The TKO expression cassette. Histone 2b or hatching enzyme cis-regulatory elements (A) employed in the cassette are modular and can easily be exchanged with any other regulatory sequence. The start of transcription (B) is set to be at least 20 bp upstream of a canonical Kozak ATG (C) and a nuclear localization sequence (D) that are best fit to sea urchin initiators and codon usage. The heavy- (E) and light-chain (G) variable regions of the anti-P3A2 scFv are separated by an encoded flexible Gly/Ser linker (F). To process, stabilize, and terminate the TKO transcripts, we inserted the SV40 large T 3′ intron (I), polyadenylation (J), and transcriptional termination (K) sequences after the stop codon (H).

Measurement of inhibitory activity of mAb with respect to specific P3A2–DNA complex formation. Relative occupancy of oligonucleotide probe (Y) is plotted against an antibody dilution series (A). The intercept, Y₀ (here Y₀ = 0.78 of total probe in the reaction) represents the probe occupancy in the absence of antibody (A = 0). The inhibitory activity is quantitatively estimated as the value of k_A, the slope; here, k_A = 1.26 × 10¹⁰ M⁻¹. The data are fit to the function Y = αk_A·A + Y₀ (Eq. 1). Here Y is occupancy of oligonucleotide probe; i.e., where PD_(A) dilution (antibody concentration was measured after purification of the antibody from the hybridoma ascites fluid) and D₀ is concentration of total probe in the reaction: Y = PD_A/D₀. In Eq. 1, α = −2P₀, where P₀ is the molar quantity of P3A2 transcription factor in the reaction; in these experiments, P₀ ranged from 2 to 5 × 10⁻¹¹ M. k_A is defined as the equilibrium constant for the reaction of a mAb molecule with two P3A2 molecules, i.e., k_A = AP₂/A · P₀². Eq. (1) follows from the assumption that all P3A2 molecules will be in complex with either the antibody or the DNA probe and that the P3A2–DNA reaction is bimolecular, i.e., PD_(A) = k_DP_(A) · D_(A), where P_(A) and D_(A) are the molar concentrations of the free P3A2 protein and the DNA probe at given antibody dilutions.

Effects of αP3A2⋅TKO on expression of CyIIIa⋅CAT, endogenous CyIIIa, and the lethal embryonic phenotype. In a–f, the H2b cis-regulatory element was used to drive expression of the injected TKO constructs, and in g–l, the hatching enzyme cis-regulatory element was used (see Materials and Methods). (a, b) Endogenous CyIIIa expression monitored by WMISH using an antisense CyIIIa probe (see Materials and Methods). (a) Embryo viewed laterally, oral side to the right; here and in following panels the arrows bracket the oral ectoderm where neither CyIIIa nor wild-type CyIIIa⋅CAT expression occurs. (b) Anal view, oral side to the top. (c, d) CyIIIa⋅CAT coinjected with αP3A2⋅TKO, driven by the H2b cis-regulatory element, lateral views. Clones of cells expressing CAT mRNA in the oral ectoderm are displayed by using WMISH. As observed when the P3A2 sites are deleted (14), these injections produce embryos displaying normal patterns of aboral ectoderm expression (i.e., embryos in which the exogenous DNA resides in the aboral ectoderm); embryos displaying both normal and ectopic oral ectoderm expression; and embryos displaying oral expression only (i.e., the exogenous DNA is only in oral ectoderm lineages); only the latter are illustrated here. (e, f) Effects of αP3A2⋅TKO on endogenous CyIIIa gene expression, monitored as in a and b. Both embryos are shown from the blastopore, and both display ectopic oral staining as well as staining in clones of archenteron cells in addition to aboral ectoderm expression. (g) Pluteus-stage (72-hr) embryo viewed from anal side. This embryo bears the αP3A2⋅TKOmis construct and is normal in morphology. (h) Gastrulation-defective phenotype at 72 hr caused by injection of the αP3A2⋅TKO construct. (i) Gastrulation-defective embryo at 48 hr viewed from blastopore, in which Endo16 transcripts are displayed by using WMISH. (j) Normal 48-hr embryo viewed laterally and bearing GFP-expressing clones of exogenous DNA in aboral ectoderm and archenteron. These clones also include the αP3A2⋅TKOmis control construct. (k, l) Gastrulation-defective embryos as in j, but bearing αP3A2⋅TKO construct present in large endodermal clones.