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Background: As the crosstalk between metabolism and antitumor immunity continues to be unraveled, we aim to develop a prognostic gene signature that integrates lipid metabolism and immune features for patients with lung adenocarcinoma (LUAD). Methods: First, differentially expressed genes (DEGs) related to lipid metabolism in LUAD were detected, and subgroups of LUAD patients were identified via the unsupervised clustering method. Based on lipid metabolism and immune-related DEGs, variables were determined by the univariate Cox and LASSO regression, and a prognostic signature was established. The prognostic value of the signature was evaluated by the Kaplan-Meier method, time-dependent ROC, and univariate and multivariate analyses. Five independent GEO datasets were employed for external validation. Gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), and immune infiltration analysis were performed to investigate the underlying mechanisms. The sensitivity to common chemotherapeutic drugs was estimated based on the GDSC database. Finally, we selected PSMC1 involved in the signature, which has not been reported in LUAD, for further experimental validation. Results: LUAD patients with different lipid metabolism patterns exhibited significant differences in overall survival and immune infiltration levels. The prognostic signature incorporated 10 genes and stratified patients into highand low-risk groups by median value splitting. The areas under the ROC curves were 0.69 (1-year), 0.72 (3-year), 0.74 (5-year), and 0.74 (10-year). The Kaplan-Meier survival analysis revealed a significantly poorer overall survival in the high-risk group in the TCGA cohort (p < 0.001). In addition, both univariate and multivariate Cox regression analyses indicated that the prognostic model was the individual factor affecting the overall survival of LUAD patients. Through GSEA and GSVA, we found that tumor progression and inflammatory and immune-related pathways were enriched in the high-risk group. Additionally, patients with high-risk scores showed higher sensitivity to chemotherapeutic drugs. The in vitro experiments further confirmed that PSMC1 could promote the proliferation and migration of LUAD cells. Conclusions: We developed and validated a novel signature incorporating both lipid metabolism and immune-related genes for all-stage LUAD patients. This signature can be applied not only for survival prediction but also for guiding personalized chemotherapy and immunotherapy regimens. [ABSTRACT FROM AUTHOR]

Background. Pancreatic adenocarcinoma (PAAD) is among the most lethal diseases and has a dismal prognosis; however, efficient treatment is currently limited. Several studies have observed epigenetic variation during tumorigenesis, suggesting the poten- tial role of RNA methylation, especially N6-methyladenosine (m6A) modification, as a novel epigenetic modification mediating PAAD prognosis. Methods. The expression levels of m6A-related genes were downloaded from The Cancer Genome Atlas-Pancreatic Adenocarcinoma (TCGA) and Genotype-Tissue Expression (GTEx) projects, and the findings were validated in four Expression Omnibus (GEO) datasets. A predictive model was constructed using a lasso regression and evaluated by a survival analysis and receiver operating characteristic curve. Consensus clustering identified two distinct subgroups with different immune activity signatures based on the expression pattern of m6A-related genes. The relationship between the mutation state of m6A-related genes and infiltration of immune cells was established and visualized using Tumor Immune Estimation Resource (https: //cistrome.shinyapps.io/timer/). Results. Fourteen of twenty-one m6A-related genes were differentially expressed between PAAD and normal tissues in TCGA-GTEx cohort. Among these genes, HNRNPC, IGF2BP2 and YTHDF1 were further validated in four GEO datasets. Moreover, an m6A-based model exhibited moderate accuracy in predicting overall survival in PAAD samples. Additionally, potential m6A modification targets were screened by selecting genes from a set of 23,391 genes that not only harbored the most m6A-modified sites but also showed a robust correlation with PAAD survival. Moreover, we correlated the expression level of m6A-related genes with the immune microenvironment of pancreatic cancer for the first time. Specifically, both arm-level gain and deletion of ALKBH5 decreased the infiltration of CD8+T cells (P <0:05 and P <0:01, respectively). Conclusion. Collectively, our findings suggest a novel anticancer strategy for restoring balanced RNA methylation in tumor cells and guide clinical physicians in developing a new practical approach for considering the impact of related genes on prognosis. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) is one of the most common and lethal types of cancer within the urinary system. Great efforts have been made to elucidate the pathogeny. However, the molecular mechanism of ccRCC is still not well understood. The aim of this study is to identify key genes in the carcinogenesis and progression of ccRCC. The mRNA microarray dataset GSE53757 was downloaded from the Gene Expression Omnibus database. The GSE53757 dataset contains tumor and matched paracancerous specimens from 72 ccRCC patients with clinical stage I to IV. The linear model of microarray data (limma) package in R language was used to identify differentially expressed genes (DEGs). The protein–protein interaction (PPI) network of the DEGs was constructed using the search tool for the retrieval of interacting genes (STRING). Subsequently, we visualized molecular interaction networks by Cytoscape software and analyzed modules with MCODE. A total of 1,284, 1,416, 1,610 and 1,185 up-regulated genes, and 932, 1,236, 1,006 and 929 down-regulated genes were identified from clinical stage I to IV ccRCC patients, respectively. The overlapping DEGs among the four clinical stages contain 870 up-regulated and 645 down-regulated genes. The enrichment analysis of DEGs in the top module was carried out with DAVID. The results showed the DEGs of the top module were mainly enriched in microtubule-based movement, mitotic cytokinesis and mitotic chromosome condensation. Eleven up-regulated genes and one down-regulated gene were identified as hub genes. Survival analysis showed the high expression of CENPE, KIF20A, KIF4A, MELK, NCAPG, NDC80, NUF2, TOP2A, TPX2 and UBE2C, and low expression of ACADM gene could be involved in the carcinogenesis, invasion or recurrence of ccRCC. Literature retrieval results showed the hub gene NDC80, CENPE and ACADM might be novel targets for the diagnosis, clinical treatment and prognosis of ccRCC. In conclusion, the findings of present study may help us understand the molecular mechanisms underlying the carcinogenesis and progression of ccRCC, and provide potential diagnostic, therapeutic and prognostic biomarkers. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the pattern of distribution of mating type (MAT) genes of Tuber indicum in ectomycorhizosphere soils from natural T. indicum-producing areas and cultivated truffle orchards and ascocarp samples from different regions. Quantitative real-time PCR and multiplex PCR were used to weight the copy numbers of MAT1-1-1 and MAT1-2-1 in natural truffle soils and cultivated orchard soils. The effect of limestone on the pattern of truffle MAT genes and the correlation between soil properties and the proportion of MAT genes were also assessed. These results indicated that an uneven and nonrandom distribution of MAT genes was common in truffle-producing areas, cultivated truffle orchards, and ascocarps gleba. The competition between the two mating type genes and the expansion of unbalanced distribution was found to be closely related to truffle fructification. Limestone treatments failed to alter the proportion of the two mating type genes in the soil. The content of available phosphorus in soil was significantly correlated with the value of MAT1-1-1/MAT1-2-1 in cultivated and natural ectomycorhizosphere soils. The application of real-time quantitative PCR can provide reference for monitoring the dynamic changes of mating type genes in soil. This study investigates the distributional pattern of T. indicum MAT genes in the ectomycorhizosphere soil and ascocarp gleba from different regions, which may provide a foundation for the cultivation of T. indicum. [ABSTRACT FROM AUTHOR]

Background: Uncovering mechanisms of epigenome evolution is an essential step towards understanding the evolution of different cellular phenotypes. While studies have confirmed DNA methylation as a conserved epigenetic mechanism in mammalian development, little is known about the conservation of tissue-specific genome-wide DNA methylation patterns. Results: Using a comparative epigenomics approach, we identified and compared the tissue-specific DNAmethylation patterns of rat against those of mouse and human across three shared tissue types. We confirmed that tissue-specific differentially methylated regions are strongly associated with tissue-specific regulatory elements. Comparisons between species revealed that at a minimum 11-37% of tissue-specific DNA methylation patterns are conserved, a phenomenon that we define as epigenetic conservation. Conserved DNA methylation is accompanied by conservation of other epigenetic marks including histone modifications. Although a significant amount of locus-specific methylation is epigenetically conserved, the majority of tissue-specific DNA methylation is not conserved across the species and tissue types that we investigated. Examination of the genetic underpinning of epigenetic conservation suggests that primary sequence conservation is a driving force behind epigenetic conservation. In contrast, evolutionary dynamics of tissue-specific DNA methylation are best explained by the maintenance or turnover of binding sites for important transcription factors. Conclusions: Our study extends the limited literature of comparative epigenomics and suggests a new paradigm for epigenetic conservation without genetic conservation through analysis of transcription factor binding sites. [ABSTRACT FROM AUTHOR]

Allelic variation in puroindoline genes determines the hardness of the important quality traits in wheat grain. In this study, a simple sequence repeat (SSR) SSR 7 was chosen from the puroindoline genome sequence. It resided at 495 bp before the start codon of Pina. The repeat number of SSR 7 was different in different types of common wheat and Ae. tauschii. Four repeat types existed in 34 wheat cultivars with the repeat numbers 16, 18, 19 and 20, respectively. Among the 24 classes of materials of Ae. tauschii, only two repeat types, 6 and 10 times, could be found. Moreover, common wheat cultivars had one or two 'AC's more than Ae. tauschii. Except for AS 61 and AS 81, a 13 bps deletion appeared at 165 bp upstream of the SSR 7 site of the rest varieties of Ae. tauschii. The segment sizes of SSR 7 PCR product in common wheat were predicted from 154-182 bps, with the segment size not linked closely to puroindoline genotype. The allelic variations existing at SSR 7 loci can provide the theory basis for marker-assisted selection breeding in wheat hardness. [ABSTRACT FROM AUTHOR]

A member of the NF-ΚB signaling pathway, PoAkirin1, was cloned from a full-length cDNA library of Japanese flounder (Paralichthys olivaceus). The full-length cDNA comprises a 5'UTR of 202 bp, an open reading frame of 564 bp encoding a 187-amino-acid polypeptide and a 521-bp 3'UTR with a poly (A) tail. The putative protein has a predicted molecular mass of 21 kDa and an isoelectric point (pI) of 9.22. Amino acid sequence alignments showed that PoAkirin1 was 99% identical to the Scophthalmus maximus Akirin protein (ADK27484). Yeast two-hybrid assays identified two proteins that interact with PoAkirin1: PoHEPN and PoC1q. The cDNA sequences of PoHEPN and PoC1q are 672 bp and 528 bp, respectively. Real-time quantitative reverse-transcriptase polymerase chain reaction analysis showed that bacteria could induce the expressions of PoAkirin1, PoHEPN and PoC1q. However, the responses of PoHEPN and PoC1q to the bacterial challenge were slower than that of PoAkirin1. To further study the function of PoAkirin1, recombinant PoAkirin1 and PoHEPN were expressed in Escherichia coli and would be used to verify the PoAkirin1-PoHEPN binding activity. These results identified two proteins that potentially interact with PoAkirin1 and that bacteria could induce their expression. [ABSTRACT FROM AUTHOR]

OBJECTIVES:The prognosis of pancreatic cancer is still very poor, and the ability to detect pancreatic cancer in high-risk groups at an early stage is therefore essential for improving its long-term survival. The purpose of this study was to explore specific biomarkers that can differentiate pancreatic cancer–associated diabetes from type 2 diabetes, for the early detection of pancreatic cancer.METHODS:From January 2006 to July 2008, 102 peripheral blood samples were collected from 25 patients diagnosed with pancreatic cancer and diabetes, 27 patients with pancreatic cancer without diabetes, 25 patients with diabetes mellitus >5 years, and 25 healthy controls. Thirty-two samples were used in microarray experiments to find differentially expressed genes specific for pancreatic cancer–associated diabetes. The results were further validated by quantitative real-time PCR for 101 blood samples. Protein expression of selected genes in serum and tissues was also detected.RESULTS:Using microarray analysis, we found 58 genes to be unique in patients with pancreatic cancer–associated diabetes, including 23 upregulated genes and 35 downregulated genes. Eleven upregulated genes were further validated by RT-PCR, and two of these genes—vanin-1 (VNN1) and matrix metalloproteinase 9 (MMP9)—were selected for logistic regression analysis. The combination of VNN1 and MMP9 showed the best discrimination of pancreatic cancer–associated diabetes from type 2 diabetes. The protein expression of MMP9 and VNN1 was in accordance with the gene expression.CONCLUSIONS:Our results indicate that the combination of VNN1 and MMP9 may be used as a novel blood biomarker panel for the discrimination of pancreatic cancer–associated diabetes from type 2 diabetes. [ABSTRACT FROM AUTHOR]

Objectives: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL). Methods: QMSP was performed in normal cervix, low-grade (L)SIL, high-grade (H)SIL, adenocarcinomas and squamous cell cervical cancers, and in corresponding cervical scrapings. Results: Only CCNA1 was never methylated in normal cervices and rarely in LSILs. All other genes showed methylation in normal cervices, with CALCA, SPARC and RAR-β2 at high levels. Methylation frequency of 6 genes (DAPK, APC, TFPI2, SPARC, CCNA1 and CADM1) increased with severity of the underlying cervical lesion. DAPK showed the highest increase in methylation frequency between LSIL and HSIL (10% vs. 40%, p<0.05), while CCNA1 and TFPI2 were most prominently methylated in cervical cancers compared to HSILs (25% vs. 52%, p<0.05, 30% vs. 58%, p<0.05). CADM1 methylation in cervical cancers was related to depth of invasion (p<0.05) and lymph vascular space involvement (p<0.01), suggesting a role in invasive potential of cervical cancers. Methylation ratios in scrapings reflected methylation status of the underlying lesions (p<0.05). Conclusion: Methylation of previously reported cervical cancer specific genes frequently occurs in normal epithelium. However, frequency of methylation increases during cervical carcinogenesis, with CCNA1 and DAPK as the best markers to distinguish normal/LSIL from HSIL/cancer lesions. [ABSTRACT FROM AUTHOR]

Background: Small noncoding RNAs (ncRNAs), including short interfering RNAs (siRNAs) and microRNAs (miRNAs), can silence genes at the transcriptional, post-transcriptional or translational level [1, 2]. Results: Here, we show that microRNA-10a (miR-10a) targets a homologous DNA region in the promoter region of the hoxd4 gene and represses its expression at the transcriptional level. Mutational analysis of the miR-10a sequence revealed that the 3' end of the miRNA sequence is the most critical element for the silencing effect. MicroRNA-10a-induced transcriptional gene inhibition requires the presence of Dicer and Argonautes 1 and 3, and it is related to promoter associated noncoding RNAs. Bisulfite sequencing analysis showed that the reduced hoxd4 expression was accompanied by de novo DNA methylation at the hoxd4 promoter. We further demonstrated that trimethylation of histone 3 lysine 27 (H3K27me3) is involved in the miR-10a-induced hoxd4 transcriptional gene silence. Conclusion: In conclusion, our results demonstrate that miR-10a can regulate human gene expression in a transcriptional manner, and indicate that endogenous small noncoding RNA-induced control of transcription may be a potential system for expressional regulation in human breast cancer cells. [ABSTRACT FROM AUTHOR]

J05 soybean was previously identified to carry 2 independent genes, Rsv1 and Rsv3, for "soybean mosaic virus" (SMV) resistance by inheritance and allelism studies. The objective of this research was to confirm the 2 genes in J05 using molecular markers so that a marker-assisted selection can be implemented. The segregation of F2 plants from J05 x Essex exhibited a good fit to a 3:1 ratio when inoculated with SMV Gi. Three simple sequence repeat (SSR) markers near Rsv1, Satt114, Satt510, and Saṯ 154, amplified polymorphic DNA fragments between J05 and Essex and were closely linked to the gene on soybean molecular linkage group (MLG) F, thus verifying the presence of Rsv1 in J05 for resistance to SMV G1. The presence of Rsv1 in J05 was confirmed by 2 closely linked SSR markers on MLG B2, Satt726 and Saṯ424, in F2:3 lines that were derived from the SMV GI-susceptible F2 plants and segregated in a 1:2:1 ratio for reaction to SMV G7. Two closely linked markers for Rsv4, Satt296 and Satt542, segregated independently of SMV resistance, indicating the absence of Rsv4 inJO5. These SSR markers for Rsvl and Rsv3 can serve as a useful molecular tool for selection and pyramiding of genes in J05 for SMV resistance. [ABSTRACT FROM AUTHOR]

The gene cluster involved in the choline-glycine betaine conversion pathway was cloned from chromosomal DNA of the Gram-positive moderate halophile Halobacillus dabanensis D-8T. Nucleotide sequence analysis revealed four genes, designated gbsT, gbsI, gbsA, and gbsB, which are clustered in a 5.1-kb fragment. After heterologous expression of gbsAB in the Escherichia coli mutant strain PD141, the transformed cells were able to grow in a selective M63 medium containing 0.7 M NaCl and 1 mM choline, in contrast to the mutant strain. Glycine betaine biosynthesis was restored and its accumulation was confirmed by using 13C nuclear magnetic resonance spectroscopy. [ABSTRACT FROM AUTHOR]

Gene families associated with the ethylene signal transduction pathway in ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) were isolated from a kiwifruit expressed sequence tag (EST) database, including five ethylene receptor genes, two CTR1-like genes, and an EIN3-like gene AdEIL1. All were differentially expressed among various kiwifruit vine tissues, and none was fruit specific. During fruit development, levels of transcripts of AdERS1a, AdETR3, and the two CTR1-like genes decreased, whereas those of AdERS1b and AdETR2 peaked at 97 d after full bloom. In ripening kiwifruit, there was a diverse response of the ethylene receptor family to internal and external ethylene. AdERS1a, AdETR2, and AdETR3 expression increased at the climacteric stage and transcripts were induced by external ethylene treatment, while AdERS1b showed no response to ethylene. AdETR1 was negatively regulated by internal and external ethylene in ripening fruit. The two CTR1-like genes also had different expression patterns, with AdCTR1 increasing at the climacteric stage and AdCTR2 undergoing little change. 1-Methylcyclopropene treatment prevented the ethylene response of all components, but transient down-regulation was only found with AdETR2 and AdCTR1. Similar gene and ethylene responses were found in both fruit flesh and core tissues. The ethylene-induced down-regulation of AdETR1 suggests that it may have a role in sensing ethylene and transmitting this response to other members of the receptor family, thus activating the signal transduction pathway. [ABSTRACT FROM PUBLISHER]

A mercaptoacetic acid (MAA)-modified cadmium sulfide (CdS) nanoparticle was synthesized in aqueous solution and used as an oligonucleotide label for the electrochemical detection of nopaline synthase (NOS) terminator gene sequence. The carboxyl groups on the surface of the CdS nanoparticle can be easily covalently linked with NH2-modified NOS oligonucleotide probe sequences. The target ssDNA sequence was fixed onto the electrode surface by covalently linking to a mercaptoethanol self-assembled gold electrode, and the DNA hybridization of target ssDNA with probe ssDNA was accomplished on the electrode surface. The CdS nanoparticles anchored on the hybrids were dissolved in the solution by the oxidation with HNO3 and further detected by a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used for monitoring the hybridization, and the NOS target sequence was satisfactorily detected in the approximate range from 8.0 × 10−12 to 4.0 × 10−9 mol L−1 with a detection limit of 2.75 × 10−12 mol L−1 (3 σ). The established method extended the nanoparticle-labeled electrochemical DNA analysis to genetically modified organisms (GMOs) specific sequence samples with higher sensitivity and selectivity. [ABSTRACT FROM AUTHOR]

Using a combination of techniques we developed, we infected zebrafish embryos using pseudotyped retroviruses and mapped the genomic locations of the proviral integrations in the F1 off- spring of the infected fish. From F1 fish, we obtained 2,045 sequences representing 933 unique retroviral integrations. A total of 599 were mappable to the current genomic assembly (Zv6), and 233 of the integrations landed within genes. By inbreeding fish carrying proviral integrations in 25 different genes, we were able to demonstrate that in ∼50% of the gene ‘hits,’ the mRNA transcript levels were reduced by ⩾70%, with the highest probability for mutation occurring if the integration was in an exon or first intron. Based on these data, the mutagenic frequency for the retrovirus is nearly one in five integrations. In addition, a strong mutagenic effect is seen when murine leukemia virus integrates specifically in the first intron of genes but not in other introns. Three of 19 gene inactivation events had embryonic defects. Using the strategy we outlined, it is possible to identify 1 mutagenic event for every 30 sequencing reactions done on the F1 fish. This is a 20- to 30-fold increase in efficiency when compared with the current resequencing approach [targeting induced local lesions in genomes (TILLING)] used in zebrafish for identifying mutations in genes. Combining this increase in efficiency with cryopreservation of sperm samples from the F1 fish, it is now possible to create a stable resource that contains mutations in every known zebrafish gene. [ABSTRACT FROM AUTHOR]

By using Southern blot hybridization and inverse polymerase chain reaction, a 5.5-kb DNA fragment was obtained from the genomic DNA of Halobacillus trueperi DSM10404T. Sequence analysis revealed that it contained a potential operon with high levels of sequence similarity to the opuA operon encoding glycine betaine transporter from Bacillus subtilis, which is a member of the ATP-binding cassette (ABC) substrate binding the protein-dependent transporter superfamily. The potential operon, designated as qatA (quaternary amine transporter), consists of three structural genes, which are predicted to encode an ATP-binding protein (QatAA), a membrane-associated protein (QatAB), and an extracellular substrate-binding protein (QatAC). Moreover, the putative promoter region of the operon was found with close homology to the σA-dependent promoter of B. subtilis. Reverse transcription (RT)–PCR analysis revealed that qatAA, qatAB, and qatAC genes were transcribed in cells of H. trueperi. Cells of Escherichia coli mutant MKH13 harboring qatA on pAY41 were able to grow on selective M9 salt medium containing glycine betaine and accumulated glycine betaine in the cytoplasm, showing that qatAA, qatAB, and qatAC genes together encode a functional glycine betaine transporter. [ABSTRACT FROM AUTHOR]

By using Southern blot hybridization and inverse polymerase chain reaction, a 5.5-kb DNA fragment was obtained from the genomic DNA of Halobacillus trueperi DSM10404T. Sequence analysis revealed that it contained a potential operon with high levels of sequence similarity to the opuA operon encoding glycine betaine transporter from Bacillus subtilis, which is a member of the ATP-binding cassette (ABC) substrate binding the protein-dependent transporter superfamily. The potential operon, designated as qatA (quaternary amine transporter), consists of three structural genes, which are predicted to encode an ATP-binding protein (QatAA), a membrane-associated protein (QatAB), and an extracellular substrate-binding protein (QatAC). Moreover, the putative promoter region of the operon was found with close homology to the σA-dependent promoter of B. subtilis. Reverse transcription (RT)–PCR analysis revealed that qatAA, qatAB, and qatAC genes were transcribed in cells of H. trueperi. Cells of Escherichia coli mutant MKH13 harboring qatA on pAY41 were able to grow on selective M9 salt medium containing glycine betaine and accumulated glycine betaine in the cytoplasm, showing that qatAA, qatAB, and qatAC genes together encode a functional glycine betaine transporter. [ABSTRACT FROM AUTHOR]

This paper describes the development of single-domain recombinant antibodies against human telomerase core protein. A His-tagged hTERT spanning main reverse-transcriptase domain of hTERT was purified from host E. coli and used to immunize BALB/c mice. The VHs (heavy chain variable region genes) were amplified by PCR from total RNA of splenocytes and further induced random mutagenesis by DNA shuffling to enrich the repertoire of VH library. All VHs were cloned into phagemid vectors and displayed to generate 4 × 1010 phage libraries. The candidates carrying VH domains against hTERT were primarily screened through three times of panning procedure on His-tagged hTERT coated microplates, and specific antibodies were further selected by West-Western blot. Two clones, designated as a3 and b8, were confirmed to interact with the target in the solid-phase assay. DNA sequencing proved their mouse VH origin. The purified single-domain antibody of b8 could not only recognize native hTERT, but also neutralize human telomerase activity on inhibitory assay and b8 showed the stronger suppressive efficacy compared with a3. The data demonstrated that the developed single-domain recombinant antibodies were hTERT-specific with high potential of binding and activity inhibition. [ABSTRACT FROM AUTHOR]

Metal-responsive transcription factor 1 (MTF-1) specifically binds to metal response elements (MREs) associated with a number of metal- and stress-responsive genes. Human MTF-1 contains a cysteine-rich cluster, -632Cys-Gln-Cys-Gln-Cys-Ala-Cys638-, conserved from pufferfish to humans far removed from the MRE-binding zinc finger domain and just C-terminal to a previously mapped serine/threonine-rich transcriptional activation domain. MTF-1 proteins containing two Cys→Ala substitutions (C632A/C634A) or a deletion in this region altogether (Δ(632-644)) are significantly impaired in their ability to induce Zn(II)- and Cd(II)-responsive transcription of a MRE-linked reporter gene in transiently transfected mouse dko7 (MTF-1-/-) cells in culture under moderate metal stress but retain the ability to drive basal levels of transcription in a MRE-dependent manner in vivo and in vitro. In addition, the mutated proteins respond to induction by Zn(II) or Cd(II) with nuclear translocation and MRE binding activities comparable with wild-type MTF-1. Attempts to rescue the Δ(632-644) deletion mutant phenotype by insetting similar Cys-rich sequences from Drosophila MTF-1 were unsuccessful, suggesting that the structure of this motif within intact human MTF-1, rather than the simple presence of multiple closely spaced Cys residues, is required for function. This cysteine cluster therefore functions at a step subsequent to nuclear translocation and MRE-binding DNA to naked promoter-containing DNA and appears to be specifically required for MTF-1 to activate transcription in the presence of inducing heavy metal ions. [ABSTRACT FROM AUTHOR]

RFX6 transcription factor is believed to play a central role in directing cell development of insulin-producing pancreatic islet. RFX6 homozygous mutations cause syndromic neonatal diabetes with hypoplastic pancreas. However, RFX6 heterozygous mutations cause maturityonset diabetes of the young (MODY) with normal pancreas development. Here, we show that RFX6 may control islet cell development and insulin production in different manners. The rfx6 knockout zebrafish generated by CRISPR/Cas9 exhibited an overt diabetes phenotype. Pancreatic islet failed to form compact structures in the knockout fish. While endocrine pancreatic islet non-ß-cells were absent, insulin-producing ß-cells were present in the knockout fish. Although insulin mRNA level was normal in the ß-cells of the knockout fish, insulin protein level was decreased. High-throughput RNA sequencing (RNAseq) showed that differentially expressed genes were enriched in the translation term in islet ß-cells from the knockout fish. Chromatin immunoprecipitation sequencing (ChIPseq) of normally developed islet ß-cells from mice demonstrated that rfx6 interacted with translation initiation factors and controlled insulin translation. Our data indicate that Rfx6 may act as a transcription factor regulating the transcription of genes involved in mRNA translation, which may represent a new mechanism and treatment strategy for diseases. [ABSTRACT FROM AUTHOR]

The gene cluster involved in the choline-glycine betaine conversion pathway was cloned from chromosomal DNA of the Gram-positive moderate halophile Halobacillus dabanensis D-8T. Nucleotide sequence analysis revealed four genes, designated gbsT, gbsI, gbsA, and gbsB, which are clustered in a 5.1-kb fragment. After heterologous expression of gbsAB in the Escherichia coli mutant strain PD141, the transformed cells were able to grow in a selective M63 medium containing 0.7 M NaCl and 1 mM choline, in contrast to the mutant strain. Glycine betaine biosynthesis was restored and its accumulation was confirmed by using 13C nuclear magnetic resonance spectroscopy. [ABSTRACT FROM AUTHOR]

Objective To investigate the H2O2-induced expression of human histone acetyltransferase-like protein (hALP), a telomerase regulation-associated gene, and its effects on the stress-triggered cellular senescence. Methods The induced expression of hALP was measured by semi-quantitative RT-PCR and immunofluorescent histochemistry after treatment of HeLa cells by H2O2. The effects of hALP expression on cellular responses to H2O2 were analyzed by MTT, flow cytometry, and SA-β-gal staining, respectively. Results hALP mRNA could be dose-dependently induced by treatments of 0. 2–1.6 mmol/L H2O2, and the induction could be observed after 6 hours and kept for 36 hours in the presence of 0.4 mmol/L H2O2. Meanwhile, the immunofluorescent staining showed marked stronger nuclear intensity of hALP protein in H2O2-treated HeLa cells. In the treatment of H2O2, the ectopic expression of hALP enhanced continuous growth and overcame G2/M arrest as well as decreased senescence-associated β-gal staining. On the contrary, the transfected clones with antisense or blank vector and original HeLa cells presented growth suppression, G2/M delay and higher percentage of SA-β-gal activities in the presence of H2O2. Conclusions The expression of hALP could be up-regulated by treatment of H2O2, and elevated expression could enhance cellular resistance to H2Oz-induced cellular senescence. The data might be of references to elucidation of basic biological function of hALP gene and its associated telomerase activity. [ABSTRACT FROM AUTHOR]