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At least 955 records · Page 53Linked to original sources
PubMed2005

LEA proteins in higher plants: structure, function, gene expression and regulation.

Late embryogenesis abundant (LEA) proteins are mainly low molecular weight (10-30 kDa) proteins, which are involved in protecting higher plants from damage caused by environmental stresses, especially drought (dehydration). These findings and the fact that the breeding of drought tolerant varieties would be of great value in agriculture, form the basis of search for anti-drought inducible genes and their characterization. LEA proteins are generally classified into six groups (families) according to their amino acid sequence and corresponding mRNA homology, which are basically localized in cytoplasm and nuclear region. LEA protein synthesis, expression and biological activities are regulated by many factors (e.g. developmental stages, hormones, ion change and dehydration), signal transduction pathways and lea genes. No tissue-specific lea gene expression has been considered as one main regulatory mechanism on the basis of extensive studies with the model plant, Arabidopsisthaliana. The study of the regulatory mechanism of lea gene expression is an important feature of modern plant molecular biology.

Arabidopsis↗
PubMed2006

Hyaluronic acid and its derivative as a multi-functional gene expression enhancer: protection from non-specific interactions, adhesion to targeted cells, and transcriptional activation.

Hyaluronic acid (HA), a natural anionic mucopolysaccharide, can be deposited onto the cationic surface of DNA/polyethyleneimine (PEI) complexes to recharge the surface potential and reduce nonspecific interactions with proteins. HA can also be used as a ligand to target specific cell receptors. Furthermore, HA-coating enhanced the transcriptional activity of the plasmid/PEI complexes, probably by loosening the tight binding between DNA and PEI, which facilitated the approach of transcription factors. Amphoteric HA derivative having spermine side chains (Spn-HA) with a structure similar to HMG protein showed higher transcription-enhancing activity than HA. Plasmid/PEI/Spn-HA ternary complex exhibited 29-fold higher transgene expression efficiency than naked plasmid/PEI complexes in CHO cells.

Animals↗
PubMed2026

Mechanism exploration of divergent partial denitrification performance under tetracycline stress: Insights from functional gene, electron transport and molecular docking.

Nitrates and antibiotics like tetracycline (TC) coexist in wastewater and inhibit nitrite (NO₂--N) accumulation during partial denitrification (PD), restricting anammox coupling. A moving bed biofilm reactor (PD-MBBR) and a sequencing batch reactor (PD-SBR) were compared under TC stress (0-8 mg/L). The PD-MBBR proved more robust, sustaining a high nitrate transformation ratio (NTR) of 95.11% and ∼53% TC removal. Metagenomic sequencing, quantitative polymerase chain reaction (qPCR), and molecular docking revealed this tolerance stemmed from physical shielding and metabolic compensation. Carrier-attached growth promoted extracellular polymeric substances (EPS) overproduction, forming a dense barrier preventing TC from binding to key denitrifying enzymes. The biofilm maintained stable nitrate reductase (NAR) activity via high narG and napA gene abundances, while nitrite reductase (NIR) was inhibited, ensuring efficient NO₂--N accumulation. This was supported by hyperactivated electron transport chain components, with complex III relative abundance increasing 15.08% and peak enzymatic activity reaching 149.02%. While IntI1-mediated horizontal gene transfer fortified community defense, concentrated antibiotic resistance genes (ARGs) within the biofilm pose a secondary dissemination risk. Thus, PD-MBBR provides an efficient pretreatment strategy for anammox, though downstream ARGs management is warranted.

Denitrification↗
PubMed2006

Genomic flank-sequencing of plasposon insertion sites for rapid identification of functional genes.

Plasposons are modified mini-Tn5 transposons for random mutagenesis of Gram-negative bacteria. Their unique design allows for the rescue cloning and sequencing of DNA that flanks insertion sites in plasposon mutants. However, this process can be laborious and time-consuming, as it involves genomic DNA isolation, restriction endonuclease treatment, subsequent religation, transformation of religated DNA into an Escherichia coli host, and re-isolation as a plasmid, which is then used as a template in sequencing reactions with primers that read from the plasposon ends into the flanking DNA regions. We describe here a method that produces flanking DNA sequences directly from genomic DNA that is isolated from plasposon mutants. By eliminating the need for rescue cloning, our protocol dramatically reduces time and effort, typically by 2 to 3 working days, as well as costs associated with digestion, ligation, transformation, and plasmid isolation. Furthermore, it allows for a high-throughput automated approach to analysis of the plasposome, i.e. the collective set of plasposon insertion sites in a plasposon mutant library. We have tested the utility of genomic flank-sequencing on three plasposon mutants of the soil bacterium Collimonas fungivorans with abolished ability to degrade chitin.

3' Flanking Region↗
PubMed2005

Maize uroporphyrinogen III methyltransferase: overexpression of the functional gene fragments in Escherichia coli and one-step purification.

S-Adenosyl-L-methionine: uroporphyrinogen III methyltransferase (SUMT), a key regulatory enzyme, converts uroporphyrinogen III to precorrin-2 in the porphinoids biosynthesis. In this study, the mature SUMT was signified that the maize SUMT precursor encoded by the open reading frame of maize SUMT cDNA was deleted the first 91 amino acids constituting the postulated signal peptide. Several mature SUMT fusion and deletion mutants were conducted. It actively expressed in Escherichia coli that the mature SUMT, or the truncated one deleting the C-terminal extra 52 amino acids based on SUMT sequence comparisons. On the contrary, it expressed as an inclusion body in E. coli that the mature SUMT fusion mutant, the SUMT precursor, or the mature SUMT deleting the N-terminal 36 amino acids including glycine-rich region involved directly in SAM binding. The purified His6-tagged mature SUMT was homodimer with a molecular weight of 34 kDa, as shown by SDS-PAGE, 52 kDa using gel-filtration chromatography, and 79 kDa by dynamic light scattering assay. Red fluorescent compounds were associated with the recombinant mature SUMT which were identified as sirohydrochlorin and trimethylpyrrocorphin by spectroscopic analysis. This association slightly altered the protein secondary structure confirmed by circular dichroism assay.

Chromatography, Gel↗
PubMed2003

Electrotransfer in differentiated myotubes: a novel, efficient procedure for functional gene transfer.

Development of reliable techniques for experimental manipulation of gene expression in multinucleated skeletal muscle fibers is critical for understanding molecular mechanisms involved in both physiology and pathophysiology. At present, viral vectors represent the only method to obtain efficient gene transfer in terminally differentiated myotubes. Here we present an in vitro procedure that relies on the application of a pulsed electric field for transferring naked DNA into differentiated myotubes seeded on coverslips. Compared with standard transfection methods, electroporation was at least 1000 times more efficient, as judged by quantitative determination of luciferase content. Percentage of transfected myotubes averaged around 45%. Moreover, we were successful in transfecting a dominant-negative ADP ribosylation factor 1 (ARF1) mutant, i.e., ARF1N126I, in myotubes, thus interfering with endoplasmic reticulum-Golgi traffic, as indicated by alterations of subcellular distribution of GM130, a cis/medial-Golgi marker. Co-transfection experiments with beta-galactosidase also showed that the ARF1 mutant appeared to inhibit myoblast fusion and could not be used before myotube formation. The present work validates the use of electroporation as a highly efficient approach for gene transfer in fully differentiated myotubes.

Animals↗
PubMed2005

In situ functional gene analysis: recognition of individual genes by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) using specific probes certainly is one of the most commonly applied molecular techniques in microbial ecology. Monitoring of community composition and dynamics can be combined with localization and identification of individual cells in situ. However, the resolution power of the method is limited by the need for high target numbers per cell. Apart from standard targets (ribosomal RNAs), mRNAs could be used successfully for in situ visualization in some cases. A new promising variant of in situ hybridization could be established that should provide access to any low copy number nucleic acid targets, such as chromosomal genes. The recognition of individual genes by FISH (RING-FISH) technology is based on target visualization mediated by polynucleotide probe network formation. The specificity of the approach is provided by intracellular probe-target hybridization. This initial hybridization apparently acts as a focal point for inter-probe hybridization within and mainly in the periphery of the cells. Probe-conferred fluorescence typically appears halo-like around the cells. RING-FISH can be used in combination with conventional oligonucleotide FISH. Thus, genetic potential can be assigned to in situ identified cells.

Escherichia coli↗
PubMed1996

The tomato Cf-2 disease resistance locus comprises two functional genes encoding leucine-rich repeat proteins.

In plants, resistance to pathogens is frequently determined by dominant resistance genes, whose products are proposed to recognize pathogen-encoded avirulence gene (Avr) products. The tomato resistance locus Cf-2 was isolated by positional cloning and found to contain two almost identical genes, each conferring resistance to isolates of tomato leaf mould (C. fulvum) expressing the corresponding Avr2 gene. The two Cf-2 genes encode protein products that differ from each other by only three amino acids and contain 38 leucine-rich repeat (LRR) motifs. Of the LRRs, 20 show extremely conserved alternating repeats. The C-terminus of Cf-2 carries regions of pronounced homology to the protein encoded by the unlinked Cf-9 gene. We suggest that this conserved region interacts with other proteins involved in activating plant defense mechanisms.

Amino Acid Sequence↗
PubMed2002

Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered, PCB-degrading Rhodococcus in soil.

A real-time PCR (RTm-PCR) assay using fluorescently labeled oligonucleotides (TaqMan probes) was used to detect and quantify the recombinant Rhodococcus sp. strain RHA1(fcb) in soil. One primer and probe set targeted a hypervariable region of the 16S rRNA gene unique to strain RHA1(fcb) and its phylogenetic relatives, and the other set targeted the recombinant 4-chlorobenzoate (4-CBA) degradation operon (fcb) and was strain-specific. The method had a 6-log dynamic range of detection (10(2)-10(7) cells ml(-1)) for both probes when DNA from pure cultures was used. Although the method was less sensitive in soil, the estimated number of cells in soil by real-time PCR corresponded to the measured number of RHA1(fcb) cells determined by colony-forming units.

Biodegradation, Environmental↗
PubMed2003

Identification of novel anti-angiogenic factors by in silico functional gene screening method.

Angiogenesis, the formation of new blood vessels out of pre-existing capillaries, occurs in a variety of pathophysiological conditions, and is regulated by a balance of angiogenic activators and inhibitors. To identify novel angiogenic factors, we developed a gene screening method by combining the prediction analysis of transcription factor (TF) binding site and the chromosomal localization analysis. First, we analyzed the promoter sequences from known angiogenesis-related factors using the MATINSPECTOR program in TRANSFAC database. Interestingly, we found that the binding site of LMO2 complex is highly conserved in the promoter regions of these factors. Second, we analyzed chromosome loci based on the hypothesis that angiogenesis-related factors might be co-localized in a specific chromosomal band. We found that angiogenesis-related factors are localized in specific 14 chromosomal bands including 5q31 and 19q13 using AngioDB and LocusLink database mining. From these two approaches, we identified 32 novel candidates that have the LMO2 complex binding site in their promoter and are located on one of 14 chromosomal bands. Among them, human recombinant troponin T and spectrin markedly inhibited the neovascularization in vivo and in vitro. Collectively, we suggest that the combination of the prediction analysis of TF binding site and the chromosomal localization analysis might be a useful strategy for gene screening of angiogenesis.

Angiogenesis Inducing Agents↗
PubMed1998

Isolation, structural analysis and mapping of the functional gene of human ribosomal protein S26.

The nucleotide sequence of the gene of human ribosomal protein S26 has been assembled from cDNA and genomic PCR-amplified DNA fragments, and its transcription start site has been determined by primer extension. The gene is composed of four exons and three introns spanning 2027bp. Like other ribosomal protein genes of vertebrates, this gene contains a short first exon corresponding exactly to the short untranslated 5'- UTR. Its transcription start site is embedded in a polypyrimidine tract. Using PCR on DNAs from hybrid cell lines with a different set of human chromosomes, the intron-containing gene of ribosomal protein S26 was mapped to human chromosome 12.

Base Sequence↗
PubMed2004

A microsatellite polymorphism in intron 2 of human Toll-like receptor 2 gene: functional implications and racial differences.

The human Toll-like receptor 2 (TLR2) mediates responses of both innate and adaptive immunity to Gram-positive bacteria, including mycobacteria. We sought functional polymorphisms in the 5'-untranslated region (UTR) of TLR2. We found a highly polymorphic (GT)n dinucleotide repeat 100 bp upstream of the TLR2 translational start site in intron 2. The numbers of GT repeats varied from 12 to 28. There were significant differences in allele distribution between African Americans and Caucasians (P=0.008) and between African Americans and Koreans (P=0.0003). The promoter activities of recombinant promoter-intron2/reporter constructs including the shortest [GT)n=12] or longest [(GT)n=28] alleles were significantly more stimulated when exposed to 200 IU ml(-1) of interferon-gamma than when exposed to 100 IU ml(-1) of GM-CSF (P<==0.03). Since TLR2 plays a critical role in the human innate immune response, this functional microsatellite polymorphism may be important in the pathogenesis of infectious and inflammatory diseases.

Alleles↗