Tetraspanin Gene Research Articles

Identification of smut-responsive genes in sugarcane using cDNA-SRAP.

Sugarcane smut, caused by the fungus Sporisorium scitamineum, is one of the main diseases that affect sugarcane worldwide. In the present study, the cDNA-SRAP technique was used to identify genes that are likely to be involved in the response of sugarcane to S. scitamineum infection. In total, 21 bands with significant differential expression during cDNA-SRAP analysis were cloned and sequenced. Real-time qPCR confirmation demonstrated that expression of 19 of these 21 differential bands was consistent with the expression observed during cDNA-SRAP analysis, with a deduced false positive rate of 9.5%. Sequence alignment indicated that 18 of 19 differentially expressed genes showed homologies from 19% to 100% to certain genes in GenBank, including the following genes: topoisomerase (EU048780), ethylene insensitive (EU048778), and tetraspanin (EU048770). A real-time qPCR assay showed that during 0-72 h after pathogen infection, expression of the topoisomerase and the ethylene insensitive genes was upregulated, whereas expression of the tetraspanin gene was downregulated, identical to the expression patterns observed under salicylic acid treatment. Therefore, all three genes are thought to play a role during S. scitamineum challenge, but with different functions. To our knowledge, this is the first report on the application of cDNA-SRAP in differential gene expression analysis of sugarcane during a sugarcane-S. scitamineum interaction. The results obtained also contribute to a better understanding of the molecular mechanisms associated with sugarcane-S. scitamineum interactions.

Insights into the mechanisms of macular degeneration associated with the R172W mutation in RDS

Mutations in the photoreceptor tetraspanin gene peripherin-2/retinal degeneration slow (PRPH2/RDS) cause both rod- and cone-dominant diseases. While rod-dominant diseases, such as autosomal dominant retinitis pigmentosa, are thought to arise due to haploinsufficiency caused by loss-of-function mutations, the mechanisms underlying PRPH2-associated cone-dominant diseases are unclear. Here we took advantage of a transgenic mouse line expressing an RDS mutant (R172W) known to cause macular degeneration (MD) in humans. To facilitate the study of cones in the heavily rod-dominant mouse retina, R172W mice were bred onto an Nrl(-/-) background (in which developing rods adopt a cone-like fate). In this model the R172W protein and the key RDS-binding partner, rod outer segment (OS) membrane protein 1 (ROM-1), were properly expressed and trafficked to cone OSs. However, the expression of R172W led to dominant defects in cone structure and function with equal effects on S- and M-cones. Furthermore, the expression of R172W in cones induced subtle alterations in RDS/ROM-1 complex assembly, specifically resulting in the formation of abnormal, large molecular weight ROM-1 complexes. Fundus imaging demonstrated that R172W mice developed severe clinical signs of disease nearly identical to those seen in human MD patients, including retinal degeneration, retinal pigment epithlium (RPE) defects and loss of the choriocapillaris. Collectively, these data identify a primary disease-causing molecular defect in cone cells and suggest that RDS-associated disease in patients may be a result of this defect coupled with secondary sequellae involving RPE and choriocapillaris cell loss.

The tetraspanin geneMaPls1contributes to virulence by affecting germination, appressorial function and enzymes for cuticle degradation in the entomopathogenic fungus,Metarhizium acridum

In most eukaryotes, tetraspanins regulate cellular activities by associating with other membrane components. In phytopathogenic fungi, the tetraspanin Pls1 controls appressorium-mediated penetration. However, regulation of Pls1 and its associated signalling pathways are not clear. In this study, the MaPls1 gene from the entomopathogenic fungus Metarhizium acridum was functionally characterized. MaPls1 was highly expressed in mycelium and appressorium, and accumulated on the plasma membrane or in the cytoplasm. Compared with a wild-type strain, the deletion mutant ΔMaPls1 had delayed germination and appressorium formation and impaired turgor pressure on locust wings, but normal germination on medium and non-host insect matrices. Bioassays showed that ΔMaPls1 had decreased virulence and hyphal body formation in haemolymph when topically inoculated, but was not different from wild type when the insect cuticle was bypassed. Moreover, the ability to grow out of the cuticle was impaired in ΔMaPls1. Digital gene expression profiling revealed that genes involved in hydrolysing host cuticle and cell wall synthesis and remodelling were downregulated in ΔMaPls1. MaPls1 participated in crosstalk with signalling pathways such as the cyclic adenosine monophosphate-dependent protein kinase A and calmodulin-dependent pathways. Taken together, these results demonstrated the important roles of MaPls1 at the early stage of infection-associated development in M. acridum.

The evolution of vertebrate tetraspanins: gene loss, retention, and massive positive selection after whole genome duplications

BackgroundThe vertebrate tetraspanin family has many features which make it suitable for preserving the imprint of ancient sequence evolution and amenable for phylogenomic analysis. So we believe that an in-depth analysis of the tetraspanin evolution not only provides more complete understanding of tetraspanin biology, but offers new insights into the influence of the two rounds of whole genome duplication (2R-WGD) at the origin of vertebrates.ResultsA detailed phylogeny of vertebrate tetraspanins was constructed by using multiple lines of information, including sequence-based phylogenetics, key structural features, intron configuration and genomic synteny. In particular, a total of 38 modern tetraspanin ortholog lineages in bony vertebrates have been identified and subsequently classified into 17 ancestral lineages existing before 2R-WGD. Based on this phylogeny, we found that the ohnolog retention rate of tetraspanins after 2R-WGD was three times as the average (a rate similar to those of transcription factors and protein kinases). This high rate didn't increase the tetrapanin family size, but changed the family composition, possibly by displacing vertebrate-specific gene lineages with the lineages conserved across deuterostomes. We also found that the period from 2R-WGD to recent time is controlled by gene losses. Meanwhile, positive selection has been detected on 80% of the branches right after 2R-WGDs, which declines significantly on both magnitude and extensity on the following speciation branches. Notably, the loss of mammalian RDS2 is accompanied by strong positive selection on mammalian ROM1, possibly due to gene loss-induced compensatory evolution.ConclusionsFirst, different from transcription factors and kinases, high duplicate retention rate after 2R-WGD didn't increase the tetraspanin family size but just reshaped the family composition. Second, the evolution of tetraspanins right after 2R-WGD had been impacted by a massive wave of gene loss and positive selection on coding sequences. Third, the lingering effect of 2R-WGD on tetraspanin gene loss and positive selection might last for 300-400 million years.

Functional variants of TSPAN8 are associated with bipolar disorder and schizophrenia

Tetraspanins affect protein trafficking and are known to influence a wide variety of physiologic processes. Recently, single nucleotide polymorphisms (SNPs) of the tetraspanin gene TSPAN8 were found among the best ranked markers of genome wide association studies on bipolar disorder (BPD) (rs1705236) and type-2 diabetes, but functional consequences remained largely unknown. In the present study, we examined 13 tagging SNPs covering the TSPAN8 gene, the intronic TSPAN8 SNP rs1705236 as well as two non-synonymous (ns) SNPs in schizophrenia (SCZ) and BPD samples. In our analysis setting, we were not able to replicate the association of rs1705236 with BPD, nor did we find an association with SCZ. In the TSPAN8 upstream transcriptional control region however, we found rs4500567 to be associated with BPD. In contrast, in SCZ the nsSNP rs3763978 was associated with disease. The significance of both associations withstood conservative Bonferroni correction. In an attempt to link the polymorphisms to functional consequences, we performed an allele-specific in silico mapping of transcription factor binding sites around rs4500567 and predicted the tolerance of the Gly73Ala exchange caused by rs3763978. The results argue for a differential promoter activity specific for the variant associated with BPD, but impaired protein functionality in SCZ. This suggests that TSPAN8 contributes to both diseases, yet with different underlying mechanisms: regulatory versus structural. Similar phenomena might also occur in other risk genes for both BPD and SCZ, providing a molecular basis for the genetic overlap of both entities.

Tetraspanin gene expression levels as potential biomarkers for malignancy of gingival squamous cell carcinoma

Accurate assessment of malignancy in oral squamous cell carcinoma is essential to optimize treatment planning. To detect a biomarker related to malignant propensity in gingival squamous cell carcinoma (GSCC), quantitative gene expression analysis of tetraspanin family genes was conducted. In 73 cases of GSCC, total RNA was extracted from carcinoma tissues, and gene expression was analyzed by quantitative real time-PCR. Six tetraspanin family genes (CD9, CD63, CD81, CD82, CD151, NAG-2) were investigated. Housekeeping genes (ACTB and GAPDH), anchor protein genes (JUP and PXN) and an integrin gene (ITGA3) were used as reference genes. Forty-five gene expression ratios were calculated from these 11 gene expression levels and were analyzed with clinical parameters using multivariate statistical methods. According to the results of the logistic regression analysis subjecting cervical lymph node metastasis as a target variable, CD9/ACTB (p = 0.013) or CD9/CD82 (p = 0.013) in addition to tumor size (p = 0.028) were detected as significant factors. In Cox proportional hazards regression analysis, delayed cervical lymph node metastasis (p = 0.039) and tumor cell positive surgical margin (p = 0.032) in addition to CD151/GAPDH (p = 0.024) were detected as significant factors for death outcome. A Kaplan-Meier survival curve presented a significantly lower survival rate of the group with a CD151/GAPDH value of 10 or more (log rank and generalized Wilcoxon tests: p = 0.0003). Results of this study present the usefulness of CD9 and CD151 expression levels as biomarkers for assessment of malignancy in GSCC. They also indicate that detection of residual tumor cells at the surgical margin and the biological malignancy of a tumor interdependently affects prognosis.

Intron Evolution: Testing Hypotheses of Intron Evolution Using the Phylogenomics of Tetraspanins

BackgroundAlthough large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several new fully sequenced genomes that cover the major lineages of the animal kingdom as well as plants, protists and fungi. The analysis of exon/intron gene structure in such an evolutionary broad set of genomes allowed us to identify ancestral intron structure in tetraspanins throughout the eukaryotic tree of life.Methodology/Principal FindingsWe performed a phylogenomic analysis of the intron/exon structure of the tetraspanin protein family. In addition, to the already characterized tetraspanin introns numbered 1 through 6 found in animals, three additional ancient, phase 0 introns we call 4a, 4b and 4c were found. These three novel introns in combination with the ancestral introns 1 to 6, define three basic tetraspanin gene structures which have been conserved throughout the animal kingdom. Our phylogenomic approach also allows the estimation of the time at which the introns of the 33 human tetraspanin paralogs appeared, which in many cases coincides with the concomitant acquisition of new introns. On the other hand, we observed that new introns (introns other than 1–6, 4a, b and c) were not randomly inserted into the tetraspanin gene structure. The region of tetraspanin genes corresponding to the small extracellular loop (SEL) accounts for only 10.5% of the total sequence length but had 46% of the new animal intron insertions.Conclusions/SignificanceOur results indicate that tests of intron evolution are strengthened by the phylogenomic approach with specific gene families like tetraspanins. These tests add to our understanding of genomic innovation coupled to major evolutionary divergence events, functional constraints and the timing of the appearance of evolutionary novelty.

The tetraspanin gene ClPLS1 is essential for appressorium-mediated penetration of the fungal pathogen Colletotrichum lindemuthianum

Conservation of the molecular mechanisms controlling appressorium-mediated penetration during evolution was assessed through a functional study of the ClPLS1 gene from Colletotrichum lindemuthianum orthologous to the MgPLS1 from Magnaporthe grisea, involved in penetration peg development. These two plant-pathogenic Pyrenomycetes differentiate appressoria to penetrate into plant tissues. We showed that ClPLS1 is a functional homologue of MgPLS1 in M. grisea. Loss of ClPLS1 function had no effect on vegetative growth, conidiation or on appressorium differentiation and maturation. However, C lpls1::hph mutants are non-pathogenic on either intact or wounded bean leaves, as a result of a defect in the formation and/or positioning of the penetration pore and consequently in the formation of the penetration peg. These observations suggest that the fungal tetraspanins control a conserved appressorial function that could be required for the correct localization of the site where the penetration peg emerges.

Increased expression of Drosophila tetraspanin, Tsp68C, suppresses the abnormal proliferation of ytr-deficient and Ras/Raf-activated hemocytes.

Tetraspanins are evolutionary conserved transmembrane proteins thought to facilitate cell proliferation, movement or fusion by acting as organizers of different signaling events. Despite their prevalence and conservation, their specific role and functions remain largely elusive, as their redundancy in various organisms has hindered loss of function studies. Here, we take a gain of function approach to study Drosophila tetraspanin Tsp68C and its effect on larval hemocytes. We recently characterized a lethal mutation in ytr, a conserved gene that encodes a nuclear arginine-rich protein of unknown function, which is accompanied by abnormal differentiation and proliferation of the larval hematopoietic tissue in flies. A hemolectin (hml)-Gal4 construct carried by hml-Gal4 transgenic flies was sufficient by itself to abrogate the hematopoietic defects in ytr mutant larvae. This rescue correlated with the overexpression of tsp68C, a tetraspanin gene nested in the hml promoter. The suppression of abnormal proliferation by the hml-Gal4 construct was not restricted to ytr-deficient hemocytes, but was also observed in hemocytes expressing the oncogenic forms of Raf or Ras proteins. However, it had no effect on overproliferation mediated by a constitutively active form of Jak. New hml-Gal4 lines, in which the tsp68C gene was silenced or deleted from the promoter, no longer rescued the hematopoietic defect in ytr mutants nor suppressed the activated Raf-induced overproliferation. Therefore, change in tetraspanin Tsp68C expression has a strong suppressor effect on abnormal proliferation and differentiation of hemocytes in the context of specific lesions, such as overactivation of the Ras/Raf/MAPK pathway.

A new class of tetraspanins in fungi

Tetraspanins are animal proteins involved in membrane complexes that are involved in cell adhesion, differentiation, and motility. The PLS1 gene from rice blast fungus Magnaporthe grisea encodes a protein (Pls1p) structurally related to tetraspanins that is required for pathogenicity. In Botrytis cinerea public sequences, we identified an EST homologous to PLS1. Using degenerated oligonucleotides, we amplified sequences homologous to PLS1 in fungi Colletotrichum lindemuthianum and Neurospora crassa. Analysis of N. crassa and M. grisea genome sequences revealed the presence of a single tetraspanin gene. Thus, fungi differ from animals, which contain between 20 and 37 paralogous tetraspanin genes. Fungal proteins encoded by BcPLS1, ClPLS1, and NcPLS1 display all the structural hallmarks of tetraspanins (predicted topology with four transmembrane domains, extra- and intracellular loops; conserved cysteine-based patterns in second extracellular loop). Phylogenetic analysis suggests that these genes define a new family of orthologous genes encoding fungal-specific tetraspanins.

Novel mental retardation-epilepsy syndrome linked to Xp21.1-p11.4.

We evaluated a kindred with X-linked mental retardation and epilepsy. Seven affected males with mild to moderate mental retardation developed seizures (primarily generalized, tonic-clonic, and atonic) that began on average at 6.8 months of age (range, 4 to 14 months). These patients did not have a history of infantile spasms. There were no dysmorphic features. Other than mental retardation, the neurological examination was unremarkable, with exception of 2 affected subjects who had mild generalized rigidity and ataxia. We identified tight linkage to a group of markers on Xp21.1-p11.4. A maximum two-point LOD score of +3.83 at straight theta = 0 was obtained for markers DXS8090, DXS1069, DXS8102, and DXS8085. This locus spans 7.7cM between DXS1049 and DXS8054 and does not overlap the locus for X-linked West syndrome. The tetraspanin gene, implicated in nonspecific mental retardation, is mapped to this region. We sequenced the tetraspanin coding sequence in subjects with X-linked mental retardation and epilepsy and did not identify disease-specific mutations. The syndrome we describe, designated X-linked mental retardation and epilepsy, is clinically and genetically distinct from X-linked West syndrome and other X-linked mental retardation-epilepsy syndromes.

Pattern of expression of tetraspanin antigen genes in Burkitt lymphoma cell lines.

Tetraspanin antigens are implicated in the prognosis of different types of tumours. In this study we determine by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) the level of 13 tetraspan messages in 21 Burkitt lymphoma (BL) cell lines. All tumour cell lines have a common pattern of tetraspanin gene expression. There are five antigens which are detected in 90% of cell lines at high levels, CD53, CD81, CD63, SAS and CD82. Another two, CD9 and CD37, were detected in 60% of cell lines, and have a very variable level of expression. The remaining antigens, A15, CoO29, KRAG, L6, TI-1 and il-TMP, are expressed at low levels in very few cell lines without any specific pattern. The level of gene expression corresponds with the level of cell surface antigen determined by flow cytometry. The average number of tetraspan proteins expressed per cell line is six. These proteins may form subunits of an oligomeric structure with 24 transmembrane domains. There are no major differences in tetraspan expression pattern among sporadic or endemic tumours, type of translocation or Epstein-Barr virus status, suggesting the original cell of these tumours is the same, probably a late pre-B cell, at the CD9 to CD37 transition point. Tetraspanin gene expression is consistent with BL being a single entity, despite variations in other parameters.