DOI: 10.5061/dryad.rv15dv4gm
title: Functional characterization of luciferase in a brittle star indicates parallel evolution influenced by genomic availability of haloalkane dehalogenase

description: Determining why convergent traits use distinct versus shared genetic
 components is crucial for understanding how evolutionary processes
 generate and sustain biodiversity. However, the factors dictating the
 genetic underpinnings of convergent traits remain incompletely understood.
 Here, we use heterologous protein expression, biochemical assays, and
 phylogenetic analyses to confirm the origin of a luciferase gene from
 haloalkane dehalogenases in the brittle star Amphiura filiformis. Through
 database searches and gene tree analyses, we also show a complex pattern
 of presence and absence of haloalkane dehalogenases across organismal
 genomes. These results first confirm parallel evolution across a vast
 phylogenetic distance, because octocorals like Renilla also use luciferase
 derived from haloalkane dehalogenases. This parallel evolution is
 surprising, even though previously hypothesized, because many organisms
 that also use coelenterazine as the bioluminescence substrate evolved
 completely distinct luciferases. The inability to detect haloalkane
 dehalogenases in the genomes of several bioluminescent groups suggests
 that the distribution of this gene family influences its recruitment as a
 luciferase. Together, our findings highlight how biochemical function and
 genomic availability help determine whether distinct or shared genetic
 components are used during the convergent evolution of traits like
 bioluminescence.
description: # Functional characterization of luciferase in a brittle star indicates
 parallel evolution influenced by genomic availability of haloalkane
 dehalogenase --- ## Directory structure and file list ```
 Brittlestar_data_code.Rmd Brittlestar_data_code.html Rmarkdown_csv_files/
 dehalogenase_data/ dehalogenase_activity_kinetics.csv
 dehalogenases_37308.csv temperature_37308.csv emission_data/
 313061_brittlestar_1_rawcal.csv 313061_brittlestar_2_rawcal.csv
 313061_brittlestar_3_rawcal.csv renilla_1_rawcal.csv renilla_2_rawcal.csv
 renilla_3_rawcal.csv gene_expression_data/ gene_expression_development.csv
 luciferase_data/ further_luciferase_testing.csv
 Marika_crude_extracts_testing.csv Oakley_crude_extracts_testing.csv
 Bioinformatic_analyses/ Fig4_data/
 queries_blastp_seq_domains_remove_seq_aligned.fa
 queries_blastp_seq_domains_remove_seq_aligned.fa.log
 queries_blastp_seq_domains_remove_seq_aligned.fa.treefile
 queries_blastp_seq_domains_remove_seq.fa Plasmid_sequences/
 Marek_pET21b_10707-1.gb Marek_pET21b_17859-1.gb Marek_pET21b_37282-1.gb
 Marek_pET21b_37332-1.gb Marek_pET21b_DhaA.gb oakley_pet21_37308-1.gb
 oakley_pet21_203026.gb oakley_pet21_224433.gb oakley_pet21_AfLuc.gb
 oakley_pet21_pyroluc.gb oakley_pet21_renilla_luc.gb ``` --- ## File
 descriptions ``` Brittle_Star_Dryad_Data/Brittlestar_data_code.Rmd: R
 markdown file containing code used for data analysis and generating
 figures. File can be opened using R. ``` ```
 Brittle_Star_Dryad_Data/Brittlestar_data_code.html: html file containing
 code and output of the R markdown file ``` ```
 Brittle_Star_Dryad_Data/Rmarkdown_csv_files/: Contains data files for the
 functional assays. All files can be opened using programs such as text
 editor software or Excel. dehalogenase_data/: Contains data files for the
 dehalogenase functional assays dehalogenase_activity_kinetics.csv: Dataset
 for the dehalogenase kinetics assay. "Sample" column refers to
 the protein tested, "Minute" column refers to the minute at
 which the sample was measured, and "RFU" column indicates the
 Relative Fluorescence Unit measured. dehalogenases_37308.csv: Dataset for
 testing DafA activity towards different substrates. "Substrate"
 column refers to the substrate tested, "Signal" and
 "Signal_SD" refers to the average and standard deviation of the
 fluorescence measurement in Relative Fluorescence Unit, and
 "Rate" and "Rate_SD" refers to the average and
 standard deviation of the calculated rate of reaction. Rate is in units of
 nmol s-1 mg-1. temperature_37308.csv: Dataset for testing DafA activity at
 different temperatures with the substrate 1,2-dibromoethane.
 "Temperature" column refers to the temperature tested in
 Celsius, "Signal" and "Signal_SD" refers to the
 average and standard deviation of the fluorescence measurement in Relative
 Fluorescence Unit, and "Rate" and "Rate_SD" refers to
 the average and standard deviation of the calculated rate of reaction.
 Rate is in units of nmol s-1 mg-1. emission_data/: Contains data files for
 the emission spectrum. For each file, "wavelength" column refers
 to the specific wavelength being measured,
 "emission_minus_background" refers to the luminescence emission
 measurement minus the background, and "calibrated_emission"
 refers to the calibrated luminescence emission measurement.
 313061_brittlestar_1_rawcal.csv: Replicate 1 of AfLuc emission spectrum
 313061_brittlestar_2_rawcal.csv: Replicate 2 of AfLuc emission spectrum
 313061_brittlestar_3_rawcal.csv: Replicate 3 of AfLuc emission spectrum
 renilla_1_rawcal.csv: Replicate 1 of RLuc emission spectrum
 renilla_2_rawcal.csv: Replicate 2 of RLuc emission spectrum
 renilla_3_rawcal.csv: Replicate 3 of RLuc emission spectrum
 luciferase_data/: Contains data files for the luciferase functional assays
 further_luciferase_testing.csv: Dataset for testing luciferase activity of
 all samples. "Concentration" column refers to the final
 concentration of luciferin used in uM, "Cycle" and
 "Time" column indicates the cycle and time the sample was
 measured in seconds, respectively, and headers of other columns are named
 for the sample in which luminescence was measured (Buffer, Bovine Serum
 Albumin (BSA), Pyrosome Luciferase (PyroLuc), AF37308 protein (B37308),
 AfLuc protein (B313061), Renilla luciferase (Rluc)). Luminescence is in
 Relative Luminescence Units. Marika_crude_extracts_testing.csv: Dataset
 for testing crude extracts for DafA, A10707.1, A17859.1, A37282.1,
 A37332.1. "Sample" column contains the name of each sample being
 measured (negative control is DhaA, a haloalkane dehalogenase from
 bacteria, positive control is Rluc, Renilla luciferase, and experimental
 samples are candidates AF10707, AF17859, AF37282, AF37308, and AF37332).
 The "Luminescence" column contains the measured luminescence
 from each sample. Luminescence is in Relative Luminescence Units.
 Oakley_crude_extracts_testing.csv: Dataset for testing crude extracts for
 AfLuc, Uni203026, Gen224433. "Well" column refers to the
 location of the sample in a 96 well sample plate, "Sample"
 column contains the name of each sample being measured (negative control
 is crude extract from bacteria transformed with an empty plasmid and
 experimental samples are candidates 224433, AfLuc (313061), and 20302.6).
 The "Luminescence" column contains the measured luminescence
 from each sample. Luminescence is in Relative Luminescence Units.
 gene_expression_data/: Contains data files for HLD/LUC gene expression in
 A. filiformis gene_expression_development.csv: Dataset for HLD/LUC gene
 expression in A. filiformis from Parey et al. 2024. "HPF" refers
 to Hours Post Fertilization. Numerical values are in log2(Transcripts Per
 Million + 1). ``` ``` Brittle_Star_Dryad_Data/Bioinformatic_analyses/:
 Contains data files for phylogenetic analysis. Files can be opened with
 text editor software, and the .treefile can be visualized using FigTree.
 Fig4_data/: Contains .fa files of unaligned and aligned hydrolase domains
 from dehalogenases, newick file of tree, and IQTREE log file
 queries_blastp_seq_domains_remove_seq_aligned.fa: Multiple sequence
 alignment of hydrolase domains from dehalogenases
 queries_blastp_seq_domains_remove_seq_aligned.fa.log: IQTREE log file
 queries_blastp_seq_domains_remove_seq_aligned.fa.treefile: Newick file of
 the maximum likelihood tree queries_blastp_seq_domains_remove_seq.fa:
 Sequences of hydrolase domains from dehalogenases ``` ```
 Brittle_Star_Dryad_Data/Plasmid_sequences/: Contains plasmid sequence .gb
 files. Files can be opened with plasmid editor tools, such as ApE.
 Marek_pET21b_10707-1.gb: Plasmid sequence for AF10707.1 with 6His tag
 Marek_pET21b_17859-1.gb: Plasmid sequence for AF17859.1 with 6His tag
 Marek_pET21b_37282-1.gb: Plasmid sequence for AF37282.1 with 6His tag
 Marek_pET21b_37332-1.gb: Plasmid sequence for AF37332.1 with 6His tag
 Marek_pET21b_DhaA.gb: Plasmid sequence for DhaA with 6His tag
 oakley_pet21_37308-1.gb: Plasmid sequence for DafA with 6His tag
 oakley_pet21_203026.gb Plasmid sequence for Uni203026 with 6His tag
 oakley_pet21_224433.gb: Plasmid sequence for Gen224433 with 6His tag
 oakley_pet21_AfLuc.gb: Plasmid sequence for AfLuc with 6His tag
 oakley_pet21_pyroluc.gb: Plasmid sequence for PyroLuc with 6His tag
 oakley_pet21_renilla_luc.gb: Plasmid sequence for RLuc with 6His tag ```
 --- NOTE: Gene sequences of proteins expressed in this paper are available
 on GenBank (Accessions: PP777633 (AfLuc), PP777634 (DafA), PP777635
 (AF10707.1), PP777636 (AF17859.1), PP777637 (AF37282.1), PP777638
 (AF37332.1), PP777639 (Gen224433), PP777640 (Uni203026), PP777641
 (PyroLuc))