ABSTRACT
The recent completion of the Caenorhabditis elegans genome has revealed that this nematode worm has 10 members of the ETS gene family. Isolation and analysis of C. elegans mutants and subsequent screens to identify interacting genes can proceed very quickly in this model organism. Molecular genetic analysis of the receptor tyrosine kinase-Ras-MAP kinase signaling pathway in C. elegans identified the ETS family transcription factor Lin-1 as a nuclear effector of this evolutionarily conserved signal transduction pathway. Here we review classical genetic approaches used to discover the role of Lin-1 in the Ras-MAP kinase signaling pathway and describe new technologies that can be applied to the analyses of signaling pathways and transcription factor regulatory networks in C. elegans.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/genetics , Genes, Helminth , MAP Kinase Signaling System/genetics , Multigene Family , Proto-Oncogene Proteins , Transcription Factors/genetics , Amino Acid Sequence , Animals , Cell Lineage , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Dimerization , Disorders of Sex Development/genetics , Evolution, Molecular , Female , Forecasting , Gene Expression Profiling , Gene Expression Regulation , Gene Targeting , Genetic Techniques , Helminth Proteins/genetics , Helminth Proteins/physiology , Humans , Larva , Male , Mice , Models, Animal , Molecular Sequence Data , Morphogenesis , Mutagenesis, Site-Directed , Oncogene Proteins/genetics , Phylogeny , Proteome , Proto-Oncogene Protein c-fli-1 , Receptor Protein-Tyrosine Kinases/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Trans-Activators/genetics , Transcription Factors/physiology , Transcriptional Regulator ERG , Vulva/cytology , Vulva/growth & development , ras Proteins/physiologyABSTRACT
The ETS related gene, ERG, is one of 20 or more genes belonging to the ETS family of transcription factors. Translocation of the ERG gene t(21;22) results in the chimeric fusion transcript seen in approximately 10% of Ewings sarcomas. In addition, recent studies have shown that a reciprocal translocation t(21;16) of ERG gives rise to two aberrant transcripts seen in some forms of acute myeloid leukaemia. In vitro studies have linked the up regulation of ERG expression with stromal cell independence in erythroleukemic clones and shown that the ERG related genes ETS1 and ETS2 have a mitogenic and transforming activity when overexpressed in NIH3T3 cells. Interestingly ERGB/FLI-1, which is also involved in Ewings sarcoma translocations and shares a very high sequence identify with ERG has been reported to be unable to transform NIH3T3 cells. In this study we investigate the effects of overexpression of ERG on cell proliferation, factor dependence, growth in soft agar and tumorigenesis in nude mice. An ERG expression construct with the human ERG2 cDNA driven by the sheep metallothionein la promoter (sMTERG) was transfected into NIH3T3 cells. Clonal cell lines overexpressing ERG were established. The cell lines became morphologically altered, grew in low serum and serum free media and gave rise to colonies in soft agar suspension. Furthermore, we demonstrate that after subcutaneous injection these clones grow as solid tumors in nude mice. These data demonstrate that c-ERG is a proto-oncogene capable of transforming NIH3T3 cells. Therefore, overexpression or inappropriate expression of ERG may contribute to oncogenesis.
Subject(s)
Cell Transformation, Neoplastic , DNA-Binding Proteins/physiology , Mitogens , Proto-Oncogene Proteins/physiology , Proto-Oncogenes , Trans-Activators , 3T3 Cells , Animals , Base Sequence , DNA Primers/chemistry , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels , Gene Expression , Humans , Mice , Mice, Nude , Molecular Sequence Data , Neoplasms, Experimental/genetics , Proto-Oncogene Mas , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Transcriptional Regulator ERGABSTRACT
Using quantitative buffy coat analysis (QBCA), rapid and accurate measurements can be made of the erythrocyte PCV, total WBC count, and platelet count, and the leukocyte population can be differentiated into total granulocytes (including quantitation of eosinophils), and lymphocytes and monocytes. The QBCA is performed by placing a blood sample (50 to 111 microliters) into a high-precision-bore microhematocrit tube that contains a freely moving, closely fitting, cylindrical plastic float. After centrifugation for 5 minutes, the buffy coat components separate by density. The plastic cylinder floats in the buffy coat, thereby expanding the lengths of the buffy coat layers. The layers are measured in a manner that is similar to that used for measuring PCV. Results of QBCA of blood samples from dogs, cats, and horses indicated that the hematologic values obtained correlated with results obtained by use of conventional methods. The accuracy and ease of use of QBCA and the availability of results while the animal is still being examined make QBCA a useful tool for hematologic evaluation of animals.
