Thursday, December 22, 2011

Large-insert cloning aids study of dinoflagellate species

Large-insert cloning products have long been a mainstay of Epicentre's product line and are critical for the study of gene expression and interactions. Recently, Jaeckisch et al. described making three kinds of libraries (cDNA, fosmid, and BAC) for characterizing the marine dinoflagellate Alexandrium ostenfeldii.

Many dinoflagellate species are notorious for the toxins they produce, as well as ecological and human health consequences associated with harmful algal blooms (HABs). One way to study these otherwise toxic compounds is to build their synthesis genes into a large-insert-capable, low-copy cloning vector, transform into a suitable host cell, and then perform the desired studies in such a way that the genes will not have a negative affect on the host. The genes for the toxins cited in the publication (macrocyclic imine toxins, described as spirolides), were inserted into the pCC1FOS vector and transfected into the TransforMAX™ EPI300 host strain for further study. Further, genomic DNA from A. ostefendii was prepared for cloning into the pIndigoBAC-5 HindIII Cloning-Ready vector and transformation into the TransforMAX EC100 host.

A total of 384 BAC clones were obtained with insert sizes ranging from 50 to 150 kb, which provided sufficient coverage to allow elucidation of the whole genome sequence and some comparative sequence data with the marine dinoflagellate H. triquetra. The authors used the sequence information obtained from the BAC and CopyControl fosmid libraries to investigate spliced leader (SL) trans-splicing and mRNA transposition mechanisms. They characterized the genome using selected clones, using a combination of transcriptomic data and random genomic clones. Examination of SL sequences revealed similar features as in other dinoflagellates, including other Alexandrium species. SL sequences in decay indicate frequent retro-transposition of mRNA species. The transposition observed in these genes probably contributes to overall genome complexity by generating additional gene copies.
The authors conclude:
The genomic sequence survey (GSS) represents the largest genomic data set of a dinoflagellate to date...The large portion of repetitive sequences and the organization within the genome is in agreement with several other studies on dinoflagellates using different approaches. It remains to be determined whether this unusual composition is directly correlated to the exceptionally [sic] genome organization of dinoflagellates with a low amount of histones and histone-like proteins.
ResearchBlogging.orgJaeckisch, N. et al. (2011). Comparative Genomic and Transcriptomic Characterization of the Toxigenic Marine Dinoflagellate Alexandrium ostenfeldii PLoS ONE, 6 (12) DOI: 10.1371/journal.pone.0028012

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