Fine Expression Profiling of Full-length Transcripts using a Size-unbiased cDNA Library Prepared with the Vector-capping Method

Recently, we have developed a vector-capping method for constructing a full-length cDNA library. In the present study, we performed in-depth analysis of the vector-capped cDNA library prepared from a single type of cell. As a result of single-pass sequencing analysis of 24 000 clones randomly isolated from the unamplified library, we identified 19 951 full-length cDNA clones whose intactness was confirmed by the presence of an additional G at their 5' end. The full-length cDNA content was >95%. Mapping these sequences to the human genome, we identified 4513 transcriptional units that include 36 antisense transcripts against known genes. Comparison of the frequencies of abundant clones showed that the expression profiles of different libraries, including the distribution of transcriptional start sites (TSSs), were reproducible. The analysis of long-sized cDNAs showed that this library contained many cDNAs with a long-sized insert up to 11 199 bp of golgin B, including multiple slicing variants for filamin A and filamin B. These results suggest that the size-unbiased full-length cDNA library constructed using the vector-capping method will be an ideal resource for fine expression profiling of transcriptional variants with alternative TSSs and alternative splicing

Diversity of Beetle Genes Encoding Novel Plant Cell Wall Degrading Enzymes

Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterostomes was inferred to be able to digest, or even synthesize, cellulose using endogenous genes, all model insects whose complete genomes have been sequenced lack genes encoding such enzymes. To establish if the apparent “disappearance” of PCWDEs from insects is simply a sampling problem, we used 454 mediated pyrosequencing to scan the gut transcriptomes of beetles that feed on a variety of plant derived diets. By sequencing the transcriptome of five beetles, and surveying publicly available ESTs, we describe 167 new beetle PCWDEs belonging to eight different enzyme families. This survey proves that these enzymes are not only present in non-model insects but that the multigene families that encode them are apparently undergoing complex birth-death dynamics. This reinforces the observation that insects themselves, and not just their microbial symbionts, are a rich source of PCWDEs. Further it emphasises that the apparent absence of genes encoding PCWDEs from model organisms is indeed simply a sampling artefact. Given the huge diversity of beetles alive today, and the diversity of their lifestyles and diets, we predict that beetle guts will emerge as an important new source of enzymes for use in biotechnology