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  • Published: Aug 15, 2010
  • Author: Steve Down
  • Channels: Proteomics
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The full genomes of an increasing number of species are being decoded, many from so-called model organisms such as the rat, mouse and fruit fly, but the vast majority remain unsequenced. The absence of genetic information strongly hinders insight into the biological behaviour of an organism and makes it difficult to apply modern molecular and proteomics techniques.

As author Matt Ridley declared in the description of his book The Autobiography of a Species in 23 Chapters, the genome is "the collective recipe for the building and running of the human body." Without it, the internal workings of the numerous non-model species are a lot more complicated to work out.

The classical way to generate the genome is whole genome sequencing. This is the most accurate procedure but is time consuming and relatively expensive. In its place, many research groups use transcriptomics to derive the expressed sequence tags (ESTs) from mitochondrial RNA. This is a more rapid, less expensive procedure which has been applied to model and non-model species.

The combined use of transcriptomics and proteomics has been exploited by scientists in China and Saudi Arabia in the first such study of a marine fouling invertebrate. Pei-Yuan Qian and co-researchers from the Hong Kong University of Science and Technology and the King Abdullah University of Science and Technology, Thuwal, examined Bugula neritina. Also known as brown bryozoan, one of the group of "moss animals," it is an erect, bushy species that colonises any underwater surface, including artificial underwater structures and vessel hulls.

The researchers reasoned that knowledge of the molecular mechanism of larva development, gained from genomic and proteomic data, might lead to the discovery of non-toxic anti-fouling agents to keep underwater surfaces clear of infestation.

The researchers chose the relatively new 454 pyrosequencing technology instead of the Sanger sequencing method, which could take several days to sequence 400-600 million base pairs. The 454 pyrosequencing is far more rapid, taking about 10 hours for the same number, with acceptably high accuracies of 99.9 or 99% for 200 and 400 base sequences, respectively.

In a two-step approach, DNA is cut into blunt ends and oligonucleotide adaptors are attached to both ends of the cut molecules. Each fragment is attached to a bead, which is then amplified via PCR to generate multiple copies of the same DNA sequence on each bead. The beads are then removed for a five-step sequencing procedure.

Adult colonies of B. neritina were collected from the rafts of a fish farm and maintained until larvae were spawned. Total RNA was extracted from four developmental stages for sequencing.

Sequencing generated 48 million base pairs in total, leading to 131,450 confident reads with an average of 347 bases. From these, 93% were assembled into 6392 unique contigs with an average length of 538 bases, which is comparable to the cDNA libraries acquired by Sanger sequencing. The data led to the prediction of 13,863 open reading frames and 6917 of these were successfully annotated.

At the same time, the proteins were also extracted and digested with trypsin. The resultant peptides were separated into 20 fractions by strong cation exchange chromatography before analysis by LC-tandem MS with electrospray ionisation.

A total of 882 proteins were identified by searching against the newly created transcriptome database. The most abundant protein was vitellogenin, which accounted for 10% of the proteins in each cell. Its discovery in this species was novel and the team suggested it served as an energy source during larval development.

Two more abundant proteins, tubulin and actin, are cytoskeletal proteins, required for maintaining larval morphology. Two other abundant proteins were hypothetical proteins attributed to the extracellular matrix, probably involved in larval attachment, although their exact functions are unknown.

From the combined data, several signalling pathways for the organism were identified and some genes which might play a role in larval settlement and metamorphosis were sidelined for further study. This was only possible due to the deep transcriptome coverage which was achieved by the sequencing method.

The combination of transcriptomics and proteomics gave an insight into the molecular biology of B. neritina. The techniques can equally be applied to other non-model organisms for which the genome has not yet been sequenced to produce large transcriptomics and proteomics data sets in a relatively short period.



The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

 

 

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