High resolution DNA fiber-fish on yeast artificial chromosomes: direct visualization of DNA replication.

Fluorescent in situ hybridization (FISH) is a powerful, direct and sensitive technique with a wide resolution range that enables the simultaneous study of multiple targets, labelled in different colours. Spreading techniques, denoted here as 'Fiber-FISH', increase FISH-resolution to the DNA fiber, using decondensed nuclear DNA as hybridization target. FISH could be a powerful analytical tool for thorough physical examination of yeast artificial chromosomes (YACs) which are often chimaeric or contain internal deletions. However, with one exception restricted to meiotic yeast chromosomes, FISH has not been used successfully on yeast/YAC DNA. We have developed a fast and simple method that can be applied routinely for compositional and structural analysis of cosmid and YAC DNA in yeast. It enables precise localization and ordering of clones, resolves overlaps and distances and gives a detailed picture of the integrity and colinearity of both probe and target. The combination of high resolution, signal abundance and short yeast cell cycle allows direct visualization of replicating DNA fibers. In a 400 kb region of the human dystrophin gene, we identified two replication origins, demonstrating that human DNA cloned in yeast is capable of initiating its own replication.

Toxic effects of ozone on murine L929 fibroblasts. Enzyme inactivation and glutathione depletion.

Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enzymes. Most sensitive to ozone exposure were glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. The activities of another cytosolic enzyme, lactate dehydrogenase, the mitochondrial enzymes glutamate dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and the activity of the lysosomal enzymes acid phosphatase and beta-glucuronidase were, initially, not or only slightly affected. The localization of the lysosomal enzymes did not change during ozone exposure. After prolonged exposure complete deterioration of the cells was observed and all enzyme activities declined. The activity of the enzymes was also monitored during ozone exposure of a sonicated cell suspension and it was shown that all these enzymes are in fact susceptible to ozone. These observations clearly demonstrate that, besides the structure and amino acid composition of an enzyme, the localization in the cell plays an important role in its susceptibility to ozone. The intracellular levels of reduced and oxidized glutathione were affected as well. The ATP content, however, proved to be insensitive to ozone exposure.

Detection of 11q13 rearrangements in hematologic neoplasias by double-color fluorescence in situ hybridization.

Rearrangements within the chromosome 11q13 region are frequent in hematologic malignancies. 50% of 75% of mantle cell lymphomas (MCLs) carry a translocation t(11;14) (q13;q32). Using Southern blot analysis, a BCL1 breakpoint can be detected in approximately 50% of MCLs. It is not known whether other MCLs harbor also breakpoints at 11q13. Breakpoints in this region not involved in t(11;14), are detected in chronic lymphocytic leukemia and acute myeloid leukemia. To detect and localize breakpoints at 11q13 more accurately, we have developed fluorescence in situ hybridization using two probe sets of differently labeled cosmids, symmetrically localized at either side of the major translocation cluster of BCL1. These probes span a region of 450 to 750 kb. We applied this assay to a series of hematologic malignancies with 11q13 abnormalities identified by classical cytogenetics. All four samples with a t(11;14) (q13;q32) showed dissociation of the differently colored signals in metaphase and interphase cells, thereby indicating a chromosomal break in the region defined by the probe sets. The frequency of abnormal metaphase and interphase cells was comparable with that observed in any of the 13 malignancies with other chromosomal 11q13 abnormalities, indicating that these chromosomal breaks occurred outside the 450- to 750-kb region covered by the probes. One patient showed triplication and one patient showed monoallelic loss of this region. The current data show that double-color fluorescence in situ hybridization is a simple and reliable method for detection of the t(11;14)(q13;q32) in interphase cell nuclei and that is can be used to distinguish this translocation from other 11q13 rearrangements in hematologic malignancies.