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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Expression of the prokaryotic gene for chloramphenicol acetyl transferase in Drosophila under the control of larval serum protein 1 gene promoters.

We have linked the protein coding region of the prokaryotic gene for chloramphenicol acetyl transferase (CAT) to the promoter region of the Drosophila genes for larval serum protein 1 ( LSP1). These regions consist of 1.65 X 10(3) and 2.25 X 10(3) base long DNA segments upstream from the LSP1 alpha and beta genes, respectively. The hybrid genes have been inserted into a P-element transformation vector and the constructs introduced into cultured Drosophila Kc cells by calcium phosphate-mediated transfection, or into the germ-lines of flies by P-element-mediated transformation. CAT expression occurs in approximately 1% of the cultured cells following transfection and the accumulation of protein is maximal three to four days after transfection; and it is dependent upon the presence of the LSP1 sequences. We have also obtained several lines of transformed flies that carry the LSP1-CAT genes in their germ-line. The onset of synthesis of functional chloramphenicol acetyl transferase in these organisms occurs in the late third larval instar, rising to a maximum at puparium formation. We continue to detect CAT until the first few hours of adulthood. Assays of CAT activity in the homogenates of dissected tissues indicated that the genes are only expressed in the fat body, as are the endogenous LSP1 genes. We have confirmed this tissue-specific localization of CAT in indirect immunofluorescence using an anti-CAT monoclonal antibody. Northern blots indicate that CAT transcripts are found only in the fat body but their abundance is an order of magnitude lower than endogenous LSP1 transcripts. Primer extension experiments show that transcription of the hybrid genes is initiated at the same nucleotide as the endogenous LSP1 genes. Taken together these data indicate that the 1.65 X 10(3) and 2.25 X 10(3) base segments of DNA upstream from the LSP1 alpha and LSP1 beta genes contain cis-acting regulatory elements necessary for correct tissue and temporal specificity of LSP1 gene expression.[1]


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