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• Identification and characterization of miRNAome in tobacco (Nicotiana tabacum) by deep sequencing combined with microarray.

  Authors: Yushuang Guo, Hanhua Liu, Zhixiao Yang, Jing Chen, Yimin Sun, Xueliang Ren

  Gene. 501(1):24-32.

  Tobacco is one of the most important economic and agricultural crops worldwide. miRNAs have been increasingly acknowledged for their important roles in different biological processes of tobacco.Tobacco is one of the most important economic and agricultural crops worldwide. miRNAs have been increasingly acknowledged for their important roles in different biological processes of tobacco. However, few miRNAs have been identified so far in tobacco impeding the development of new tobacco strains with better properties. In this study, high-throughput sequencing technology was employed to identify novel tobacco miRNAs. A total of 84 potential miRNAs were obtained in tobacco, including 33 conserved and 51 novel miRNAs. Tissue-specific and topping-related miRNAs were identified. A tobacco miRNA microarray was also constructed to investigate miRNA expression patterns in different tissues, and their expression patterns were further validated by qRT-PCR and Northern Blot. Finally, the potential targets of these miRNAs were predicted based on a sequence homology search. Thus, in the current study, we have performed the comprehensive analysis of tobacco miRNAs, including their identification, expression pattern and target prediction. Our study opens a new avenue for further elucidation for their roles underlying the regulation of diversity of physiological processes.

• Analyses of chromosome copy number and expression level of four genes in the ciliate Chilodonella uncinata reveal a complex pattern that suggests epigenetic regulation.

  Authors: Laure Bellec, Laura A Katz

  Gene.

  Chilodonella uncinata, like all ciliates, contains two distinct nuclei in every cell: a germline micronucleus and a somatic macronucleus. The macronucleus develops from the zygotic nucleus through aChilodonella uncinata, like all ciliates, contains two distinct nuclei in every cell: a germline micronucleus and a somatic macronucleus. The macronucleus develops from the zygotic nucleus through a series of chromosomal rearrangements. Macronuclear development in C. uncinata yields a nucleus with highly amplified gene-sized chromosomes. The macronucleus is transcriptionally active during vegetative growth while there is no expression in the micronucleus except during a brief period following conjugation. Gene family evolution in ciliates occurs through complex processes including gene duplication and an alternative processing of scrambled genes. Here we use quantitative PCR to compare relative expression levels of eight genes (SSU-rDNA, Actin, α-Tubulin and five β-Tubulin sequences) to their abundance as macronuclear chromosomes. We show that three strains of the morphospecies C. uncinata share similar patterns across all loci. For example, we find an inverse correlation among five β-tubulin genes whereby the more abundant macronuclear chromosomes have lower levels of expression compared to less abundant chromosomes. We discuss implication of ours finding, which suggest an epigenetic mechanism maintains chromosome copy number in C. uncinata.

• First BAFF gene cloned from an aquatic mammal.

  Authors: Fengtao You, Wenhua Ren, Shasha Gu, Wenqian Wang, Lidan Zhou, Yijun Zhang, Weifeng Gan, Mingxing Chen

  Gene.

  The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species. Research into the immune system of the finless porpoise is essential to the protection of this species, but,The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species. Research into the immune system of the finless porpoise is essential to the protection of this species, but, to date, no genes coding for proteins from the tumor necrosis factor family (TNF family) have yet been reported from finless porpoises. The TNF B cell activating factor (BAFF) is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. It acts through its three receptors, BAFF-R, BCMA, and TACI. In the present study, the full-length cDNA of BAFF (designated NpBAFF) from the finless porpoise was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques, and its biological activities have been characterized. To our knowledge, this is the first report of any BAFF gene being cloned from an aquatic mammal. The full-length cDNA of NpBAFF consists of 1502 bases including an 852bp open reading frame encoding 283 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known BAFF homologues. Sequence comparison indicated that the amino acid sequence of NpBAFF was very similar to its bovine (87.68%), porcine (76.33%), hircine (87.68%) and canine (82.19%) counterparts. The predicted three-dimensional (3D) structure of the NpsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its human counterpart. Phylogenetic analysis indicated that NpBAFF showed a notable homology with artiodactyla BAFFs. The SUMO-NpsBAFF was efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that NpsBAFF could bind to its receptors on B cells. In vitro, MTT assays indicated that SUMO-NpsBAFF could promote the survival or proliferation of mouse splenic B cells grown with anti-mouse IgM. These findings indicate that NpBAFF plays an important role in the survival or proliferation of B cells and has functional cross-reactivity among cetaceans and other mammals. The present findings may provide valuable information for research into the immune system of the finless porpoise.

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

  Authors: Andrey Shirak, Shay Reicher, Shai Uliel, Tevie Mehlman, Alla Shainskaya, Micha Ron, Eyal Seroussi

  Gene.

  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 fromIn 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 O. 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, 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.

• Investigation on BRCA1 SNPs and its effects on mastitis in Chinese commercial cattle.

  Authors: Zheng Rong Yuan, Jun Ya Li, Jiao Li, Xue Gao, Hui Jiang Gao, Shang Zhong Xu

  Gene.

  The main object of this study was to investigate whether the bovine breast cancer 1 (BRCA1) gene was associated with mastitis resistance in Chinese commercial cattle. A total of 51 SNPs were screenedThe main object of this study was to investigate whether the bovine breast cancer 1 (BRCA1) gene was associated with mastitis resistance in Chinese commercial cattle. A total of 51 SNPs were screened from public data resources and DNA sequencing. Three SNPs (c.5682G>C,c.26198C>T and c.46126G>T) were genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Created Restriction Site PCR (CRS-PCR) methods and twenty-one combinations of these SNPs were observed. The single SNP and their genetic effects on somatic cell score (SCS) were evaluated and a significant association with SCS was found in c.46126G>T. The mean SCS of individuals with genotype KK was significantly lower than those of genotypes KL and LL. The results of combined genotypes analysis of three SNPs showed that HHLLNN genotype with the highest SCS were easily for the mastitis susceptibility, whereas GGKKMM genotype with the lowest SCS were favorable for the mastitis resistance. The information provided in the present study will be very useful for improving mastitis resistance in dairy cattle by marker-assisted selection.

• Overexpression of the Qc-SNARE gene OsSYP71 enhances tolerance to oxidative stress and resistance to rice blast in rice (Oryza sativa L.).

  Authors: Yong-Mei Bao, Shu-Jing Sun, Meng Li, Li Li, Wen-Lei Cao, Jia Luo, Hai-Juan Tang, Ji Huang, Zhou-Fei Wang, Jian-Fei Wang, Hong-Sheng Zhang

  Gene.

  OsSYP71 is an oxidative stress and rice blast response gene that encodes a Qc-SNARE protein in rice. Qc-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factorOsSYP71 is an oxidative stress and rice blast response gene that encodes a Qc-SNARE protein in rice. Qc-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which function as important components of the vesicle trafficking machinery in eukaryotic cells. In this paper, twelve Qc-SNARE genes were isolated from rice, and expression patterns of nine genes were detected in various tissues and in seedlings challenged with oxidative stresses and inoculated with rice blast. The expression of OsSYP71 was clearly up-regulated under these stresses. Overexpression of OsSYP71 in rice showed more tolerance to oxidative stress and resistance to rice blast than wild-type plants. These results indicate that Qc-SNAREs play an important role in rice response to environmental stresses, and OsSYP71 is useful in engineering crop plants with enhanced tolerance to oxidative stress and resistance to rice blast.

• Array-CGH characterization of a de novo t(X;Y)(p22;q11) in a female with short stature and mental retardation.

  Authors: Chiara Palka Bayard de Volo, Stefania De Marco, Valentina Chiavaroli, Melissa Alfonsi, Giuseppe Calabrese, Francesco Chiarelli, Angelika Mohn

  Gene.

  We report the clinical and molecular investigations in a girl with 46,X,-X,+der(X)t(X;Y)(p22;q11) de novo karyotype who presented an intricate phenotype characterized by mental retardation and facialWe report the clinical and molecular investigations in a girl with 46,X,-X,+der(X)t(X;Y)(p22;q11) de novo karyotype who presented an intricate phenotype characterized by mental retardation and facial dysmorphisms in combination with short stature. The structure of the derivative X chromosome was studied using BAC array-CGH which disclosed the Xp22 breakpoint between the STS and the VCX3A gene and the presence of the Yq11.1qter chromosome. It is common that females with Xp;Yq translocations present only short stature and are normal in every other aspect. Thus, this would be the first case in which a girl with Xp;Yq translocation presents an unusual phenotype with intermediate male clinical features with Xp;Yq translocations. The risk of developing gonadoblastoma in females with Y chromosome material is also discussed and, to this effect, different explanations related to this apparent variation are also presented.

• Association study of MicroRNA polymorphisms with hepatocellular carcinoma in Korean population.

  Authors: Won Hee Kim, Kyung Tae Min, Young Joo Jeon, Chang-Il Kwon, Kwang Hyun Ko, Pil Won Park, Sung Pyo Hong, Kyu Seong Rim, Sung Won Kwon, Seong Gyu Hwang, Nam Keun Kim

  Gene.

  BACKGROUND: Recent studies have suggested that common genetic polymorphisms alter the processing of microRNA (miRNA) and may be associated with the development and progression of cancer. PATIENTS ANDBACKGROUND: Recent studies have suggested that common genetic polymorphisms alter the processing of microRNA (miRNA) and may be associated with the development and progression of cancer. PATIENTS AND METHODS: The association of miRNA polymorphisms with HCC survival were analyzed in 159 HCC patients and 201 controls by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The risk of HCC was significantly lower for the miR-499A>G, AG+GG in HCC patients (AOR=0.603, 95% CI=0.370-0.984) and hepatitis B virus (HBV)-related HCC patients (AOR=0.561, 95% CI 0.331-0.950). In addition, the risk of HCC was significantly lower for the miR-149C>T, CT and CT+CC in HCC patients (CT; AOR=0.542, 95% CI=0.332-0.886, CT+CC; AOR=0.536, 95% CI=0.335-0.858) and HBV-related HCC patients (CT: AOR=0.510, 95% CI 0.305-0.854, CT+CC: AOR=0.496, 95% CI 0.302-0.813). The miR-149C>T polymorphism was also associated with survival rate of HCC patients in OKUDA II stage. CONCLUSIONS: miR-149C>T and miR-499A>G were associated with HBV-related HCC. Further studies on larger populations will need to be conducted to confirm these results.

• Influence of functional genetic polymorphism (-590C/T) in non-small-cell lung cancer (NSCLC) development: The paradoxal role of IL-4.

  Authors: Mónica Gomes, Ana Coelho, António Araújo, Ana Luísa Teixeira, Raquel Catarino, Rui Medeiros

  Gene.

  Lung cancer is the leading cause of death by cancer in the world, originating about 17.5% of total deaths from cancer (1.18 million). Inflammation plays an important role in the pathogenesis of lungLung cancer is the leading cause of death by cancer in the world, originating about 17.5% of total deaths from cancer (1.18 million). Inflammation plays an important role in the pathogenesis of lung cancer. IL-4 is an anti-inflammatory cytokine, which reduces the production of proinflammatory cytokines by monocytes and with direct antiproliferative effects in some tumors. The polymorphism -590C/T SNP is a C to T transition in the -590 position of the promoter region of the IL-4 gene. The aim of this study was to evaluate the influence of this polymorphism in the susceptibility to NSCLC. DNA was extracted from peripheral blood of 1060 individuals (391 patients diagnosed with NSCLC and a control group of 669 individuals without cancer). The characterization of IL-4 -590C/T genotypes was performed by PCR-RFLP (BsmFI). The -590C/T polymorphism genotypes were classified as low (CC) and high expression (TT). The frequencies obtained for the CC and TT genotypes were 90.1% and 9.9%, respectively, in the control group and 92.9% and 7.1%, respectively, in the case group. The analysis of the TT and CC genotype frequencies in the two groups, showed a statistically significant difference in its distribution, indicating a protection of 80% for the development of NSCLC, type epidermoid in individuals with the TT genotype when compared with individuals with CC genotype (P=0.024, OR=0.221: 95% CI=0.053-0.928). We present for the first time that increased expression of IL-4 associated with the TT genotype may contribute to immune surveillance during NSCLC development.

• Association of genetic variation in co-stimulatory molecules genes with outcome of liver transplant in Iranian patients.

  Authors: Mohammad Hossein Karimi, Maryam Motazedian, Fariba Abedi, Ramin Yaghobi, Bita Geramizadeh, Saman Nikegbalian

  Gene.

  CD28, cytotoxic T-lymphocyte associated antigen 4 (CTLA4), inducible costimulator (ICOS) and programmed cell death 1 are closely-linked genes located on chromosome 2q and encode co-stimulatoryCD28, cytotoxic T-lymphocyte associated antigen 4 (CTLA4), inducible costimulator (ICOS) and programmed cell death 1 are closely-linked genes located on chromosome 2q and encode co-stimulatory molecules, which are T-cell activity regulators. The principal assignment of T-cell mediated immune response in allograft rejection is an interesting topic of multiple studies. Although the variation in these genes may influence the graft survival and the amount of immunosuppression needed, the studies so far have been restricted solely to the CTLA4 gene. In 145 patients who underwent liver allograft transplantation, 10 single nucleotide polymorphisms of CD28, CTLA4, ICOS, and PD.1 genes were defined. To distinguish the polymorphisms of all 10 SNPs, PCR-RFLP method was used and according to the standard criteria, acute rejection episodes were determined. CTLA4-1661, AA genotype was significantly more frequent in the patients with acute rejection and AG genotype was significantly more frequent in the patients without rejection. Frequencies of CTLA4+49 AG A allele and CTLA4-1661AG A allele were significantly higher than those of CTLA4+49 AG and CTLA4-1661AG, G allele in the patients with acute rejection. ICOS+693, GG genotype and G allele were significantly less frequent in the patients with acute rejection and CD28 CT genotype was significantly more in patients with acute rejection. The present results demonstrate that potentially functional genetic variation in T-cell co-stimulatory molecules including ICOS, CTLA4 and CD28 can influence liver transplant outcome.

• Comparative analysis of the Hrp pathogenicity island of Rubus- and spiraeoideae-infecting Erwinia amylovora strains identifies the IT region as a remnant of an integrative conjugative element.

  Authors: Rachel A Mann, Jochen Blom, Andreas Bühlmann, Kim M Plummer, Steven V Beer, Joanne E Luck, Alexander Goesmann, Jürg E Frey, Brendan C Rodoni, Brion Duffy, Theo H M Smits

  Gene.

  The Hrp pathogenicity island (hrpPAI) of Erwinia amylovora encodes a type III secretion system (T3SS) and other genes required for pathogenesis on host plants, but also includes the so-called islandThe Hrp pathogenicity island (hrpPAI) of Erwinia amylovora encodes a type III secretion system (T3SS) and other genes required for pathogenesis on host plants, but also includes the so-called island transfer (IT) region, a region that originates from an integrative conjugative element (ICE). Comparative genomic analysis of the IT regions of two Spiraeoideae- and three Rubus-infecting strains revealed that the regions in Spiraeoideae-infecting strains were syntenic and highly conserved in length and genetic information, but that the IT regions of the Rubus-infecting strains varied in gene content and length, showing a mosaic structure. None of the ICE in E. amylovora strains were complete, as conserved ICE genes and the left border were missing, probably due to reductive genome evolution. Comparison of the hrpPAI region of E. amylovora strains to syntenic regions from other Erwinia spp. indicates that the hrpPAI and the IT regions are the result of several insertion and deletion events that have occurred within the ICE. It also suggests that the T3SS was present in a common ancestor of the pathoadapted Erwinia spp. and that insertion and deletion events in the IT region occurred during speciation.

• Presenilin-2 polymorphisms and risk of sporadic AD: Evidence from a meta-analysis.

  Authors: Chunhai Chen, Zhou Zhou, Maoquan Li, Mingyue Qu, Qinlong Ma, Min Zhong, Yanwen Zhang, Zhengping Yu

  Gene.

  Association studies of presenilin-2 (PSEN2) polymorphisms and sporadic Alzheimer's disease (AD) have yielded inconsistent results, possibly because single studies often lack sufficient statisticalAssociation studies of presenilin-2 (PSEN2) polymorphisms and sporadic Alzheimer's disease (AD) have yielded inconsistent results, possibly because single studies often lack sufficient statistical power. In this study, we performed a meta-analysis to evaluate the association of the two most extensively studied PSEN2 polymorphisms, rs8383 and 5'indel, with the risk of sporadic AD. We systematically reviewed relevant studies retrieved by Medline, Pubmed, Embase, AlzGene, and CNKI. Data were analyzed using the Stata (v11.0) software package. The fixed effects model or random-effects model were applied depending on between-study heterogeneity. Publication bias was eveluated using Egger's test and Begg's funnel plots. Overall, the meta-analysis included 6 case-control studies for each polymorphism with 2,186 confirmed AD cases and 2,507 healthy controls in total. Analysis suggested a significant association between SNP rs8383 polymorphism and AD risk with no evidence of between-study heterogeneity or publication bias. In contrast, we found no evidence for an association between the 5'indel polymorphism and AD risk. Further stratified analyses by apolipoprotein ε4 status or ethnicity also failed to reveal a statistically significant association between the 5'indel polymorphism of PSEN2 and AD risk. Our analysis supports the hypothesis that the PSEN2 rs8383 polymorphism is associated with an enlarged risk of sporadic AD. However, larger scale association studies are necessary to further validate the association of PSEN2 polymorphisms with sporadic AD risk and to define potential gene-gene interactions.

• Evolutionary growth process of highly conserved sequences in vertebrate genomes.

  Authors: Minaka Ishibashi, Akiko Ogura Noda, Ryuichi Sakate, Tadashi Imanishi

  Gene.

  Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved amongGenome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100 percent identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage.

• Molecular characterization and transcription regulation analysis of type I IFN gene in grass carp (Ctenopharyngodon idella).

  Authors: Dongming Li, Wanlong Tan, Meisheng Ma, Xinjian Yu, Qinan Lai, Zhiqiang Wu, Gang Lin, Chengyu Hu

  Gene.

  Type I interferons and interferon regulatory factor 7 (IRF7), which are crucial for innate immunity against viral infection, have been identified in many teleost fishes in recent years. In thisType I interferons and interferon regulatory factor 7 (IRF7), which are crucial for innate immunity against viral infection, have been identified in many teleost fishes in recent years. In this study, the complete genomic sequence of grass carp (Ctenopharyngodon idella) type I interferon (termed CiIFN) (GU139255) and the full-length IRF7 cDNA sequence of grass carp (termed CiIRF7) (GQ141741) were cloned and characterized. CiIFN consists of 3368bp, retaining the characteristic 5-exon/4-intron gene organization in fish type I IFNs. The CiIFN spans 5 exons and encodes a polypeptide of 180 amino acids, with the first 22 amino acids representing a putative signal peptide. The CiIFN promoter sequence was found to be 760bp, which can be divided into a proximal region (from -1 to -140bp) and a distal region (from -400 to -700bp). The cDNA of CiIRF7 was found to be 1808bp in full length, with an ORF of 1293bp that encodes a putative protein of 430 amino acids. The putative amino acid sequence of CiIRF7 possesses a DNA-binding domain (DBD) in the N-terminal region. Real-time PCR analysis revealed that CiIFN displayed a low constitutive expression in all the tissues tested. After stimulation by polyinosinic:polycytidylic acid (Poly I:C), the expression of CiIFN was significantly up-regulated in most tissues of grass carp, with a relatively strong expression in spleen, kidney and intestine. The recombinant polypeptides of CiIRF7 and CiIRF7-nDBD were analyzed in gel mobility shift assays, along with the PCR amplification products of the proximal region (CiIFNP2), the distal region (CiIFNP6) and the full-length (CiIFNP7) of CiIFN promoter sequence. The results revealed that CiIRF7 could bind to the distal region as well as to the proximal region of CiIFN promoter sequence in vitro. Subsequently, the CiIFNPs (CiIFNP7/2/6) were cloned into pGL3-Basic vectors and CiIRF7 was subcloned into pcDNA3.1 vectors, then pGL3-CiIFNPs were separately transiently transfected or co-transfected with pcDNA3.1-CiIRF7 into the mouse myeloma cell lines (MMCL) SP2/0 and the grass carp kidney cell lines (CIK), and the impact of CiIRF7 on CiIFN promoter activity was measured by luciferase assays in the transfected cells. These results demonstrated that CiIRF7 acted as a positive regulator on the transcription of CiIFN.

• In silico structural, functional and pathogenicity evaluation of a novel mutation: An overview of HSD3B2 gene mutations.

  Authors: Bahareh Rabbani, Nejat Mahdieh, Mohammad Tahgi Haghi Ashtiani, Aria Sotoudeh, Ali Rabbani

  Gene.

  Mutations of 3 beta hydroxysteroid dehydrogenase type II (HSD) gene result in different clinical consequences. We explain a patient who demonstrated salt wasting form of 3B-HSD deficiency in infancy.Mutations of 3 beta hydroxysteroid dehydrogenase type II (HSD) gene result in different clinical consequences. We explain a patient who demonstrated salt wasting form of 3B-HSD deficiency in infancy. Signs of hyponatremia and hyperkalemia were recognized in the infant with ambiguous genitalia and perineal hypospadias. The 46,XY male was genotyped by direct sequencing of HSD3B2 gene. Steroid profiles showed elevated concentration of 17 hydorxyprogestrone, and decrease in concentration of cortisol, and testosterone. Dehydroepiandrotone (DHEA) to androstenedione ratio had 6 fold increases. Direct sequencing of the patient revealed homozygous missense A82P mutation in exon 3. This mutation was confirmed by segregation analysis of the parents. Bioinformatic tools were used for in silico structural and functional analyses. Also, the pathological effect of the mutation was validated by different software. Alanine is a conserved amino acid in the membrane binding domain of the enzyme and proline substitution was predicted to destabilize the protein. This report may highlight the importance of the screening programs of the disorder in Iran.

• Gene expression profiles reveal significant differences between rat liver cancer and liver regeneration.

  Authors: Gaiping Wang, Cunshuan Xu, Jia Zhi, Yunpeng Hao, Lianxing Zhang, Cuifang Chang

  Gene.

  Rapid cell proliferation and growth occur in both liver cancer (LC) and liver regeneration (LR). Does it imply that LC and LR share some similar molecular mechanisms? To elucidate the intrinsicRapid cell proliferation and growth occur in both liver cancer (LC) and liver regeneration (LR). Does it imply that LC and LR share some similar molecular mechanisms? To elucidate the intrinsic similarities and differences between above two processes at transcriptional level, rat models of diethylnitrosamine-induced LC and 2/3 partial hepatectomy-induced LR were separately established. Then Rat Genome 230 2.0 Array was used to detect gene expression profiles of livers tissues obtained from above two models, and bioinformatics methods, such as hierarchical clustering, k-means clustering and Expression Analysis Systematic Explorer (EASE), were applied to uncover the correlation between gene expression changes and physiological activities in LC and LR. Subsequently expression changes of six selected genes were confirmed by real-time quantitative RT-PCR. As a result, the expressions of 909 genes were found significantly changed during LC occurrence and 948 genes in LR. The expression profiles of above two events were extremely different, and their expression patterns were classified into 6 clusters. Based on the correlation between expression patterns and functional profiles, we found that drug/toxin metabolism and oxidation reduction were induced in LC, but decreased in LR at the transcription level, while lipid, steroid and chemical homeostasis were remarkably repressed in LC but induced in LR; Inflammation/immune response and apoptosis were not obvious or weaker in LC than in LR, whereas the activities of angiogenesis and cell adhesion/migration in LC were much stronger.

• Cloning and expression of neuron-specific enolase in the corpus luteum of dairy goats.

  Authors: Xia Meng, Feihu Li, Shulin Chen, Caiyan Tang, Wenhua Zhang, Zhonghui Wang, Shanting Zhao

  Gene.

  Neuron-specific enolase (NSE) is the key molecular marker for diffuse neuroendocrine system (DNES) cells, its expression in the pregnant corpus luteum (CL) of dairy goats was studied by theNeuron-specific enolase (NSE) is the key molecular marker for diffuse neuroendocrine system (DNES) cells, its expression in the pregnant corpus luteum (CL) of dairy goats was studied by the immunofluorescence method and the ultra structural characteristics of luteal cells were detected by the electron microscopy to identify the existence of DNES cells in the pregnant CL of dairy goats. Besides, the coding sequence of dairy goats NSE gene was cloned and its biological information was analyzed. Results revealed that NSE immunopositive cells exhibited widespread cytoplasmic staining throughout the whole pregnant CL. In addition, these cells showed typical characteristics of DNES cells in the electron microscopy. These results suggested that many DNES cells exist in the pregnant CL of dairy goats. Meanwhile, we identified the coding sequence of dairy goats NSE (GenBank accession No. JN887466). Its nucleotide sequence homology was found to be 97.9%, 89.3%, 90% and 92.6%, respectively, compared with that of Bos taurus, Rattus norvegicus, Mus musculus and Homo sapiens, while the amino acid sequence homology was 99.1%,97%,97.2% and 98.2% respectively. These results first showed that the functional amino acids coded by the NSE gene were highly conserved in Caprine, Bos taurus, Rattus norvegicus, Mus musculus and Homo sapiens. It was implied that the gene NSE in dairy goats had close homology to that of NSE of other species. Our findings demonstrated the possible existence of DNES cells in pregnant CL, providing new clue for further understanding of interactions between the neuroendocrine and reproductive system. Characterization of gene sequence of dairy goats NSE will enable us to synthesize interference RNA for further study on the role of NSE on the formation, function and apotosis of pregnant CL in dairy goats.

• Characterization of the Atlantic salmon (Salmo salar) brain-type fatty acid binding protein (fabp7) genes reveals the fates of teleost fabp7 genes following whole genome duplications.

  Authors: Yvonne Y Y Lai, Krzysztof P Lubieniecki, Ben F Koop, William S Davidson

  Gene.

  Whole genome duplications (WGDs) are considered to have been a driving force in the generation of evolutionary diversity that is characteristic of higher eukaryotes. The ancestor of salmonidsWhole genome duplications (WGDs) are considered to have been a driving force in the generation of evolutionary diversity that is characteristic of higher eukaryotes. The ancestor of salmonids underwent two additional WGDs compared to mammals, one (3R) at the base of the teleost radiation and another (4R) in the common ancestor of extant salmonids. We have chosen the fatty acid binding protein (fabp) gene family as a model to study the fate of duplicated genes in teleosts following WGDs. As previously described for zebrafish, we identified two copies (fabp7a and fabp7b) of the brain-type fabp gene in several fish including rainbow smelt, but there was only a single transcript in northern pike, the closest relative of the salmonids, and two rather than the expected four fabp7 genes in Atlantic salmon, rainbow trout and grayling. A phylogenetic analysis revealed that a loss of the fabp7a gene occurred in the common ancestor of the northern pike and salmonids after it had diverged from the rainbow smelt, and that the 4R WGD then gave rise to the fabp7bI and fabp7bII observed in salmonids. This is supported by genetic mapping that placed the Atlantic salmon duplicated fabp7b genes on homeologous chromosomes. There was no evidence of neo-functionalization in the salmonid fabp7bI and fabp7bII genes based on dN/dS ratios and an examination of amino acid substitutions. Atlantic salmon fabp7bI and fabp7bII genes are both expressed broadly like fabp7b expression in northern pike. However, only Atlantic salmon fabp7bII, like its counterpart in northern pike and zebrafish, was expressed in the liver. A comparison of ~2000bp upstream of Atlantic salmon fabp7b gene duplicates revealed an insertion of 62bp in fabp7bI relative to fabp7bII. The presence of predicted transcription factor binding sites in this insertion sequence may explain the differential expression of the fabp7b gene duplicates in Atlantic salmon liver.

• Molecular characterization and transcriptional analysis of fad24 in pigs.

  Authors: Minzhang Cheng, Huijun Yang, Huan Long, Yan Zhang, Xiaodong Chen, Zaiqing Yang, Ting Lei

  Gene.

  Fad24 is a novel gene involved in initiating adipogenesis. Most of the current information on fad24 is limited to mice, and the transcriptional regulation of fad24 remains unknown. Here we revealedFad24 is a novel gene involved in initiating adipogenesis. Most of the current information on fad24 is limited to mice, and the transcriptional regulation of fad24 remains unknown. Here we revealed that porcine fad24 encodes a protein structurally homologous to its human and rodent orthologs. Like its mouse counterpart, pig fad24 had positive effect on the adipogenic differentiation of mesenchymal stem cells (MSCs). Distinct fad24 expression patterns were observed during development of skeletal muscle and subcutaneous fat in pigs. The proximal promoter region of porcine fad24 was predicted to contain a number of transcription factor binding motifs related to both adipogenesis and myogenesis. Among them, a putative binding site for krüppel-like factor (KLF)-15 was chosen for further analysis because the role of KLF15 in early adipogenesis is unclear, although its expression in preadipocytes is up-regulated shortly after adipogenic induction. We demonstrated that expression of fad24 and klf15 are concomitantly up-regulated at the onset of adipogenesis in MSCs. Functional KLF15 significantly enhanced the promoter activity of fad24, whereas dominant negative KLF15 had no such effects. Transactivation of fad24 by KLF15 was further confirmed by site-directed mutation and chromatin immunoprecipitation assays. Taken together, our findings provide additional information on fad24 that may contribute to understanding the molecular events involved in early adipocyte differentiation.

• Association between the Pro12Ala polymorphism of PPAR-γ gene and the polycystic ovary syndrome: A meta-analysis of case-control studies.

  Authors: Hong Zhang, Yan Bi, Changjun Hu, Weiping Lu, Dalong Zhu

  Gene.

  Several studies have been conducted to examine the association between PPAR-γ2 Pro12Ala polymorphism and polycystic ovary syndrome (PCOS), but the results remain inconsistent. To make a more preciseSeveral studies have been conducted to examine the association between PPAR-γ2 Pro12Ala polymorphism and polycystic ovary syndrome (PCOS), but the results remain inconsistent. To make a more precise estimation of the relationship, a meta-analysis was performed. In the current meta-analysis, a total of 17 case-control studies, including 2176 cases and 2373 controls, were selected. Odds ratios (ORs) and 95% confidence intervals (CIs) for Pro/Ala+Ala/Ala versus Pro/Pro genotype in all population and different nationality groups, and homeostasis model assessment-insulin resistance (HOMA-IR) of different genotype were evaluated. In the overall analysis, significant association between PPAR-γ2 Pro12Ala polymorphism and reduced risk of PCOS was observed (OR=0.75; 95%CI, 0.62-0.91; p=0.003). Stratified analysis showed that significantly strong association was presented only in Europeans (OR=0.74; 95%CI, 0.60-0.90; p=0.003), but not in Asians (OR=0.86; 95%CI, 0.51-1.43; p=0.56). Additionally, carrying the Ala12 allele was not associated with HOMA-IR in PCOS patients (OR=-0.29; 95%CI, -0.82-0.24; p=0.29). This meta-analysis supported that PPAR-γ2 Pro12Ala polymorphism was capable of reducing polycystic ovary syndrome risk in Europeans, but not in Asians.

• Transactivation of the human NME5 gene by Sp1 in pancreatic cancer cells.

  Authors: Fu Li, Zhenzhou Jiang, Ke Wang, Jingjing Guo, Gang Hu, Lixin Sun, Tao Wang, Xuzhen Tang, Ling He, Jincheng Yao, Danyi Wen, Xiaoran Qin, Luyong Zhang

  Gene.

  Non-metastatic cells 5 (NME5), a recently found gene belonging to the NDPK-like molecules gene family, is highly expressed in testis and some types of human cancer. Current studies have revealedNon-metastatic cells 5 (NME5), a recently found gene belonging to the NDPK-like molecules gene family, is highly expressed in testis and some types of human cancer. Current studies have revealed diverse potential functions of NME5 and we have reported that NME5 is associated with innate resistance to gemcitabine in human pancreatic cancer cells in previous study. However, the mechanism underlying the transcriptional regulation of NME5 has not been elucidated yet. In this study, we analyzed the 5'-flanking region of the human NME5 gene and revealed its transcription start site (TSS) at -35bp relative to its translation start codon ATG. Using 5' unidirectional deletion analysis, we demonstrated that the proximal promoter of NME5 is located within -1051bp to +35bp. Two functional GC-boxes (-300bp and -323bp) were identified within the promoter region. Mutation of either GC-box led to significant reduction in NME5 promoter activity, whereas overexpression of Sp1 activated NME5 promoter activity in MIA PaCa-2 and 293T cells. In silico analysis predicted that transcription factor Sp1 binds to both GC-boxes, which were confirmed by EMSA and ChIP. In addition, we found that compared with MIA PaCa-2, Sp1 was highly expressed in PAXC002, a well characterized human pancreatic cancer cell line with innate gemcitabine resistance where NME5 was reported to be highly expressed, indicating that Sp1 induces NEM5 expression in PAXC002 cells. In conclusion, our study characterized for the first time the human NME5 promoter which is controlled by Sp1 transcription factor in pancreatic cancer.

• The evolution of three types of indoleamine 2,3 dioxygenases in fungi with distinct molecular and biochemical characteristics.

  Authors: Hajime J Yuasa, Helen J Ball

  Gene.

  Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme and known as a mammalian immunosuppressive molecule. In fungi, the primary role of IDO is to be supply of nicotinamide adenineIndoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme and known as a mammalian immunosuppressive molecule. In fungi, the primary role of IDO is to be supply of nicotinamide adenine dinucleotide (NAD(+)) via the kynurenine pathway. We previously reported that the koji-mold, Aspergillus oryzae has two IDO genes, IDOα and IDOβ. In the present study, we found that A. oryzae also has the third IDO, IDOγ. These three-types of IDOs are widely distributed among the Pezizomycotina fungi, although the black truffle, Tuber melanosporum has only one corresponding gene to IDOα/IDOβ. The yeast, Saccharomyces cerevisiae has a single IDO gene. Generally, Pezizomycotina IDOα showed similar enzymatic properties to the yeast IDO, suggesting that the IDOα is a functional homologue of the S. cerevisiae IDO. In contrast to IDOα, the K(m) value of IDOβ is higher. However, the reaction velocity of IDOβ is very fast, resulting in comparable or higher catalytic efficiency than IDOα. Thus IDOβ may functionally substitute for IDOα in fungal L-Trp metabolism. The enzymatic activity of IDOγ was comparatively very low with the values of enzymatic parameters comparable to vertebrate IDO2 enzymes. IDOα and IDOβ have similar gene structures, suggesting they were generated by gene duplication which occurred rather early in Pezizomycotina evolution, although the timing of the duplication remains debatable. In contrast, the phylogenetic trees suggest that IDOγs form an evolutionarily distinct group of IDO enzymes, with a closer relationship to group I bacterial IDOs than other fungal IDOs. The ancestor of the IDOγ family is likely to have diverged from other eukaryotic IDOs at a very early stage of eukaryotic evolution.

• Insulin-Like Growth Factor I (IGF-1) Gene Polymorphism in Patients with Non-Metastatic Breast Cancer.

  Authors: Arzu Yaren, Sebahat Turgut, Ceylan Ayada, Raziye Akcilar, Serkan Degirmencioglu, Gamze Gokoz Dogu

  Gene.

  We aimed to assess the association between IGF-I gene (CA repeats) polymorphism in breast cancer patients and their clinicopathological features, as well as disease recurrence and survival.We aimed to assess the association between IGF-I gene (CA repeats) polymorphism in breast cancer patients and their clinicopathological features, as well as disease recurrence and survival. Seventy-six non-metastatic breast cancer patients were enrolled in the present study. The IGF-I (CA) repeats were studied with polymerase chain reaction by using proper primers belonging to these gene areas from DNA samples. Results show that the non 19- non 19 homozygote were more common in patients without lymph node involvement (p=0.04), with low histological grade (p=0.04), with positive hormone receptor status (p=0.01), and in patients without recurrence (p=0.06). These results suggest that the non 19-non 19 carriers have some favorable prognostic factors, and IGF-I gene polymorphism (CA repeats) may affect disease recurrence and overall survival.

• Genome sequence of the phage c1P1, which infects the beer spoilage bacterium Pediococcus damnosus.

  Authors: David Kelly, Orla O'Sullivan, Susan Mills, Olivia McAuliffe, R Paul Ross, Horst Neve, Aidan Coffey

  Gene.

  Pediococcus damnosus (P. damnosus) bacteriophage (phage) clP1 is a novel virulent phage isolated from a municipal sewage sample collected in Southern Ireland. This phage infects the beer spoilagePediococcus damnosus (P. damnosus) bacteriophage (phage) clP1 is a novel virulent phage isolated from a municipal sewage sample collected in Southern Ireland. This phage infects the beer spoilage strain P. damnosus P82 which was isolated from German breweries. Sequencing of the phage has revealed a linear double stranded DNA genome of 38,013 base pairs (bp) with an overall GC content of 47.6%. Fifty seven open reading frames (ORFs) were identified of which 30 showed homology to previously sequenced proteins, and as a consequence 20 of these were assigned predicted functions. The majority of genes displayed homology with genes from the Lactobacillus plantarum phage ΦJL-1. All genes were located on the same coding strand and in the same orientation. Morphological characterization placed phage clP1 as a member of the Siphoviridae family with an isometric head (59 nm diameter) and non-contractile tail (length 175 nm; diameter 10 nm. Interestingly, the phage clP1 genome was found to share very limited identity with other phage genome sequences in the database, and was hence considered unique. This was highlighted by the genome organisation which differed slightly to the consensus pattern of genomic organisation usually found in Siphoviridae phages. With the genetic machinery present for a lytic lifecycle and the absence of potential endotoxin factors, this phage may have applications in the biocontrol of beer spoilage bacteria. To our knowledge, this study represents the first reported P. damnosus phage genome sequence.

• Structure, tissue distribution and estrogen regulation of splice variants of the sea bream estrogen receptor α gene.

  Authors: P I S Pinto, R Teodósio, S Socorro, D M Power, A V M Canário

  Gene.

  Estrogen actions are mainly mediated by specific nuclear estrogen receptors (ERs), for which different genes and a diversity of transcript variants have been identified, mainly in mammals. In thisEstrogen actions are mainly mediated by specific nuclear estrogen receptors (ERs), for which different genes and a diversity of transcript variants have been identified, mainly in mammals. In this study, we investigated the presence of ER splice variants in the teleost fish gilthead sea bream (Sparus auratus), by comparison with the genomic organization of the related species Takifugu rubripes. Two exon2-deleted ERα transcript variants were isolated from liver cDNA of estradiol-treated fish. The ΔE2 variant lacks ERα exon 2, generating a premature termination codon and a putative C-terminal truncated receptor, while the ΔE2,3* variant contains an in-frame deletion of exon 2 and part of exon 3 and codes for a putative ERα protein variant lacking most of the DNA-binding domain. Both variants were expressed at very low levels in several female and male sea bream tissues, and their expression was highly inducible in liver by estradiol-17β treatment with a strong positive correlation with the typical wild-type (wt) ERα response in this tissue. These findings identify novel estrogen responsive splice variants of fish ERα, and provide the basis for future studies to investigate possible modulation of wt-ER actions by splice variants.

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