Comparative mapping of mouse and rat chromosomes by fluorescence in situ hybridization.

Comparative fluorescence in situ hybridization mapping using DNA libraries from flow-sorted mouse chromosomes and region-specific mouse BAC clones on rat chromosomes reveals chromosomal homologies between mouse (Mus musculus, MMU) and rat (Rattus norvegicus, RNO). Each of the MMU 2, 3, 4, 6, 7, 9, 12, 14, 15, 16, 18, 19, and X chromosomes paints only a single rat chromosome or chromosome segment and, thus, the chromosomes are largely conserved between the two species. In contrast, the painting probes for MMU chromosomes 1, 5, 8, 10, 11, 13, and 17 produce split hybridization signals in the rat, disclosing evolutionary chromosome rearrangements.

Sequestration of mammalian Rad51-recombination protein into micronuclei.

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after gamma irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication.

DNA nicks and increased sensitivity of DNA to fluorescence in situ end labeling during functional spermiogenesis.

Terminal transferase can be used to quantitate DNA strand breaks in situ by labeling free 3'-hydroxyl ends with exogenous nucleotides. Endogenous nicks in DNA temporally appear and disappear during functionally significant structural rearrangements of chromatin. Fluorescence in situ end labeling of mouse and rat testicular cells demonstrated that functional spermiogenesis is associated with abundant DNA nicks that occur in elongating spermatids, most likely as a result of nucleoprotein changes during terminal differentiation.

Orangutan alpha-satellite monomers are closely related to the human consensus sequence.

Alpha-satellite is a family of tandemly repeated DNA found at the centromeric regions of all human and primate chromosomes. Human alpha-satellite subsets are largely chromosome-specific and have been further grouped into four suprachromosomal families (SFs), each characterized by a unique set of monomeric types. Although chimpanzee and gorilla alpha-satellites share sufficient sequence similarity to fit the established SFs, the assumption that the derived human alpha-satellite consensus and monomeric types represent the sequence of ancestral repeats remains unestablished.

Chromosome-specific alpha-satellite DNA from the centromere of chimpanzee chromosome 4.

The centromeric regions of human and primate chromosomes are characterized by diverged subsets of tandemly repeated alpha-satellite DNA. Comparison of the alpha-satellites on known homologous chromosomes in human and chimpanzee provides insight into the very rapid evolution of satellite DNA sequences and the mechanisms that shape complex genomes. By using oligonucleotide primers specific for a conserved region of human alpha-satellite DNA, we have amplified a chromosome-specific alpha-satellite subset from the chimpanzee genome by the polymerase chain reaction.

Analysis of replication timing of ribosomal RNA genes by fluorescence in situ hybridization.

Fluorescence in situ hybridization has been used to study the replication timing of various repeat DNA families in the short arms of human acrocentric chromosomes. In interphase nuclei, unreplicated DNA segments show singlet hybridization signals whereas replicated loci have doublet signals. The distribution of these two patterns in unsynchronized cell cultures revealed that the rRNA gene clusters replicate earlier than the closely juxtaposed alpha- and beta-satellite DNA sequences.

Complex FISH probes for the subtelomeric regions of all human chromosomes: comparative hybridization of CEPH YACs to chromosomes of the Old World monkey Presbytis cristata and great apes.

We have generated a human subtelomere probe panel, utilizing well characterized CEPH YACs, for the investigation of human chromosome pathology and evolution through fluorescent in situ hybridization (FISH). Region-specific FISH probes will be extremely valuable for detecting cytogenetically cryptic telomere abnormalities. Here, we present the first comparative mapping study (with 29 subtelomere probes and 6 chromosome paints) to the Old World monkey Presbytis cristata, followed by hybridizations to the great apes, gorilla and orangutan, when rearrangements were detected.

High-resolution analysis of DNA replication in released chromatin fibers containing 5-bromodeoxyuridine.

A strategy has been devised to physically map replication sites in released chromatin of mammalian cells. When added to the culture medium, 5-bromodeoxyuridine (BrdU) is incorporated into replicating DNA, partially replacing thymidine. BrdU pulses as short as one minute can be visualized on preparations of straightened chromatin fibers from protein-extracted nuclei by means of monoclonal anti-BrdU antibody.

Region-specific YAC banding and painting probes for comparative genome mapping: implications for the evolution of human chromosome 2.

To date, several hundred nonchimeric yeast artificial chromosomes (YACs) from the Centre d'Etude du Polymorphisme Humain containing polymorphic sequence-tagged sites have been mapped by fluoresence in situ hybridization (FISH) on human metaphase chromosomes. Because they carry an average of 1 Mb of human genomic DNA, CEPH YACs generate high-intensity in situ hybridization signals. The available set of cytogenetically and genetically anchored YACs, approximately one every 5-10 cM evenly spaced over almost the entire human genome, provides complex region-specific probes for molecular cytogenetics.

Inhibition of RNA polymerase II transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains.

Fluorescence in situ hybridization and immunofluorescence have been used to visualize specific genomic DNA sequences and proteins in interphase nuclei treated with transcriptional inhibitors. The adenosine analog 5,6-dichloro-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin selectively inhibit transcription by RNA polymerase II at low doses. Upon exposure to DRB or alpha-amanitin the fibrillar components of the normally compact nucleolus unravel into necklace-like structures which represent highly extended linear arrays of ribosomal (r)RNA genes.

Chromosomal localization of long trinucleotide repeats in the human genome by fluorescence in situ hybridization.

Trinucleotide microsatellites are widespread in the human and other mammalian genomes. Expansions of unstable trinucleotide repeats have been associated so far with a number of different genetic diseases including fragile X, myotonic dystrophy (DM) and Huntington disease. While ten possible trinucleotides can occur at the DNA level, only CTG and CCG repeats are involved in the disorders described so far.

Evolution of the gonosomal heterochromatin of Microtus agrestis: rapid amplification of a large, multimeric, repeat unit containing a 3.0-kb (GATA)11-positive, middle repetitive element.

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have cloned and characterized a 2,999-bp (GATA)n-positive sequence, following HaeIII digestion, that is confined to the noncentromeric heterochromatin of the X chromosome. The cloned element exhibits an accumulation of certain oligomers, which are scattered throughout its entire length, and several copies of Chi-related sequence motifs, which are thought to be implicated in recombination.