Authors: Slawomir Kumala, Yasmina Hadjsahraoui, Joanna Rzeszowskawolny, Ronald Hancock
    Abstract:

    The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a ∼170 kb circular Minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the Minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and γ-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized Minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular Minichromosome is linearized and results in its DNA becoming inaccessible to probes.

Christian H. Haering - One of the best experts on this subject based on the ideXlab platform.

  • Condensin structures chromosomal DNA through topological links
    Nature Structural and Molecular Biology, 2011
    Co-Authors: Sara Cuylen, Jan Metz, Christian H. Haering
    Abstract:

    The multisubunit condensin complex is essential for the structural organization of eukaryotic chromosomes during their segregation by the mitotic spindle, but the mechanistic basis for its function is not understood. To address how condensin binds to and structures chromosomes, we have isolated from Saccharomyces cerevisiae cells circular Minichromosomes linked to condensin. We find that either linearization of Minichromosome DNA or proteolytic opening of the ring-like structure formed through the connection of the two ATPase heads of condensin's structural maintenance of chromosomes (SMC) heterodimer by its kleisin subunit eliminates their association. This suggests that condensin rings encircle chromosomal DNA. We further show that release of condensin from chromosomes by ring opening in dividing cells compromises the partitioning of chromosome regions distal to centromeres. Condensin hence forms topological links within chromatid arms that provide the arms with the structural rigidity necessary for their segregation.

Antoun Toubaji - One of the best experts on this subject based on the ideXlab platform.

  • immunohistochemical expression of Minichromosome maintenance complex protein 2 predicts biochemical recurrence in prostate cancer a tissue microarray and digital imaging analysis based study of 428 cases
    Human Pathology, 2012
    Co-Authors: Antoun Toubaji, Siobhan Sutcliffe, Alcides Chaux, Kristen Lecksell, Jessica L Hicks, Angelo M De Marzo, Elizabeth A Platz, George J Netto
    Abstract:

    Prostate cancer remains a major health problem in the United States. Established clinicopathologic parameters such as Gleason score, T stage, and prostate-specific antigen levels are currently the guiding tools for prognostication and disease management. The addition of biomarkers could increase the accuracy of these parameters for predicting disease progression, response to therapy, and survival. In this regard, the goal of this study was to evaluate Minichromosome maintenance complex protein 2 and Ki-67 immunohistochemical expression as predictors of outcome in prostate cancer. For this purpose, 11 tissue microarrays were constructed using tumor and nontumor samples from 428 patients. Patients were divided into short-term (mean, 2.9 years) and long-term (mean, 14.1 years) follow-up groups. End points were biochemical recurrence for the short-term follow-up group and prostate cancer–related death for the long-term follow-up group. All men in the long-term follow-up group had biochemical recurrence at the time of recruitment. Expression of both markers was higher in tumor than in nontumor glands. Percentage of Minichromosome maintenance complex protein 2 was associated with Gleason score in both groups. Percentage of Ki-67 was associated with Gleason score and pathologic stage only in the short-term follow-up group. Higher Minichromosome maintenance complex protein 2 percentages were associated with biochemical recurrence in the short-term follow-up group. In the long-term follow-up group, neither Minichromosome maintenance complex protein 2 nor Ki-67 levels predicted prostate cancer death. In conclusion, our results suggest that in patients treated by radical prostatectomy for clinically localized prostate cancer, immunohistochemistry for Minichromosome maintenance complex protein 2 expression could be used to predict biochemical recurrence, independent of other known clinicopathologic factors.

Mitsuhiro Yanagida - One of the best experts on this subject based on the ideXlab platform.

  • A large circular Minichromosome of Schizosaccharomyces pombe requires a high dose of type II DNA topoisomerase for its stabilization
    Molecular and General Genetics MGG, 1995
    Co-Authors: Shin Murakami, Mitsuhiro Yanagida, Osami Niwa
    Abstract:

    We have constructed circular Minichromosomes, ranging in size from 36 to 110 kb, containing the centromeric repeats of Schizosaccharomyces pombe cen3 . Comparison of their mitotic stability showed that the circular Minichromosomes became more unstable with increasing in size, however, a linear cen3 Minichromosome, which is almost the same size as the largest circular one tested, does not show such instability. High levels of expression of the top2 ^+ (type II DNA topoisomerase; topo II) but not top1 ^+ gene (type I DNA topoisomerase) suppressed the instability of the largest circular Minichromosome, whereas partial inactivation of topo II dramatically destabilized the Minichromosome. A mutant topo II, defective in nuclear localization but still retaining its in vitro relaxation activity, did not stabilize the circular Minichromosome. These results indicate that endogenous type II DNA topoisomerase is insufficient for accurate segregation of the circular Minichromosome. In addition, the replication of the minichromosomal DNA appears to proceed normally, because the presence of the unstable Minichromosome did not cause G2 delay. A likely cause of the instability is intertwining of the Minichromosome DNA possibly occuring after DNA replication. An interaction between topo II and the centromeric repeats is implied by the finding that multiple copies of the centromeric repeat, dg-dh, affect stability of the Minichromosome similarly to top2 ^+ gene dosage.

  • fission yeast Minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality
    Molecular Biology of the Cell, 1994
    Co-Authors: Kohta Takahashi, Hiroshi Yamada, Mitsuhiro Yanagida
    Abstract:

    Abstract Precise chromosome transmission in cell division cycle is maintained by a number of genes. The attempt made in the present study was to isolate temperature-sensitive (ts) fission yeast mutants that display high loss rates of Minichromosomes at permissive or semipermissive temperature (designated mis). By colony color assay of 539 ts strains that contain a Minichromosome, we have identified 12 genetic loci (mis1-mis12) and determined their phenotypes at restrictive temperature. Seven of them are related to cell cycle block phenotype at restrictive temperature, three of them in mitosis. Unequal distribution of regular chromosomes in the daughters is extensive in mis6 and mis12. Cells become inviable after rounds of cell division due to missegregation. The phenotype of mis5 is DNA replication defect and hypersensitivity to UV ray and hydroxyurea. mis5+ encodes a novel member of the ubiquitous MCM family required for the onset of replication. The mis5+ gene is essential for viability and functionally distinct from other previously identified members in fission yeast, cdc21+, nda1+, and nda4+. The mis11 mutant phenotype was the cell division block with reduced cell size. Progression of the G1 and G2 phases is blocked in mis11. The cloned mis11+ gene is identical to prp2+, which is essential for RNA splicing and similar to a mammalian splicing factor U2AF65.