Figure 1.
RT-PCR validation of transcriptome data.
A) AAEL008851, an example of an EZG gene where both RT-PCR and transcriptome sequencing show expression at the 2–3 hr time range. B) AAEL001543, a zygotic gene with a similar profile as AAEL008851 but which has less expression in the 2–4 hr time range (note very faint band from RT-PCR at 2–3 hr was present that may not be discernable in image). C) AAEL008722, a zygotic gene having expression detected later at 4–5 hr by PCR. Normalized expression values of each transcript from the transcriptome data are at the bottom of each figure. Note that AAEL008722 is not included in our “EZG set” as it is not present until the 4–8 hr time range in the transcriptome data.
Figure 2.
Ae. aegypti EZGs have more intronless genes.
The percentage of intronless genes was compared between EZGs, 4–8 hr, 8–12 hr and all Ae. aegypti genes. P-values calculated by the Chi-squared test are shown for two comparisons. See File S2 for numbers of genes with and without introns for each group. See File S3 for transcript IDs for the 2–4, 4–8, and 8–12 hr groups.
Figure 3.
Discovery of a motif involved in early zygotic gene transcription.
Analysis of 400 bp upstream sequence from 61 EZGs by SCOPE. A) Snapshot of SCOPE output showing the top 10 motifs identified. The top 5 are color-labeled and correspond to the motif locations in Figure 3C. Note that the highest-ranking motif TACCYVB is the complement of VBRGGTA. “Count” is the total motif occurrences on both strands, “Sig Value” is the negative log of the E value, “Coverage” is the % of sequences in the dataset that contain the motif, and “Algorithm” shows which of the three algorithms identified the motif. B) Sequence logos and PWM for VBRGGTA. C) Schematic from SCOPE output showing 400 bp upstream sequence and locations of 5 highest scoring motifs in Fig. 3A. Red blocks correspond to VBRGGTA. The gene IDs are shown to the right of each sequence with the prefix “AAEL0” omitted.
Table 1.
Comparison of the Ae. aegypti EZG motif with Drosophila TAGteam motifs using STAMP.
Figure 4.
A 38 bp region of the KLC2.1 promoter containing two VBRGGTA motifs is necessary and sufficient to direct early zygotic transcription.
A) Duplication of the 38 bp fragment that contains the motifs enhances early zygotic transcriptional activity. Schematic shows constructs and upstream sequences tested with positions given relative to the TSS (+1). The 38 bp fragment containing 2 VBRGGTA (4 GGTA/TACC motifs shaded in gray) is underlined and labeled “m”. Primer locations used for PCR are depicted by arrows and labeled lines show sequence regions used for luciferase assay. Construct F10R3 doesn't contain the 38 bp fragment. F8R3 contains the 38 bp fragment. F8R3+m has a duplication of the 38 bp fragment. F1R3 is a positive control that contains the full 1 kb upstream/promoter sequence and is used hereafter as a positive control. B) Simultaneous mutations M1 and M4 (F1R3+M) in the 38 bp fragment obliterates activity. C) Addition of the 38 bp fragment confers early zygotic activity to a heterologous AgCecA promoter from a divergent mosquito species. CecA refers to the construct with the unmodified AgCecA promoter. CecA+m refers to the CecA construct with the addition of the 38 bp fragment. CecA+M refers to the CecA construct that contains the 38 bp fragment with mutations in all 4 positions indicated as in Figure 4B. A one-tailed T-test revealed significant differences between both CecA+m/CecA+M (*1, P = 0.019) and CecA+m/CecA (*2, P = 0.018). D) Mutational analysis of each individual motif in the 38 bp fragment. Each mutant is labeled M1–M4 and mutated bases are indicated in red. In all panels the positions of the VBRGGTA motif are indicated, and the ratio of firefly luciferase/renilla luciferase represents the mean of triplicate injections +/− the standard error.