Tonghai Dou

Genome-wide prediction of G protein-coupled receptors in Verticillium spp.

G protein-coupled receptors (GPCRs) are critical factors in regulating morphogenesis, mating, infection and virulence in fungi. In this study, various computational strategies were applied to identify GPCR-like proteins from the genomes of both Verticillium dahliae and Verticillium albo-atrum. The putative GPCRs were distributed over 13 classes, and significantly, three of those represented novel classes of GPCR-like proteins in fungi. The three novel GPCRs had high levels of identity to their counterparts in higher eukaryotes, including Homo sapiens. The numbers of GPCR-like proteins in the two Verticillium spp. were similar to those seen in other filamentous fungi, such as Magnaporthe grisea, Neurospora crassa and Fusarium graminearum. Additionally, the carbon/amino acid receptors were divided into three different subclasses, indicating that differences among the GPCRs existed not only among different classes but also within classes. In conclusion, the identification and classification of GPCRs and their homology to some well-studied fungi will be an important starting point for future research in Verticillium spp.

The evolution of alternative splicing exons in vascular endothelial growth factor A.

The C-terminus alternative splicing in VEGFA (vascular endothelial growth factor A) is known for its impact on physiological and pathological angiogenesis. Based on our prediction and RT-PCR verification, we identified anti-angiogenic VEGFA165b isoforms in mouse and rabbit for the first time. We also found that the relative expression level of VEGFA165b isoform had been increasing from rodents to human, and exon8b may have experienced a minor-to-major form exon conversion, possibly correlated with its gain-of-function. It is suggested that introduction of alternative splicing exons (esp. exon6 and exon8b) made important contributions to the transcriptional diversity of VEGFA and played a crucial role in the evolution of its regulatory mechanism.

Isolation and Characterization of Ubiquitin-activating Enzyme E1-domain Containing 1, UBE1DC1

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Activated by other proteins, ubiquitin and ubiquitin-like proteins can covalently modify target proteins. The enzymes responsible for the activation of this modification have been known to include UBA1, SAE2, UBA3, SAE1 and ULA1. Here we report a new ubiquitin activating enzyme like cDNA, named ubiquitin activating enzyme E1-domain containing 1 (UBE1DC1), whose cDNA is 2654 base pairs in length and contains an open reading frame encoding 404 amino acids. The UBE1DC1 gene consists of 12 exons and is located at human chromosome 3q22. The result of RT-PCR showed that UBE1DC1 is expressed in most of human tissues.

Hox genes from the parasitic flatworm Schistosoma japonicum.

Hox genes are characterized by a highly conserved peptide domain and contribute to antero-posterior axis patterning during embryogenesis. These genes have been widely studied in a variety of animal species due to their central role in evolutionary developmental biology. Based on the published genome assembly and unpublished re-sequencing project data, we present the first genome-wide characterization and comparative genomic analysis of the Hox gene family within Schistosoma japonicum. Eight Hox genes were identified and validated in our investigation. Phylogenetic analysis revealed that these genes are distributed among seven orthology groups of the Hox gene family. Our study further suggested that differences in the Lox5 gene copy number existed between the two closely related species, S. japonicum and Schistosoma mansoni. Semi-quantitative real-time polymerase chain reaction experiments revealed that Lox5 and Hox4 gene expression was high in the schistosomulum stage, and all four genes investigated showed highest expression within the eggs.

Cloning and Characterization of a novel splice variant of human Rab18 gene (RAB18)

Rab GTPase proteins are a kind of small GTP-binding proteins, which functions mainly focus on regulating interacellular trafficking pathways during vesicular transport. To date, 60 distinct human RAB proteins have been identified. RAB18 gene is discovered from endothelial cells. Its function is considered as endosomes and plasma membrane recycling. Research indicates RAB18 may relate to inflammation and some kinds of tumor. Here we report a splice variant of RAB18, which is 2571 bp in length and has an open reading frame coding a predicted 235 amino-acids protein. RT-PCR shows that the cDNA has different expression pattern with RAB18 and is highly expressed in testis.

Cloning and characterization of a novel splice variant of human U2AF1L3 gene.

Pre-mRNA splicing allows individual genes to produce multiple protein isoforms with diverse functions. Recognition of functional splice sites in pre-mRNAs is very important in this splicing process and requires some protein auxiliary factors such as U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF35, encoded by U2AF1). By its RNA binding domains, U2AF35 interacts with U2AF65 to bind 3′ splice site of pre-mRNA and initiates splicing. Another protein, which is named as U2AF1-like3 (U2AF1L3), shows high similarity with U2AF35 and may have related function in pre-mRNA splicing. Here, we report a splice variant of U2AF1L3, which is 767 bp in length and has an open reading frame (ORF) coding a predicted 181 amino acids protein. Reverse transcription-PCR (RT-PCR) shows that this isoform has different expression pattern with U2AF1L3 and is highly expressed in heart, brain and lung.

Optimized Exon-Exon Junction Library and its Application on Rodents' Brain Transcriptome Analysis

ABSTRACT Background: Alternative splicing (AS), which plays an important role in gene expression and functional regulation, has been analyzed on genome-scale by various bioinformatic approaches based on RNA-seq data. Compared with the huge number of studies on mouse, the AS researches approaching the rat, whose genome is intermedia between mouse and human, were still limited. To enrich the knowledge on AS events in rodents’ brain, we perfomed a comprehensive analysis on four transcriptome libraries (mouse cerebrum, mouse cerebellum, rat cerebrum, and rat cerebellum), recruiting high-throughput sequencing technology. An optimized exon-exon junction library approach was introduced to adapt the longer RNA-seq reads and to improve mapping efficiency. Results: In total, 7,106 mouse genes and 2,734 rat genes were differentially expressed between cerebrum and cerebellum, while 7,125 mouse genes and 1,795 rat genes exhibited varieties on transcript variant level. Only half of the differentially expressed exon-exon junctions could be reflected at gene expression level. Functional cluster analysis showed that 32 pathways in mouse and 9 pathways in rat were significantly enriched, and 6 of them were in both. Interestingly, some differentially expressed transcript variants did not show difference on gene expression level, such as

Signatures of positive selection at hemopexin (PEX) domain of matrix metalloproteinase-9 (MMP-9) gene

Matrix metalloproteinases-9 (MMP-9) is an important cancer-associated, zinc-dependent endopeptidase. To investigate the natural selection hypothesis of MMP-9, the orthologous sequences from 12 vertebrates were compared and a molecular evolution analysis was performed. Results suggest that amino acid residues present in the middle region of the protein are more selectively constrained, whereas amino acid residues in the C-terminal region of the MMP-9 protein including exon 13 showed lowest conservation level in non-primate species, suggesting that it is an exon with fast evolving rate compared to the others analyzed. InterProScan analysis shows that exon 13 was located in hemopexin (PEX) domain of MMP-9. Positive selection was detected in PEX domain of MMP-9 protein between human and other species, which indicates that selective pressure may play a role in shaping the function of MMP-9 in the course of evolution.

Signatures of positive selection in LY96 gene in vertebrates

As a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4), Lymphocyte Antigen 96 (LY96), also called myeloid differentiation 2 (MD2), is required for the activation of TLR4 by lipopolysaccharide (LPS) and plays an important role in innate immunity, which is the first line of defence against microbial infections. Previous studies have proposed that mammalian toll-like receptors (TLRs) have evolved under diversifying selection due to their role in pathogen detection. Given the fact that LY96 is highly functionally linked to TLR4, it would be interesting to test whether LY96 is under the intense pressure of natural selection. To investigate the natural selection hypothesis, we compared the coding sequences from 13 vertebrates and evaluated the molecular evolution of LY96 gene in these species. Result shows that natural selection at exon 4 has indeed played a role in shaping the function of LY96 in the course of evolution. In addition to the study of Nakajima, we found the two branch nodes with Ka/Ks ratios greater than 1: the one leading to cow and pig and the other to rabbit and the primates.

Cloning and characterization of a novel splice variant of human U2AF1L3 gene: Full Length Research Paper

Pre-mRNA splicing allows individual genes to produce multiple protein isoforms with diverse functions. Recognition of functional splice sites in pre-mRNAs is very important in this splicing process and requires some protein auxiliary factors such as U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF35, encoded by U2AF1). By its RNA binding domains, U2AF35 interacts with U2AF65 to bind 3′ splice site of pre-mRNA and initiates splicing. Another protein, which is named as U2AF1-like3 (U2AF1L3), shows high similarity with U2AF35 and may have related function in pre-mRNA splicing. Here, we report a splice variant of U2AF1L3, which is 767 bp in length and has an open reading frame (ORF) coding a predicted 181 amino acids protein. Reverse transcription-PCR (RT-PCR) shows that this isoform has different expression pattern with U2AF1L3 and is highly expressed in heart, brain and lung.