Experiment / E93MU2SQZOther

Natural enhancer-allele activity in dual-luciferase reporters

Influence of genetic polymorphism on transcriptional enhancer activity in the malaria vector Anopheles coluzzii

Naturally segregating enhancer haplotypes amplified from Ngousso and Fd colonies were cloned into pGL-Gateway-DSCP and assayed for allele-dependent enhancer activity in A. coluzzii 4a3A cells. The panel covers six validated enhancer loci and includes the sequence of each tested allele from Supplementary File S1.

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Organism: NCBITaxon:1518534
Taxonomy ID: NCBITaxon:1518534
Biosample: CVCL:Z622
Reference genome: AgamP4
Design focus: Variant-focused
Region of interest: 3R:22609939-22611138,X:1888505-1890055,2L:41302647-41303886,2L:30333431-30334787,2R:3485436-3486583,2L:25382828-25384253

Perturbation & assay details

Basal / Untreated; 24 h after reporter transfection

At least two genetically distinct alleles per enhancer were selected from colonies initiated in Cameroon, Mali, or Burkina Faso. Six-fold replication across at least two independent plates yielded n = 12 per allele; Kruskal–Wallis with post-hoc pairwise comparisons supplied the compact-letter groups.

Episomal dual-luciferase natural-allele assay: pGL-Gateway-DSCP firefly reporter plus pRL-ubi-63E Renilla internal control; normalized RLU readout, not barcode-count MPRA.

Processed data

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Column dictionary · 21 definitions

element_id: Enhancer locus tested.
proximal_gene: Nearest annotated gene used to name the locus.
gene_id: Gene identifier from the paper.
chromosome: Chromosome arm in PEST AgamP4 coordinates.
coordinate_start: 1-based inclusive candidate interval start.
coordinate_end: 1-based inclusive candidate interval end.
interval_length_bp: Inclusive candidate interval length.
allele_label: Allele name shown on the Figure 4 x-axis.
source_colony: A. coluzzii laboratory colony from which the cloned haplotype was obtained.
colony_origin: Colony initiation population/year reported in Methods.
source_fasta_file: Supplementary File S1 FASTA filename retained inside raw_data/supplementary_dataset_1.zip.
source_fasta_record: Exact FASTA record used for the sequence column.
sequence_5to3: Ungapped nucleotide sequence reconstructed from the aligned S1 FASTA record, written 5′ to 3′.
sequence_length_bp: Length of the ungapped sequence supplied in the table.
sequence_md5: MD5 checksum of the ungapped sequence string, for row-level sequence identity checks.
relative_luciferase_activity_median_approx: Approximate Figure 4 median of Renilla-corrected firefly activity normalized to DLX; digitized from the plotted median bar.
n_measurements: Figure 4 reports n = 12 wells for each allele condition.
statistical_group: Compact-letter group printed above the Figure 4 distribution; shared letters indicate no reported post-hoc difference.
background_relation: Specific background interpretation stated in the paper for the low ACE1 and LRIM1 alleles; blank means not explicitly assigned in the paper.
source_figure: Publication figure supporting the activity value.
qc_pass: All source-tested allele constructs retained; low activity was not used as an exclusion because low/null function is the biological result of interest.

Quality control

Applied the paper’s source QC: cloned inserts were resequenced, Renilla correction and DLX normalization were used, and each displayed condition had n = 12. Low/null alleles were retained as valid biological observations, not filtered as technical failures.

Curation notes

Activity values are plot-derived approximations from Figure 4 because no numeric luciferase source table was deposited. S1 aligned FASTAs contain gap characters; sequence_5to3 removes alignment gaps and retains the actual nongap sequence. The parent raw_data zip preserves every original S1 record.