Centromere repeat

Repetitive-sequence DNA can be broadly classified as moderately repetitive or as highly repetitive. The highly repetitive sequences consist of 5-500 base pair lengths repeated many times in tandem. These sequences are usually clustered in centromeres and telomeres of the chromosome and are present in about 1-10 milHon copies per haploid genome. These sequences are transcriptionally inactive and may play a strucmral role in the chromosome (see Chapter 40). 

The centromeric probe for human chromosomes is a cloned seqnence, p82H, of the alphoid-repeated DNA family. The cloning and characterization of this sequence are described in (4). 

Dnmt3a De novo DNA methyltransferase Adult/Embryo DNMT3a mice die at 4 weeks ES cells from DNMT3a mice are viable and capable of de novo methylation DNMT3a embryos and ES cells exhibit demethylation of centromeric satellite repeats... 

For repetitive DNA sequences, such as alphoid repeats, especially for identification of centromeric regions on chromosomes, as well as in mterphase nuclei, I routinely prepare labeled probes by PCR using a centromeric DNA-specific set of primers. For single-copy gene mapping by FISH, it is essential to use DNA cloned into either cosmids or in yeast artificial chromosome vectors (YAC) m order to obtain the level of sensitivity that will enable visualization of signal with a fluorescence microscope... 

The method described here is ideally suited for detection of both alphoid repeats at the centromeric regions of all chromosomes, and moderately repetitive DNA sequences within the arms and telomeric regions... 

Mewes HW, Albermann K, Bahr M, Frishman D, Gleissner A, Hani J, Heuman K, Kleine K, Maierl A, Oliver SG, Pfeifer F, Zollner A Overview of the yeast genome. Nature 1997 387 7-65. Mewes HW, Albermann K, Heumann K, Liebl S, Pfeifer F MIPS A database for protein sequences, homology data and yeast genome information. Nucleic Acid Res 1997 35 28-30. Clarke L, Baum MP Functional analysis of a centromere from fission yeast A role for centromere-specific repeated DNA sequences. Mol Cell Biol 1990 10 1863-1872. 

DNA methylation also is present within tandem-repetitive DNA sequences in the heterochromatin. They also are thought to inhibit recombination between homologous repeats, which could lead to genomic instability (46). In fact, mutations of the DNMT3b cause centromeric instability. In addition, HDACs lie at the heart of heterochromatin pathways and can be recruited by transcriptional repressor or by methyl-DNA binding proteins. 

Centromere—constricted region of the chromosome joining two sister chromatids. The centromere is composed of highly repeated DNA sequences approximately 220 units in length. 

Mitchell, A. R, Gosden, J. R., and Miller, D. A. (1985) A cloned sequence, p82H, of the alphoid repeated DNA family found at the centromeres of all human chromosomes. Chromosoma 92,369-377. 

Formamide hybridization mix For repeat sequence probes (e.g., centromeric probes), 2X SSC, 500 ug/mL SSDNA, 10% dextran sulphate, 70% formamide. 

R192 D. J. Patel, S. Bouaziz, A. Kettani and Y. Wang, Structures of Guanine-Rich and Cytosine-Rich Quadruplexes Formed in Vitro by Telomeric, Centromeric, and Triplet Repeat Disease DNA Sequences , p. 389... 

Most satellite DNA is composed of repeats of 14-500 base pairs in tandem repeats of 20-100 kb. In situ hybridization studies with metaphase chromosomes have localized these satellite DNAs to specific chromosomal regions. In most mammals, much of this satellite DNA lies near centromeres, the discrete chromosomal regions that attach to spindle microtubules during mitosis and meiosis. Satellite DNA is also located at telomeres, the ends of chromosomes, and at specific locations within chromosome arms in some organisms. These latter sequences can be useful for identifying particular chromosomes by fluorescence in situ hybridization (FISH), as Illustrated in Figure 10-5. 

Simple-sequence DNA located at centromeres may assist in attaching chromosomes to spindle microtubules during mitosis. As yet, however, there is little clear-cut experimental evidence demonstrating any function for most simple-sequence DNA, with the exception of the short repeats at the very ends of chromosomes discussed in a later section. 

Simple-sequence DNA, which consists largely of quite short sequences repeated in long tandem arrays, is preferentially located in centromeres, telomeres, and specific locations within the arms of particular chromosomes. 

Very rapidly reassociating sequences come from a simple sequence that is repeated many times (up to a million times per cell) in clusters of tandem repeats of the same sequence. Such DNA is also called satellite DNA since it forms separate, satellite peaks in density gradients due to its uniform base composition. One satellite DNA is found at the telomeres (ends) of chromosomes another is at the centromeres. 

The ultimate job of the SAC is to ensure that cells enter anaphase only after all chromosomes have acquired a bipolar (i.e., amphitelic) attachment to the spindle. As described earlier, the checkpoint signal is initiated at kinetochores that either are not attached to the spindle or display a loss of tension. The kineto-chore is composed of many specific proteins that play two crucial roles (1) coordinate attachment to microtubules, and (2) send signals when attachments are incorrect. Kinetochores are assembled by a complicated process at the centromere, a specific region of the chromosome characterized by the presence of nucleotide repeats. Thus, it is the DNA sequence that ultimately determines where the kinetochore will assemble. Centromeric chromatin is also characterized by the presence of a variant histone, CENP-A, which takes the place of histone H3 in the nucleosome core. 

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