A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.T he role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome. Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5-11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells. In addition, because FISH is applied to fixed cells, it can reveal the positioning of chromosomes relative to nuclear, cytoplasmic, and even tissue structures. FISH can also be used to visualize RNA, permitting the simultaneous assessment of gene expression, chromosome position, and protein localization.FISH probes are typically derived from cloned genomic regions or flow-sorted chromosomes, which are labeled directly via nick translation or PCR in the presence of fluorophore-conjugated nucleotides or labeled indirectly with nucleotide-conjugated haptens, such as biotin and digoxigenin, and then visualized with secondary detection reagents. Probe DNA is often fragmented into ∼150-to 250-bp pieces to facilitate its penetration into fixed cells (12) and, as many genomic clones contain repetitive sequences that occur abundantly in the genome, hybridization is typically performed in the presence of unlabeled repetitive DNA (13). Another limitation to clone-based probes is that the genomic regions that can be visualized with them are restricted by the availability of clones and the size of ...

Transcriptome profiling studies have recently uncovered a large number of noncoding RNA transcripts (ncRNAs) in eukaryotic organisms, and there is growing interest in their role in the cell. For example, in haploid Saccharomyces cerevisiae cells, the expression of an overlapping antisense ncRNA, referred to here as RME2 (Regulator of Meiosis 2), prevents IME4 expression. In diploid cells, the a1-␣2 complex represses the transcription of RME2, allowing IME4 to be induced during meiosis. In this study we show that antisense transcription across the IME4 promoter region does not block transcription factors from binding and is not required for repression. Mutational analyses found that sequences within the IME4 open reading frame (ORF) are required for the repression mediated by RME2 transcription. These results support a model where transcription of RME2 blocks the elongation of the full-length IME4 transcript but not its initiation. We have found that another antisense transcript, called RME3, represses ZIP2 in a cell-type-specific manner. These results suggest that regulated antisense transcription may be a widespread mechanism for the control of gene expression and may account for the roles of some of the previously uncharacterized ncRNAs in yeast.One of the main paradigms for the control of gene expression is that regulatory proteins bind to the promoter regions of genes to activate or repress transcription. However, it is now clear that noncoding RNAs (ncRNAs) also play important roles in gene regulation. For example, RNA interference (RNAi)-mediated regulation controls gene expression in Caenorhabditis elegans, Arabidopsis thaliana, humans, and many other organisms (16,24). However, a large number of ncRNAs do not appear to be involved in RNAimediated regulation. For example, more than 900 ncRNAs are expressed in the yeast Saccharomyces cerevisiae (11,15,33,40,45). Several of these ncRNAs act to regulate gene expression in yeast (2,5,18,26). However, S. cerevisiae lacks the enzymes Dicer and Argonaute, which are required for RNAi, and therefore, it must utilize different mechanisms for ncRNA-mediated regulation (12). In this paper we investigate how two antisense ncRNAs regulate the expression of genes required for meiosis in yeast.Under starvation conditions, diploid yeast undergoes meiosis and sporulation to form four haploid spores. This process involves the expression of more than 500 genes that are highly regulated in a coordinated manner (7,28). Entry into the meiotic pathway is controlled by the expression of IME1, the master initiator of meiosis (20, 29). There are two signals that regulate IME1 expression (Fig. 1A). One signal relates to the nutritional status of the cell, activating IME1 expression when the cell is starved of both nitrogen and a fermentable carbon source (14, 37). The second signal operates through cell-typespecific regulation, which allows the expression of meiotic genes only in a/␣ diploid cells. Cell-type-specific regulation is controlled by the a1-␣2 repressor complex, which reg...

Homolog pairing, which plays a critical role in meiosis, poses a potential risk if it occurs in inappropriate tissues or between nonallelic sites, as it can lead to changes in gene expression, chromosome entanglements, and loss-of-heterozygosity due to mitotic recombination. This is particularly true in Drosophila, which supports organismal-wide pairing throughout development. Discovered over a century ago, such extensive pairing has led to the perception that germline pairing in the adult gonad is an extension of the pairing established during embryogenesis and, therefore, differs from the mechanism utilized in most species to initiate pairing specifically in the germline. Here, we show that, contrary to long-standing assumptions, Drosophila meiotic pairing in the gonad is not an extension of pairing established during embryogenesis. Instead, we find that homologous chromosomes are unpaired in primordial germ cells from the moment the germline can be distinguished from the soma in the embryo and remain unpaired even in the germline stem cells of the adult gonad. We further establish that pairing originates immediately after the stem cell stage. This pairing occurs well before the initiation of meiosis and, strikingly, continues through the several mitotic divisions preceding meiosis. These discoveries indicate that the spatial organization of the Drosophila genome differs between the germline and the soma from the earliest moments of development and thus argue that homolog pairing in the germline is an active process as versus a passive continuation of pairing established during embryogenesis.

Oligopaint probes are fluorescently-labeled, single-stranded DNA oligonucleotides that can be used to visualize genomic regions ranging in size from tens of kilobases to many megabases. This unit details how Oligopaint probes can be synthesized using basic molecular biological techniques as well as provides protocols for FISH, 3D-FISH, and sample preparation.

Sensorineural hearing loss is frequently reported in young patients with chronic renal failure having haemodialysis. The effect of a single session of haemodialysis on hearing acuity was assessed prospectively in nine children with end-stage renal disease using pure-tone audiometry (PTA) and distortion-product otoacoustic emissions (DPOAEs). Results were compared with those obtained from nine audiologically normal healthy children also tested with PTA and DPOAEs twice during a 4-h interval. Sensorineural hearing loss of unknown aetiology was found in 55.5% of renal patients, mainly in the higher frequencies. Patients on HD had mean PTA thresholds significantly poorer than those of the control group in the frequency range 1000-12 000Hz (P < 0.05). Their mean DPOAE amplitudes were significantly lower in all frequencies > 1184 Hz (P < 0.05). Furthermore, patients' ears with normal PTA thresholds between 250 and 4000 Hz also had decreased DPOAE amplitudes. No significant changes in PTA thresholds or DPOAE amplitudes were encountered in renal patients before and after a HD session (P > 0.05). Changes in PTA thresholds or DPOAE amplitudes were not significantly different than those in the control group (P > 0.05). In conclusion, sensorineural hearing loss of unknown origin, especially in high frequencies, is frequent in young renal patients under HD and single HD sessions do not seem to alter the hearing acuity of these patients. DPOAEs seem to be more sensitive to incipient cochlear damage than behaviour thresholds in monitoring renal patients.