Methods
Somatic cell genetic studies gave the first proof that functional tumor suppressor genes exist in mammalian genomes. Initial studies showed that whole-cell hybrids between tumorigenic mouse or human cell lines and their normal counterparts became nontumorigenic upon inoculation into animals. However, identification of the operative tumor suppressor gene proved difficult due to the presence of the...
Fluorescencein situhybridization (FISH) applied to metaphase chromosomes provides a mapping resolution of 1 to 3 Mb. FISH applied to interphase nuclei has a resolution of 50 kb and ranges 1–2 Mb. This better resolution is attributed to the higher degree of chromatin decondensation. Here, we describe FISH applied to naked DNA fibers (fiber FISH) and show that with such fully decondensed chromatin a...
Genotyping methods based on nonradioactive detection of PCR products and suitable for large-scale mapping projects are described. Two alternative techniques are proposed for the genotyping of polymorphic short tandem repeats or microsatellite markers. The first is designed for investigators who do not have access to automatic sequencing machines. This technique uses multiplex analysis of PCR products...
Primedin situlabeling (PRINS) has become a routine technique for the microscopical staining of specific DNA sequences in cells and nuclei. For this purpose, the technique has the general advantage of being fast, simple, and sensitive. Furthermore, the reaction characteristics of the technique enable it to discriminate efficiently among closely related sequences—sometimes even when these differ by...
Chicken cell lines derived from avian leukosis virus-induced B-cell tumors are highly recombination-proficient. In these cells, homologous recombination between transfected DNA sequences and the corresponding chromosomal alleles occurs at high frequency, and targeting efficiencies of 10–90% can be obtained. We previously described a chromosome shuttle approach in which these cells can be used to modify...
Microcell-mediated chromosome transfer has been used to study genetic loci that can either activate or repress tissue-specific gene expression. We have used microcell hybrids to study activation of the muscle phenotype, as well as to identify a genetic locus responsible for inhibiting expression of the myogenic determination gene MyoD in primary fibroblasts. We have shown that microcell transfer of...
Genetic isolates represent exceptional resources in the identification of disease loci. Not only have specific monogenic diseases become enriched and families frequent enough to be utilized in linkage analyses, but also the vast majority of patients carry the same mutation and share identical chromosomal haplotypes at significant distances. Additionally, regional isolates often reveal an increased...
The combination of synthetic oligonucleotide probes and DNA ligases is central to several recently developed genetic assays. Among the advantages of ligase-mediated gene detection is that ligation of probe pairs provides highly specific detection of unique DNA sequences in genomic samples. The technique also allows for convenient distinction between sequence variants, since mismatched bases at the...
Genes expressed from a defined genomic interval can be cloned using interspecific microcell hybrids and subtractive hybridization. This approach involves identifying a pair of interspecific hybrids that contain nearly isogenic fragments of a relevant chromosome, except for a region of nonoverlap encompassing the locus of interest. Radiolabeled cDNAs are synthesized from the hybrid cell line that contains...
Microcell fusion is a technology that involves the transfer of single or small clusters of intact chromosomes from one cell to another. The transferred chromosome can be stably retained in the recipient cell background under dominant selective pressure. Hybrid clones generated by this method result in karyotypically simple and homogeneous populations that are excellent resources for physical mapping...
Microcell hybrids are useful resources for the mapping of human chromosomes. The procedure of microcell-mediated chromosome transfer often causes fragmentation of the donor chromosome. These fragment-containing microcell hybrids frequently contain a limited region around the locus used in selecting for retention of the chromosome in the hybrids, as well as other fragments from the donor chromosome...
It is now possible to start at a cytogenetically defined position in any eucaryotic genome and proceed toward isolation and identification of candidate genes known to map to that position, taking advantage of the new PCR amplification technology to produce position-specific DNA. The starting material for this very useful exercise is DNA microdissected from standard cytogenetic preparations. Here we...
Conventional genome mapping and sequencing involves the analysis and processing of individual samples and pieces of experimental data. Although these methods work, it is quite clear that more efficient and less expensive methods are needed. Our top down physical mapping experiments have focused on the parallel processing of information from multiple samples at one time. This approach has aided the...
For any given gene, the identification of exon boundaries and the corresponding flanking intron sequences is the essential prerequisite for asking interesting questions related to its structural organization, spanning from the study of its evolution to the molecular mechanisms underlying possible alternative splicing. In addition, this type of knowledge allows us to carry out mutation screenings for...
A rate-limiting step in the analysis of large segments of genomic DNA is the generation of a set of representative short single-copy sequences that can be used for development of fine-structure maps. In direct response to this need, we have developed interspersed repetitive DNA sequence (IRS)-bubble PCR. IRS-bubble PCR was designed to amplify the human DNA content of somatic cell hybrids, yeast artificial...
Fluorescencein situhybridization (FISH) with gene- and locus-specific probes provides a rapid means to assess copy numbers of specific sequences in individual interphase nuclei. Recent technical improvements have made FISH applicable to the analysis of both fresh and archival tissue specimens in research as well as in diagnostic laboratories. FISH is limited to analysis of one or a few loci at a time,...
Microcell-mediated chromosome transfer (MMCT) offers a unique method for introducing tagged individual human chromosomes from mouse/human monochromosomal hybrids into cell lines displaying recessive mutant phenotypes. Functional analysis of the resultant microcell hybrids bearing different tagged individual human chromosomes permits identification of the complementing chromosome. Using this approach,...
The FK506 binding protein (FKBP12) is the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. Recently, we have shown that FKBP12 copurifies with the ryanodine receptor (RyR), a 565,000-Da protein with four subunits that form the intracellular calcium release channels of the sarcoplasmic reticulum and endoplasmic reticulum. To identify the cellular function of FKBP12, in the absence...
The rapidly expanding use of implants for reconstruction and repair of diseased or traumatized tissues and organs has fueled the search for biomaterials that are stable, nontoxic, and biologically inert. Unfortunately, implants frequently cause acute or chronic inflammation resulting in tissue damage and rejection. Inflammation usually occurs at the biomaterial–tissue interface and reflects surface...