Abstract
BackgroundUnderstanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins – representing the vertebrate secretome – are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown.ResultsWe identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%–3% estimate from random gene mutagenesis studies.Conclusion(s)This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.
Highlights
The increasing availability of genomic and EST sequence data for model genetic organisms has greatly facilitated genome-wide approaches for gene discovery and analysis
Morpholino phosphorodiamidate oligonucleotides (MOs), neutrally charged nucleic acid analogs created by replacing the ribose sugar with a morpholine moiety and the phosophodiester backbone with a phosphorodiamidate linkage[4], were used to target the putative 585 co-translationally translocated (CTT) proteins identified for loss-of-function studies in zebrafish embryos[1]
A MO reverse-genetic approach in zebrafish embryos does not suffer from these drawbacks and has distinct advantages associated with F0 screening and a priori knowledge of the gene sequence
Summary
The increasing availability of genomic and EST sequence data for model genetic organisms has greatly facilitated genome-wide approaches for gene discovery and analysis. We used a morpholino-based gene ‘knockdown’ strategy (Figure 1) to assess the role of members of the secretome in vertebrate development and function[1]. A software pipeline (Figure 2) for comparative genomic data mining was developed to identify CTT proteins en route to the endoplasmic reticulum, cell membranes, or external regulatory sites[2,3]. Utilizing the TargetP and SignalP algorithms for signal peptide and cleavage site prediction, reference CTT protein sets were created from completed genome projects (H. sapiens, F. rubripes, and M. musculis) and compared to sequence data from the TIGR Zebrafish Gene Indices (versions 6.1, 8, 12, and 16) and the Zebrafish Genome Project (Sanger, build Zv2 – Zv6) to identify putative CTT proteins for reverse genetic analysis. We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome
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