Ubiquitin-related Protein Research Articles

Comparative Proteomic Analysis of Proteins in Response to Simulated Acid Rain in Arabidopsis

A proteomic study using 2-D gel electrophoresis and MALDI-TOF MS was performed to characterize the responses of Arabidopsis thaliana plants to simulated acid rain (SiAR) stress, which is a global environmental problem and has become a serious issue in China in recent years. The emphasis of the present study was to investigate the overall protein expression changes when exposed to SiAR. Out of over 1000 protein spots reproducibly resolved, 50 of them changed their abundance by at least 2-fold. Analysis of protein expression patterns revealed that a set of proteins associated with energy production, metabolism, cell rescue, cell defense and protein folding, etc., could play important roles in mediating plant response to SiAR. In addition to this, some proteins involved in stress responses and jasmonic acid pathway are also involved in plant response to SiAR. More interestingly, the expression of several ubiquitination-related proteins changed dramatically after 32-h SiAR treatment, suggesting that they may act as a molecular marker for the injury phenotype caused by SiAR. Based on our results, we proposed a schematic model to explain the mechanisms associated with the systematic response of Arabidopsis plants to SiAR.

Incipient intranuclear inclusion body disease in a 78-year-old woman

We report an incipient case of intranuclear inclusion body disease (INIBD) in a 78-year-old woman. No apparent neurological symptoms were noticed during the clinical course. Post mortem examination revealed widespread occurrence of eosinophilic intranuclear inclusions in neuronal and glial cells of the central and peripheral nervous systems, as well as in parenchymal cells of the visceral organs. The inclusions were observed more frequently in glial cells than in neuronal cells. Ultrastructurally, the inclusions consisted of granular and filamentous material. Immunohistochemically, the inclusions were positive for ubiquitin, ubiquitin-related proteins (NEDD8 ultimate buster 1, small ubiquitin modifier-1, small ubiquitin modifier-2 and p62), promyelocytic leukemia protein and abnormally expanded polyglutamine. Consistent with previous studies, the vast majority of inclusion-bearing glial cells were astrocytes. Furthermore, p25α-positive oligodendrocytes rarely contained intranuclear inclusions. These findings suggest that INIBD may occur in non-demented elderly individuals and that oligodendrocyte is also involved in the disease process of INIBD.

Tumor Suppressor Function of CYLD in Nonmelanoma Skin Cancer

Ubiquitin and ubiquitin-related proteins posttranslationally modify substrates, and thereby alter the functions of their targets. The ubiquitination process is involved in various physiological responses, and dysregulation of components of the ubiquitin system has been linked to many diseases including skin cancer. The ubiquitin pathways activated among skin cancers are highly diverse and may reflect the various characteristics of the cancer type. Basal cell carcinoma and squamous cell carcinoma, the most common types of human skin cancer, are instances where the involvement of the deubiquitination enzyme CYLD has been recently highlighted. In basal cell carcinoma, the tumor suppressor protein CYLD is repressed at the transcriptional levels through hedgehog signaling pathway. Downregulation of CYLD in basal cell carcinoma was also shown to interfere with TrkC expression and signaling, thereby promoting cancer progression. By contrast, the level of CYLD is unchanged in squamous cell carcinoma, instead, catalytic inactivation of CYLD in the skin has been linked to the development of squamous cell carcinoma. This paper will focus on the current knowledge that links CYLD to nonmelanoma skin cancers and will explore recent insights regarding CYLD regulation of NF-κB and hedgehog signaling during the development and progression of these types of human tumors.

SnapShot: The SUMO System

SUMO (small ubiquitin-related modifier) is a highly conserved eukaryotic member of the family of ubiquitin-related proteins. Despite limited sequence similarity, SUMO and ubiquitin have highly homologous structures (i.e., with a root-mean-square deviation [rmsd] difference of 2.1 A). Like ubiquitin, SUMO is covalently attached to other proteins (SUMOylation) and thus functions as a posttranslational modifier. Although a major function of ubiquitination is to promote protein degradation, SUMOylation does not usually trigger proteolyis of the conjugated protein. Instead, a main function of SUMOylation is to foster—and occasionally disrupt—protein-protein interactions.Although less frequent than ubiquitination, SUMOylation regulates numerous processes and has many substrates in the cytosol and the nucleus. Lower eukaryotes possess only one SUMO form, but higher eukaryotes express several, perhaps functionally distinct, SUMO variants. In most organisms, SUMO is essential for viability. This SnapShot depicts the reactions involved in SUMO ligation, the effects of SUMOylation on target proteins, and cellular processes regulated by SUMOylation.

Growth and progression of melanoma and non‐melanoma skin cancers regulated by ubiquitination

Basal cell carcinomas (BCC), squamous cell carcinoma (SCC), and melanomas are the major types of skin tumors. Despite being skin cancers, the characteristics of each cancer are widely varied. BCCs often do not proliferate rapidly, and rarely metastasize. Squamous cell carcinomas are more malignant and a certain subtype of SCC is highly metastatic. Melanomas are highly proliferative and invasive, and are most frequently metastatic. Ubiquitin and ubiquitin-related proteins post-translationally modify proteins and thereby alter the functions of their target proteins. The ubiquitination process is involved in various physiological responses, including cell growth, cell death, and DNA damage repair. Accumulating evidence suggests that ubiquitin pathways are involved in different types of cancers, including skin cancers. This review describes the major ubiquitin pathways in BCC, SCC, and melanoma. The ubiquitin pathways that are activated among the skin cancers are highly diverse, which might reflect the various characteristics of these three cancer types. Meanwhile, there are also common pathways between BCC, SCC, and melanoma. Therefore, examining the ubiquitin pathways will reveal the mechanisms of these three major skin cancer types and will suggest treatment options.

Abstract 1582: Rho GDP dissociation inhibitor 2 (RhoGDI2) plays a functionally important role on EGFR signaling pathway in adenocarcinoma cells of lung cancer

Abstract It has been known that Rho GDP dissociation inhibitor 2 (RhoGDI2) is a regulator of Rho family GTPase. We identified a RhoGDI2 protein through the protein expression profile and then investigated its functional role on EGFR downstream signal in NSCLC (non-small cell lung cancer). Three adenocarcinoma cell lines were selected according to the EGFR and K-RAS mutation status; PC9 (E476-A750 del in exon 19, wild type K-RAS), H1975 (T790M in exon 20 and L858R in exon 21, wild type K-RAS), A549 (wild type EGFR, K-RAS mutation in codon 12). Among 342 protein spots detected on the 2D gel, 134 spots were initially selected as differentially expressed proteins using proteome analysis. It was noteworthy that the ubiquitination-related proteins were highly expressed in EGFR TKIs-sensitive PC9 cells. On the other hand, the EGFR TKIs-resistant H1975 cells harboring both T790M and L858R mutations expressed the signal transduction-related proteins such as Zinc finger protein, G protein-coupled receptor and Ras oncogene family protein. We observed that p-EGFR (Y1047), p-AKT, and p-ERK were highly activated in PC9 cells in contrast to no or weak activation of these signaling proteins in A549 and H1975 cells. In PC9 cell, the expression of RhoGDI2 was significantly reduced after gefitinib treatment. On the contrary, its expression was enhanced by gefitinib in H1975 cells. Accordingly, RhoGDI2 of the final six protein spots was selected for further study. In six NSCLC cell lines, RhoGDI2 mRNA and protein expressions were evaluated using RT-PCR and Western blot analysis, respectively. The highest expression level was observed in PC-9 cells. When RhoGDI2 siRNA was transfected in PC9 cells, it was shown that RhoGDI2 protein expression was markedly reduced. Additionally, in this transfected PC-9 cells, no changes in the expression of p-ERK and truncated caspase-3 were detected, but total EGFR and p-AKT expression were increased. When gefitinib was added in the PC9 cells transfected with RhoGDI siRNA, the expression level of total EGFR was less decreased compared to that of PC-9 cells transfected with GAPDH (positive control). The RhoGDI2 siRNA transfected PC-9 cells treated with gefitinib showed more increased expression level of actin compared to that of positive control. We observed that the metastatic potential of the RhoGDI2 siRNA transfected PC-9 cells with lack of its expression was significantly increased in transwell migration assay. Taken together, it is suggested that RhoGDI2 protein is an important down-stream molecule of EGFR signaling pathway in PC9 cells harboring EGFR sensitive mutation, and plays a functional role in cell migration and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1582.

The COP9 signalosome and its role in plant development

The COP9 signalosome (CSN) is an evolutionarily conserved multiprotein complex with a role in the regulation of cullin-RING type E3 ubiquitin ligases (CRLs). CSN exerts its function on E3 ligases by deconjugating the ubiquitin-related protein NEDD8 from the CRL cullin subunit. Thereby, CSN has an impact on multiple CRL-dependent processes. In recent years, advances have been made in understanding the structural organisation and biochemical function of CSN: Crystal structure analysis and mass spectrometry-assisted studies have come up with first models of the pair-wise and complex interactions of the 8 CSN subunits. Based on the analysis of mutant phenotypes, it can now be taken as an accepted fact that – at least in plants –the major biochemical function of CSN resides in its deneddylation activity, which is mediated by CSN subunit 5 (CSN5). Furthermore, it could be demonstrated that CSN function and deneddylation are required but not essential for CRL-mediated processes, and models for the role of neddylation and deneddylation in controlling CRL activity are emerging. Significant advances have also been made in identifying pathways that are growth restricting in the Arabidopsis csn mutants. Recently it has been shown that a G2 phase arrest, possibly due to genomic instability, restricts growth in Arabidopsis csn mutants. This review provides an update on recent advances in understanding CSN structure and function and summarises the current knowledge on its role in plant development and cell cycle progression.

A heavyweight joins the fray

Tagging of DNA-damage-associated proteins by ubiquitin is key to coordinating the DNA-damage response. The ubiquitin-related protein SUMO is revealed as a crucial regulator of ubiquitylation in DNA repair.

‘Two-Stage Double-Technique Hybrid (TSDTH)’ Identification Strategy for the Analysis of BMP2-Induced Transdifferentiation of Premyoblast C2C12 Cells to Osteoblast

Transdifferentiation offers new opportunities in the area of cell replacement therapy; however, the molecular mechanism by which transdifferentiation occurs is not fully understood. Our understanding about the sophisticated regulations of transdifferentiation is limited yet since their comprehensive proteome regulations have not been fully elucidated. Studies on bone morphogenic protein-2 (BMP2)-induced transdifferentiation of murine C2C12 cells, a myogenic lineage committed premyoblast, to osteogenic cells can provide a full picture of the dynamic events that occur at the level of protein activity and/or expression. Here, we investigated the overall dynamic regulatory proteome associated with BMP2-induced osteoblast transdifferentiation in premyoblast C2C12 cells using a novel Two-Stage Double-Technique Hybrid (TSDTH) strategy for proteomic analysis. Here, we took the approach of a TSDTH involving phosphoproteomic analysis after a short-term treatment (stage one, 30 min) and a long-term treatment (stage two, 3 days); SILAC (Stable isotope labeling with amino acids in cell culture)-proteomics was used to map the proteins. In these experiments, a total of 1321 potential phosphoproteins were identified in stage one analysis and 433 proteins were quantified in stage two analysis. Among them, 374 BMP2-specific phosphoproteins and 54 up- or down-regulated proteins were selected. In first stage analysis, several deubiquitination enzymes including Uch-l3 as well as ubiquitination related proteins were newly identified, and its inhibitor reduced the stability of phosphorylated Smad1, and the BMP2-induced ALP levels of C2C12 cells were detected. In second stage analysis, Thrombospondin1 was identified as the highest up-regulated protein by BMP2-long time stimulation and this was confirmed with immunoblot analysis. Furthermore, pathway enrichment and network analyses revealed that insulin-like growth factor (IGF) and calcium signaling pathways as well as TGFbeta/BMP signaling proteins are found to be potentially involved in the early and long-term actions of BMP2. Collectively, our TSDTH is a useful simple strategy to obtain comprehensive molecular mechanism of cellular processes such as transdifferentiation.

Isolation and identification of ubiquitin-related proteins from Arabidopsis seedlings

The majority of proteins in eukaryotic cells are modified according to highly regulated mechanisms to fulfill specific functions and to achieve localization, stability, and transport. Protein ubiquitination is one of the major post-translational modifications occurring in eukaryotic cells. To obtain the proteomic dataset related to the ubiquitin (Ub)-dependent regulatory system in Arabidopsis, affinity purification with an anti-Ub antibody under native condition was performed. Using MS/MS analysis, 196 distinct proteins represented by 251 distinct genes were identified. The identified proteins were involved in metabolism (23.0%), stress response (21.4%), translation (16.8%), transport (6.7%), cell morphology (3.6%), and signal transduction (1.5%), in addition to proteolysis (16.8%) to which proteasome subunits (14.3%) is included. On the basis of potential ubiquitination-targeting signal motifs, in-gel mobilities, and previous reports, 78 of the identified proteins were classified as ubiquitinated proteins and the rest were speculated to be associated proteins of ubiquitinated proteins. The degradation of three proteins predicted to be ubiquitinated proteins was inhibited by a proteasome inhibitor, suggesting that the proteins were regulated by Ub/proteasome-dependent proteolysis.

Expression and Purification of Glutathione Transferase-Small Ubiquitin-Related Modifier-Metallothionein Fusion Protein and Its Neuronal and Hepatic Protection against d-Galactose-Induced Oxidative Damage in Mouse Model

The present study aimed to produce and pathophysiologically evaluate the metallothionein (MT) fusion protein. A recombinant plasmid containing DNA segment coding the pET-glutathione transferase (GST)-small ubiquitin-related modifier (SUMO)-MT fusion protein was inserted into Escherichia coli for expression. The expression level of the fusion protein was very high, reaching to 38.4% of the total supernatant proteins from the organism. Subsequent filtration through glutathione Sepharose 4B gel and Sephadex G-25 yielded an MT fusion protein with purity more than 95%. When exposed to metals, E. coli containing the GST-SUMO-MT fusion protein showed an increased accumulation of Cd(2+), Zn(2+), or Cu(2+) at approximately 4.2, 4.0, or 1.6 times higher, respectively, than those containing the control protein. Administration of GST-SUMO-MT to mice that were also treated with D-galactose to induce neuronal and hepatic damage showed a significant improvement of animal learning and memory capacity, which was depressed in mice treated by D-galactose alone. Administration of MT fusion protein also prevented D-galactose-increased malondialdehyde contents and histopathological changes in the brain and liver. Furthermore, supplement of the fusion protein significantly prevented D-galactose-increased nitric oxide contents and -decreased superoxide dismutase activity in the brain, liver, and serum. The fusion protein was also able to prevent ionizing radiation-induced DNA damage of the mouse thymus. The present study indicates that GST-SUMO-MT has a normal metal binding feature and also significantly protects the multiple tissues against oxidative damage in vivo caused by chronic exposure to D-galactose and by ionizing radiation. Therefore, GST-SUMO-MT may be a potential candidate to be developed for the clinical application.

Ginkgolic Acid Inhibits Protein SUMOylation by Blocking Formation of the E1-SUMO Intermediate

Protein modification by small ubiquitin-related modifier proteins (SUMOs) controls diverse cellular functions. Dysregulation of SUMOylation or deSUMOylation processes has been implicated in the development of cancer and neurodegenerative diseases. However, no small-molecule inhibiting protein SUMOylation has been reported so far. Here, we report inhibition of SUMOylation by ginkgolic acid and its analog, anacardic acid. Ginkgolic acid and anacardic acid inhibit protein SUMOylation both in vitro and in vivo without affecting in vivo ubiquitination. Binding assays with a fluorescently labeled probe showed that ginkgolic acid directly binds E1 and inhibits the formation of the E1-SUMO intermediate. These studies will provide not only a useful tool for investigating the roles of SUMO conjugations in a variety of pathways in cells, but also a basis for the development of drugs targeted against diseases involving aberrant SUMOylation.

PLIC proteins or ubiquilins regulate autophagy‐dependent cell survival during nutrient starvation

Ubiquilins (UBQLNs) are adaptor proteins thought to deliver ubiquitinated substrates to proteasomes. Here, we show a role for UBQLN in autophagy: enforced expression of UBQLN protects cells from starvation-induced death, whereas depletion of UBQLN renders cells more susceptible. The UBQLN protective effect requires the autophagy-related genes ATG5 and ATG7, two essential components of autophagy. The ubiquitin-associated domain of UBQLN mediates both its association with autophagosomes and its protective effect against starvation. Depletion of UBQLN delays the delivery of autophagosomes to lysosomes. This study identifies a new role for UBQLN in regulating the maturation of autophagy, expanding the involvement of ubiquitin-related proteins in this process.

Midbody ring disposal by autophagy is a post-abscission event of cytokinesis

At the end of cytokinesis, the dividing cells are connected by an intercellular bridge, containing the midbody along with a single, densely ubiquitylated, circular structure called the midbody ring (MR). Recent studies revealed that the MR serves as a target site for membrane delivery and as a physical barrier between the prospective daughter cells. The MR materializes in telophase, localizes to the intercellular bridge during cytokinesis, and moves asymmetrically into one cell after abscission. Daughter cells rarely accumulate MRs of previous divisions, but how these large structures finally disappear remains unknown. Here, we show that MRs are discarded by autophagy, which involves their sequestration into autophagosomes and delivery to lysosomes for degradation. Notably, autophagy factors, such as the ubiquitin adaptor p62 (Refs 4, 5) and the ubiquitin-related protein Atg8 (ref. 6), associate with the MR during abscission, suggesting that autophagy is coupled to cytokinesis. Moreover, MRs accumulate in cells of patients with lysosomal storage disorders, indicating that defective MR disposal is characteristic of these diseases. Thus our findings suggest that autophagy has a broader role than previously assumed, and that cell renovation by clearing from superfluous large macromolecular assemblies, such as MRs, is an important autophagic function.

Ubiquitin-related protein FAT10 and hepatocellular carcinoma

Ubiqitin/ubiqitin-related system is a significant pathway of protein degradation in vi-vo. Plenty of researches suggest that the functional disorder of this system will lead to the expression abnormality of oncogene and anti-oncogene. It plays an important role in the development of tumors. FAT10 is one of the members of ubiqitin-related proteins, which is discovered in recent years. The present research reveals that the expression of FAT10 is highly upregulated in malignant tumors,especially in hepatocellular carcinoma. FAT10 may be become an epige-netic marker for liver preneoplasia. Key words: Liver Neoplasms; Ubiquitin; FAT10

Regulation of Mitotic Spindle Asymmetry by SUMO and the Spindle-Assembly Checkpoint in Yeast

During mitosis, the kinetochore microtubules capture and segregate chromosomes, and the astral microtubules position the spindle within the cell. Although the spindle is symmetric, proper positioning of the spindle in asymmetrically dividing cells generally correlates with the formation of morphologically and structurally distinct asters [1]. In budding yeast, the spindle-orientation proteins Kar9 and dynein decorate only one aster of the metaphase spindle and direct it toward the bud [2, 3]. The mechanisms controlling the distribution of Kar9 and dynein remain unclear. Here, we show that SUMO regulates astral-microtubule function in at least two ways. First, Kar9 was sumoylated in vivo. Sumoylation and Cdk1-dependent phosphorylation of Kar9 independently promoted Kar9 asymmetry on the spindle. Second, proper regulation of kinetochore function by SUMO was also required for Kar9 asymmetry. Indeed, activation of the spindle-assembly checkpoint (SAC) due to SUMO and kinetochore defects promoted symmetric redistribution of Kar9 in a Mad2-dependent manner. The control of Kar9 distribution by the SAC was independent of Kar9 sumoylation and phosphorylation. Together, our data reveal that three independent mechanisms contribute to Kar9 asymmetry: Cdk1-dependent phosphorylation, sumoylation, and SAC signaling. Hence, the two seemingly independent spindle domains, kinetochores and astral microtubules, function in a tightly coordinated fashion.

Modification of Drosophila p53 by SUMO Modulates Its Transactivation and Pro-apoptotic Functions

Conjugation to SUMO is a reversible post-translational modification that regulates several transcription factors involved in cell proliferation, differentiation, and disease. The p53 tumor suppressor can be modified by SUMO-1 in mammalian cells, but the functional consequences of this modification are unclear. Here, we demonstrate that the Drosophila homolog of human p53 can be efficiently sumoylated in insect cells. We identify two lysine residues involved in SUMO attachment, one at the C terminus, between the DNA binding and oligomerization domains, and one at the N terminus of the protein. We find that sumoylation helps recruit Drosophila p53 to nuclear dot-like structures that can be marked by human PML and the Drosophila homologue of Daxx. We demonstrate that mutation of both sumoylation sites dramatically reduces the transcriptional activity of p53 and its ability to induce apoptosis in transgenic flies, providing in vivo evidence that sumoylation is critical for Drosophila p53 function.

Transcriptome Analysis of Trichothecene-Induced Gene Expression in Barley

Fusarium head blight, caused primarily by Fusarium graminearum, is a major disease problem on barley (Hordeum vulgare L.). Trichothecene mycotoxins produced by the fungus during infection increase the aggressiveness of the fungus and promote infection in wheat (Triticum aestivum L.). Loss-of-function mutations in the TRI5 gene in F. graminearum result in the inability to synthesize trichothecenes and in reduced virulence on wheat. We examined the impact of pathogen-derived trichothecenes on virulence and the transcriptional differences in barley spikes infected with a trichothecene-producing wild-type strain and a loss-of-function tri5 trichothecene nonproducing mutant. Disease severity, fungal biomass, and floret necrosis and bleaching were reduced in spikes inoculated with the tri5 mutant strain compared with the wild-type strain, indicating that the inability to synthesize trichothecenes results in reduced virulence in barley. We detected 63 transcripts that were induced during trichothecene accumulation, including genes encoding putative trichothecene detoxification and transport proteins, ubiquitination-related proteins, programmed cell death-related proteins, transcription factors, and cytochrome P450s. We also detected 414 gene transcripts that were designated as basal defense response genes largely independent of trichothecene accumulation. Our results show that barley exhibits a specific response to trichothecene accumulation that can be separated from the basal defense response. We propose that barley responds to trichothecene accumulation by inducing at least two general responses. One response is the induction of genes encoding trichothecene detoxification and transport activities that may reduce the impact of trichothecenes. The other response is to induce genes encoding proteins associated with ubiquitination and cell death which may promote successful establishment of the disease.

Nitric Oxide Destabilizes Pias3 and Regulates Sumoylation

Small ubiquitin-related protein modifiers (SUMO) modification is an important mechanism for posttranslational regulation of protein function. However, it is largely unknown how the sumoylation pathway is regulated. Here, we report that nitric oxide (NO) causes global hyposumoylation in mammalian cells. Both SUMO E2 conjugating enzyme Ubc9 and E3 ligase protein inhibitor of activated STAT3 (Pias3) were targets for S-nitrosation. S-nitrosation did not interfere with the SUMO conjugating activity of Ubc9, but promoted Pias3 degradation by facilitating its interaction with tripartite motif-containing 32 (Trim32), a ubiquitin E3 ligase. On the one hand, NO promoted Trim32-mediated Pias3 ubiquitination. On the other hand, NO enhanced the stimulatory effect of Pias3 on Trim32 autoubiquitination. The residue Cys459 of Pias3 was identified as a target site for S-nitrosation. Mutation of Cys459 abolished the stimulatory effect of NO on the Pias3-Trim32 interaction, indicating a requirement of S-nitrosation at Cys459 for positive regulation of the Pias3-Trim32 interplay. This study reveals a novel crosstalk between S-nitrosation, ubiquitination, and sumoylation, which may be crucial for NO-related physiological and pathological processes.

Identification of transcribed derived fragments involved in self-incompatibility in perennial ryegrass (Lolium perenne L.) using cDNA-AFLP

Self-incompatibility (SI) is a mechanism that prevents self-pollination and inbreeding in many flowering plant species. SI in Lolium perenne is controlled by two multi-allelic loci, S and Z. SI has important consequences for L. perenne breeding as it prevents the efficient production of inbred lines and hybrids. In this study, the cDNA-AFLP approach was used to identify key components of the SI response, S and Z, as well as genes involved in the signaling cascade triggered by a SI response in L. perenne. A total of 169 transcripts displaying an allele-specific expression profile were identified. Several of these transcripts displayed homology to genes involved in general cellular functions. In other cases, interesting homologies to proteins, such as kinases, known to be involved in SI in other plant families were found. A genome-wide expression analysis, on the other hand, allowed us to identify 515 transcript derived fragments as putatively related to SI in L. perenne. All the expression profiles were quantified using AFLP-QuantarProTM and clustered using hierarchical and AQBC clustering methods. A subset of these genes was selected for sequencing and assigned into 10 functional categories. Homologies were found to proteins known to be involved in fertilization and SI processes in other plant families such as ubiquitin-related and calcium-related proteins.