Gene expression assay. Range of X-Gal staining intensities observed in individual tailbud embryos following zygote electroporation () of CiTnI/nuclear β-galactosidase reporter constructs: (A) +++, (B) ++, (C) +, (D) undetectable. DNA constructs used were CiTnI(−836/−335)nZ (A, B) and CiTnI(−836/−571)nZ (C, D). Construct gene expression levels are reported in and as the percentage of normal-appearing embryos showing detectable staining, a population parameter that correlated well with individual embryo X-Gal staining intensities (see ‘Materials and Methods’ section).

Localization of CiTnI transcriptional elements through large-scale end-deletion analysis. The figure schematically depicts CiTnI gene DNA segments. ATG start codon-based coordinates are shown at the top, with the TSS mapped in the present study indicated by a curved arrow. The XbaI site at −645 to −640 is indicated. Each DNA region tested (black bars) was linked at the right end to a promoterless nuclear β-galactosidase reporter gene (dashed lines indicate the deleted CiTnI DNA downstream of the region being tested). Recombinant reporter constructs were assayed as in . The column ‘construct’ lists the construct name or the precise range of the CiTnI DNA segment being tested and the column ‘expression’ gives the percentage of normally-developed embryos that showed detectable X-Gal staining (absolute numbers in brackets). CiTnI DNA segments −638/−335 and −836/−623 were tested with and without the HrMA4 enhancer (white bar), as indicated.

CiTnI TSS identification by experimental molecular genetics and by high-throughput 5′-RACE sequence analysis of minor transcripts present in normal tailbud embryo RNA. Panel (A) TSS mapping by experimental molecular genetics. The panel shows, on the second sequence line, CiTnI (Atlantic allele) DNA in the region −550 to −491, and beneath it the very similar sequence of the Pacific allele (dots indicate identitity, and note that the ATG-based coordinates are shifted by 2 nt). The topmost sequence line shows the 5′-terminal sequence of a ∼500 bp CiTnI-LacZ mRNA 5′-RACE product generated from embryos expressing construct CiTnI(−819/−383)nZ. Four independent 5′-RACE clones all gave the same sequence, as shown (arbitrary anchor/oligo(dG) sequences introduced during the 5′-RACE procedure have been trimmed). The 5′-end of all four products mapped precisely to CiTnI nucleotide −523 (Atlantic allele, corresponding to −521 Pacific allele). Panels (B and C) CiTnI TSS mapping by high-throughput sequencing of random-primed oligocapping 5′-RACE products from naturally occurring transcripts present in normal tailbud embryo RNA. The panels show the distribution of 5′-reads mapping uniquely to CiTnI (Pacific allele) region −550 to −490 (B) or region −900 to −1 (C). Panel B corresponds spatially to Panel A, with each histogram bar representing a single nucleotide position. The spatial relationships of Panels B and C are indicated by dashed lines. In Panel C each histogram bar represents a 10-nt bin. The genomic position mapped for each read corresponds to the first nucleotide of the read for non-trans-spliced reads (black bars), and the first nt following the 16-nt SL sequence for trans-spliced reads (white bars). Note the logarithmic scale of the read count axes; all positions/bins in the region with no bars shown had zero reads mapped, and the fact that several bins in Panel C contained a single mapped read is shown, arbitrarily, by the shortest black bars visible. All other bars shown represent two or more mapped reads. Exact numbers for peak counts of non-trans-spliced and trans-spliced reads are indicated.

Smaller-scale 3′ deletion analysis of the transcriptionally-active region of CiTnI and TATA element mutation. Conventions as in . Site-directed mutation of the TATA element located at −555 to −546 is symbolized by a diagonally-lined square.

The CiTnI TSS is located between highly conserved sequence blocks. The upper sequence shows the C. intestinalis CiTnI gene (Atlantic allele) in the −556 to −481 regions including the TSS at −523 (arrow). Atlantic and Pacific CiTnI alleles are identical throughout the region, except for the two bases shown in lower case, where the Pacific allele matches the CsTnI sequence. The lower sequence is that of the C. savignyi orthologue CsTnI, with identical nucleotides indicated by dots, and gaps by dashes. Highly-conserved sequence blocks CH3 and CH4, identified by Johnson et al (), are boxed. The lower arrow marks the NNPP second-choice TSS prediction for the CsTnI gene region −821 to −354, i.e., position −515, with the question mark denoting the absence of experimental data to confirm or refute the use of this predicted TSS in the CsTnI gene.

Similar features in the core promoter regions of CiTnI and the H. roretzi actin genes HrMA4a and HrMA1a. Sequences (from GenBank AF237978.2, S76735.1 and D29014.1) are aligned on the common CATC sequence that includes the experimentally-identified TSSs (bolded nucleotide) (,). TATA elements and TSS CATC motifs are shown in upper case.