Application of yeast artificial chromosomes in fluorescence in situ hybridization.
ABSTRACT In addition to the well-known applications of yeast artificial chromosomes (YACs) in classical molecular genetics, they also are used for molecular cytogenetic studies. YACs, as well as other locus-specific probes like DNA, plasmids, cosmids, P1-clones, or bacterial artificial chromosomes can be labeled with fluorochromes and applied in fluorescence in situ hybridization (FISH) experiments. Various applications are possible, such as gene mapping, FISH banding, determination of chromosomal breakpoints, characterization of derivative chromosomes, studies on the interphase architecture, or karyotypic evolution studies. This chapter outlines the basic principle of how YACs can be hybridized in situ on chromosome preparations. Moreover, an overview is given on possible questions to be processed using YACs as FISH probes.
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ABSTRACT: Soluble sulfotransferases (SULTs) generate electrophilically reactive metabolites from numerous food-borne compounds, environmental contaminants and drugs, often resulting in mutagenicity and carcinogenicity. Substrate specificity, regulation and tissue distribution of SULTs show large interspecies differences. In humans, therefore, SULTs may be involved in the induction of cancer in different tissues than in standard animal models. To construct a rodent model taking some species differences into account, we transferred a 68.5 kb human (h) genomic sequence that comprised the transcribed and long flanking regions of SULT1A1 and 1A2 into murine oocytes. This approach resulted in several mouse lines expressing these human genes in a copy number-dependent manner with a tissue distribution similar to that in humans. In previous in vitro studies, we had demonstrated that human SULT1A1 and 1A2 efficiently catalyze the terminal activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to a mutagen. The transgenic mice were used to study the hSULT1A1/1A2-mediated activation. Tissue distribution and levels of DNA adducts were determined in hSULT1A1/1A2 transgenic and wild-type mice after an oral dosage of PhIP. Transgenic mice exhibited significantly elevated PhIP-DNA adduct levels compared with the wild-type in liver (13-fold), lung (3.8-fold), colon (2-fold), kidney (1.6-fold) and cecum (1.5-fold). Moreover, among the eight tissues examined, liver was the one with the lowest and highest adduct levels in wild-type and transgenic mice, respectively. Hence, expression of hSULT1A1/1A2 not only enhanced the genotoxicity but also substantially changed the organotropism of PhIP.Carcinogenesis 09/2011; 32(11):1734-40. · 5.64 Impact Factor
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ABSTRACT: Background: Fluorescence in situ hybridization (FISH) assays are indispensable in diagnostics and research. Routine application of this so-called molecular cytogenetic technique on human chromosomes started in 1986. Since then, a huge variety of different approaches for chromosomal differentiation based on FISH has been described. It was established to characterize marker chromosomes identified in conventional banding analysis as well as cryptic rearrangements not resolved by standard cytogenetics. Objective/method: Even though molecular cytogenetics, like banding cytogenetics for almost 40 years, is often called dead now, it offers unique possibilities of single cell analysis. Thus, a review is presented here on the available diagnostic-relevant FISH methods and probe sets applied in routine pre- and postnatal clinical as well as tumor cytogenetics. Conclusion: Molecular cytogenetics is a fast, straightforward and reliable tool that is indispensable in cytogenetic diagnostics. It is and will continue to be of high clinical impact in diagnostics, especially in the overwhelming majority of routine cytogenetic laboratories that cannot afford and do not need high-throughput chip-based platforms for their daily work.Expert Opinion on Medical Diagnostics 07/2009; 3(4):453-60.