			Home About us Contact

FISH Signals (fish + signal)

Distribution by Scientific Domains

Life Sciences	75%

Selected Abstracts

Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries

ENVIRONMENTAL MICROBIOLOGY, Issue 11 2002
Andreas Schramm
Summary A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated Td values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries. [source]

Co-amplification of 8p12 and 11q13 in breast cancers is not the result of a single genomic event

GENES, CHROMOSOMES AND CANCER, Issue 5 2007
Anna L. Paterson
Epithelial cancers frequently have multiple amplifications, and particular amplicons tend to occur together. These co-amplifications have been suggested to result from amplification of pre-existing junctions between two chromosomes, that is, translocation junctions. We investigated this hypothesis for two amplifications frequent in breast cancer, at 8p12 and 11q13, which had been reported to be associated in Southern blot studies. We confirmed that both genomic amplification and expression of genes was correlated between the frequently-amplified regions of 8p and 11q, in array CGH and microarray expression data, supporting the importance of co-amplification. We examined by FISH the physical structure of co-amplifications that we had identified by array CGH, in five breast cancer cell lines (HCC1500, MDA-MB-134, MDA-MB-175, SUM44, and ZR-75-1), four breast tumors, and a pancreatic cancer cell line (SUIT2). We found a variety of arrangements: amplification of translocation junctions; entirely independent amplification of the two regions on separate chromosomes; and separate amplification of 8p and 11q sequences in distinct sites on the same rearranged chromosome. In this last arrangement, interphase nuclei often showed intermingling of FISH signals from 8p12 and 11q13, giving a false impression that the sequences were interdigitated. We conclude that co-amplification of the main 8p and 11q amplicons in breast tumors is not usually the result of a preceding translocation event but most likely reflects selection of clones that have amplified both loci. This article contains supplementary material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2007 Wiley-Liss, Inc. [source]

Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2005
B.P.B. Rambags
Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n,=,22) or in vitro (IVP: n,=,20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring system in which extra FISH signals were taken to indicate increases in ploidy and "missing" signals were assumed to be "false negatives," 98% of the cells were scored as diploid and the majority of embryos (30/42: 71%) were classified as exclusively diploid. However, one IVP embryo was recorded as entirely triploid and a further seven IVP and four in vivo embryos were classified as mosaics containing diploid and polyploid cells, such that the incidence of apparently mixoploid embryos tended to be higher for IVP than in vivo embryos (P,=,0.118). When the number of FISH signals per nucleus was examined in more detail for 11 of the embryos, the classification as diploid or polyploid was largely supported because 2,174 of 2,274 nuclei (95.6%) contained equal numbers of signals for the two chromosomes. However, the remaining 100 cells (4.4%) had an uneven number of chromosomes and, while it is probable that many were artefacts of the FISH procedure, it is also likely that a proportion were the result of other types of aneuploidy (e.g., trisomy, monosomy, or nullisomy). These results demonstrate that chromosomally abnormal cells are present in morphologically normal equine conceptuses and suggest that IVP may increase their likelihood. Definitive distinction between polyploidy, aneuploidy and FISH artefacts would require the use of more than one probe per chromosome and/or probes for more than two chromosomes. © 2005 Wiley-Liss, Inc. [source]