scholarly journals A recombineering pipeline to clone large and complex genes in Chlamydomonas

2020 ◽  
Author(s):  
Tom Emrich-Mills ◽  
Gary Yates ◽  
James Barrett ◽  
Irina Grouneva ◽  
Chun Sing Lau ◽  
...  
Keyword(s):  
Success Rate ◽  
Fluorescent Protein ◽  
Gc Content ◽  
Research Community ◽  
Mass Spectrometry Data ◽  
Mutant Library ◽  
Parallel Cloning ◽  
Mutant Complementation ◽  
Protein Tagging ◽  
Genomic Gc Content

AbstractThe ability to clone genes has driven fundamental advances in cell and molecular biology, enabling researchers to introduce precise mutations, generate fluorescent protein fusions for localization and to confirm genetic causation by mutant complementation. Most gene cloning is PCR or DNA synthesis dependent, which can become costly and technically challenging as genes increase in size and particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, with a high percentage of genes containing complex sequence structures, an average genomic GC content of 64% and gene expression requiring regular introns for stable transcription. Here we overcome these challenges via the development of a recombineering pipeline that enables the rapid parallel cloning of genes from a Chlamydomonas BAC collection. We show the method can successfully retrieve large and complex genes that PCR-based methods have previously failed to clone, including genes as large as 23 kilobases, thus making previously technically challenging genes to study now amenable to cloning. We initially applied the pipeline to 12 targets with a 92% cloning success rate. We then developed a high-throughput approach and targeted 191 genes relating to the Chlamydomonas CO2 concentrating mechanism (CCM) with an overall cloning success rate of 77% that is independent of gene size. Localization of a subset of CCM targets has confirmed previous mass spectrometry data and identified new pyrenoid components. To expand the functionality of our system, we developed a series of localization vectors that enable complementation of Chlamydomonas Library Project mutants and enable protein tagging with a range of fluorophores. Vectors and detailed protocols are available to facilitate the easy adoption of this method by the Chlamydomonas research community. We envision that this technology will open up new possibilities in algal and plant research and be complementary to the Chlamydomonas mutant library.

scholarly journals A recombineering pipeline to clone large and complex genes in Chlamydomonas

2021 ◽  
Author(s):  
Tom Z Emrich-Mills ◽  
Gary Yates ◽  
James Barrett ◽  
Philipp Girr ◽  
Irina Grouneva ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bacterial Artificial Chromosome ◽  
Molecular Biology ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Research Community ◽  
Mass Spectrometry Data ◽  
Parallel Cloning ◽  
Mutant Complementation ◽  
Biology Research

Abstract The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is PCR or DNA synthesis dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kilobases. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.

scholarly journals An orange fluorescent protein tagging system for real-time pollen tracking

2013 ◽  
Vol 6 (1) ◽  
Author(s):  
J Hollis Rice ◽  
Reginald J Millwood ◽  
Richard E Mundell ◽  
Orlando D Chambers ◽  
Laura L Abercrombie ◽  
...  
Keyword(s):  
Real Time ◽  
Fluorescent Protein ◽  
Orange Fluorescent Protein ◽  
Protein Tagging ◽  
Tagging System

scholarly journals Coupling Between Protein Level Selection and Codon Usage Optimization in the Evolution of Bacteria and Archaea

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Wenqi Ran ◽  
David M. Kristensen ◽  
Eugene V. Koonin
Keyword(s):  
Codon Usage ◽  
Protein Level ◽  
Codon Usage Bias ◽  
Protein Sequence ◽  
Gc Content ◽  
Protein Sequences ◽  
Microbial Evolution ◽  
Fine Tuning ◽  
Selection For ◽  
Genomic Gc Content

ABSTRACT The relationship between the selection affecting codon usage and selection on protein sequences of orthologous genes in diverse groups of bacteria and archaea was examined by using the Alignable Tight Genome Clusters database of prokaryote genomes. The codon usage bias is generally low, with 57.5% of the gene-specific optimal codon frequencies (F opt ) being below 0.55. This apparent weak selection on codon usage contrasts with the strong purifying selection on amino acid sequences, with 65.8% of the gene-specific dN/dS ratios being below 0.1. For most of the genomes compared, a limited but statistically significant negative correlation between F opt and dN/dS was observed, which is indicative of a link between selection on protein sequence and selection on codon usage. The strength of the coupling between the protein level selection and codon usage bias showed a strong positive correlation with the genomic GC content. Combined with previous observations on the selection for GC-rich codons in bacteria and archaea with GC-rich genomes, these findings suggest that selection for translational fine-tuning could be an important factor in microbial evolution that drives the evolution of genome GC content away from mutational equilibrium. This type of selection is particularly pronounced in slowly evolving, “high-status” genes. A significantly stronger link between the two aspects of selection is observed in free-living bacteria than in parasitic bacteria and in genes encoding metabolic enzymes and transporters than in informational genes. These differences might reflect the special importance of translational fine-tuning for the adaptability of gene expression to environmental changes. The results of this work establish the coupling between protein level selection and selection for translational optimization as a distinct and potentially important factor in microbial evolution. IMPORTANCE Selection affects the evolution of microbial genomes at many levels, including both the structure of proteins and the regulation of their production. Here we demonstrate the coupling between the selection on protein sequences and the optimization of codon usage in a broad range of bacteria and archaea. The strength of this coupling varies over a wide range and strongly and positively correlates with the genomic GC content. The cause(s) of the evolution of high GC content is a long-standing open question, given the universal mutational bias toward AT. We propose that optimization of codon usage could be one of the key factors that determine the evolution of GC-rich genomes. This work establishes the coupling between selection at the level of protein sequence and at the level of codon choice optimization as a distinct aspect of genome evolution.

scholarly journals Selection Maintains Low Genomic GC Content in Marine SAR11 Lineages

2015 ◽  
Vol 32 (10) ◽  
pp. 2738-2748 ◽  
Author(s):  
Haiwei Luo ◽  
Luke R. Thompson ◽  
Ulrich Stingl ◽  
Austin L. Hughes
Keyword(s):  
Gc Content ◽  
Genomic Gc Content

Fluorescent Protein Tagging of Arabidopsis MAPKs for In Vivo Localization Studies

2014 ◽  
pp. 131-145
Author(s):  
Anna Doskočilová ◽  
Ivan Luptovčiak ◽  
Veronika Smékalová ◽  
Jozef Šamaj
Keyword(s):  
Fluorescent Protein ◽  
Protein Tagging

scholarly journals Molecular Characterization and Antimicrobial Susceptibilities of Nocardia Species Isolated from the Soil; A Comparison with Species Isolated from Humans

2020 ◽  
Vol 8 (6) ◽  
pp. 900
Author(s):  
Gema Carrasco ◽  
Sara Monzón ◽  
María San Segundo ◽  
Enrique García ◽  
Noelia Garrido ◽  
...  
Keyword(s):  
16S Rrna ◽  
Topoisomerase Ii ◽  
Gc Content ◽  
Amino Acid Identity ◽  
Beta Lactams ◽  
Nocardia Species ◽  
Antimicrobial Susceptibilities ◽  
The Difference ◽  
Subunit B ◽  
Genomic Gc Content

Nocardia species, one of the most predominant Actinobacteria of the soil microbiota, cause infection in humans following traumatic inoculation or inhalation. The identification, typing, phylogenetic relationship and antimicrobial susceptibilities of 38 soil Nocardia strains from Lara State, Venezuela, were studied by 16S rRNA and gyrB (subunit B of topoisomerase II) genes, multilocus sequence analysis (MLSA), whole-genome sequencing (WGS), and microdilution. The results were compared with those for human strains. Just seven Nocardia species with one or two strains each, except for Nocardia cyriacigeorgica with 29, were identified. MLSA confirmed the species assignments made by 16S rRNA and gyrB analyses (89.5% and 71.0% respectively), and grouped each soil strain with its corresponding reference and clinical strains, except for 19 N. cyriacigeorgica strains found at five locations which grouped into a soil-only cluster. The soil strains of N. cyriacigeorgica showed fewer gyrB haplotypes than the examined human strains (13 vs. 17) but did show a larger number of gyrB SNPs (212 vs. 77). Their susceptibilities to antimicrobials were similar except for beta-lactams, fluoroquinolones, minocycline, and clarithromycin, with the soil strains more susceptible to the first three (p ≤ 0.05). WGS was performed on four strains belonging to the soil-only cluster and on two outside it, and the results compared with public N. cyriacigeorgica genomes. The average nucleotide/amino acid identity, in silico genome-to-genome hybridization similarity, and the difference in the genomic GC content, suggest that some strains of the soil-only cluster may belong to a novel subspecies or even a new species (proposed name Nocardia venezuelensis).

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