An in vivo titration of regulatory factors required for expression of a fusion gene in transgenic sea urchin embryos.
We report that endogenous regulatory factors mediating expression of a lineage-specific sea urchin embryo gene can be titrated in vivo by introduction of a sufficient molar excess of DNA-binding sites. Thus we obtain an estimate of the quantity of limiting factor(s) required for developmental activation and transcriptional expression, which can be compared with estimates of factor prevalence obtained by measurements in vitro carried out under equilibrium conditions. A fusion construct in which the bacterial gene for chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2.3.1.28) is controlled by cis-regulatory elements of the CyIIIa cytoskeletal actin gene (CyIIIa-CAT) was introduced in varying numbers of copies into sea urchin eggs. The activity of the CyIIIa-CAT fusion gene in 24-hr blastula-stage embryos was shown to saturate as the number of exogenous genes was increased. The mean number of CyIIIa-CAT fusion genes per nucleus at which half saturation was obtained was 105 +/- 40 (mean +/- SD). This result suggests that equilibrium parameters measured earlier in vitro may apply, at least approximately, within the embryo nuclei.
Livant
D L
DL
Division of Biology, California Institute of Technology, Pasadena 91125.
Cutting
A E
AE
Britten
R J
RJ
Davidson
E H
EH
D000199:Actins / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D008845:Microinjections; D012045:Regulatory Sequences, Nucleic Acid; D012617:Sea Urchins / Q000196:embryology; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D000199:Actins; D014157:Transcription Factors; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase