Comparative and evolutionary studies of mammalian arylsulfatase and sterylsulfatase genes and proteins encoded on the X-chromosome

At least 19 sulfatase genes have been reported on the human genome, including four arylsulfatase (ARS) genes (ARSD; ARSE; ARSF; ARSH) and a sterylsulfatase (STS) gene located together on the X-chromosome. Bioinformatic analyses of mammalian genomes were undertaken using known human STS and ARS amino acid sequences to study the evolution of these genes and proteins encoded on eutherian and marsupial genomes. Several domain regions and key residues were conserved including signal peptides, active site residues, metal (Ca²⁺) and substrate binding sequences, transmembranes and N-glycosylation sites. Phylogenetic analyses describe the relationships and potential origins of these genes during mammalian evolution. Primate ARSH enzymes lacked signal peptide sequences which may influence their biological functions. CpG117 and CpG92 were detected within the 5′ region of the human STS and ARSD genes, respectively, and miR-205 within the 3′-UTR for the human STS gene, using bioinformatic methods A proposal is described for a primordial invertebrate STS-like gene serving as an ancestor for unequal cross over events generating the gene complex on the eutherian mammalian X-chromosome.     

Cellulose synthase (CesA) genes in algae and seedless plants

Microfibril structure is determined largely by the organization of arrays of integral plasma membrane protein particles known as “terminal complexes”, which include cellulose synthase catalytic subunits encoded by CesA genes. Although the CesA genes of plants and bacteria share conserved regions, variations in terminal complex and microfibril structure presumably result from sequence differences. Thus, the CesA domains that influence terminal complex assembly may be revealed by examining the differences between CesA genes from green algae in which terminal complex structure ranges from rosettes (plant-like) to linear (bacteria-like). This report describes a second CesA gene that has been cloned from Mesotaenium caldariorum, a unicellular green alga from the order Zygnematales, which have rosette terminal complexes. Both McCesA1 and McCesA2 are similar to seed plant CesAs in domain structure and intron position. Seed plants have multiple CesAs and CesA-like (Csl) genes, some of which appear to be expressed specifically during cell expansion, secondary cell wall deposition in vascular tissue, or tip growth. Diversification of the CesA and Csl gene families can be explored by comparing these genes in mosses, which lack vascular tissue with secondary cell walls, and early divergent vascular plants such as ferns. Degenerate primers were used to amplify and clone five unique CesA and Csl fragments from genomic DNA isolated from Physcomitrella patens. Probes derived from the cloned fragments were used to isolate several clones from a Physcomitrella genomic library. One Csl fragment was amplified from genomic DNA isolated from the fern Ceratopteris richardii. Phylogenetic analysis supports the presence of CslD genes in both mosses and ferns, but does not support the presence of secondary cell wall specific CesA orthologs in mosses.     

Molecular evolution and structural variations in nuclear encoded chloroplast localized heat shock protein 26 (sHSP26) from genetically diverse wheat species

Heat shock proteins are an important class of molecular chaperones known to impart tolerance under high temperature stress. sHSP26, a member of small heat shock protein subfamily is specifically involved in protecting plant’s photosynthetic machinery. The present study aimed at identifying and characterizing sequence and structural variations in sHSP26 from genetically diverse progenitor and non-progenitor species of wheat. In silico analysis identified three paralogous copies of TaHSP26 to reside on short arm of chromosome 4A while one homeologue each was localized on long arm of chromosome 4B and 4D of cultivated bread wheat. Wild DD-genome donor Aegilops tauschii carried an additional sHSP26 gene (AET4Gv20569400) which was absent in the cultivated DD genome of bread wheat. In vitro amplification of this novel gene in wild accessions of Ae. tauschii and synthetic hexaploid wheat but not in cultivated bread wheat validated this finding. Further, significant length polymorphism could be identified in exon1 from diverse sHSP26 sequences. Multiple sequence alignment of procured sequences revealed numerous sSNPs and nsSNPs. D3A, P125 L, Q242 K were designated as homeolog specific- while A49 G as non-progenitor specific amino acid replacements. A 9-bp indel in TmHSP26-1(GA) translated into a deletion of SPM amino acid segment in chloroplast specific conserved consensus region III. High degree of divergence in nucleotide sequence between cultivated and wild species appeared in the form of higher ω values (Ka/Ks >1) indicating positive selection during the course of evolution. Phylogenetic analysis elucidated ancestral relationships between wheat sHSP26 proteins and orthologous proteins across plant kingdom. Overall, data mining approach may be employed as an effective pre-breeding strategy to identify and mobilize novel stress responsive genes and distinct allelic variants from wider germplasm collections of wheat to enhance climate resilience of present day elite wheat cultivars.     

Multiplexed and amplified chemiluminescence resonance energy transfer (CRET) detection of genes and microRNAs using dye-loaded hemin/G-quadruplex-modified UiO-66 metal–organic framework nanoparticles

Dye-loaded UiO-66 metal–organic framework nanoparticles (NMOFs) modified with catalytic hemin/G-quadruplex DNAzyme labels act as functional hybrid modules for the chemiluminescence resonance energy transfer (CRET) analysis of miRNAs (miRNA-155 or miRNA-21) or genes (p53 or BRCA1). The dye-loaded NMOFs (dye = fluorescein (Fl) or rhodamine 6G (Rh 6G)) are modified with hairpin probes that are engineered to include in their loop domains recognition sequences for the miRNAs or genes, and in their stem regions caged G-quadruplex domains. In the presence of the analytes miRNAs or genes, the hairpin structures are opened, leading, in the presence of hemin, to the self-assembly of hemin/G-quadruplex DNAzyme labels linked to the dye-loaded NMOFs. In the presence of luminol and H₂O₂, the hemin/G-quadruplex DNAzyme labels catalyze the generation of chemiluminescence that provides radiative energy to stimulate the process of CRET to the dye loaded in the NMOFs, resulting in the luminescence of the loaded dye without external excitation. The resulting CRET signals relate to the concentrations of the miRNAs or the genes and allow the sensitive analysis of miRNAs and genes. In addition, the DNA hairpin-functionalized dye-loaded NMOF sensing modules were further applied to develop amplified miRNA or gene CRET-based sensing platforms. The dye-loaded NMOFs were modified with hairpin probes that include in their loop domain the recognition sequences for miRNA-155 or miRNA-21 or the recognition sequences for the p53 or BRCA1 genes. Subjecting the hairpin-modified NMOFs to the respective miRNAs or genes, in the presence of two hairpins H_i and H_j that include in their stem regions caged G-quadruplex subunit domains, results in the analyte-triggered opening of the probe hairpin linked to the NMOFs, and the opened hairpin tethers induce the cross-opening of the hairpins H_i and H_j by the hybridization chain reaction, HCR, resulting in the assembly of G-quadruplex wires tethered to the NMOFs. The binding of hemin to the HCR-generated chains yields hemin/G-quadruplex DNAzyme wires that enhance, in the presence of luminol/H₂O₂, the CRET processes in the hybrid nanostructures. These amplification platforms lead to the amplified sensing of miRNAs and genes. By mixing the Fl- and Rh 6G-loaded hairpin-functionalized UiO NMOFs, the multiplexed CRET detection of miRNA-155, miRNA-21 and the p53 and BRCA1 genes is demonstrated.

Hemin/G-quadruplex DNAzyme-modified metal–organic framework nanoparticles act as functional hybrids for the catalyzed oxidation of luminol by H₂O₂, causing chemiluminescence and activation of chemiluminescence resonance energy transfer to the dye loads.     

Global expression analysis of CESA and CSL genes in Arabidopsis

We have used Affymetrix gene chips to measure the expression of 10 CESA and 29 CSL genes of Arabidopsis in different developmental stages or organs. These measurements reveal that many of the genes exhibit different levels of expression in the various organs. While several CESA genes are highly expressed in all the tissues examined, very few CSL genes approach such high levels of expression. This suggests that the CSL genes either encode enzymes for the synthesis of minor components of cell walls or are expressed only in specific cell types. The expression data also highlights the potential importance of the CESA genes for primary and secondary cell wall formation during different developmental stages and in the different organs examined.     

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas in conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.     

Computational algorithmic and molecular dynamics study of functional and structural impacts of non-synonymous single nucleotide polymorphisms in human DHFR gene

Human dihydrofolate reductase (DHFR) is a conserved enzyme that is central to folate metabolism and is widely targeted in pathogenic diseases as well as cancers. Although studies have reported the fact that genetic mutations in DHFR leads to a rare autosomal recessive inborn error of folate metabolism and drug resistance, there is a lack of an extensive study on how the deleterious non-synonymous SNPs (nsSNPs) disrupt its phenotypic effects. In this study, we aim at discovering the structural and functional consequences of nsSNPs in DHFR by employing a combined computational approach consisting of ten recently developed in silico tools for identification of damaging nsSNPs and molecular dynamics (MD) simulation for getting deeper insights into the magnitudes of damaging effects. Our study revealed the presence of 12 most deleterious nsSNPs affecting the native phenotypic effects, with three (R71T, G118D, Y122D) identified in the co-factor and ligand binding active sites. MD simulations also suggested that these three SNPs particularly Y122D, alter the overall structural flexibility and dynamics of the native DHFR protein which can provide more understandings into the crucial roles of these mutants in influencing the loss of DHFR function.     

Identification of key genes and expression profiles in osteoarthritis by co-expressed network analysis

BackgroundThe underlying molecular characteristics of osteoarthritis (OA), a common age-related joint disease, remains elusive. Here, we aimed to identify potential early diagnostic biomarkers and elucidate underlying mechanisms of OA using weighted gene co-expression network analysis (WGCNA).Material and methodsWe obtained the gene expression profile dataset GSE55235, GSE55457, and GSE55584, from the Gene Expression Omnibus. WGCNA was used to investigate the changes in co-expressed genes between normal and OA synovial membrane samples. Modules that were highly correlated to OA were subjected to functional enrichment analysis using the R clusterProfiler package. Differentially expressed genes (DEGs) between the two samples were screened using the “limma” package in R. A Venn diagram was constructed to intersect the genes in significant modules and DEGs. RT -PCR was used to further verify the hub gene expression levels between normal and OA samples.ResultsThe preserved significant module was found to be highly associated with OA development and progression (P < 1e-200, correlation = 0.92). Functional enrichment analysis suggested that the antiquewhite4 module was highly correlated to FoxO signaling pathway, and the metabolism of fatty acids and 2-oxocarboxylic acid. A total of 13 hub genes were identified based on significant module network topology and DEG analysis, and RT-PCR confirmed that these genes were significantly increased in OA samples compared with that in normal samples.ConclusionsWe identified 13 hub genes correlated to the development and progression of OA, which may provide new biomarkers and drug targets for OA.     

Genome-wide analysis of magnesium transporter genes in Solanum lycopersicum

Magnesium (Mg) is an important micronutrient for various physiological processes in plants. In this study, putative Magnesium Transporter (MGT) genes have been identified in Solanum lycopersicum, Solanum tuberosum, Brachypodium distachyon, Fagaria vesca, Brassica juncea and were classified into 5 distinct groups based on their sequence homology. MGT genes are very diverse and possess very low sequence identity within its family. However, the Gly-Met-Asn (GMN) signature motif is present in most of the genes which are believed to be essential for Mg²⁺ recognition. In S. lycopersicum, different physiological root growth pattern was observed in both Mg excess and deficient conditions. Quantitative RT-PCR gene expression study shows that most of the SlMGT genes were upregulated in response to Mg deficient condition.     

HER-2/neu and topoisomerase IIalpha--simultaneous drug targets in cancer

In solid tumors the predominant genetic mechanism for oncogene activation is through amplification of genes. The HER-2 (also known as ErbB2/c-erbB2/HER-2/neu) oncogene is the most frequently amplified oncogene in breast cancer and is also commonly amplified in other forms of cancer. Alongside its important role in tumor induction, growth and progression, HER-2 is also a target for a new form of chemotherapy. Since 1998, breast cancer patients have been treated with considerable success with Herceptin (trastuzumab), a recombinant antibody designed to block signaling through the HER-2 receptor. In addition to Herceptin, a large number of various HER-2 directed immunological and genetic approaches, either targeting the HER-2 receptor, its signaling pathways or both HER-2 and epidermal growth factor receptor (EGFR) together, have demonstrated promising pre-clinical potential towards HER-2 amplified carcinomas. Moreover, the HER-2 amplicon contains other genes with altered copy numbers that could be used as targets for chemotherapy. The topoisomerase IIalpha (topoIIalpha) gene (TOP2A) is located adjacent to the HER-2 oncogene at the chromosome location 17q12-q21 and is either amplified or deleted, with equal frequency, in almost 90% of HER-2 amplified primary breast tumors. Recent data suggest that amplification or deletion of TOP2A may account for both sensitivity or resistance to topoII-inhibitor-chemotherapy, depending on the specific genetic defect at the TOP2A locus. The understanding of HER-2 amplification and its role in the pathogenesis of cancer is expanding. The number of therapeutic strategies targeting HER-2 signaling pathways will most probably be introduced in the treatment of HER-2 amplified tumors within the next few years. Combining HER-2 targeting therapies with conventional forms of cytotoxic chemotherapy, where additional diagnostics tests such as those ascertaining topoIIalpha status, may be helpful for the ideal selection of patients for the combination therapy of a HER-2 targeting drug together with a cytotoxic drug. The clinical and therapeutic importance of the HER-2 and TOPO2A status of tumor cells in cancer management will only increase within the next few years.     

Genomic assays for Epstein–Barr virus-positive gastric adenocarcinoma

A small set of gastric adenocarcinomas (9%) harbor Epstein–Barr virus (EBV) DNA within malignant cells, and the virus is not an innocent bystander but rather is intimately linked to pathogenesis and tumor maintenance. Evidence comes from unique genomic features of host DNA, mRNA, microRNA and CpG methylation profiles as revealed by recent comprehensive genomic analysis by The Cancer Genome Atlas Network. Their data show that gastric cancer is not one disease but rather comprises four major classes: EBV-positive, microsatellite instability (MSI), genomically stable and chromosome instability. The EBV-positive class has even more marked CpG methylation than does the MSI class, and viral cancers have a unique pattern of methylation linked to the downregulation of CDKN2A (p16) but not MLH1. EBV-positive cancers often have mutated PIK3CA and ARID1A and an amplified 9p24.1 locus linked to overexpression of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2). Multiple noncoding viral RNAs are highly expressed. Patients who fail standard therapy may qualify for enrollment in clinical trials targeting cancer-related human gene pathways or promoting destruction of infected cells through lytic induction of EBV genes. Genomic tests such as the GastroGenus Gastric Cancer Classifier are available to identify actionable variants in formalin-fixed cancer tissue of affected patients.