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PubMed2004

Interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 is important for actin-dependent endocytic internalization.

A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.

Actins↗

PubMed1998

Automated cell count in flow cytometry: a valuable tool to assess CD4 absolute levels in peripheral blood.

The enumeration of lymphocyte subsets in absolute counts has long relied on different methods applied separately to whole blood cell count, lymphocyte differential appreciation, and flow cytometric evaluation of lymphocyte subsets percentages. The development of multicolor labeling methods inflow cytometry now allows a more homogeneous appreciation of several cell subsets among gated lymphocytes. The use of internal calibrators, such as microbead suspensions, also permits a direct appreciation of subsets in absolute counts in a single-platform method. These methods were compared with a traditional multiplatform method of assessing absolute counts of lymphocyte subsets in a pilot study in which all manipulations were performed by 1 person and in a full-scale larger study performed in the normal working conditions of a hospital laboratory. Microspheres seem to be a reliable tool to perform absolute count enumeration inflow cytometry, but several precautions in the sample preparation and flow cytometric analysis are required.

Antibodies, Monoclonal↗

PubMed1998

Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry.

Immunophenotyping by flow cytometry has not been widely applied to cerebrospinal fluid (CSF) analysis. We attempted to optimize flow cytometric detection of malignant lymphoma in CSF samples by the routine use of 3- and 4-color flow cytometry, with specific selection of lymphoid cells by fluorescence vs 90 degrees light scatter gating. Thirty-six consecutive CSF samples were immunophenotyped by flow cytometry, and the results were compared with those of standard microscopic examination. Lymphoid events were adequate for analysis in 27 of the 36 samples. Each of the 9 unsuccessful samples was more than 24 hours old at analysis or contained fewer than 1 x 10(4) total cells (< or =1 cell/microL). Lymphoma was detected in 10 of the remaining 27 cases. Six lymphomas were detected by morphology and flow cytometry, 1 only by morphologic examination, and 3 only by flow cytometry. Therefore, the combination of flow cytometry and morphologic examination enhanced the detection by 43% over morphologic examination alone. Flow cytometry permitted the detection of lymphoid clones totaling less than 1% of total cells. Multicolor flow cytometry is a rapid and sensitive technique that enhances detection of lymphoma in paucicellular CSF samples. Given the great sensitivity of flow cytometry, future studies will be necessary to assess the significance of detecting small lymphoid clones in this setting.

Antigens, CD↗

PubMed2005

Experimental design for three-color and four-color gene expression microarrays.

MOTIVATION: Three-color microarrays, compared with two-color microarrays, can increase design efficiency and power to detect differential expression without additional samples and arrays. Furthermore, three-color microarray technology is currently available at a reasonable cost. Despite the potential advantages, clear guidelines for designing and analyzing three-color experiments do not exist. RESULTS: We propose a three- and a four-color cyclic design (loop) and a complementary graphical representation to help design experiments that are balanced, efficient and robust to hybridization failures. In theory, three-color loop designs are more efficient than two-color loop designs. Experiments using both two- and three-color platforms were performed in parallel and their outputs were analyzed using linear mixed model analysis in R/MAANOVA. These results demonstrate that three-color experiments using the same number of samples (and fewer arrays) will perform as efficiently as two-color experiments. The improved efficiency of the design is somewhat offset by a reduced dynamic range and increased variability in the three-color experimental system. This result suggests that, with minor technological improvements, three-color microarrays using loop designs could detect differential expression more efficiently than two-color loop designs. AVAILABILITY: http://www.jax.org/staff/churchill/labsite/software SUPPLEMENTARY INFORMATION: Multicolor cyclic design construction methods and examples along with additional results of the experiment are provided at http://www.jax.org/staff/churchill/labsite/pubs/yong.

Algorithms↗

PubMed1995

Synergistic increase in chromosomal breakage within the euchromatin induced by an interaction of the benzene metabolites phenol and hydroquinone in mice.

The hematopoietic and carcinogenic effects of benzene may result from an interaction of various benzene metabolites. Following the co-administration of phenol and hydroquinone, a synergistic increase in myelotoxicity and genotoxicity has been observed in the bone marrow of mice. To understand the mechanisms underlying these synergistic genotoxic effects we have studied the origin of micronuclei (MN) formed in bone marrow erythrocytes following the co-administration of these two metabolites. Phenol and hydroquinone were administered to male CD-1 mice by i.p. injection three times at 24 h intervals. The frequency of MN was evaluated in bone marrow cells harvested 24 h following the final dose. A marked increase in MN was observed in mice co-administered phenol and hydroquinone, which was significantly greater than that observed with the individual metabolites. Labeling with the CREST antibody and multicolor fluorescence in situ hybridization with the mouse major and minor satellite probes indicated that both chromosomal loss and breakage and occurred. The major increase in MN induced by the phenol and hydroquinone combination originated from breakage in the euchromatic region of the mouse chromosomes. The origin of MN in mice co-administered phenol and hydroquinone differed substantially from that induced by hydroquinone alone, but was almost identical to that seen in MN from benzene-treated mice. These results strongly support the hypothesis that interactive effects among benzene metabolites play an important role in the genotoxic and carcinogenic effects of benzene.

Animals↗

PubMed1999

Increased chromosomal instability in peripheral lymphocytes and risk of human gliomas.

Brain tumors exhibit considerable chromosome instability (CIN), suggesting that genetic susceptibility may contribute to brain tumorigenesis. To test this hypothesis, in this pilot study, we examined for CIN in short-term lymphocyte cultures from 25 adult glioma patients and 28 age-, sex- and ethnicity-matched healthy controls (all Caucasian). We evaluated CIN by a multicolor fluorescence in situ hybridization assay using two probes: a classic satellite probe for a large heterochromatin breakage-prone region of chromosome 1 and an alpha satellite probe for a smaller region adjacent to the heterochromatin probe. Our results showed a significant increase in the mean number of spontaneous breaks per 1000 cells in glioma patients (mean +/- SD, 2.4+/-0.8) compared with controls (1.4+/-0.9; P < 0.001). By using the median number of breaks per 1000 cells in the controls as the cutoff value, we observed a crude odds ratio (OR) of 8.5 [95% confidence interval (CI) = 2.05-34.9, P < 0.001] for spontaneous breaks and brain tumor risk. After adjustment for age, sex and smoking status, the adjusted OR was 15.3 (95% CI, 2.71-87.8). A significant increase in cells with chromosome 1 aneuploidy (in the form of hyperdiploidy) (P < 0.001) was also observed in the glioma cases, with an adjusted OR of 6.6 (95% CI = 1.5-30, P < 0.05). These findings suggest that CIN can be detected in the peripheral blood lymphocytes of brain tumor patients and may be a marker for identifying individuals at risk.

Aneuploidy↗

PubMed1992

High-resolution in situ hybridization using DNA halo preparations.

To improve DNA resolution of fluorescence in situ hybridization we have adapted a nuclear extraction technique, resulting in highly extended DNA loops arranged around the nuclear matrix in a halo-like structure. In situ hybridization signals from alphoid and cosmid DNAs appear as beads-on-a-string, which, according to preliminary experiments, results from the association of individual probe fragments. By multicolor hybridizations we have been able to determine relative map position and to easily detect 10 kb overlap between individual cosmid clones, each of which shows linear beaded signals of ca. 10 microns, suggesting that the DNA is essentially linearized in our protocol. The map configuration can be typically derived from analysis of 5-10 cells only. The resolution range of the technique is at least 10-200 kb, and probably as little as a few kb, thus greatly extending the abilities of the existing FISH methodologies. This novel technique is much more efficient and practicable than pronuclei hybridizations, another method for high resolution FISH, and readily produces results with probes of a variety of genomic origin. In conclusion the DNA halo technique should be able to contribute significantly to the assessment of cosmid and YAC overlaps as well as to the sizing of gaps between adjacent contigs generated in genome projects.

Cells, Cultured↗

PubMed1993

FISH ordering of reference markers and of the gene for the alpha 5 subunit of the gamma-aminobutyric acid receptor (GABRA5) within the Angelman and Prader-Willi syndrome chromosomal regions.

We have established a probe order within the Angelman/Prader-Willi chromosomal regions by multicolor fluorescence in situ hybridization (FISH). The probe [locus] order extending distally from the centromere is 34[D15S9]-IR4-3R[D15S11]-189-1[D15S13]-PW71++ + [D15S63]-3-21[D15S10]-28 beta 3-H3[GABRB3]-IR10-1 [D15S12]. This order agrees with that recently reported (1) with the exception of PW71 [D15S63]. In addition, a second gamma-aminobutyric acid (GABAA) receptor, the alpha 5 subunit, has been localized within the reference map to between GABRB3 and D15S12. The locus order was further confirmed by DNA hybridization analysis of two patients, one with Angelman syndrome and one with Prader-Willi syndrome, with different unbalanced translocations and molecular extents of deletion. Our results provide a framework map of chromosome 15q11-q13 into which additional markers can be oriented and allow a further differentiation of the critical genetic regions of the two syndromes.

Amino Acid Sequence↗

PubMed1998

Chromosome painting: a useful art.

Chromosome 'painting' refers to the hybridization of fluorescently labeled chromosome-specific, composite probe pools to cytological preparations. Chromosome painting allows the visualization of individual chromosomes in metaphase or interphase cells and the identification of both numerical and structural chromosomal aberrations in human pathology with high sensitivity and specificity. In addition to human chromosome-specific probe pools, painting probes have become available for an increasing range of different species. They can be applied to cross-species comparisons as well as to the study of chromosomal rearrangements in animal models of human diseases. The simultaneous hybridization of multiple chromosome painting probes, each tagged with a specific fluorochrome or fluorochrome combination, has resulted in the differential color display of human (and mouse) chromosomes, i.e. color karyotyping. In this review, we will summarize recent developments of multicolor chromosome painting, describe applications in basic chromosome research and cytogenetic diagnostics, and discuss limitations and future directions.

Animals↗

PubMed2006

Variation in MLH1 distribution in recombination maps for individual chromosomes from human males.

Meiotic recombination is essential for the segregation of homologous chromosomes and the formation of normal haploid gametes. Little is known about patterns of meiotic recombination in human germ cells or the mechanisms that control these patterns. Documentation of the normal range of variability of recombination distribution over the genome among individuals is an essential prerequisite for understanding abnormal recombination patterns, which may be associated with non-disjunction and chromosome rearrangements. In this article, variation in recombination maps for individual chromosomes among 10 normal human males is examined for the first time. An immunocytogenetic approach allowed analysis of pachytene cells, using antibodies to detect the mature synaptonemal complex (SCP1/SCP3), the centromere (CREST) and sites of crossing over (MLH1). Individual bivalents were identified with centromere-specific multicolor fluorescence in situ hybridization. Significant heterogeneity in MLH1 focus frequency across donors was observed for larger chromosome arms (P<0.05, one-way ANOVA). Significant inter-donor variation in the overall crossover frequency per cell was also found (P<0.0001, one-way ANOVA). Furthermore, several chromosome arms showed significant differences in crossover distribution along the SCs among donors. Inter-individual variation in interference distances was observed for all chromosomes. The significance of altered recombination patterns among individuals and the role of interference are discussed.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Fluorescence in situ hybridization landmarks for chromosomes of culicoides variipennis (Diptera: Ceratopogonidae).

Because the three chromosomes of Culicoides variipennis (Coquillett) are morphologically indistinguishable, physical landmarks were needed so that the chromosomes can be identified uniquely and oriented unambiguously before initiating the construction of a physical map based on FISH (fluorescence in situ hybridization). When repetitive sequence clones p1887 and K8.1a8 were labeled with digoxigenin and probe K8a.2G2 was labeled with biotin-11-dUTP and digoxigenin, the 3 chromosomes could be differentiated unambiguously when visualized with specific band-pass filters. This provided the basis for C. variipennis FISH landmark probes that enabled the identification and orientation of all 3 pairs of chromosomes. Using this multicolor FISH labeling strategy, probes that provide unique landmarks for the C. variipennis FISH physical map have been found and may be used in all FISH reactions where an unknown probe is placed to metaphase chromosomes of C. variipennis. A physical map of the C. variipennis genome will provide the foundation for map-based positional cloning of the gene(s) that control vector competence for the bluetongue viruses.

Animals↗

PubMed1995

A method for the rapid generation of alpha- and classical satellite probes for human chromosome 9 by polymerase chain reaction using genomic DNA and their application to detect chromosomal alterations in interphase cells.

Fluorescence in situ hybridization (FISH) using chromosome-specific DNA probes is a technique which has recently become widely used for the analysis of chromosome alterations in interphase and metaphase cells. In this report, a polymerase chain reaction (PCR)-based method is described for simultaneously amplifying and labelling probes targeting the alpha- and classical satellite regions of chromosome 9 using either plasmid or genomic DNA. Chromosome-specific probes were generated using readily obtainable plasmid DNA and genomic DNA from a hybrid cell line containing human chromosome 9 in a hamster cell background. The utility of these probes to detect and quantify structural and numerical aberrations in interphase cells was demonstrated using a new multicolor FISH strategy by comparing the frequencies of hyperdiploidy and chromosome breakage affecting the regions targeted by the probes in interphase and metaphase human lymphocytes irradiated during culture. The irradiated cells exhibited a significantly higher frequency of tetrasomy and breakage effecting the centromeric/pericentric region of chromosome 9 as compared with non-exposed cells. In general, similar frequencies of breakage and hyperdiploidy were observed in the interphase and metaphase preparations. These results show that DNA probes for the repetitive sequences in human chromosomes can be easily generated from genomic DNA and that these probes can be effectively used to detect chromosome breakage and aneuploidy in interphase and metaphase lymphocytes in vitro.

Aneuploidy↗

PubMed1999

Noscapine hydrochloride disrupts the mitotic spindle in mammalian cells and induces aneuploidy as well as polyploidy in cultured human lymphocytes.

Noscapine hydrochloride is a centrally acting antitussive opium derivative widely used in cough suppressants. Recent studies have reported that noscapine is a potent inducer of polyploidy but not of aneuploidy in vitro. To obtain more comprehensive information about the cytogenetic effects of this compound, we treated cultured human lymphocytes (HPL) and Chinese hamster ovary (CHO) cells with various concentrations of noscapine hydrochloride. Using a differential staining technique noscapine was shown to disrupt the mitotic spindle at concentrations < 5 micrograms/ml in both cell types. The use of multicolor fluorescence in situ hybridization (FISH) on noscapine-treated human lymphocytes showed a dose-dependent induction of hyperdiploidy of chromosome 1 but not of chromosomal breakage in the 1cen-q12 region under in vitro conditions, indicating that noscapine is specifically inducing numerical chromosomal aberrations. FISH with probes targeting different chromosomes revealed that noscapine is capable of inducing both polyploidy and true hyperdiploidy. Our results show that noscapine, by disrupting the function of the mitotic spindle, has the ability to induce aneuploidy and not uniquely polyploidy as previously reported. By using these types of molecular cytogenetic techniques, it should be possible to evaluate the ability of noscapine to induce aneuploidy in the upper intestinal tract in vivo.

Aneuploidy↗

PubMed2003

Detection by dual color fluorescence in situ hybridization of in vivo chromosome damage in rat hepatocyte nuclei.

Our previous study using a multicolor fluorescence in situ hybridization (FISH) technique revealed that region-specific DNA probes for rat chromosome 1 enabled the detection of structural chromosome damage in rat interphase nuclei from peripheral blood and bone marrow cells. In the present study, this FISH technique was modified for application to a non-hematopoietic organ (liver) and the usefulness of the system was tested using diethylnitrosamine (DEN) as a model hepatocarcinogen. Male Sprague-Dawley rats were orally treated once with DEN at 200 mg/kg. Their livers were removed at 4, 7 or 14 days after treatment and homogenized with a tissue grinder to isolate hepatocyte nuclei. The nucleus suspension was fixed in methanol:acetic acid and air dried. Dual color FISH with two probes, one labeled with tetramethylrhodamine and one with digoxigenin, demonstrated that the maximum increase in the frequency of nuclei with spatially abnormal signals was observed 7 days after treatment. A dose-response relationship for induction of abnormal nuclei was observed. This improved dual color FISH system is potentially valuable for assessing in vivo clastogenicity in all rat organs.

Animals↗

PubMed1992

Sensitive detection of RNAs in single cells by flow cytometry.

A rapid and sensitive fluorescent in situ hybridization method has been developed to probe RNA contents of individual cells by flow cytometry. Fixed cells in suspension were hybridized with 5' end-fluorophore-labeled oligodeoxynucleotides complementary to defined regions of the RNA of interest and analyzed by flow cytometry. With this method, we monitored combinations of histone H4 mRNA, 18S rRNA and 28S rRNA levels in synchronized HeLa S3 cells by multicolor analysis. A fluorescence signal equivalent to 1800 copies of histone H4 mRNA per cell was detected with signal-to-background ratio of 5.4. If non-specific binding of the fluorophore-labeled probe can be reduced, as few as 100 copies of mRNA of the size of H4 could be detected in individual cells by flow cytometry.

Base Sequence↗

PubMed1994

Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.

Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps.

Base Sequence↗

PubMed2001

Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection.

SSCP and heteroduplex analysis (HA) continue to be the most popular methods of mutation detection due to their simplicity, high sensitivity and low cost. The advantages of these methods are most clearly visible when large genes, such as BRCA1 and BRCA2, are scanned for scattered unknown mutations and/or when a large number of DNA samples is screened for specific mutations. Here we describe a novel combined SSCP/duplex analysis adapted to the modern capillary electrophoresis (CE) system, which takes advantage of multicolor labeling of DNA fragments and laser-induced fluorescence detection. In developing this method, we first established the optimum conditions for homoduplex and heteroduplex analysis by CE. These were determined based on comprehensive analysis of representative Tamra-500 markers and BRCA1 fragments at different concentrations of sieving polymer and temperatures in the presence or absence of glycerol. The intrinsic features of DNA duplex structures are discussed in detail to explain differences in the migration rates between various types of duplexes. When combined SSCP/duplex analysis was carried out in single conditions, those found to be optimal for analysis of duplexes, all 31 BRCA1 and BRCA2 mutations, polymorphisms and variants tested were detected. It is worth noting that the panel of analyzed sequence variants was enriched in base substitutions, which are usually more difficult to detect. The sensitivity of mutation detection in the SSCP portion alone was 90%, and that in the duplex portion was 81% in the single conditions of electrophoresis. As is also shown here, the proposed combined SSCP/duplex analysis by CE has the potential of being applied to the analysis of pooled genomic DNA samples, and to multiplex analysis of amplicons from different gene fragments. These modifications may further reduce the costs of analysis, making the method attractive for large scale application in SNP scanning and screening.

BRCA1 Protein↗

PubMed2002

Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides.

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3'-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3'-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.

Biotinylation↗