A single module type I polyketide synthase directs de novo macrolactone biogenesis during galbonolide biosynthesis in Streptomyces galbus.

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-(13)C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed ß-keto group modifications.

OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice.

Jasmonates play important roles in development, stress responses and defense in plants. Here, we report the results of a study using a functional genomics approach that identified a rice basic helix-loop-helix domain gene, OsbHLH148, that conferred drought tolerance as a component of the jasmonate signaling module in rice. OsbHLH148 transcript levels were rapidly increased by treatment with methyl jasmonate (MeJA) or abscisic acid, and abiotic stresses including dehydration, high salinity, low temperature and wounding. Transgenic over-expression of OsbHLH148 in rice confers plant tolerance to drought stress. Expression profiling followed by DNA microarray and RNA gel-blot analyses of transgenic versus wild-type rice identified genes that are up-regulated by OsbHLH148 over-expression. These include OsDREB and OsJAZ genes that are involved in stress responses and the jasmonate signaling pathway, respectively. OsJAZ1, a rice ZIM domain protein, interacted with OsbHLH148 in yeast two-hybrid and pull-down assays, but it interacted with the putative OsCOI1 only in the presence of coronatine. Furthermore, the OsJAZ1 protein was degraded by rice and Arabidopsis extracts in the presence of coronatine, and its degradation was inhibited by MG132, a 26S proteasome inhibitor, suggesting 26S proteasome-mediated degradation of OsJAZ1 via the SCF(OsCOI1) complex. The transcription level of OsJAZ1 increased upon exposure of rice to MeJA. These results show that OsJAZ1 could act as a transcriptional regulator of the OsbHLH148-related jasmonate signaling pathway leading to drought tolerance. Thus, our study suggests that OsbHLH148 acts on an initial response of jasmonate-regulated gene expression toward drought tolerance, constituting the OsbHLH148-OsJAZ-OsCOI1 signaling module in rice.

Complete genome sequence analysis of Leuconostoc kimchii IMSNU 11154.

Leuconostoc kimchii IMSNU 11154, isolated from kimchi, a traditional Korean fermented food, is known to be an important antimicrobial lactic acid bacterium with probiotic potential. Here we announce the complete genome sequence of L. kimchii IMSNU 11154 consisting of a 2,101,787-bp chromosome and five plasmids. The strain has genes for dextran formation from sucrose and for mannitol formation from fructose. Antimicrobial and antioxidative functions of L. kimchii IMSNU 11154 could be attributed to a leucosin B-like peptide and multiple enzymes to reduce hydrogen peroxide and oxidized thiols, respectively.

The histone deacetylase OsHDAC1 epigenetically regulates the OsNAC6 gene that controls seedling root growth in rice.

We have previously isolated a rice gene encoding a histone deacetylase, OsHDAC1, and observed that its transgenic overexpression increases seedling root growth. To identify the transcriptional repression events that occur as a result of OsHDAC1 overexpression (OsHDAC1(OE)), a global profiling of root-expressed genes was performed on OsHDAC1(OE) or HDAC inhibitor-treated non-transgenic (NT) roots, in comparison with untreated NT roots. We selected 39 genes that are induced and repressed in HDAC inhibitor-treated NT and OsHDAC1(OE) roots, compared with NT roots, respectively. Interestingly, OsNAC6, a member of the NAM-ATAF-CUC (NAC) family, was identified as a key component of the OsHDAC1 regulon, and was found to be epigenetically repressed by OsHDAC1 overexpression. The root phenotype of OsNAC6 knock-out seedlings was observed to be similar to that of the OsHDAC1(OE) seedlings. Conversely, the root phenotype of the OsNAC6 overexpressors was similar to that of the OsHDAC1 knock-out seedlings. These observations indicate that OsHDAC1 negatively regulates the OsNAC6 gene that primarily mediates the alteration in the root growth of the OsHDAC1(OE) seedlings. Chromatin immunoprecipitation assays of the OsNAC6 promoter region using antibodies specific to acetylated histones H3 and H4 revealed that OsHDAC1 epigenetically represses the expression of OsNAC6 by deacetylating K9, K14 and K18 on H3 and K5, K12 and K16 on H4.

Complete genome sequence of Burkholderia glumae BGR1.

Burkholderia glumae is the causative agent of grain and seedling rot in rice and of bacterial wilt in many field crops. Here, we report the complete genome sequence of B. glumae BGR1 isolated from a diseased rice panicle in Korea.

Quadruple 9-mer-based protein binding microarray with DsRed fusion protein.

BACKGROUND: The interaction between a transcription factor and DNA motif (cis-acting element) is an important regulatory step in gene regulation. Comprehensive genome-wide methods have been developed to characterize protein-DNA interactions. Recently, the universal protein binding microarray (PBM) was introduced to determine if a DNA motif interacts with proteins in a genome-wide manner. RESULTS: We facilitated the PBM technology using a DsRed fluorescent protein and a concatenated sequence of oligonucleotides. The PBM was designed in such a way that target probes were synthesized as quadruples of all possible 9-mer combinations, permitting unequivocal interpretation of the cis-acting elements. The complimentary DNA strands of the features were synthesized with a primer and DNA polymerase on microarray slides. Proteins were labeled via N-terminal fusion with DsRed fluorescent protein, which circumvents the need for a multi-step incubation. The PBM presented herein confirmed the well-known DNA binding sequences of Cbf1 and CBF1/DREB1B, and it was also applied to elucidate the unidentified cis-acting element of the OsNAC6 rice transcription factor. CONCLUSION: Our method demonstrated PBM can be conveniently performed by adopting: (1) quadruple 9-mers may increase protein-DNA binding interactions in the microarray, and (2) a one-step incubation shortens the wash and hybridization steps. This technology will facilitate greater understanding of genome-wide interactions between proteins and DNA.

GBParsy: a GenBank flatfile parser library with high speed.

BACKGROUND: GenBank flatfile (GBF) format is one of the most popular sequence file formats because of its detailed sequence features and ease of readability. To use the data in the file by a computer, a parsing process is required and is performed according to a given grammar for the sequence and the description in a GBF. Currently, several parser libraries for the GBF have been developed. However, with the accumulation of DNA sequence information from eukaryotic chromosomes, parsing a eukaryotic genome sequence with these libraries inevitably takes a long time, due to the large GBF file and its correspondingly large genomic nucleotide sequence and related feature information. Thus, there is significant need to develop a parsing program with high speed and efficient use of system memory. RESULTS: We developed a library, GBParsy, which was C language-based and parses GBF files. The parsing speed was maximized by using content-specified functions in place of regular expressions that are flexible but slow. In addition, we optimized an algorithm related to memory usage so that it also increased parsing performance and efficiency of memory usage. GBParsy is at least 5-100x faster than current parsers in benchmark tests. CONCLUSION: GBParsy is estimated to extract annotated information from almost 100 Mb of a GenBank flatfile for chromosomal sequence information within a second. Thus, it should be used for a variety of applications such as on-time visualization of a genome at a web site.

Growth retardation and death of rice plants irradiated with carbon ion beams is preceded by very early dose- and time-dependent gene expression changes.

The carbon-ion beam (CIB) generated by the heavy-ion medical accelerator in Chiba (HIMAC) was targeted to 7-day-old rice. Physiological parameters such as growth, and gene expression profiles were examined immediately after CIB irradiation. Dose-dependent growth suppression was seen three days post-irradiation (PI), and all the irradiated plants died by 15 days PI. Microarray (Agilent rice 22K) analysis of the plants immediately after irradiation (iai) revealed effects on gene expression at 270 Gy; 353 genes were up-regulated and 87 down-regulated. Exactly the same set of genes was affected at 90 Gy. Among the highly induced genes were genes involved in information storage and processing, cellular processes and signaling, and metabolism. RT-PCR analysis confirmed the microarray data.

Microarray detection of food-borne pathogens using specific probes prepared by comparative genomics.

In order to design a method for the accurate detection and identification of food-borne pathogens, we used comparative genomics to select 70-mer oligonucleotide probes specific for 11 major food-borne pathogens (10 overlapping probes per pathogen) for use in microarray analysis. We analyzed the hybridization pattern of this constructed microarray with the Cy3-labeled genomic DNA of various food-borne pathogens and other bacteria. Our microarray showed a highly specific hybridization pattern with the genomic DNA of each food-borne pathogen; little unexpected cross-hybridization was observed. Microarray data were analyzed and clustered using the GenePix Pro 6.0 and GeneSpring GX 7.3.1 programs. The analyzed dendrogram revealed the discriminating power of constructed microarray. Each food-borne pathogen clustered according to its hybridization specificity and non-pathogenic species were discriminated from pathogenic species. Our method can be applied to the rapid and accurate detection and identification of food-borne pathogens in the food industry. In addition, this study demonstrates that genome sequence comparison and DNA microarray analysis have a powerful application in epidemiologic and taxonomic studies, as well as in the food safety and biodefense fields.

A WUSCHEL Homeobox Transcription Factor, OsWOX13, Enhances Drought Tolerance and Triggers Early Flowering in Rice.

Plants have evolved strategies to cope with drought stress by maximizing physiological capacity and adjusting developmental processes such as flowering time. The WOX13 orthologous group is the most conserved among the clade of WOX homeodomain-containing proteins and is found to function in both drought stress and flower development. In this study, we isolated and characterized OsWOX13 from rice. OsWOX13 was regulated spatially in vegetative organs but temporally in flowers and seeds. Overexpression of OsWOX13 (OsWOX13-ov) in rice under the rab21 promoter resulted in drought resistance and early flowering by 7-10 days. Screening of gene expression profiles in mature leaf and panicles of OsWOX13-ov showed a broad spectrum of effects on biological processes, such as abiotic and biotic stresses, exerting a cross-talk between responses. Protein binding microarray and electrophoretic mobility shift assay analyses supported ATTGATTG as the putative cis-element binding of OsWOX13. OsDREB1A and OsDREB1F, drought stress response transcription factors, contain ATTGATTG motif(s) in their promoters and are preferentially expressed in OsWOX13-ov. In addition, Heading date 3a and OsMADS14, regulators in the flowering pathway and development, were enhanced in OsWOX13-ov. These results suggest that OsWOX13 mediates the stress response and early flowering and, thus, may be a regulator of genes involved in drought escape.

Genome-wide identification of grain filling genes regulated by the OsSMF1 transcription factor in rice.

BACKGROUND: Spatial- and temporal-specific expression patterns are primarily regulated at the transcriptional level by gene promoters. Therefore, it is important to identify the binding motifs of transcription factors to better understand the networks associated with embryogenesis. RESULTS: Here, we used a protein-binding microarray (PBM) to identify the binding motifs of OsSMF1, which is a basic leucine zipper transcription factor involved in the regulation of rice seed maturation. OsSMF1 (previously called RISBZ1 or OsbZIP58) is known to interact with GCN4 motifs (TGA(G/C)TCA) to regulate seed storage protein synthesis, and it functions as a key regulator of starch synthesis. Quadruple 9-mer-based PBM analysis and electrophoretic mobility shift assay revealed that OsSMF1 bound to the GCN4 (TGA(G/C)TCA), ACGT (CCACGT(C/G)), and ATGA (GGATGAC) motifs with three different affinities. We predicted 44 putative OsSMF1 target genes using data obtained from both the PBM and RiceArrayNet. Among these putative target genes, 18, 21, and 13 genes contained GCN4, ACGT, and ATGA motifs within their 1-kb promoter regions, respectively. Among them, six genes encoding major grain filling proteins and transcription factors were chosen to confirm the activation of their expression in vivo. OsSMF1 was shown to bind directly to the promoters of Os03g0168500 (GCN4 motif), patatin-like gene (GCN4 motif), α-globulin (ACGT motif), rice prolamin box-binding factor (RPBF) (ATGA motif), and ONAC024 (GCN4 and ACGT motifs) and to regulate their expression. CONCLUSIONS: The results of this study suggest that OsSMF1 is one of the key transcription factors that functions in a wide range of seed developmental processes with different specific binding affinities for the three DNA-binding motifs.

Analysis of Genes with Alternatively Spliced Transcripts in the Leaf, Root, Panicle and Seed of Rice Using a Long Oligomer Microarray and RNA-Seq.

Pre-mRNA splicing further increases protein diversity acquired through evolution. The underlying driving forces for this phenomenon are unknown, especially in terms of gene expression. A rice alternatively spliced transcript detection microarray (ASDM) and RNA sequencing (RNA-Seq) were applied to differentiate the transcriptome of 4 representative organs of Oryza sativa L. cv. Ilmi: leaves, roots, 1-cm-stage panicles and young seeds at 21 days after pollination. Comparison of data obtained by microarray and RNA-Seq showed a bell-shaped distribution and a co-lineation for highly expressed genes. Transcripts were classified according to the degree of organ enrichment using a coefficient value (CV, the ratio of the standard deviation to the mean values): highly variable (CVI), variable (CVII), and constitutive (CVIII) groups. A higher index of the portion of loci with alternatively spliced transcripts in a group (IAST) value was observed for the constitutive group. Genes of the highly variable group showed the characteristics of the examined organs, and alternatively spliced transcripts tended to exhibit the same organ specificity or less organ preferences, with avoidance of 'organ distinctness'. In addition, within a locus, a tendency of higher expression was found for transcripts with a longer coding sequence (CDS), and a spliced intron was the most commonly found type of alternative splicing for an extended CDS. Thus, pre-mRNA splicing might have evolved to retain maximum functionality in terms of organ preference and multiplicity.

RapaNet: A Web Tool for the Co-Expression Analysis of Brassica rapa Genes.

Accumulated microarray data are used for assessing gene function by providing statistical values for co-expressed genes; however, only a limited number of Web tools are available for analyzing the co-expression of genes of Brassica rapa. We have developed a Web tool called RapaNet (http://bioinfo.mju.ac.kr/arraynet/brassica300k/query/), which is based on a data set of 143 B rapa microarrays compiled from various organs and at different developmental stages during exposure to biotic or abiotic stress. RapaNet visualizes correlated gene expression information via correlational networks and phylogenetic trees using Pearson correlation coefficient (r). In addition, RapaNet provides hierarchical clustering diagrams, scatterplots of log ratio intensities, related pathway maps, and cis-element lists of promoter regions. To ascertain the functionality of RapaNet, the correlated genes encoding ribosomal protein (L7Ae), photosystem II protein D1 (psbA), and cytochrome P450 monooxygenase in glucosinolate biosynthesis (CYP79F1) were retrieved from RapaNet and compared with their Arabidopsis homologues. An analysis of the co-expressed genes revealed their shared and unique features.

Deep and comparative analysis of the mycelium and appressorium transcriptomes of Magnaporthe grisea using MPSS, RL-SAGE, and oligoarray methods.

BACKGROUND: Rice blast, caused by the fungal pathogen Magnaporthe grisea, is a devastating disease causing tremendous yield loss in rice production. The public availability of the complete genome sequence of M. grisea provides ample opportunities to understand the molecular mechanism of its pathogenesis on rice plants at the transcriptome level. To identify all the expressed genes encoded in the fungal genome, we have analyzed the mycelium and appressorium transcriptomes using massively parallel signature sequencing (MPSS), robust-long serial analysis of gene expression (RL-SAGE) and oligoarray methods. RESULTS: The MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray. CONCLUSION: The comprehensive and deep transcriptome analysis by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and function of these genes in fungal growth, development and pathogenesis in the host plants.

Quantitative measurement of serum allergen-specific IgE on protein chip.

Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease inflicting more than quarter of the world population. In order to identify allergen sources, skin provocation test and IgE serology was performed using allergen extracts. Such process identifies allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. Recently, microarray technology has been developed for allergen-specific IgE detection using rolling circle amplification. This study was carried out to evaluate protein chip technology for the quantitative measurement and limits of sensitivity of multiple allergen-specific IgE by an immunofluorescence assay. Significance of positive calibrators was tested using purified human IgE. Dermatophagoides pteronyssinus (Dp), egg white, milk, soybean, and wheat were used as allergens and human serum albumin as negative control. Sensitivity and clinical efficacy of protein chip were evaluated using allergy immune serum for Dp. The fluorescent intensities for purified human IgE as calibrator were well correlated with the concentrations of human IgE. Two-fold dilution of serum allowed an optimal reaction with Dp (1 mg/ml) at which serum Dp-specific IgE levels by protein chip were compatible with those by UniCap. The sensitivity of protein chip in this study was found at level of 1 IU/ml of IgE. Dp-specific IgE levels by protein chip correlated well with those of UniCap by comparing 10 atopic dermatitis. Additional 18 sera were tested for above multiple antigens other than Dp and significant results were obtained for many antigens as well as Dp. These results indicated that spotting of heterogeneous protein mixture on protein chip and the quantitative measurement of serum allergen-specific IgE levels using immunofluorescence assay can be successfully applied in the clinical laboratory for the diagnosis of allergy and could be applied to diagnosis of autoimmune and infectious diseases

A pepper MSRB2 gene confers drought tolerance in rice through the protection of chloroplast-targeted genes.

BACKGROUND: The perturbation of the steady state of reactive oxygen species (ROS) due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 (responsive to ABA protein 21) promoter with/without a selection marker, the bar gene. RESULTS: A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol (DTT). An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. CONCLUSIONS: Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice.

Abiotic stress responsive rice ASR1 and ASR3 exhibit different tissue-dependent sugar and hormone-sensitivities.

The expression of the six rice ASR genes is differentially regulated in a tissue-dependent manner according to environmental conditions and reproductive stages. OsASR1 and OsASR3 are the most abundant and are found in most tissues; they are enriched in the leaves and roots, respectively. Coexpression analysis of OsASR1 and OsASR3 and a comparison of the cis-acting elements upstream of OsASR1 and OsASR3 suggested that their expression is regulated in common by abiotic stresses but differently regulated by hormone and sugar signals. The results of quantitative real-time PCR analyses of OsASR1 and OsASR3 expression under various conditions further support this model. The expression of both OsASR1 and OsASR3 was induced by drought stress, which is a major regulator of the expression of all ASR genes in rice. In contrast, ABA is not a common regulator of the expression of these genes. OsASR1 transcription was highly induced by ABA, whereas OsASR3 transcription was strongly induced by GA. In addition, OsASR1 and OsASR3 expression was significantly induced by sucrose and sucrose/glucose treatments, respectively. The induction of gene expression in response to these specific hormone and sugar signals was primarily observed in the major target tissues of these genes (i.e., OsASR1 in leaves and OsASR3 in roots). Our data also showed that the overexpression of either OsASR1 or OsASR3 in transgenic rice plants increased their tolerance to drought and cold stress. Taken together, our results revealed that the transcriptional control of different rice ASR genes exhibit different tissue-dependent sugar and hormone-sensitivities.

Transcriptome analysis of leaf and root of rice seedling to acute dehydration.

BACKGROUND: Water deficiency is one of the most serious worldwide problems for agriculture. Recently, it has become more serious and outspread, which urgently requires the production of drought-tolerant plants. Microarray experiments using mRNA from air-dried leaves and roots of rice were performed in an attempt to study genes involved in acute dehydration response. RESULTS: Set of 10,537 rice genes was significantly up- or down-regulated in leaves or roots under the treatment. Gene Ontology analysis highlighted gene expression during acute dehydration response depending on organ types and the duration of stress. Rice responded by down-regulating many processes which are mainly involved in inhibiting growth and development. On the other hand, phytohormones (ABA, cytokinin, brassinosteroid) and protective molecules were induced to answer to multiple stresses. Leaves induced more genes than roots but those genes were scattered in various processes, most significantly were productions of osmoprotectants and precursors for important pathways in roots. Roots up-regulated fewer genes and focused on inducing antioxidants and enhancing photosynthesis. Myb, zf-C3HC4, and NAM were most strongly affected transcription factors with the dominance of leaf over root. CONCLUSIONS: Leaf and root tissues shared some common gene expression during stress, with the purpose of enhancing protective systems. However, these two tissues appeared to act differently in response to the different level of dehydration they experience. Besides, they can affect each other via the signaling and transportation system.

An integrated PCR microfluidic chip incorporating aseptic electrochemical cell lysis and capillary electrophoresis amperometric DNA detection for rapid and quantitative genetic analysis.

A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm(2) area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 µl min(-1). For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare.

Convenient determination of protein-binding DNA sequences using quadruple 9-mer-based microarray and DsRed-monomer fusion protein.

Protein-binding DNA microarray (PBM) is one of the high-throughput methods to define DNA sequences which potentially bind to a given DNA-binding protein. Quadruple 9-mer-based protein-binding DNA microarray, named Q9-PBM, is designed in such a way that target probes are synthesized as quadruples of all possible 9-mer combinations. Also, recombinant proteins fused with DsRed-monomer fluorescent protein are conveniently constructed. Q9-PBM confirms the well-known DNA-binding sequences of Cbf1 and CBF1/DREB1B transcription factors, and also identifies the adjacent sequences. Moreover, Q9-PBM is applied to elucidate the unidentified cis-acting element of the OsNAC6 rice transcription factor. This technology will facilitate greater understanding of genome-wide interactions between proteins and DNA.