Katula, Karen S. and Hough-Evans, Barbara R. and Britten, Roy J. and Davidson, Eric H. (1987) Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs. Development, 101 (3). pp. 437-447. ISSN 0950-1991. http://resolver.caltech.edu/CaltechAUTHORS:KATdev87
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The 5' terminus of the CyI actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2.5 kb of 5' flanking sequence, the transcribed leader sequence, and the first few codons of the CyI gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was micro-injected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous CyI gene first appear. The exogenous CyI.CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient CyI.CAT genes. The amounts of CAT enzyme produced by embryos bearing CyI.CAT deletions that lack various regions of the CyI sequence were measured. As little as 254 nucleotides of upstream CyI sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining less than 850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete CyI.CAT fusion construct.
|Additional Information:||Copyright © 1987 by Company of Biologists. (Accepted 10 July 1987) We thank Michael Graham, an undergraduate research assistant, for his work on this project. Dr Constantin Flytzanius constructed the clone pClpN5 and we are grateful to him for making it available to us. This research was supported by NIH grant GM-02927. K.S.K. was supported by an NIH postdoctoral training grant (HD-07257).|
|Subject Keywords:||gene transfer, cytoskeletal actin, developmental regulation, sea urchin, Cyl|
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|Deposited On:||12 Nov 2008 06:10|
|Last Modified:||26 Dec 2012 10:30|
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