Identification of chromosomal fusion sites in Arabidopsis mutants using sequential bicolour BAC-FISH

Genome ◽  
2006 ◽  
Vol 49 (8) ◽  
pp. 1036-1042 ◽  
Author(s):  
Petr Mokros ◽  
Jan Vrbsky ◽  
Jiri Siroky
Keyword(s):  
In Situ Hybridization ◽  
Ccd Camera ◽  
Repair System ◽  
End Joining ◽  
Anaphase Bridges ◽  
Dicentric Chromosomes ◽  
Hybridization Procedure ◽  
Chromosomal Breaks ◽  
Chromosome Fusions

Double stranded chromosomal breaks are repaired by homologous recombination or nonhomologous end joining (NHEJ). When broken chromosome ends are fused together by NHEJ, the resulting dicentric chromosomes can be detected as anaphase bridges during the subsequent mitosis. Telomeres in the absence of functional telomerase shorten, became unprotected, and are eventually recognized by the cell repair system as double stranded breaks. As result, chromosomes of Arabidopsis thaliana plants that are deficient in the gene for telomerase reverse transcriptase (TERT) are prone to chromosome fusions. We use Arabidopsis tert–/– mutants as a model system for analyzing terminal chromosome fusions. Here we report a novel and sensitive cytogenetic assay for the identification and characterization of chromosome-terminal fusion events by employing fluorescence in situ hybridization (FISH) with multiple probes and a repeated hybridization approach. A mixture of chromosome-specific subtelomeric probes is applied successively in 3 FISH reactions to the slides containing mitotic anaphase figures with anaphase bridges. Each figure is registered by a CCD camera after each in situ hybridization procedure. By comparing the signals presented on the bridge in successive images the assessment of the particular chromosome arms involved in fusion is possible. This experimental setup enables unambiguous identification of individual chromosome ends employed in fusion events.Key words: Arabidopsis; BAC probes; AtTERT gene; bicolour FISH; anaphase.

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

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1512-1519 ◽  
Author(s):  
LJ Coignet ◽  
E Schuuring ◽  
RE Kibbelaar ◽  
TK Raap ◽  
KK Kleiverda ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Hematologic Malignancies ◽  
Current Data ◽  
Lymphocytic Leukemia ◽  
Cell Nuclei ◽  
Interphase Cell ◽  
Interphase Cells ◽  
Chromosomal Breaks

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.

scholarly journals Comparison of 35S and chemiluminescence for HPV in situ hybridization in carcinoma cell lines and on human cervical intraepithelial neoplasia.

1996 ◽  
Vol 44 (7) ◽  
pp. 665-671 ◽  
Author(s):  
P Lorimier ◽  
L Lamarcq ◽  
A Negoescu ◽  
C Robert ◽  
F Labat-Moleur ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Ccd Camera ◽  
Intraepithelial Neoplasia ◽  
Hpv 16 ◽  
Human Carcinoma ◽  
Carcinoma Cell Lines ◽  
Biopsy Specimens

For in situ hybridization (ISH), development of sensitive, nontoxic alternatives to the use of radioactivity is a constant concern. In this trend, and close to chromogenes and fluorophores, chemiluminescence appears an attractive method. A first positive experience in immunocytochemistry and in ISH, by using the enhanced luminol as luminogene substrate for horseradish peroxidase (HRP) led us to compare the sensitivity of 35S autoradiography and chemiluminescence. For this purpose, we used three human carcinoma cell lines, CaSki [400-600 copies of human papilloma virus (HPV) 16], HeLa (10-50 copies of HPV 18), and SiHa (1-5 copies of HPV 16), and 40 biopsy specimens of human cervical preneoplastic and neoplastic lesions. We performed ISH by using HPV cDNA biotin-labeled probes, detected by a two-step immunocytochemical reaction, the secondary antibodies being either 35S-labeled for autoradiography or HRP-labeled for chemiluminescence. An intensified CCD camera allowed acquisition of the luminescent signal. After only 10 min of photon accumulation, on cell line smears as well as on serial tissue sections, chemiluminescence gave comparable results to those obtained by a 3-week exposure for 35S autoradiography. A quantitative approach on cervical biopsy specimens confirmed this similar level of sensitivity by measuring the area of 35S- or chemiluminescence-stained nuclei. Our results indicate that chemiluminescence is a credible and perfectible alternative to radioisotopes for in situ detection of nucleic acids by hybridization.

scholarly journals Expression of MsLEC1 Transgenes in Alfalfa Plants Causes Symbiotic Abnormalities

2004 ◽  
Vol 17 (1) ◽  
pp. 16-26 ◽  
Author(s):  
Laurence M. Brill ◽  
Nancy A. Fujishige ◽  
Cheryl A. Hackworth ◽  
Ann M. Hirsch
Keyword(s):  
In Situ Hybridization ◽  
Sinorhizobium Meliloti ◽  
Northern Blot Analysis ◽  
Root Tips ◽  
Mrna Accumulation ◽  
Lectin Gene ◽  
Hybridization Procedure ◽  
Sense Orientation ◽  
Legume Lectins

Legume lectins have been proposed to have important symbiotic roles during Rhizobium-legume symbioses. To test this hypothesis, the symbiotic responses of transgenic alfalfa plants that express a portion of the putative alfalfa lectin gene MsLEC1 or MsLEC2 in either the antisense or sense orientation were analyzed following inoculation with wild-type Sinorhizobium meliloti 1021. MsLEC1-antisense (LEC1AS) plants were stunted, exhibited hypernodulation, and developed not only abnormally large nodules but also numerous small nodules, both of which senesced prematurely. MsLEC2-antisense plants were intermediate in growth and nodule number compared with LEC1AS and vector control plants. The symbiotic abnormalities of MsLEC1-sense transgene plants were similar to but milder than the responses shown by the LEC1AS plants, whereas MsLEC2-sense transgene plants exhibited symbiotic responses that were identical to those of vector and nontransgenic control plants. MsLEC1 mRNA accumulation was not detected in nodule RNA by Northern blot analysis but was localized to alfalfa nodule meristems and the adjacent cells of the invasion zone by in situ hybridization; transcripts were also detected in root meristems. A similar spatial pattern of MsLEC2 expression was found by using a whole-mount in situ hybridization procedure. Moreover, mRNAs for an orthologous lectin gene (MaLEC) were detected in white sweetclover (Melilotus alba) nodules and root tips.

Detection of hepatitis delta virus RNA by a nonradioactive in situ hybridization procedure

1992 ◽  
Vol 23 (5) ◽  
pp. 557-561 ◽  
Author(s):  
Donatella Pacchioni ◽  
Francesco Negro ◽  
Elisabetta Chiaberge ◽  
Mario Rizzetto ◽  
Ferruccio Bonino ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Hepatitis Delta Virus ◽  
Hepatitis Delta ◽  
Virus Rna ◽  
Hybridization Procedure ◽  
Nonradioactive In Situ Hybridization ◽  
Delta Virus

Localization of low abundance DNA sequences in tissue sections by in situ hybridization

1986 ◽  
Vol 81 (1) ◽  
pp. 143-162 ◽  
Author(s):  
C.W. Lo
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequences ◽  
Detection System ◽  
Mouse Tissue ◽  
Globin Genes ◽  
Beta Globin ◽  
Tissue Sections ◽  
Specific Hybridization ◽  
Hybridization Procedure

Specific DNA sequences were loaclized in the nuclei of paraffin-embedded mouse tissue sections with in situ hybridization using a biotinylated globin probe in conjunction with a streptavidin-alkaline phosphatase detection system. Globin inserts were clearly detected in the tissue sections of transgenic mice containing 1000, 120 or 5 copies of the exogenously introduced beta-globin genes. In addition, specific hybridization signal was also obtained for the endogenous complement of beta-globin genes in the tissue sections of normal mice. These results demonstrate that this hybridization procedure is very sensitive and should be useful for characterizing the distribution of low abundance DNA sequences in cells and tissue sections.

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