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At least 577 records · Page 32

Conserved gene clusters in bacterial genomes provide further support for the primacy of RNA

Five complete bacterial genome sequences have been released to the scientific community. These include four (eu)Bacteria, Haemophilus influenzae, Mycoplasma genitalium, M. pneumoniae, and Synechocystis PCC 6803, as well as one Archaeon, Methanococcus jannaschii. Features of organization shared by these genomes are likely to have arisen very early in the history of the bacteria and thus can be expected to provide further insight into the nature of early ancestors. Results of a genome comparison of these five organisms confirm earlier observations that gene order is remarkably unpreserved. There are, nevertheless, at least 16 clusters of two or more genes whose order remains the same among the four (eu)Bacteria and these are presumed to reflect conserved elements of coordinated gene expression that require gene proximity. Eight of these gene orders are essentially conserved in the Archaea as well. Many of these clusters are known to be regulated by RNA-level mechanisms in Escherichia coli, which supports the earlier suggestion that this type of regulation of gene expression may have arisen very early. We conclude that although the last common ancestor may have had a DNA genome, it likely was preceded by progenotes with an RNA genome.

Non-NASA Center↗

Data and programs in support of network analysis of genes and their association with diseases

The network-based approaches that were employed in order to depict the relationships between human genetic diseases and their associated genes are described. Towards this direction, monopartite disease-disease and gene-gene networks were constructed from bipartite gene-disease association networks. The latter were created by collecting and integrating data from three diverse resources, each one with different content, covering from rare monogenic disorders to common complex diseases. Moreover, topological and clustering graph analyses were performed. The methodology and the programs presented in this article are related to the research article entitled “Network analysis of genes and their association with diseases”.

Disease-disease networks↗

Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

NASA Discipline Radiation Health↗

Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

Abstract Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1 : sgRNA design Basic Protocol 2 : Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors Basic Protocol 3 : Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4 : ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales Support Protocol 1 : Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2 : Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3 : Transformation of E. coli by electroporation Support Protocol 4 : Transformation of CaCl 2 ‐competent E. coli

59 BASIC BIOLOGICAL SCIENCES↗

In–context promoter bashing of the Sorghum bicolor gene models functionally annotated as bundle sheath cell preferred expressing phosphoenolpyruvate carboxykinase and alanine aminotransferase

In-context promoter bashing via genome editing is a route to identify and characterize critical regulatory regions that govern expression of genes of interest. The outcomes of in-context promoter bashing can be used to inform editing strategies to modulate the expression of selected gene models in a desired fashion. Here, we employed in-context promoter bashing to characterize the proximal upstream regulatory regions of sorghum genes encoding phosphoenolpyruvate carboxykinase bundle sheath (SbPEPCK.BS, SbiTx430.01G455400) and alanine aminotransferase bundle sheath (SbAlaAT.BS, SbiTx430.02G006600), two proteins involved in the PCK C 4 pathway. Characterized germinal edits within the targeted regions upstream of these two genes ranged in size from 138 up to 1790 bp. A 138 bp within the SbPEPCK.BS upstream region and a 1643 bp element within the SbAlaAT.BS upstream region were determined to be important for maintenance of transcription levels. No change in development or various physiological parameters was observed in characterized lineages carrying promoter edits. However, significant changes in seed reserves and a reduction in 100-seed weight were consistently observed, under both greenhouse and field environments, in plants carrying an edit in the promoter of SbPEPCK.BS gene, which were significantly reduced in transcript accumulation for this gene.

60 APPLIED LIFE SCIENCES↗

Functional analysis of Salix purpurea genes support roles for ARR17 and GATA15 as master regulators of sex determination

Abstract The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regulating sex in the genus Salix have been limited to genome resequencing and differential gene expression, which are mostly descriptive in nature, and functional validation of candidate sex determination genes has not yet been conducted. Here, we used Arabidopsis to functionally characterize a suite of previously identified candidate genes involved in sex determination and sex dimorphism in the bioenergy shrub willow Salix purpurea . Six candidate master regulator genes for sex determination were heterologously expressed in Arabidopsis, followed by floral proteome analysis. In addition, 11 transcription factors with predicted roles in mediating sex dimorphism downstream of the SDR were tested using DAP‐Seq in both male and female S. purpurea DNA. The results of this study provide further evidence to support models for the roles of ARR17 and GATA15 as master regulator genes of sex determination in S. purpurea , contributing to a regulatory system that is notably different from that of its sister genus Populus . Evidence was also obtained for the roles of two transcription factors, an AP2 / ERF family gene and a homeodomain‐like transcription factor, in downstream regulation of sex dimorphism.

60 APPLIED LIFE SCIENCES↗

A high-capacity gene stacking toolkit for the oleaginous microalga, Nannochloropsis oceanica CCMP1779

Nannochloropsis oceanica CCMP1779 is an oleaginous heterokont microalga that is particularly amenable to synthetic biology approaches. The underlying complex genetic engineering often requires the expression of multiple transgenes (gene stacking). We integrated several techniques into a vector toolkit for multi-gene expression, including: multiple resistance marker genes, bidirectional promoters, and assembly of multiple expression cassettes into a single vector. We developed a series of gateway entry and destination vectors for assembly of two bidirectional promoter expression cassettes, which facilitates combinatorial high-capacity gene stacking. Several endogenous bidirectional promoters were demonstrated to be effective for driving transgene expression, with a dual-luciferase reporter system used to monitor transgene expression under different environmental conditions. The bidirectional promoter between the nitrate reductase and the nitrate transporter genes drives nitrogen source dependent transcription, enabling conditional transgene expression. Lethal concentrations of the antibiotics, Blasticidin, G418, and Nourseothricin, were determined and the corresponding marker genes used for genomic transformation selection.

59 BASIC BIOLOGICAL SCIENCES↗

A tomato proline-, lysine-, and glutamic-rich type gene SpPKE1 positively regulates drought stress tolerance

Highlights: • Overexpression of SpPKE1 confers tomato and tobacco tolerance to drought stress. • SpPKE1 modulate the expressions of stress-responsive genes. • SpPKE1 interacts with an F-box protein in yeast. Plant abiotic resistance in cultivated species features limited variability. Using genes of wild species serves as a valid approach for improving abiotic resistance of cultivated plants. In this study, we uncovered a previously uncharacterized proline-, lysine-, and glutamic-rich protein gene (SpPKE1), which was isolated from drought-resistant wild tomato species Solanum pennellii (LA0716). When M82, which is a drought-sensitive tomato cultivar, was engineered to overexpress SpPKE1, its tolerance under drought stress was significantly improved by the accumulation of more chlorophyll, proline, and limited malondialdehyde compared with that in RNA interference (RNAi)-suppression lines, which were more sensitive than the wild-type plants. Several ion transporter genes, abiotic-related transcriptional factors, and reactive oxygen species-scavenging genes were upregulated in PKE1 overexpression (OE) lines but downregulated in RNAi plants. OE of SpPKE1 enhanced drought tolerance in tobacco. Screening results of yeast two-hybrid protein–protein interaction revealed that SpPKE1 can bind to an F-box protein that plays an important role in plant drought resistance. We posited that PKE1 enhanced drought tolerance by modulating the expressions of stress-responsive genes and interacting with the F-box protein.

60 APPLIED LIFE SCIENCES↗

Role of acid responsive genes in the susceptibility of Escherichia coli to ciclopirox

Highlights: • Ciclopirox antibacterial activity was analysed by genome-wide expression profiling. • Ciclopirox affects expression of glutamate-dependent acid resistance pathway genes. • Down-regulation of hns transcription likely increases ciclopirox activity. • Susceptibility to ciclopirox accompanies increased cell size and decreased motility. • The findings identify probable pathways for ciclopirox action against E. coli. Antibiotic resistance poses a huge threat to the effective treatment of bacterial infections. To circumvent the limitations in developing new antibiotics, researchers are attempting to repurpose pre-developed drugs that are known to be safe. Ciclopirox, an off-patent antifungal agent, inhibits the growth of Gram-negative bacteria, and genes involved in galactose metabolism and lipopolysaccharide (LPS) biosynthesis are plausible antibacterial targets for ciclopirox, since their expression levels partially increase susceptibility at restrictive concentrations. In the present study, to identify new target genes involved in the susceptibility of Escherichia coli to ciclopirox, genome-wide mRNA profiling was performed following ciclopirox addition at sublethal concentrations, and glutamate-dependent acid resistance (GDAR) genes were differentially regulated. Additional susceptibility testing, growth analyses and viability assays of GDAR regulatory genes revealed that down-regulation of evgS or hns strongly enhanced susceptibility to ciclopirox. Further microscopy and phenotypic analyses revealed that down-regulation of these genes increased cell size and decreased motility. Our findings could help to maximise the efficacy of ciclopirox against hard-to-treat Gram-negative pathogens.

60 APPLIED LIFE SCIENCES↗

Long noncoding RNA HOTTIP cooperates with CCCTC-binding factor to coordinate HOXA gene expression

Highlights: • HOTTIP is required for activation of distal HOXA genes but its role is restricted by CTCF boundary. • HOTTIP and CTCF coordinate to specify the pattern of histone modifications on HOXA genes in foreskin fibroblasts. • HOTTIP RNA acts as a new interaction partner of CTCF. The spatiotemporal control of HOX gene expression is dependent on positional identity and often correlated to their genomic location within each loci. Maintenance of HOX expression patterns is under complex transcriptional and epigenetic regulation, which is not well understood. Here we demonstrate that HOTTIP, a lincRNA transcribed from the 5′ edge of the HOXA locus, physically associates with the CCCTC-binding factor (CTCF) that serves as an insulator by organizing HOXA cluster into disjoint domains, to cooperatively maintain the chromatin modifications of HOXA genes and thus coordinate the transcriptional activation of distal HOXA genes in human foreskin fibroblasts. Our results reveal the functional connection of HOTTIP and CTCF, and shed light on lincRNAs in gene activation and CTCF mediated chromatin organization.

60 APPLIED LIFE SCIENCES↗

Effect of cisplatin on the clock genes expression in the liver, heart and kidney

Highlights: • Cisplatin-induced nephrotoxicity showed a circadian rhythm. • Cisplatin altered the clock genes expression in a tissue- and gene-specific manner. • Dbp was consistently suppressed in peripheral tissues after cisplatin treatment. Cisplatin is a platinum-based chemotherapy drug that is widely used to treat various types of malignancies. Although the involvement of circadian clock in cisplatin metabolism and excretion has been reported, the effect of cisplatin on circadian rhythm remains unclear. In the present study, we investigated the effects of cisplatin on clock genes expression in mouse peripheral tissues. Cisplatin induced severe nephrotoxicity, as revealed by the significant increase of blood urea nitrogen and serum creatinine levels. Moreover, cisplatin circadian time-dependently induced p21 expression in the liver, heart and kidney, with the highest increase during the dark phase. In addition, cisplatin altered the clock genes expression in the liver, heart and kidney in a tissue- and gene-specific manner. Interesting, the expression of D site of the albumin promoter binding protein (Dbp), a gene involved in detoxification and drug metabolism, was consistently suppressed in the liver, heart and kidney after cisplatin treatment, implying a role of DBP in the toxicity of cisplatin.

60 APPLIED LIFE SCIENCES↗

BAF53A regulates androgen receptor-mediated gene expression and proliferation in LNCaP cells

The actin-like protein of the SWI/SNF complex, BAF53A, regulates gene expression by the gene-specific chromatin remodeling of target genes. However, the function of BAF53A in the androgen receptor pathway in prostate cancer cells remains unclear. Here, we demonstrated that BAF53A positively regulates the expression of endogenous AR target genes (e.g. PSA, TMPRSS2, FKBP5, and KLK2) in LNCaP cells. It functions as a coactivator in AR-mediated transcription by interacting with other nuclear receptor coactivators, such as p300 and FLII, and is associated with AR in the presence of dihydrotestosterone (DHT). The DHT-induced recruitment of BAF53A to the proximal and distal androgen response elements (AREs) of the PSA gene in the presence of BRG1 (but not BRM) was inhibited by an AR antagonist, suggesting the coactivator function of BAF53A in the SWI/SNF complex. Depletion of BAF53A in LNCaP cells resulted in a significant decrease in growth rate. Furthermore, the expression of BAF53A in prostate cancer tissue was significantly elevated, compared to that in normal prostate tissue, and correlated with the expression of AR, and BRG1, but not BRM. Therefore, our results suggested that BAF53A plays an important role in the expression of AR target genes in prostate cancer, and can be used clinically for the treatment of prostate cancer.

60 APPLIED LIFE SCIENCES↗

Expression profile analysis of differentially expressed genes in ruptured intracranial aneurysms: In search of biomarkers

Highlights: • Bioinformatics can identify potential biomarkers of ruptured intracranial aneurysm. • BASP1, CD74, CEBPB, ECHDC2, GZMK, HLA-DRB3, KLHL3, SLC2A3 & SOCS3 affect IA rupture. • DEGs are involved in antigen processing and presentation and immune response. • Vascular brain injury affects gene expression in IA tissue. Intracranial aneurysms (IAs) result from the bulging of arterial walls secondary to several factors such as flow, vessel morphology, and genetics. Subarachnoid hemorrhage occurs when such walls rupture, leading to high disability and mortality. Despite numerous investigations pertaining to the relationship between geometric characteristics and IA rupture, only a few have obtained consistent results. This study aimed to further identify the potential genes associated with the pathogenesis of IAs, which may provide novel molecular biomarkers. We downloaded and reanalyzed six datasets, which were divided into four groups. IA walls and blood samples were screened for differentially expressed genes (DEGs); then, functional and pathway enrichment analyses were conducted. In total, 158 common DEGs were identified from Groups 1–3 and 396 genes (187 upregulated and 209 downregulated genes) were differentially expressed in Group 4. The functional analysis revealed that the DEGs were mainly associated with the major histocompatibility complex class II protein complex and antigen processing and presentation. Finally, we identified nine key genes, both in aneurysm tissue samples and blood samples, of which three were mostly associated with the progression and rupture of IAs. Bioinformatics was used to analyze the datasets of the ruptured IAs and identify potential biomarkers, which may provide information for the early detection and treatment of IAs.

60 APPLIED LIFE SCIENCES↗

Genetics of gene expression in the aging human brain reveal TDP-43 proteinopathy pathophysiology

Genetic variants that control distant gene expression are highly relevant for disease mechanisms but remain poorly charaterized. Here, leveraging the modular structure of gene coexpression in the aging human brain, we performed genome-wide association studies of gene coexpression modules and identified TMEM106B and RBFOX1 as module quantitative trait loci (modQTLs). The TMEM106B modQTL is a known risk locus of neurodegenerative TDP-43 proteinopathy and has a large-scale dominant transcriptomic effect including lysosomal and splicing dysregulation, recapitulating TDP-43 pathophysiology. Further, a locus in another TDP-43 proteinopathy risk gene, GRN, shows similar transcriptomic effects. TMEM106B and APOE/amyloid-ß converge on altered myelination/lysosomal gene expression, and jointly promote limbic-predominant age-related TDP-43 encephalopathy neuropathological change. Thus, genetic architecture of distant gene expression in the aging human brain elucidates TDP-43 proteinopathy pathophysiology.

Yang, Hyun-Sik↗

Assessment of global and gene-specific DNA methylation in rat liver and kidney in response to non-genotoxic carcinogen exposure

Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC–MS/MS in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity. - Highlights: • Studied non-genotoxic carcinogens caused no change on global DNA hypomethylation. • d-Limonene, DCB and chloroform did not show any genome-wide methylation changes. • Some genes were differentially methylated in response to MPY, TCDD and OTA. • Protein kinase activity and mTOR cell signaling are involved in OTA carcinogenicity. • Our data highlight Cpne4 may be a potential biomarker for non-genotoxic carcinogens.

60 APPLIED LIFE SCIENCES↗

Identification of the UBA2-WTIP fusion gene in acute myeloid leukemia

Identifying and targeting oncogenic fusion genes have revolutionized the treatment of leukemia, such as PML-RARα fusion gene in acute promyelocytic leukemia. Here we identified an intrachromosomal fusion gene located on chromosome 19q.13 between UBA2 and WTIP gene in a case of acute myeloid leukemia. The UBA2-WTIP fusion gene contains the N-terminal E1{sub e}nzyme{sub f}amily, VAE{sub U}bl domains of UBA2, and the C-terminal LIM domains of WTIP. The UBA2-WTIP fusion was detected by reverse transcriptase polymerase chain reaction and Sanger sequencing in 19 of 56 acute myeloid leukemia samples (33.9%). Ectopic expression of the UBA2-WTIP fusion in human acute myeloid leukemia KG-1a cells showed enhanced cell proliferation both in vitro and in vivo. The UBA2-WTIP fusion induced phosphorylation of STAT3, STAT5 and ERK1/2, and abrogates WTIP-mediated mammalian processing body formation. Finally, triptolide displayed selective cytotoxicity against KG-1a cells harboring the UBA2-WTIP fusion. Collectively, our findings suggest that the UBA2-WTIP fusion is an oncogenic fusion gene, as well as a promising therapeutic target for the treatment of acute myeloid leukemia.

60 APPLIED LIFE SCIENCES↗

MAGI: A Method for Metabolite Annotation and Gene Integration

Metabolomics is a widely used technology for obtaining direct measures of metabolic activities from diverse biological systems. However, ambiguous metabolite identifications are a common challenge and biochemical interpretation is often limited by incomplete and inaccurate genome-based predictions of enzyme activities (that is, gene annotations). Metabolite Annotation and Gene Integration (MAGI) generates a metabolite-gene association score using a biochemical reaction network. This is calculated by a method that emphasizes consensus between metabolites and genes via biochemical reactions. To demonstrate the potential of this method, we applied MAGI to integrate sequence data and metabolomics data collected from Streptomyces coelicolor A3(2), an extensively characterized bacterium that produces diverse secondary metabolites. Our findings suggest that coupling metabolomics and genomics data by scoring consensus between the two increases the quality of both metabolite identifications and gene annotations in this organism. MAGI also made biochemical predictions for poorly annotated genes that were consistent with the extensive literature on this important organism. Furthermore, this limited analysis suggests that using metabolomics data has the potential to improve annotations in sequenced organisms and also provides testable hypotheses for specific biochemical functions.

59 BASIC BIOLOGICAL SCIENCES↗

Seasonal Patterns in Mobile Colistin Resistance Gene Variants in Wastewater Bioaerosols and Liquid Sludge

To address the growing threat of global antimicrobial resistance, a one-health approach is needed to understand the complex socioecological cycling of antibiotic resistance genes. In this study, a metagenomics approach using DNA shotgun sequencing, metagenome assembly, and antibiotic resistance gene (ARG) annotation was used to examine seasonal patterns in the abundance of mobile colistin resistance (mcr) gene variants in bioaerosols and liquid sludge in three wastewater treatment plants (WWTPs). ARGs represented 0.2–0.8 and 0.1–0.2% of the bioaerosol and liquid sludge metagenomes, respectively, while mcr genes represented 0–0.3 and 0–0.5% of the identified ARGs in bioaerosol and liquid sludge metagenomes. Seven of the ten known mcr variants were detected in wastewater bioaerosol and liquid samples, with mcr-5 and mcr-8 being the most prevalent across all seasons and sites. Furthermore, additional functional and taxonomic annotation of mcr-containing metagenomic contigs showed that mcr genes were often located on contigs with other co-occurring ARGs and mobile genetic elements and may be harbored by opportunistic human pathogens and other bacterial taxa not previously associated with mcr genes. Atmospheric dispersion modeling showed that mcr-containing bioaerosols can be transported kilometers away from the WWTPs, resulting in the possible dissemination of these ARGs into surrounding environments and communities.

Antimicrobial agents↗