RESUMO
Numerical chromosome aberrations are incompatible with normal human development. Our laboratories develop hybridization-based screening tools that generate a maximum of cytogenetic information for each polar body or blastomere analyzed. The methods are developed considering that the abnormality might require preparation of case-specific probes and that only one or two cells will be available for diagnosis, most of which might be in the interphase stage. Furthermore, assay efficiencies have to be high, since there is typically not enough time to repeat an experiment or reconfirm a result prior to fertilization or embryo transfer. Structural alterations are delineated with breakpoint-spanning probes. When screening for numerical abnormalities, we apply a Spectral Imaging-based approach to simultaneously score as many as ten different chromosome types in individual interphase cells. Finally, DNA micro-arrays are under development to score all of the human chromosomes in a single experiment and to increase the resolution with which micro-deletions can be delineated.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos/diagnóstico , Hibridização in Situ Fluorescente/métodos , Interfase/genética , Análise de Sequência com Séries de Oligonucleotídeos , Diagnóstico Pré-Implantação/métodos , Blastômeros , Sondas de DNA , Feminino , Humanos , Processamento de Imagem Assistida por Computador/métodos , Cariotipagem , Programas de Rastreamento , GravidezRESUMO
This study targeted the development of a novel microarray tool to allow rapid determination of the expression levels of 58 different tyrosine kinase (tk) genes in small tumor samples. The goals were to define a reference probe for multi-sample comparison and to investigate the variability and reproducibility of the image acquisition and RT-PCR procedures. The small number of tk genes on our arrays enabled us to define a reference probe by artificially mixing all genes on the arrays. Such a probe provided contrast reference for comparative hybridization of control and sample DNA and enabled cross-comparison of more than two samples against one another. Comparison of signals generated from multiple scanning eliminated the concern of photo bleaching and scanner intrinsic noise. Tests performed with breast, thyroid, and prostate cancer samples yielded distinctive patterns and suggest the feasibility of our approach. Repeated experiments indicated reproducibility of such arrays. Up- or downregulated genes identified by this rapid screening are now being investigated with techniques such as in situ hybridization.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Neoplasias da Glândula Tireoide/metabolismo , Feminino , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Proteínas Tirosina Quinases/genética , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The microarray format of RNA transcript analysis should provide new clues to carcinogenic processes. Because of the complex and heterogeneous nature of most tumor samples, histochemical techniques, particularly RNA fluorescent in situ hybridization (FISH), are required to test the predictions from microarray expression experiments. Here we describe our approach to verify new microarray data by examining RNA expression levels of five to seven different transcripts in a very few cells via FISH. (J Histochem Cytochem 49:925-926, 2001)
Assuntos
Biomarcadores Tumorais/metabolismo , Hibridização in Situ Fluorescente/métodos , Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biomarcadores Tumorais/genética , Análise de Fourier , Humanos , Hibridização in Situ Fluorescente/instrumentação , Microscopia de Fluorescência , Microscopia de Interferência , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , RNA Neoplásico/metabolismo , Processamento de Sinais Assistido por Computador , Transdução de Sinais , Espectrometria de FluorescênciaRESUMO
Abnormal expression of tyrosine kinase (TK) genes is common in tumors, in which it is believed to alter cell growth and response to external stimuli such as growth factors and hormones. Although the etiology and pathogenesis of carcinomas of the thyroid or breast remain unclear, there is evidence that the expression of TK genes, such as receptor tyrosine kinases, or mitogen-activated protein kinases, is dysregulated in these tumors, and that overexpression of particular TK genes due to gene amplification, changes in gene regulation, or structural alterations leads to oncogenic transformation of epithelial cells. We developed a rapid scheme to measure semiquantitatively the expression levels of 50-100 TK genes. Our assay is based on RT-PCR with mixed based primers that anneal to conserved regions in the catalytic domain of TK genes to generate gene-specific fragments. PCR products are then labeled by random priming and hybridized to DNA microarrays carrying known TK gene targets. Inclusion of differently labeled fragments from reference or normal cells allows identification of TK genes that show altered expression levels during malignant transformation or tumor progression. Examples demonstrate how this innovative assay might help to define new markers for tumor progression and potential targets for disease intervention. (J Histochem Cytochem 49:673-674, 2001)
Assuntos
Neoplasias/metabolismo , Proteínas Tirosina Quinases/genética , Transdução de Sinais , Neoplasias da Mama/metabolismo , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais CultivadasRESUMO
Rapid construction of high-resolution physical maps requires accurate information about overlap between DNA clones and the size of gaps between clones or clone contigs. We recently developed a procedure termed 'quantitative DNA fiber mapping' (QDFM) to help construct physical maps by measuring the overlap between clones or the physical distance between non-overlapping contigs. QDFM is based on hybridization of non-isotopically labeled probes onto DNA molecules that were bound to a solid support and stretched homogeneously to approximately 2.3 kb/microm. In this paper, we describe the design of probes that bind specifically to the cloning vector of DNA recombinants to facilitate physical mapping. Probes described here delineate the most frequently used cloning vectors such as BACs, P1s, PACs and YACs. As demonstrated in representative hybridizations, vector-specific probes provide valuable information about molecule integrity, insert size and orientation as well as localization of hybridization domains relative to specifically-marked vector sequences.
Assuntos
Mapeamento Físico do Cromossomo/métodos , Cromossomos Artificiais de Levedura , Cromossomos Bacterianos , Clonagem Molecular , DNA/genética , DNA/metabolismo , Primers do DNA , Sondas de DNA , Vetores Genéticos , Humanos , Hibridização in Situ Fluorescente , Plasmídeos , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Balanced reciprocal translocations are known to interfere with homolog pairing in meiosis. Many individuals carrying such chromosomal abnormalities suffer from reduced fertility or spontaneous abortions and seek help in the form of assisted reproductive technology. Although most translocations are relatively easy to detect in metaphase cells, the majority of embryonic cells biopsied in the course of in vitro fertilization (IVF) procedures are in interphase. These nuclei are, thus, unsuitable for analysis by chromosome banding or painting using fluorescence in situ hybridization (FISH). Our assay, based on FISH detection of breakpoint-spanning DNA probes, identifies translocations in interphase nuclei by microscopic inspection of hybridization domains. Probes are selected that span the breakpoint regions on normal homologs. The probes should hybridize to several hundred kilobases of DNA flanking the breakpoint. The two breakpoint-spanning DNA probes for the translocation chromosomes are labeled in separate colors (e.g., red and green). The translocation event producing two fused red/green hybridization domains can then be detected in interphase cell nuclei using a fluorescence microscope. We applied this scheme to analyze somatic and germ cells from 21 translocation patients, each with distinct breakpoints. Here, we summarize our experience and provide a description of strategies, cost estimates, as well as typical time frames.
Assuntos
Sondas de DNA , Interfase , Translocação Genética , Blastômeros , Feminino , Genótipo , Humanos , Hibridização in Situ Fluorescente , MasculinoAssuntos
Cromossomos Humanos Par 2/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA/genética , Humanos , Hibridização in Situ Fluorescente , c-Mer Tirosina QuinaseRESUMO
Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle "on" vs. "off" reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. "Male-lethal" (SxlM) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, SxlM haplo-X animals (chromosomal males) die because of improper dosage compensation, and SxlM chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Five independent spontaneous SxlM alleles were shown previously to be transposon insertions into what was subsequently found to be the region of regulated sex-specific Sxl RNA splicing. We show that these five alleles represent three different mutant types: SxlM1, SxlM3, and SxlM4. SxlM1 is an insertion of a roo element 674 bp downstream of the translation-terminating male-specific exon. SxlM3 is an insertion of a hobo transposon (not 297 as previously reported) into the 3' splice site of the male exon, and SxlM4 is an insertion of a novel transposon into the male-specific exon itself. We show that these three gain-of-function mutants differ considerably in their ability to bypass the sex determination signal, with SxlM4 being the strongest and SxlM1 the weakest. This difference is also reflected in effects of these mutations on sex-specific RNA splicing and on the rate of appearance of SXL protein in male embryos. Transcript analysis of double-mutant male-viable SxlM derivatives in which the SxlM insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these SxlM alleles is a consequence of an alteration of the structure of the pre-mRNA that allows some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of SxlM alleles appears to depend on the mutant alleles' responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos/fisiologia , Hormônios de Inseto/genética , Splicing de RNA , Proteínas de Ligação a RNA , Diferenciação Sexual/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA , Mecanismo Genético de Compensação de Dose , Drosophila melanogaster/embriologia , Feminino , Genes Letais/fisiologia , Genes de Troca , Masculino , Dados de Sequência Molecular , Mutação/fisiologia , Precursores de RNA/análise , RNA Mensageiro/análiseRESUMO
In addition to controlling somatic sexual development in Drosophila melanogaster, the Sex-lethal (Sxl) gene is required for proper differentiation of female germ cells. To investigate its role in germ-line development, we have examined the expression of Sxl in wild-type ovaries and ovaries that are defective in early steps of germ cell differentiation. As in the soma, the basic mechanism for on/off regulation of Sxl relies on sex-specific processing of its transcripts in germ cells. One class of female-sterile mutations, which includes fs(1)1621 and the tumorous-ovary-producing allele of the ovarian tumor gene, otu1, is defective in the splicing process. These mutants have germ lines with high amounts of Sxl RNA spliced in the male mode and a severe reduction of protein levels in the germ cells. Another class of female-sterile mutations produces a phenotype similar to that seen in fs(1)1621 and otu1 but appears to express normal levels of Sxl protein in the germ cells. However, this second class does not show the changes in protein distribution normally observed in wild-type germ cells. In the wild-type germarium, the non-differentiated germ cells show a strong cytoplasmic accumulation of Sxl protein followed, as the germ cells differentiate, by a dramatic reduction and redistribution of the protein into nuclear foci. Interestingly, two female-sterile alleles of Sxl, Sxlf4 and Sxlf5 belong to the second class, which shows persistent cytoplasmic accumulation of Sxl protein. These Sxl female-sterile mutants encode an altered protein indicating that Sxl regulates processes that eventually lead to the changes in Sxl protein distribution. Lastly, we demonstrate that during the final stages of oogenesis several mechanisms must operate to prevent the progeny from inheriting Sxl protein. Conceivably, this regulation safeguards the inadvertent activation of the Sxl autoregulatory feedback loop in the male zygote.