R. Espinosa

Sequence-independent amplification and labeling of yeast artificial chromosomes for fluorescence in situ hybridization

We have developed a method that allows reliable construction of high quality FISH probes from yeast artificial chromosomes (YACs) based on the separation of YACs by pulse-field gel electrophoresis and a rapid sequence-independent amplification procedure (SIA). These probes can be used to localize YACs on metaphase chromosomes and also with high efficiency, in interphase nuclei.

Chromosomal instability in chromosome band 12p13: multiple breaks leading to complex rearrangements including cytogenetically undetectable sub-clones.

During fluorescence in situ hybridization (FISH) analysis of metaphase cells from 70 patients with lymphoid and myeloid hematologic malignancies and chromosomal rearrangements involving band 12p13, we identified nine patients (four with lymphoid malignancies, four with myeloid malignancies and one with biphenotypic leukemia) who showed more complicated rearrangements than we had expected from conventional cytogenetic study. In six patients, multiple breaks occurred in small segments of 12p with subsequent translocations and insertions of these segments into other chromosomes, sometimes to unexpected regions. In three patients additional chromosome breaks resulted in a sub-clone which was cytogenetically indistinguishable from the main clone in each patient based on the cytogenetic analysis. These subtle molecular events were detected exclusively in a region covering TEL/ETV6 and KIP1/CDKN1B. Seven of nine had a previous history of chemo/radiotherapy; all the patients showed complex karyotypes, even though they were newly diagnosed with leukemia. Survival data were available in five patients, and all survived less than 6 months. These findings suggest that the 12p13 region, especially the above-mentioned region, is genetically unstable and fragile. It is likely that multiple chromosome breaks were induced through mutagens used in chemo/ radiotherapy, and are associated with a sub-group of patients with an extremely bad prognosis.

Physical and genetic map of 5q31: use of fluorescence in situ hybridization data to identify errors in the CEPH database

Chromosome 5, band q31, contains the genes responsible for a number of interesting genetic and malignant diseases, as well as many cloned genes. To prepare a high-resolution map of this region, eight

Localization of the gene encoding insulin-degrading enzyme to human chromosome 10, bands q23→q25

Insulin-degrading enzyme (IDE) is a cytosolic proteinase involved in the cellular processing of insulin. Using somatic cell hybrid analysis and in situ chromosomal hybridization, we have localized the gene encoding IDE to human chromosome 10, bands q23----q25. The murine Ide gene was previously mapped to Chromosome 19; together, these results suggest that the IDE gene is a member of a conserved syntenic group on human chromosome 10, bands q23----q25 and mouse Chromosome 19.

A physical and genetic linkage map of the distal long arm of human chromosome 5

By in situ hybridization of probes for three cloned genes and eight genetically-linked polymorphic DNA markers, we have prepared a physical map of the distal long arm of chromosome 5. These results, together with the localizations of 11 genes and the genetic linkage map reported previously by us and by other investigators, represent a map that spans 55 cM.

Physical localization of 70 polymorphic markers to human chromosome 5 by fluorescence in situ hybridization

We used fluorescence in situ hybridization (FISH) to prepare a cytogenetic framework map of 21 polymorphic markers that had been used previously to construct a genetic linkage anchor map of chromosome 5. In addition, we localized 49 other markers that have been genotyped on CEPH families. This study demonstrates that FISH can be used to confirm genetic linkage data, and that it can provide a means of determining the cytogenetic locations and relative order of markers whose order could not be assigned by genetic linkage analysis alone. The cytogenetic map prepared by FISH may help to identify probes of interest for regional mapping studies.