Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries
TLDR
A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported and should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.Abstract:
A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.read more
Citations
More filters
Journal ArticleDOI
A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome
Mark E. Curran,Igor Splawski,Katherine W. Timothy,Vincent Gm,Eric D. Green,Keating Mt,Keating Mt +6 more
TL;DR: In this article, the authors investigated patients with long QT syndrome (LQT), an inherited disorder causing sudden death from a ventricular tachyarrythmia, torsade de pointes.
Journal ArticleDOI
Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.
Michael J. Hendzel,Y Wei,Michael A. Mancini,A. Van Hooser,Tamara A. Ranalli,Bill R. Brinkley,David P. Bazett-Jones,C D Allis +7 more
TL;DR: It is proposed that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that “drive” chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M- phase.
Journal ArticleDOI
Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9
Thomas Wagner,J. Wirth,Jobst Meyer,Bernhard Zabel,Marika Held,J. Zimmer,Juan J. Pasantes,Juan J. Pasantes,Franca Dagna Bricarelli,Jürgen Keutel,Elisabeth Hustert,Ulrich Wolf,Niels Tommerup,Werner Schempp,Gerd Scherer +14 more
TL;DR: Inactivating mutations on oneSOX9 allele identified in nontranslocation CMPD1-SRA1 cases point to haploinsufficiency for SOX9 as the cause for both campomelic dysplasia and autosomal XY sex reversal.
Journal ArticleDOI
High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones.
Peter Lichter,Chieh-Ju Chang Tang,Katherine M. Call,Gary G. Hermanson,Glen A. Evans,David E. Housman,David C. Ward +6 more
TL;DR: The results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization and show that by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally.
Journal ArticleDOI
Pml Is Critical for Nd10 Formation and Recruits the Pml-Interacting Protein Daxx to This Nuclear Structure When Modified by Sumo-1
Alexander M. Ishov,Alexey G. Sotnikov,Dmitri Negorev,Olga Vladimirova,Norma F Neff,Tetsu Kamitani,Edward T.H. Yeh,Jerome F. Strauss,Gerd G. Maul +8 more
TL;DR: The findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML.
References
More filters
Book
Molecular Cloning: A Laboratory Manual
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Book
Basic methods in molecular biology
TL;DR: General methods bacterial strains and cloning vectors enzymes that modify DNA and RNA in vitro amplification of DNA using the polymerase chain reaction (PCR) and the thermostable Taq DNA polymerase, introduction DNA restriction fragment analysis and preparation.
Journal ArticleDOI
Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization.
TL;DR: The use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations is described and chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic DNA was used as a probe.
Journal ArticleDOI
Repeated Sequences in DNA
Roy J. Britten,D. E. Kohne +1 more
TL;DR: Hundreds of thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms and used in medicine, science, and engineering.