Double-color Fluorescence Research Articles

Differential Detection of Bioavailable Mercury and Cadmium Based on a Robust Dual-Sensing Bacterial Biosensor.

Genetically programmed biosensors have been widely used to monitor bioavailable heavy metal pollutions in terms of their toxicity to living organisms. Most bacterial biosensors were initially designed to detect specific heavy metals such as mercury and cadmium. However, most available biosensors failed to distinguish cadmium from various heavy metals, especially mercury. Integrating diverse sensing elements into a single genetic construct or a single host strain has been demonstrated to quantify several heavy metals simultaneously. In this study, a dual-sensing construct was assembled by employing mercury-responsive regulator (MerR) and cadmium-responsive regulator (CadR) as the separate sensory elements and enhanced fluorescent protein (eGFP) and mCherry red fluorescent protein (mCherry) as the separate reporters. Compared with two corresponding single-sensing bacterial sensors, the dual-sensing bacterial sensor emitted differential double-color fluorescence upon exposure to 0–40 μM toxic Hg(II) and red fluorescence upon exposure to toxic Cd(II) below 200 μM. Bioavailable Hg(II) could be quantitatively determined using double-color fluorescence within a narrow concentration range (0–5 μM). But bioavailable Cd(II) could be quantitatively measured using red fluorescence over a wide concentration range (0–200 μM). The dual-sensing biosensor was applied to detect bioavailable Hg(II) and Cd(II) simultaneously. Significant higher red fluorescence reflected the predominant pollution of Cd(II), and significant higher green fluorescence suggested the predominant pollution of Hg(II). Our findings show that the synergistic application of various sensory modules contributes to an efficient biological device that responds to concurrent heavy metal pollutants in the environment.

Detection of Bioavailable Cadmium by Double-Color Fluorescence Based on a Dual-Sensing Bioreporter System.

Cadmium (Cd) is carcinogenic to humans and can accumulate in the liver, kidneys, and bones. There is widespread presence of cadmium in the environment as a consequence of anthropogenic activities. It is important to detect cadmium in the environment to prevent further exposure to humans. Previous whole-cell biosensor designs were focused on single-sensing constructs but have had difficulty in distinguishing cadmium from other metal ions such as lead (Pb) and mercury (Hg). We developed a dual-sensing bacterial bioreporter system to detect bioavailable cadmium by employing CadC and CadR as separate metal sensory elements and eGFP and mCherry as fluorescent reporters in one genetic construct. The capability of this dual-sensing biosensor was proved to simultaneously detect bioavailable cadmium and its toxic effects using two sets of sensing systems while still maintaining similar specificity and sensitivity of respective signal-sensing biosensors. The productions of double-color fluorescence were directly proportional to the exposure concentration of cadmium, thereby serving as an effective quantitative biosensor to detect bioavailable cadmium. This novel dual-sensing biosensor was then validated to respond to Cd(II) spiked in environmental water samples. This is the first report of the development of a novel dual-sensing, whole-cell biosensor for simultaneous detection of bioavailable cadmium. The application of two biosensing modules provides versatile biosensing signals and improved performance that can make a significant impact on monitoring high concentration of bioavailable Cd(II) in environmental water to reduce human exposure to the harmful effects of cadmium.

The Prognostic Impact of t(14;16) in Multiple Myeloma: A Multicenter Retrospective Study of 213 Patients. Is It Time to Revise the Revised ISS?

The Prognostic Impact of t(14;16) in Multiple Myeloma: A Multicenter Retrospective Study of 213 Patients. Is It Time to Revise the Revised ISS?

The Prognostic Impact of t(14;20) in Multiple Myeloma - a Multicenter Retrospective Study of 26 Patients

IntroductionThe prognosis in multiple myeloma (MM) is based upon complex biological and clinical features. One of the strongest predictors of outcome are the intrinsic genetic abnormalities in the malignant plasma cells. t(14;20), as other chromosome 14 abnormalities, deregulates the c-musculoaponeurotic fibrosarcoma (c-MAF) oncogene. Due to the relative rarity of t(14;20) [<1.5% of newly diagnosed MM; slightly higher in monoclonal gammopathies of unknown significance- about 5%], there are no large databases which provide the natural history of this abnormality. Its meaning remains controversial due to the small sample sizes in previously published reports in contrast to t(4;14) and 17p- abnormalities which have been described independently in the literature.MethodsWe retrospectively analyzed the clinical features and outcomes of patients t(14;20) from 5 clinical centers in Germany, Italy and the United States. Newly diagnosed MM patients according to the International Myeloma Working Group (IMWG) criteria were enrolled. The t(14;20) was detected by double color fluorescence in situ hybridization (FISH) using bone marrow samples. Baseline characteristics at diagnosis, comorbidities, patient treatment regimens and clinical outcomes were collected using unified case report forms. Clinical responses were assessed according to the IMWG uniform response criteria. Overall survival (OS) was defined as the period between the date of initial diagnosis and the date of death or the date of the last observation. Progression-free survival (PFS) was defined as the period between the date of initial diagnosis and either the date of the first relapse or death of any causes or the date of the last observation. Cox proportional hazard regression analysis was applied to assess risk factors of death. Survival curves were plotted by the Kaplan-Meier method and compared using log-rank and Breslow-Gehan-Wilcoxon tests.Results26 patients were included in analysis including 11 males (42.3%). Median age at MM diagnosis was 68.5 years (range 56-71). Immunoglobulin isotype included: IgG (n=14, 53.8%), IgA (n=9, 34.6%), light chain (n=3, 11.5%). R-ISS stage at diagnosis included: stage III (n=8, 30.8%); stage II (n=9, 34.6%) and stage I (n=1, 3.8%)- the staging for remaining 8 patients is unknown. Anemia (<10g/dl) was observed in 13 (50%), impaired renal function (creatinine clearance <40 mL per minute or serum creatinine >2 mg/dl) in 3 (11.5%). In 14 (53.8%) cases osteolytic lesions were present. In 15 patients (57.7%) the t(14;20) was associated with other cytogenetic aberrations, including del17p in 2 patients (7.7%), t(4;14) in 5 patients (19.2%) and t(14;16) in 4 patients (15.4%). First line treatment for MM included mostly PIs + IMIDs in 8 patients (30.8%), PIs + IMIDs + chemotherapy in 7 patients (26.9%) or PIs alone in 3 cases (11.5%). Auto-PBSCT was performed in 8 cases (30.7%).Median PFS was 30 months (CI 95% 17.8 months; Figure 1, panel A). Median OS was not reached (Figure 1, panel B). 5-year OS from MM diagnosis was 77.5% (±10.6%).ConclusionIn this study of 26 patients with t(14;20) the median PFS and 5-year OS were 30 months and not reached, respectively. Historically, the presence of a t(14;20) is considered to be a very poor prognostic factor-this was not observed in this multi-institutional retrospective analysis. In comparison to the recent studies (Ross et al, Haematologica, 2010; n=27; median survival 14.4 months), the PFS and OS of patients with t(14;20) in our analysis are better than expected. It should be highlighted that most probably in the Ross et al. study patients were not treated with novel drug therapies. Nonetheless, this controversy remains to be elucidated in the future studies. DisclosuresCastillo:Genentech: Consultancy; Pharmacyclics: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Beigene: Consultancy, Research Funding. Hari:Celgene: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding; Spectrum: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Kite Pharma: Consultancy, Honoraria; Sanofi: Honoraria, Research Funding; Janssen: Honoraria. Goldberg:COTA Inc.: Employment, Equity Ownership.

Chromosomal Translocation (14;16)-Positive Multiple Myeloma Shows Negativity for CD56 Expression and Unfavorable Outcome Even in the Era of Novel Drugs

IntroductionMultiple myeloma (MM) is an incurable plasma cell neoplasm developing through long-term multistep genetic events. Biological and clinical features of the MM are known to be associated in part with relatively early genetic aberrations such as chromosomal translocations involving IGH. The t(14;16)(q32;q23) involving c-MAF oncogene locus is an important chromosomal aberration observed in approximately 5 percent of newly diagnosed MM. Various studies have suggested that MM carrying t(14;16) is associated with specific clinical characteristics. However, these studies were not definitive, since the number of the patients analyzed was relatively small. The aim of this study is to clarify the clinical features of patients with newly diagnosed MM harboring t(14;16) detected by double-color fluorescence in situ hybridization (FISH) in Japan.MethodsClinical and laboratory features of t(14;16)-positive MM diagnosed between 2002 and 2013 were collected retrospectively as a nationwide study in Japan after approval by each institutional ethical committee. The t(14;16) translocation was detected by FISH analysis using bone marrow or peripheral blood samples from all patients. Expression of surface antigens such as CD56 and CD20 was detected by flow cytometric analysis (FCM) and defined as positive when more than 20% of the CD38-positive plasma cells were positive.To compare t(14;16)-positive and t(14;16)-negative MM, we also assessed 132 patients with newly diagnosed symptomatic MM and without c-MAF mRNA expression, as confirmed by global RQ/RT-PCR using purified plasma cells (Tajima E, et al.:Haematologica 2005; 90: 559, Inagaki A et al.: Leuk Res 2013; 37: 1648) at Nagoya City University Hospital.ResultsIn total, 37 patients carrying t(14;16)-positive MM were enrolled from 19 institutions. Median ages of the MM patients with or without t(14;16) at diagnosis were 62 and 68, respectively. Regarding the cell surface phenotype, none of the t(14;16)-positive MM cells was positive for CD56 (Fig. 1), whereas 82 of 118 (69%) t(14;16)-negative ones were positive. Positivity for CD20 antigen was more common in t(14;16)-positive MM cells (11/23, 48%) than in t(14;16)-negative ones (16/115, 14%)(p= 0.001). The proportion of patients with additional chromosome aberrations other than t(14;16), determined by G-banded karyotyping, was higher in patients with t(14;16) (16/30, 53%) than in those without (19/131 cases, 15%) (p< 0.001). Moreover, MM patients with t(14;16) showed higher frequencies of IgG subtype M protein, leukocytosis (p= 0.001), thrombocytopenia (p< 0.001) and hyperproteinemia (p= 0.001), and a lower frequency of hypercalcemia (p= 0.001), compared to those without t(14;16).Overall survival (OS) of the patients with t(14;16) was significantly shorter than that of those without t(14;16) even though the patients received one or more lines of treatment containing novel drugs such as bortezomib, thalidomide and lenalidomide (p= 0.014) (Fig. 2). Poor PS (PS ≥ 2-4), low PLT count (<100x103/ƒÊL), or high LDH levels (>1.0N) were significantly unfavorable prognostic factors for OS in patients with t(14;16)-positive MM, whereas they were not in those without t(14;16). Progression-free survival (PFS) of the patients with t(14;16) was also significantly shorter than those without t(14;16) (p= 0.002)ConclusionThe t(14;16)-positive MM comprises a specific category in MM, which is featured by negativity for CD56, higher positivity for CD20 and unfavorable outcome, even in the novel drug era. Unraveling biological characteristics of this specific disease category will lead us to establish novel treatment strategies. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Double-color fluorescence in situ hybridization (FISH) for the detection of Bacillus anthracis spores in environmental samples with a novel permeabilization protocol

For anti-bioterrorism measures against the use of Bacillus anthracis, a double-color fluorescence in situ hybridization (FISH) is proposed, for the rapid and specific detection of B. anthracis. The probes were designed based on the differences in 16S and 23S rRNA genes of B. cereus group. A new permeabilization protocol was developed to enhance the permeability of FISH probes into B. anthracis spores. The highest detection rate (90.8±0.69) of B. anthracis spores by FISH was obtained with successive incubation steps with 50% ethanol at 80°C, a mixture of SDS/DTT solution (10mg/ml SDS, 50mM DTT) at 65°C and finally in a lysozyme solution (20mg/ml) at 37°C for 30min each. This protocol was evaluated for the detection of B. anthracis spores in soil and air samples after adding formalin-fixed spores at different densities. The results have proven the success of double-color FISH in detecting B. anthracis spores in air samples in the range of 103 spores/m3 and above. Conversely, for detecting B. anthracis spores in a soil sample, the lowest detection limit was found to be 107 spores/g dry soils. These results confirm the applicability of the developed permeabilization protocol, combined with the double-color FISH technique in specific detection of B. anthracis in soil and air samples.

Identification of IGHCδ–BACH2 fusion transcripts resulting from cryptic chromosomal rearrangements of 14q32 with 6q15 in aggressive B‐cell lymphoma/leukemia

In B-cell malignancies, genes implicated in B-cell differentiation, germinal center formation, apoptosis, and cell cycle regulation are juxtaposed to immunoglobulin loci through chromosomal translocations. In this study, we identified the BTB and CNC homology 2 (BACH2) gene as a novel translocation partner of the immunoglobulin heavy chain (IGH) locus in a patient with IGH-MYC-positive, highly aggressive B-cell lymphoma/leukemia carrying der(14)t(8;14) and del(6)(q15). Fluorescence in situ hybridization analysis using an IGH/MYC probe detected an IGH-MYC fusion signal on der(14) and IGH signal on del(6). Genome copy number analysis showed a deletion in the 6q15-25 region and a centromeric breakpoint within the BACH2 gene. cDNA bubble polymerase chain reaction using BACH2 primers revealed that the first exon of Cδ was fused to the 5'-untranslated region of BACH2 exon 2. The Cδ-BACH2 fusion transcript consisted of exon 1 of Cδ and exons 2 to 9 of BACH2, encompassing the entire BACH2 coding region, and the BACH2 was highly expressed in this patient. These results indicate that Cδ-BACH2 fusion may cause constitutive activation of BACH2. Although additional screening of 47 samples of B-cell non-Hodgkin's lymphoma (B-NHL) patients and 29 cell lines derived from B-cell malignancies by double-color fluorescence in situ hybridization analysis detected a split signal with deletion of centromeric region of BACH2 only in a patient with follicular lymphoma, BACH2 was highly expressed in lymphoma cells of the patient and B-NHL cell lines with IGH-MYC translocation. These findings suggest that BACH2 plays a critical role in B-cell lymphomagenesis, especially related to IGH-MYC translocation in some way.

EINE DIPLOIDE FORM AUS DER TETRAPLOIDEN OENOTHERA GIGANTEA

To determine whether genetic material in 5q13.3 breakpoint region changed in the course of establishing the cell line with a constitutional chromosomal inversion inv(5)(p13.1q13.3) associated with hairy cell leukemia, double color fluorescence in situ hybridization (FISH) on metaphase, interphase and DNA fibers were performed in cells of the cell line with the cosmids cCI5-216 and cCI5-267 DNA probes labeled by either biotinylate or digoxigenin. It showed that the cell line gave the same results as those of the original cells, which indicated that no change of the genetic material at 5q13.3 breakpoint region occurred. The cell line is valuable to reveal the molecular pathogenesis of hairy cell leukemia.

Inactivation and mechanisms of chlorine dioxide on Nosema bombycis

Biological tests demonstrated that the inactivation of Nosema bombycis ( N. bombycis) spores by chlorine dioxide (ClO 2) occurs very fast and is highly sensitive. The lowest effective inactivation dosage and time was 15 mg/mL for 30 min. The inactivation of spores was additionally verified by using double color fluorescence stain and spore germination testing. A series of biological changes, including a large number of substrates that were leaked out from the spores included proteins, DNA, polysaccharide, K +, and Ca 2+, occurred a short time after N. bombycis spores were treated with ClO 2. In addition, the lipid of spores was disrupted and ATPase activity was inhibited, which resulted in the destruction of the inner structure of the spores.

Chromosomal localization of the NotI clones from human chromosome 3 on quail microchromosomes

For the purpose of comparative mapping of quail (Coturnix c. japonica) and human (Homo sapiens) genomes, DNA fragments from human chromosome 3 (HSA3p14-21 and HSA3q13-23) were localized on quail mitotic chromosomes. Using the method of double-color fluorescence DNA-DNA in situ hybridization, these fragments were mapped to two different microchromosomes. Earlier, similar studies were performed using chicken mitotic chromosomes. There it was demonstrated that the clones of interest were distributed among three microchromosomes (GGA12, GGA14, and GGA15). Thus, interspecific difference in the location of human chromosome 3 DNA fragments in the genomes of closely related avian species was discovered. A new confirmation of the hypothesis on the preferable localization of the gene-rich human chromosome regions on avian microchromosomes was obtained. At the same time, a suggestion on the localization of some orthologous genes in the genome of the organism under study was made: ARF4, SCN5A, PHF7, ABHD6, ZDHHC3, MAPKAPK3, ADSYNA (homolog of chicken chromosome 12), DRD2, PP2C-ETA, RAB7, CCKAR, and PKD1 (homolog of chicken chromosome 15). However, localization of the corresponding quail genes needs to be confirmed, as far as the sequences used were only the orthologs of the corresponding chicken genes.

FISH analysis of 45S rDNA and 5S rDNA genes in Triticum poloni-cum L. and T. turgidum L. cv. Ailanmai

Using the method of double color fluorescence in situ hybridization (FISH), we had analyzed Triticum polonicum L. and T. turgidum L. cv. Ailanmai with the probes of 45S rDNA and 5S rDNA. The results indicated that there were highly consistent in T. polonicum L. High and T. turgidum L. cv. Ailanmai, both having four 45S rDNA loci and six 5S rDNA loci. In T. polonicum L. Dwarf, there were also four 45S rDNA loci, the same as that in T. polonicum L. High and T. turgidum L. cv. Ailanmai, but there were eight 5S rDNA loci. At the same time, we discussed the reason of interspecific and intraspecific variation of the two types of rDNA in locus number and location between T. polonicum L. and T. turgidum L. cv. Ailanmai.

Chromosomal localization of seven HSA3q13→q23 NotI linking clones on chicken microchromosomes: orthology of GGA14 and GGA15 to a gene-rich region of HSA3

Double-color fluorescence in situ hybridization was performed on chicken chromosomes using seven unique clones from the human chromosome 3-specific NotI linking libraries. Six of them (NL1-097, NL2-092, NL2-230, NLM-007, NLM-118, and NLM-196) were located on the same chicken microchromosome and NL1-290 on another. Two chicken microchromosome GGA15-specific BAC clones, JE024F14 containing the IGVPS gene and JE020G17 containing the ALDH1A1 gene, were cytogenetically mapped to the same microchromosome that carried the six NotI linking clones, allowing identification of this chromosome as GGA15. Two GGA14-specific clones, JE027C23 and JE014E08 containing the HBA gene cluster, were co-localized on the same microchromosome as NL1-290, suggesting that this chromosome was GGA14. The results indicated that the human chromosomal region HSA3q13→q23 is likely to be orthologous to GGA15 and GGA14. The breakpoint of evolutionary conservation of human and chicken chromosomes was detected on HSA3q13.3→q23 between NL1-290, on the one hand, and six other NotI clones, on the other hand. Considering the available chicken-human comparative mapping data, another breakpoint appears to exist between the above NotI loci and four other genes, TFRC, EIF4A2, SKIL and DHX36 located on HSA3q24→qter and GGA9. Based on human sequences within the NotI clones, localization of the six new chicken coding sequences orthologous to the human/rodent genes was suggested to be on GGA15 and one on GGA14. Microchromosomal location of seven NotI clones from the HSA3q21 T-band region can be considered as evidence in support of our hypothesis about the functional analogy of mammalian T-bands and avian microchromosomes.

Polysomy 17 in HER-2/neu Status Elaboration in Breast Cancer: Effect on Daily Practice

To assess the effect of chromosome 17 copy number on HER-2/neu status determination in breast cancers. HER-2/neu gene copy and chromosome 17 centromere numbers were evaluated on 893 breast carcinomas using double color fluorescence in situ hybridization (FISH). The net and chromosome 17 corrected (ratio) HER-2/neu copy numbers were compared and related to immunohistochemistry done according to the Food and Drug Administration (FDA)-approved scoring system (0, 1+, 2+, and 3+) as a first screening step in 584 cases. When a ratio > or = 2 was considered as criterion for FISH positivity, 49.3% (440 of 893) of cases showed amplification versus 56.2% (502 of 893) by using a net HER-2/neu gene copy number >4 as a alternative criterion; 14.8% (67 of 453) of cases having a ratio <2 had a net HER-2/neu gene copy number >4 and 1.1% (5 of 440) with a ratio > or = 2 had a net HER-2/neu gene copy number <4. Among discordant cases, 88.8% (64 of 72) were polysomic (>2.25 chromosomes 17/cell) and among polysomic cases, 12.8% (40 of 312) of the low polysomic (2.26-3.75 chromosomes 17/cell) and 36.9% (24 of 65) of the highly polysomic (>3.75 chromosomes 17/cell) cases showed discordance. In cases with a ratio <2, polysomy 17 incidences were 85.7% (6 of 7) in IHC 3+, 42.4% (79 of 186) in IHC 2+, 33.3% (15 of 45) in IHC 1+, and 29.1% (16 of 55) in IHC 0. A net increase in HER-2/neu gene copy number consecutive to polysomy 17 in the absence of specific gene amplification might lead to a strong protein overexpression in a small subset of breast carcinomas. HER-2/neu status determination by FISH is dependent on the criterion considered for positivity in clinical practice.

An additional segment at 1p36 derived from der(18)t(14;18) in patients with diffuse large B-cell lymphomas transformed from follicular lymphoma

The particular translocation in follicular lymphomas (FLs) is a t(14;18)(q32;q21), recombining the immunoglobulin heavy chain (IgH) gene on chromosome 14 with the B-cell leukemia/lymphoma 2 (BCL2) gene on chromosome 18. Some low-grade FLs are aggressively transformed into diffuse large B-cell lymphomas, presumably by acquisition of secondary chromosomal changes, including chromosomal band 1p36. A common example is add(1)(p36). Because it is difficult to identify the origin of add(1)(p36) even on high-resolution G-banding analysis, we used spectral karyotyping (SKY) and double-color fluorescence in situ hybridization (DC-FISH) to define the t(14;18) and the extra band at 1p36 in two cases of diffuse large B-cell lymphoma (DLBCL). SKY revealed that the extra chromosomal segment on 1p36 was derived from chromosome 18. DC-FISH defined BCL2/IgH fusion signals at 1p36 in addition to t(14;18), suggesting that BCL2/IgH fusion at 1p36 was an evolutionary alteration following the primary BCL2/IgH translocation on der(18) in both cases. Our results indicate that IgH alleles, implicated in chromosomal rearrangement, may themselves frequently be targets for secondary translocations, suggesting that multiple IgH translocations and insertions are associated with the progression of FL.

The occurrence of graft-versus-host disease is the major predictive factor for response to donor lymphocyte infusions in multiple myeloma

AbstractThe graft-versus-myeloma (GVM) effect of donor lymphocyte infusions (DLIs) is well established. We now report the outcome of DLI in 54 patients with relapsed myeloma following allogeneic transplantation. Twenty-eight patients (52%) responded, 19 patients (35%) with a partial response and 9 patients (17%) with a complete response. Progression-free and overall survival were 19 and 23 months, respectively. We found that acute and chronic graft-versus-host disease (GVHD) observed in 57% and 47% of patients, respectively, following DLI were the strongest predictors for response. This suggests that targets for GVHD and GVM are identical. In a subgroup analysis, deletion of chromosome 13, as determined by double-color fluorescence in situ hybridization (FISH), had no impact on outcome, indicating that these patients are candidates for early allogeneic transplantation followed by DLI, in case of insufficient response. (Blood. 2004;103:4362-4364)

Sexing river buffalo (Bubalus bubalis L.), sheep (Ovis aries L.), goat (Capra hircus L.), and cattle spermatozoa by double color FISH using bovine (Bos taurus L.) X- and Y-painting probes.

River buffalo, sheep, and goat spermatozoa were cross-hybridized using double color fluorescence in situ hybridization (FISH) with bovine Xcen- and Y-chromosome painting probes, prepared by DOP-PCR of laser-microdissected-catapulted chromosomes, to investigate the possibility of using bovine probes for sexing sperm of other members of the family Bovidae. Before sperm analysis, the probes were hybridized on metaphase chromosomes of each species, as control. Frozen-thawed spermatozoa of cattle, river buffalo, sheep, and goat were decondensed in suspension with 5 mM DTT. Sperm samples obtained from three individuals of each species were investigated, more than 1,000 spermatozoa were scored in each animal. FISH analysis of more than 12,000 sperm revealed high level of sperm with X- or Y-signals in all of the species investigated, indicating FISH efficiency over 99%. Significant interspecific differences were detected in the frequency of aberrant spermatozoa (aneuploid and diploid) between goat (0.393%) and sheep (0.033%) (P < 0.01), goat and cattle (0.096%) (P < 0.5), as well as between river buffalo (0.224%) and sheep (P < 0.5). There was no significant difference between river buffalo and cattle. The present study demonstrated that it is possible to use bovine X-Y painting probes for sexing and analyzing sperm of other species of the family, thus facilitating future studies on the incidence of chromosome abnormalities in sperm as well as on sex predetermination of embryos for the livestock industry. Mol. Reprod. Dev. 67: 108-115, 2004.

Interphase detection of immunoglobulin heavy chain gene translocations with specific oncogene loci in 173 patients with B-cell lymphoma

To detect immunoglobulin heavy chain ( IGH) gene translocations with specific oncogene loci, we established an interphase cytogenetic approach using double-color fluorescence in situ hybridization (DC-FISH), which we used to analyze 173 patients with B-cell lymphoma. DC-FISH using the IGH gene (14q32.3) in combination with c-MYC (8q24.1), BCL1 (11q13.3), BCL2 (18q21.3), BCL6 (3q27), and PAX-5 (9p13) gene probes detected IGH translocations in 70 (40.5%) of 173 patients. The partner genes involved in IGH translocations were identified in 56 (80%) of 70 patients, and fusion of the IGH gene with specific oncogenes was detected in 53 of 56 patients, particularly in interphase nuclei of 28 patients for whom cytogenetic analysis was not informative. The most common partner gene was BCL2 (19 patients; 27% of IGH translocation-positive patients), followed by BCL6 (16; 23%), BCL1 (11; 16%), c-MYC (7; 10%), and PAX-5 (2; 3%). These oncogenes were closely associated with subtypes of B-cell lymphoma. The other partners were 19q13 ( BCL3), 6p25 ( MUM1/IRF4), 1q36, and chromosome 8 identified in one patient each. Six of the nine patients with add(14)(q32) showed a BCL6/ IGH translocation. Double translocations of the IGH gene were found in three patients; c-MYC+ BCL1, c-MYC+ BCL2, and c-MYC+ BCL6 in each one. Interphase FISH using specific IGH-translocation probes is valuable for defining clinically meaningful subgroups of B-cell lymphoma.

Ectopic expression of MAFB gene in human myeloma cells carrying (14;20)(q32;q11) chromosomal translocations.

Chromosome 14q+, which represents a chromosomal rearrangement involving the immunoglobulin heavy chain gene (IgH) locus, is a genetic hallmark of human multiple myeloma (MM). Here, we report the identification of (14;20)(q32;qll) chromosomal translocations found in MM cells. Double color fluorescence in situ hybridization analyses pinpointed the breakpoints at the 20qll locus in two MM cell lines within a length of at most 680 kb between the KIAA0823 and MAFB gene loci. Among the transcribed sequences in the vicinity of the breakpoints, an ectopic expression of the MAFB gene, which is located at 450‐680 kb telomeric to one of the breakpoints and encodes a member of the MAF family basic region/leucine zipper transcription factor, was demonstrated to be associated with t(14;20). This finding, together with that of a previous study describing its transforming activity, suggests that the MAFB gene may be one of the targets deregulated by regulatory elements of the IgH gene as a result of t(14;20).

Autosomal aneuploidy rate in spermatozoa: a comparison between normospermic and oligospermic males, using a double-color FISH

Aneuploidy rate for chromosomes 3, 7 and 12 in spermatozoa from normospermic (> 20M/ml) and from oligospermic samples (≤ 20M/ml) have been studied with a double-color fluorescence in situ hybridization. In the normospermic samples, the disomy rate for chromosomes 3 and 12 were similar, with the same mean of 0.17%. The disomy frequency for chromosome 7 was significantly higher (z = 2.4, p<0-.05) with a mean of 0.28 %. In the oligospermic samples, there was no significant difference in the disomy rate for chromosomes 3, 7 and 12 (0.24 %; 0.24 %; 0.28 %). Comparing the two semen populations, the rate of disomy 7 was higher in normospermic males while the rate of disomy 12 was significantly higher in oligospermic males. The frequency of diploid sperm was not significantly different between the two groups. Work is in progress to compare moderate and severe oligospermic patients.

MOZ is fused to p300 in an acute monocytic leukemia with t(8;22).

We report on the fusion of the monocytic leukemia zinc finger protein (MOZ) gene to the adenoviral E1A-associated protein p300 (p300) gene in acute monocytic leukemia M5 associated with a t(8;22)(p11;q13) translocation. We studied two patients with double-color fluorescence in situ hybridization (FISH) using the yeast artificial chromosome 176C9 and the bacterial artificial chromosome clone H59D10 specific to the MOZ and p300 genes, respectively. Both probes were split in the patients' chromosome metaphase cells, and the two derivative chromosomes were each labeled with both probes. We showed by Southern blot the rearrangement of the MOZ gene, and cloned the fusion transcripts in one patient carrying the t(8;22) by reverse transcription-polymerase chain reaction using MOZ- and p300-specific primers. Both fusion transcripts were expressed. This result defines a novel reciprocal translocation involving two acetyltransferases, MOZ and p300, resulting in an abnormal transcriptional co-activator that could play a critical role in leukemogenesis.