Gender and sexual behavior modulate the composition of serum lipocalins in Nile tilapia (Oreochromis niloticus).

In tilapia species, plasma lipoproteins with high electrophoretic mobility function in intra- and intergender communication. Blood samples taken at onset and peak of daily sexual activity from dominant and subordinate Oreochromis niloticus males and females were fractionated by native gel electrophoresis and the fast-migrating proteins were subjected to mass spectrometry. Mining the sequence data of the Cichlid Genome Consortium, we identified 11 proteins from the lipocalin super-family and characterized their genes' structures. Phylogenetic and structural analyses subdivided these genes into two classes: (I) 3-coding-exon apolipoproteins and (II) more complex 6-coding-exon sulfide-bond-containing lipocalins. Five apolipoproteins and PTGDSL1, TBTBP, and MSP proteins were modulated by gender and sexual behavior. PTGDSL1 protein was only observed in the plasma serum of dominant males. However, the cysteine residue in the position that is crucial for synthetase activity in mammalian prostaglandin D synthetases was not conserved in PTGDSL1 or PTGDSL2 proteins. In line with previous reports suggesting their involvement in male functions as pheromone transporters, TBTBP and MSP proteins were not detected in females at the onset of daily activity. Their increasing amount in males was concordant with the increase in apolipoproteins AFP4L, APOA4a, APOA4b, APO14kD and APOC2, which were detected exclusively in dominant males, indicating a possible role in mobilization of the energy required to maintain their social hierarchy.

Microarray analysis revealed upregulation of nitrate reductase in juvenile cuttings of Eucalyptus grandis, which correlated with increased nitric oxide production and adventitious root formation.

The loss of rooting capability following the transition from the juvenile to the mature phase is a known phenomenon in woody plant development. Eucalyptus grandis was used here as a model system to study the differences in gene expression between juvenile and mature cuttings. RNA was prepared from the base of the two types of cuttings before root induction and hybridized to a DNA microarray of E. grandis. In juvenile cuttings, 363 transcripts were specifically upregulated, enriched in enzymes of oxidation/reduction processes. In mature cuttings, 245 transcripts were specifically upregulated, enriched in transcription factors involved in the regulation of secondary metabolites. A gene encoding for nitrate reductase (NIA), which is involved in nitric oxide (NO) production, was among the genes that were upregulated in juvenile cuttings. Concomitantly, a transient burst of NO was observed upon excision, which was higher in juvenile cuttings than in mature ones. Treatment with an NO donor improved rooting of both juvenile and mature cuttings. A single NIA gene was found in the newly released E. grandis genome sequence, the cDNA of which was isolated, overexpressed in Arabidopsis plants and shown to increase NO production in intact plants. Therefore, higher levels of NIA in E. grandis juvenile cuttings might lead to increased ability to produce NO and to form adventitious roots. Arabidopsis transgenic plants constantly expressing EgNIA did not exhibit a significantly higher lateral or adventitious root formation, suggesting that spatial and temporal rather than a constitutive increase in NO is favorable for root differentiation.

High-throughput marker discovery in melon using a self-designed oligo microarray.

BACKGROUND: Genetic maps constitute the basis of breeding programs for many agricultural organisms. The creation of these maps is dependent on marker discovery. Melon, among other crops, is still lagging in genomic resources, limiting the ability to discover new markers in a high-throughput fashion. One of the methods used to search for molecular markers is DNA hybridization to microarrays. Microarray hybridization of DNA from different accessions can reveal differences between them--single-feature polymorphisms (SFPs). These SFPs can be used as markers for breeding purposes, or they can be converted to conventional markers by sequencing. This method has been utilized in a few different plants to discover genetic variation, using Affymetrix arrays that exist for only a few organisms. We applied this approach with some modifications for marker discovery in melon. RESULTS: Using a custom-designed oligonucleotide microarray based on a partial EST collection of melon, we discovered 6184 putative SFPs between the parents of our mapping population. Validation by sequencing of 245 SFPs from the two parents showed a sensitivity of around 79%. Most SFPs (81%) contained single-nucleotide polymorphisms. Testing the SFPs on another mapping population of melon confirmed that many of them are conserved. CONCLUSION: Thousands of new SFPs that can be used for genetic mapping and molecular-assisted breeding in melon were discovered using a custom-designed oligo microarray. A portion of these SFPs are conserved and can be used in different breeding populations. Although improvement of the discovery rate is still needed, this approach is applicable to many agricultural systems with limited genomic resources.

Role of protein translocation pathways across the endoplasmic reticulum in Trypanosoma brucei.

The translocation of secretory and membrane proteins across the endoplasmic reticulum (ER) membrane is mediated by co-translational (via the signal recognition particle (SRP)) and post-translational mechanisms. In this study, we investigated the relative contributions of these two pathways in trypanosomes. A homologue of SEC71, which functions in the post-translocation chaperone pathway in yeast, was identified and silenced by RNA interference. This factor is essential for parasite viability. In SEC71-silenced cells, signal peptide (SP)-containing proteins traversed the ER, but several were mislocalized, whereas polytopic membrane protein biogenesis was unaffected. Surprisingly trypanosomes can interchangeably utilize two of the pathways to translocate SP-containing proteins except for glycosylphosphatidylinositol-anchored proteins, whose level was reduced in SEC71-silenced cells but not in cells depleted for SRP68, an SRP-binding protein. Entry of SP-containing proteins to the ER was significantly blocked only in cells co-silenced for the two translocation pathways (SEC71 and SRP68). SEC63, a factor essential for both translocation pathways in yeast, was identified and silenced by RNA interference. SEC63 silencing affected entry to the ER of both SP-containing proteins and polytopic membrane proteins, suggesting that, as in yeast, this factor is essential for both translocation pathways in vivo. This study suggests that, unlike bacteria or other eukaryotes, trypanosomes are generally promiscuous in their choice of mechanism for translocating SP-containing proteins to the ER, although the SRP-independent pathway is favored for glycosylphosphatidylinositol-anchored proteins, which are the most abundant surface proteins in these parasites.

Elucidating the role of C/D snoRNA in rRNA processing and modification in Trypanosoma brucei.

Most eukaryotic C/D small nucleolar RNAs (snoRNAs) guide 2'-O methylation (Nm) on rRNA and are also involved in rRNA processing. The four core proteins that bind C/D snoRNA in Trypanosoma brucei are fibrillarin (NOP1), NOP56, NOP58, and SNU13. Silencing of NOP1 by RNA interference identified rRNA-processing and modification defects that caused lethality. Systematic mapping of 2'-O-methyls on rRNA revealed the existence of hypermethylation at certain positions of the rRNA in the bloodstream form of the parasites, suggesting that this modification may assist the parasites in coping with the major temperature changes during cycling between their insect and mammalian hosts. The rRNA-processing defects of NOP1-depleted cells suggest the involvement of C/D snoRNA in trypanosome-specific rRNA-processing events to generate the small rRNA fragments. MRP RNA, which is involved in rRNA processing, was identified in this study in one of the snoRNA gene clusters, suggesting that trypanosomes utilize a combination of unique C/D snoRNAs and conserved snoRNAs for rRNA processing.

Genome-wide analysis of C/D and H/ACA-like small nucleolar RNAs in Leishmania major indicates conservation among trypanosomatids in the repertoire and in their rRNA targets.

Small nucleolar RNAs (snoRNAs) are a large group of noncoding RNAs that exist in eukaryotes and archaea and guide modifications such as 2'-O-ribose methylations and pseudouridylation on rRNAs and snRNAs. Recently, we described a genome-wide screening approach with Trypanosoma brucei that revealed over 90 guide RNAs. In this study, we extended this approach to analyze the repertoire of the closely related human pathogen Leishmania major. We describe 23 clusters that encode 62 C/Ds that can potentially guide 79 methylations and 37 H/ACA-like RNAs that can potentially guide 30 pseudouridylation reactions. Like T. brucei, Leishmania also contains many modifications and guide RNAs relative to its genome size. This study describes 10 H/ACAs and 14 C/Ds that were not found in T. brucei. Mapping of 2'-O-methylations in rRNA regions rich in modifications suggests the existence of trypanosomatid-specific modifications conserved in T. brucei and Leishmania. Structural features of C/D snoRNAs, such as copy number, conservation of boxes, K turns, and intragenic and extragenic base pairing, were examined to elucidate the great variation in snoRNA abundance. This study highlights the power of comparative genomics for determining conserved features of noncoding RNAs.

The Trypanosoma brucei signal recognition particle lacks the Alu-domain-binding proteins: purification and functional analysis of its binding proteins by RNAi.

Trypanosomes are protozoan parasites that have a major impact on human health and that of livestock. These parasites represent a very early branch in the eukaryotic lineage, and possess unique RNA processing mechanisms. The trypanosome signal recognition particle (SRP) is also unusual in being the first signal recognition particle described in nature to be comprised of two RNA molecules, the 7SL RNA and a tRNA-like molecule. In this study, we further elucidated the unique properties of this particle. The genes encoding three SRP proteins (SRP19, SRP72 and SRP68) were identified by bioinformatics analysis. Silencing of these genes by RNAi suggests that the SRP-mediated protein translocation pathway is essential for growth. The depletion of SRP72 and SRP68 induced sudden death, most probably as a result of toxicity due to the accumulation of the pre-SRP in the nucleolus. Purification of the trypanosome particle to homogeneity, by TAP-tagging, identified four SRP proteins (SRP72, SRP68, SRP54 and SRP19), but no Alu-domain-binding protein homologs. This study highlights the unique features of the trypanosome SRP complex and further supports the hypothesis that the tRNA-like molecule present in this particle may replace the function of the Alu-domain-binding proteins present in many eukaryotic SRP complexes.

A genome-wide analysis of C/D and H/ACA-like small nucleolar RNAs in Trypanosoma brucei reveals a trypanosome-specific pattern of rRNA modification.

Small nucleolar RNAs (snoRNAs) constitute newly discovered noncoding small RNAs, most of which function in guiding modifications such as 2'-O-ribose methylation and pseudouridylation on rRNAs and snRNAs. To investigate the genome organization of Trypanosoma brucei snoRNAs and the pattern of rRNA modifications, we used a whole-genome approach to identify the repertoire of these guide RNAs. Twenty-one clusters encoding for 57 C/D snoRNAs and 34 H/ACA-like RNAs, which have the potential to direct 84 methylations and 32 pseudouridines, respectively, were identified. The number of 2'-O-methyls (Nms) identified on rRNA represent 80% of the expected modifications. The modifications guided by these RNAs suggest that trypanosomes contain many modifications and guide RNAs relative to their genome size. Interestingly, approximately 40% of the Nms are species-specific modifications that do not exist in yeast, humans, or plants, and 40% of the species-specific predicted modifications are located in unique positions outside the highly conserved domains. Although most of the guide RNAs were found in reiterated clusters, a few single-copy genes were identified. The large repertoire of modifications and guide RNAs in trypanosomes suggests that these modifications possibly play a central role in these parasites.

Small nucleolar RNAs that guide modification in trypanosomatids: repertoire, targets, genome organisation, and unique functions.

Small nucleolar RNAs constitute a family of newly discovered non-coding small RNAs, most of which function in guiding RNA modifications. Two prevalent types of modifications are 2'-O-methylation and pseudouridylation. The modification is directed by the formation of a canonical small nucleolar RNA-target duplex. Initially, RNA-guided modification was shown to take place on rRNA, but recent studies suggest that small nuclear RNA, mRNA, tRNA, and the trypanosome spliced leader RNA also undergo guided modifications. Trypanosomes contain more modifications and potentially more small nucleolar RNAs than yeast, and the increased number of modifications may help to preserve ribosome function under adverse environmental conditions during the cycling between the insect and mammalian host. The genome organisation in clusters carrying the two types of small nucleolar RNAs, C/D and H/ACA-like RNAs, resembles that in plants. However, the trypanosomatid H/ACA RNAs are similar to those found in Archaea and are composed of a single hairpin that may represent the primordial H/ACA RNA. In this review we summarise this new field of trypanosome small nucleolar RNAs, emphasising the open questions regarding the number of small nucleolar RNAs, the repertoire, genome organisation, and the unique function of guided modifications in these protozoan parasites.

Identification and functional characterization of lsm proteins in Trypanosoma brucei.

RNA interference of Sm proteins in Trypanosoma brucei demonstrated that the stability of the small nuclear RNAs (U1, U2, U4, U5) and the spliced leader RNA, but not U6 RNA, were affected upon Sm depletion (Mandelboim, M., Barth, S., Biton, M., Liang, X. H., and Michaeli, S. (2003) J. Biol. Chem. 278, 51469-51478), suggesting that Lsm proteins that bind and stabilize U6 RNA in other eukaryotes should exist in trypanosomes. In this study, we identified seven Lsm proteins (Lsm2p to Lsm8p) and examined the function of Lsm3p and Lsm8p by RNA interference silencing. Both Lsm proteins were found to be essential for U6 stability and mRNA decay. Silencing was lethal, and cis- and trans-splicing were inhibited. Importantly, silencing also affected the level of U4.U6 and the U4.U6/U5 tri-small nuclear ribonucleoprotein complexes. The presence of Lsm proteins in trypanosomes that diverged early in the eukaryotic lineage suggests that these proteins are highly conserved in both structure and function among eukaryotes. Interestingly, however, Lsm1p that is specific to the mRNA decay complex was not identified in the genome data base of any kinetoplastidae, and the Lsm8p that in other eukaryotes exclusively functions in U6 stability was found to function in trypanosomes also in mRNA decay. These data therefore suggest that in trypanosomes only a single Lsm complex may exist.

Scaling law in sizes of protein sequence families: from super-families to orphan genes.

It has been observed that the size of protein sequence families is unevenly distributed, with few super families with a large number of members and many "orphan" proteins that do not belong to any family. Here it is shown that the distribution of sizes of protein families in different databases and classifications (Protomap, Prodom, Cog) follows a power-law behavior with similar scaling exponents, which is characteristic of self-organizing systems. Since large databases are used in this study, a more detailed analysis of the data than in previous studies was possible. Hence, it is shown that the size distribution is governed by two exponents, different for the super families and the orphan proteins. A simple model of protein evolution is proposed, in which proteins are dynamically generated and clustered into families. The model yields a scaling behavior very similar to the distribution observed in the actual sequence databases, including the two distinct regimes for the large and small families, and thus suggests that the existence of "super families" of proteins and "orphan" proteins are two manifestations of the same evolutionary process.

RNA interference of signal peptide-binding protein SRP54 elicits deleterious effects and protein sorting defects in trypanosomes.

Trypanosomes are protozoan parasites that have a major impact on health. This family diverged very early from the eukaryotic lineage and possesses unique RNA processing mechanisms such as trans-splicing and RNA editing. The trypanosome signal recognition particle (SRP) has a unique composition compared with all known SRP complexes, because it contains two RNA molecules, the 7SL RNA and a tRNA-like molecule. RNA interference was utilized to elucidate the essentiality of the SRP pathway and its role in protein translocation in Trypanosoma brucei. The production of double stranded RNA specific for the signal peptide-binding protein SRP54 induced the degradation of the mRNA and a loss of the SRP54 protein. SRP54 depletion elicited inhibition in growth and cytokinesis, suggesting that the SRP pathway is essential. The translocation of four signal peptide-containing proteins was examined. Surprisingly, the proteins were translocated to the endoplasmic reticulum and properly processed. However, the surface EP procyclin, the lysosomal protein p67, and the flagellar pocket protein CRAM were mislocalized and accumulated in megavesicles, most likely because of a secondary effect on protein sorting. The translocation of these proteins to the endoplasmic reticulum under SRP54 depletion suggests that an alternative pathway for protein translocation exists in trypanosomes.