Transcriptome annotation using tandem SAGE tags

Analysis of several million expressed gene signatures (tags) revealed an increasing number of different sequences, largely exceeding that of annotated genes in mammalian genomes. Serial analysis of gene expression (SAGE) can reveal new Poly(A) RNAs transcribed from previously unrecognized chromosomal regions. However, conventional SAGE tags are too short to identify unambiguously unique sites in large genomes. Here, we design a novel strategy with tags anchored on two different restrictions sites of cDNAs. New transcripts are then tentatively defined by the two SAGE tags in tandem and by the spanning sequence read on the genome between these tagged sites. Having developed a new algorithm to locate these tag-delimited genomic sequences (TDGS), we first validated its capacity to recognize known genes and its ability to reveal new transcripts with two SAGE libraries built in parallel from a single RNA sample. Our algorithm proves fast enough to experiment this strategy at a large scale. We then collected and processed the complete sets of human SAGE tags to predict yet unknown transcripts. A cross-validation with tiling arrays data shows that 47% of these TDGS overlap transcriptional active regions. Our method provides a new and complementary approach for complex transcriptome annotation

Of mice and men: divergence of gene expression patterns in kidney.

Since the development of methods for homologous gene recombination, mouse models have played a central role in research in renal pathophysiology. However, many published and unpublished results show that mice with genetic changes mimicking human pathogenic mutations do not display the human phenotype. These functional differences may stem from differences in gene expression between mouse and human kidneys. However, large scale comparison of gene expression networks revealed conservation of gene expression among a large panel of human and mouse tissues including kidneys. Because renal functions result from the spatial integration of elementary processes originating in the glomerulus and the successive segments constituting the nephron, we hypothesized that differences in gene expression profiles along the human and mouse nephron might account for different behaviors. Analysis of SAGE libraries generated from the glomerulus and seven anatomically defined nephron segments from human and mouse kidneys allowed us to identify 4644 pairs of gene orthologs expressed in either one or both species. Quantitative analysis shows that many transcripts are present at different levels in the two species. It also shows poor conservation of gene expression profiles, with less than 10% of the 4644 gene orthologs displaying a higher conservation of expression profiles than the neutral expectation (p<0.05). Accordingly, hierarchical clustering reveals a higher degree of conservation of gene expression patterns between functionally unrelated kidney structures within a given species than between cognate structures from the two species. Similar findings were obtained for sub-groups of genes with either kidney-specific or housekeeping functions. Conservation of gene expression at the scale of the whole organ and divergence at the level of its constituting sub-structures likely account for the fact that although kidneys assume the same global function in the two species, many mouse "models" of human pathologies do not display the expected phenotype

Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system

Background Biofloc technology (BFT), a rearing method with little or no water exchange, is gaining popularity in aquaculture. In the water column, such systems develop conglomerates of microbes, algae and protozoa, together with detritus and dead organic particles. The intensive microbial community presents in these systems can be used as a pond water quality treatment system, and the microbial protein can serve as a feed additive. The current problem with BFT is the difficulty of controlling its bacterial community composition for both optimal water quality and optimal shrimp health. The main objective of the present study was to investigate microbial diversity of samples obtained from different culture environments (Biofloc technology and clear seawater) as well as from the intestines of shrimp reared in both environments through high-throughput sequencing technology. Results Analyses of the bacterial community identified in water from BFT and “clear seawater” (CW) systems (control) containing the shrimp Litopenaeus stylirostris revealed large differences in the frequency distribution of operational taxonomic units (OTUs). Four out of the five most dominant bacterial communities were different in both culture methods. Bacteria found in great abundance in BFT have two principal characteristics: the need for an organic substrate or nitrogen sources to grow and the capacity to attach to surfaces and co-aggregate. A correlation was found between bacteria groups and physicochemical and biological parameters measured in rearing tanks. Moreover, rearing-water bacterial communities influenced the microbiota of shrimp. Indeed, the biofloc environment modified the shrimp intestine microbiota, as the low level (27 %) of similarity between intestinal bacterial communities from the two treatments. Conclusion This study provides the first information describing the complex biofloc microbial community, which can help to understand the environment-microbiota-host relationship in this rearing system

Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach

Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~ 400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~ 7 kDa) and two proteins in the 25 and 50. kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery

Scatter-plot of tag distribution in OMCD of mouse and human.

<p>This diagram plots the abundance of gene orthologous-specific tags in the OMCD of the two species. Data correspond to 66,374 and 70,524 tags in human and mouse kidneys respectively. The size of the spots corresponds to the number of different transcripts and their colour to the p value, as indicated in the inset. In this logarithmic scale, null abundances were plotted at a value of one.</p

Comparison of structural and functional features in human and mouse kidneys.

<p>Data for body and kidney weights (BW & KW), glomerular filtration rate (GFR), number of nephrons (for both kidneys), urine volume (or water excretion) and sodium intake are commonly accepted values for humans. For mice, KW and GFR are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046876#pone.0046876-Cheval2" target="_blank">[10]</a>; the GFR is calculated by multiplying the single nephron GFR of 10 nl/min <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046876#pone.0046876-Schnermann1" target="_blank">[27]</a> by the number of nephrons; water and sodium intake are unpublished data from our laboratory.</p

Comparison of gene expression profiles in human and mouse nephron.

<p><b>A.</b> The columns show the histogram of distribution of the Euclidean distances d of the 4644 pairs of orthologs genes in the HMKS database and of random associated pairs of the same genes. <b>B.</b> Cumulative distribution of Euclidean distances d for pairs of orthologous genes and random-paired genes. The inset shows that 9.3% of orthologous transcripts displayed an Euclidean distance smaller than that of the 5% random pairs of transcripts with the lowest Euclidean distances.</p

Comparison of gene expression in human and mouse kidney as determined by SAGE and RT-PCR.

<p>Expression of selected genes was determined in the glomerulus (triangles), the S1 segment (circles), the cortical thick ascending limb of Henle’s loop (squares) or the cortical collecting duct (lozenges) by either SAGE or RT-PCR. In both cases, data were normalized to Rplp1 expression. Results were calculated as the human-to-mouse ratio of expression levels (for calculations, a tag occurrence of 0.01 was taken when the tag was not detected). Data are presented according to a base 2 log scale; they were arbitrarily distributed in two panels for legibility purposes.</p