Showing papers on "Complementary DNA published in 2003"

Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses

Abstract: To identify cold-, drought-, high-salinity-, and/or abscisic acid (ABA)-inducible genes in rice (Oryza sativa), we prepared a rice cDNA microarray including about 1,700 independent cDNAs derived from cDNA libraries prepared from drought-, cold-, and high-salinity-treated rice plants. We confirmed stress-inducible expression of the candidate genes selected by microarray analysis using RNA gel-blot analysis and finally identified a total of 73 genes as stress inducible including 58 novel unreported genes in rice. Among them, 36, 62, 57, and 43 genes were induced by cold, drought, high salinity, and ABA, respectively. We observed a strong association in the expression of stress-responsive genes and found 15 genes that responded to all four treatments. Venn diagram analysis revealed greater cross talk between signaling pathways for drought, ABA, and high-salinity stresses than between signaling pathways for cold and ABA stresses or cold and high-salinity stresses in rice. The rice genome database search enabled us not only to identify possible known cis-acting elements in the promoter regions of several stress-inducible genes but also to expect the existence of novel cis-acting elements involved in stress-responsive gene expression in rice stress-inducible promoters. Comparative analysis of Arabidopsis and rice showed that among the 73 stress-inducible rice genes, 51 already have been reported in Arabidopsis with similar function or gene name. Transcriptome analysis revealed novel stress-inducible genes, suggesting some differences between Arabidopsis and rice in their response to stress.

Collection, Mapping, and Annotation of Over 28,000 cDNA Clones from japonica Rice

Abstract: We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

An Abundance of Bidirectional Promoters in the Human Genome

Abstract: The alignment of full-length human cDNA sequences to the finished sequence of the human genome provides a unique opportunity to study the distribution of genes throughout the genome. By analyzing the distances between 23,752 genes, we identified a class of divergently transcribed gene pairs, representing more than 10% of the genes in the genome, whose transcription start sites are separated by less than 1000 base pairs. Although this bidirectional arrangement has been previously described in humans and other species, the prevalence of bidirectional gene pairs in the human genome is striking, and the mechanisms of regulation of all but a few bidirectional genes are unknown. Our work shows that the transcripts of many bidirectional pairs are coexpressed, but some are antiregulated. Further, we show that many of the promoter segments between two bidirectional genes initiate transcription in both directions and contain shared elements that regulate both genes. We also show that the bidirectional arrangement is often conserved among mouse orthologs. These findings demonstrate that a bidirectional arrangement provides a unique mechanism of regulation for a significant number of mammalian genes.

Highly Expressed Genes in Pancreatic Ductal Adenocarcinomas: A Comprehensive Characterization and Comparison of the Transcription Profiles Obtained from Three Major Technologies

Abstract: When using gene expression profiling to understand human tumors, one is often confronted with long lists of genes that need to be further categorized into meaningful data. We performed a comprehensive evaluation and comparison of gene expression profiles obtained from pancreatic cancers to determine those genes most differentially expressed and thus with the most promise for translation into clinically useful targets. cDNA was prepared from 50 samples of normal pancreas or duodenal mucosal tissues, 7 samples of chronic pancreatitis, and 39 samples of pancreas cancer tissues or cancer cell lines and hybridized to the complete Affymetrix Human Genome U133 GeneChip set (arrays U133A and U133B) for simultaneous analysis of 45,000 fragments corresponding to 33,000 known genes and 6,000 expressed sequence tags. Genes expressed at levels at least 3-fold greater in the pancreatic cancers as compared with nonneoplastic tissues were identified. Three hundred seventy-seven Affymetrix fragments were identified as having ≥3-fold expression levels in pancreas cancer specimens as compared with nonneoplastic tissues, corresponding to 234 known genes. Serial analysis of gene expression libraries (http://www.ncbi.nlm.nih.gov/SAGE/) of two normal pancreatic ductal cell cultures (HX and H126) were used to exclude 17 genes with high expression levels in the normal duct epithelium (more than five tags/library). Of the remaining 217 known genes, 75 have been previously reported as highly expressed in pancreatic cancers, while the remaining 142 genes are novel. We used principal components analysis (PCA) to identify the genes among these 217 identified as the most differentially expressed and specific to pancreatic cancer tissues or cell lines. Among the most differentially expressed genes identified by PCA were Mesothelin, Muc4, Muc5A/C, Kallikrein 10, Transglutaminase 2, Fascin, TMPRSS3 and stratifin. The differential expression identified by PCA for these genes indicates they are among the more attractive targets for novel therapeutic targets, tumor markers, or as a means of screening pancreatic cancer samples for information regarding tumor classification or potential therapeutic responses. Our findings were also compared in detail to the previously reported findings of highly expressed genes in other studies of global gene expression in pancreatic cancers. We found that robust changes in gene expression were most often identified by more than one gene expression platform. Forty genes were identified by more than one method (U133 oligonucleotide arrays, cDNA arrays or serial analysis of gene expression), and 6 of these genes were identified by all three methods. Our findings identify a novel set of genes as highly expressed in pancreatic cancer, validate the differential expression of previously reported genes, and provide additional support for those genes most differentially expressed to be translated into clinically useful targets.

The Secreted Protein Discovery Initiative (SPDI), a Large-Scale Effort to Identify Novel Human Secreted and Transmembrane Proteins: A Bioinformatics Assessment

Abstract: A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.

Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways.

Abstract: We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.

The Use of Real-Time Reverse Transcriptase PCR for the Quantification of Cytokine Gene Expression

Abstract: Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is becoming a widely used method to quantify cytokines from cells, tissues, or tissue biopsies. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in a single step. Using TaqMan chemistry (Applied Biosystems, Foster City, CA) and the ABI Prism 7700 Sequence Detection System (Applied Biosystems), we validated a large panel of murine and human cytokines, as well as other factors playing a role in the immune system, such as chemokines and apoptotic markers. Although the method allows fast, sensitive, and accurate quantification, different control assays are necessary for the method to be reliable. By construction of complementary DNA (cDNA) plasmid clones, standard curves are generated that allow direct quantification of every unknown sample. Furthermore, the choice of a reliable housekeeping gene is very important. Finally, co-amplification of contaminating genomic DNA is avoided by designing sets of primers located in different exons or on intron–exon junctions. In conclusion, the real-time RT-PCR technique is very accurate and sensitive, allows high throughput, and can be performed on very small samples. The development of real-time RT-PCR has resulted in an exponential increase in its use over the last couple of years, and the method has undoubtedly become the standard for quantifying cytokine patterns, clarifying many functional properties of immune cells and their associated diseases.

Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus

Abstract: A previously undescribed coronavirus (CoV) is the etiologic agent responsible for severe acute respiratory syndrome (SARS). Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoV Urbani strain, and have rescued molecularly cloned SARS viruses (infectious clone SARS-CoV) that contained the expected marker mutations inserted into the component clones. Recombinant viruses replicated as efficiently as WT virus and both were inhibited by treatment with the cysteine proteinase inhibitor (2S,3S)-transepoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester. In addition, subgenomic transcripts were initiated from the consensus sequence ACGAAC in both the WT and infectious clone SARS-CoV. Availability of a SARS-CoV full-length cDNA provides a template for manipulation of the viral genome, allowing for the rapid and rational development and testing of candidate vaccines and therapeutics against this important human pathogen.

Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male‐sterile Kosena radish

Abstract: Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.

Juvenile hormone acid methyltransferase: a key regulatory enzyme for insect metamorphosis.

Abstract: Juvenile hormone (JH) acid methyltransferase (JHAMT) is an enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis pathway in insects By fluorescent mRNA differential display, we have cloned a cDNA encoding JHAMT from the corpora allata (CA) of the silkworm, Bombyx mori (BmJHAMT) The BmJHAMT cDNA encodes an ORF of 278 aa with a calculated molecular mass of 32,544 Da The predicted amino acid sequence contains a conserved S-adenosyl-l-methionine (SAM) binding motif found in the family of SAM-dependent methyltransferases Purified N-terminal 6×His-tagged recombinant BmJHAMT protein expressed in Escherichia coli catalyzed conversion of farnesoic acid and JH acids I, II, and III to their cognate methyl esters in the presence of SAM, confirming that this cDNA encodes a functional JHAMT Putative orthologs, DmJHAMT and AgJHAMT, were identified from the genome sequence of the fruit fly Drosophila melanogaster, and a malaria vector, Anopheles gambiae, respectively Northern blot and quantitative RT-PCR analyses revealed that the BmJHAMT gene was expressed specifically in the CA throughout the third and fourth instar At the beginning of the last (fifth) instar, the expression level of BmJHAMT declined rapidly and became undetectable by day 4 and remained so until pupation Correlation of the BmJHAMT gene expression and the JH biosynthetic activity in the CA suggests that the transcriptional suppression of the BmJHAMT gene is crucial for the termination of JH biosynthesis in the CA, which is a prerequisite for the initiation of metamorphosis

Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane.

Abstract: To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged.

Monitoring Expression Profiles of Arabidopsis Gene Expression during Rehydration Process after Dehydration Using ca. 7000 Full-Length cDNA Microarray

Abstract: Plants respond and adapt to drought stress in order to survive under stress conditions. Several genes that respond to drought at the transcriptional level have been described, but there are few reports on genes involved in the recovery from dehydration. Analysis of rehydration-inducible genes should help not only to understand the molecular mechanisms of stress responses in higher plants, but also to improve the stress tolerance of crops by gene manipulation. We used a full-length cDNA microarray containing ca. 7000 Arabidopsis full-length cDNAs and identified 152 rehydration-inducible genes. Venn diagram analysis showed relationship of the rehydration-inducible genes to proline-inducible and water-treatment-inducible genes. Among the 152 rehydration-inducible genes, 58 genes contained the ACTCAT sequence involved in proline- and hypoosmolarity-inducible gene expression in their promoter regions, suggesting that ACTCAT sequence is a major cis-acting element involved in rehydration-inducible gene expression, and that some novel cis-acting elements are involved in rehydration-inducible gene expression. Functional analysis of rehydration-inducible and rehydration-repressed genes revealed their functions not only in the release from a stressed status but also in the recovery of growth in plants.

15000 unique zebrafish EST clusters and their future use in microarray for profiling gene expression patterns during embryogenesis.

Abstract: A total of 15590 unique zebrafish EST clusters from two cDNA libraries have been identified. Most significantly, only 22% (3437) of the 15590 unique clusters matched 2805 (of 15200) clusters in the Danio rerio UniGene database, indicating that our EST set is complementary to the existing ESTs in the public database and will be invaluable in assisting the annotation of genes based on the upcoming zebrafish genome sequence. Blast search showed that 7824 of our unique clusters matched 6710 known or predicted proteins in the nonredundant database. A cDNA microarray representing approximately 3100 unique zebrafish cDNA clusters has been generated and used to profile the gene expression patterns across six different embryonic stages (cleavage, blastula, gastrula, segmentation, pharyngula, and hatching). Analysis of expression data using K-means clustering revealed that genes coding for muscle-specific proteins displayed similar expression patterns, confirming that the coordinate gene expression is important for myogenesis. Our results demonstrate that the combination of microarray technology with the zebrafish model system can provide useful information on how genes are coordinated in a genetic network to control zebrafish embryogenesis and can help to identify novel genes that are important for organogenesis.

Atlantic salmon interferon genes: cloning, sequence analysis, expression, and biological activity.

Abstract: In this work, we report cDNA cloning of two type I interferons (IFNs) from the head kidney of Atlantic salmon, called SasaIFN- a1 (829 bp) and SasaIFN- a2 (1290 bp). Both translate into 175 amino acid precursor molecules showing 95% amino acid sequence identity. The precursors have a putative 23 amino acid signal peptide, which suggests that the mature Atlantic salmon IFNs contain 152 amino acids (18.2 kDa). Salmon IFN appears to have five a-helices, similar to mammalian and avian type I IFNs, and showed 45% sequence identity with zebrafish IFN, up to 29% identity with mammalian IFN- a sequences, and 17%‐ 18% sequence identity with mammalian IFN- b and chicken type I IFNs. Human embryonic kidney 293 cells transfected with the SasaIFN-a1 cDNA gene produced high titers of acid-stable antiviral activity, which protected salmonid cells against infectious pancreatic necrosis virus (IPNV) and also induced Mx protein in the cells. Poly(I)-poly(C) induced two IFN transcripts in head kidney of Atlantic salmon. Genomic IFN sequences contained four introns and five exons, which is different from the intronless type I IFN genes of birds and mammals.

Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array

Abstract: Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule. To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver. We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes. cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases. Twenty-nine cDNAs, including a putative vacuolar (H+)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a. fiber cells when compared to that in 0 d.p.a. ovules. Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period. This report constitutes the first systematic analysis of genes involved in cotton fiber development. Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.

Global analysis of Borrelia burgdorferi genes regulated by mammalian host-specific signals.

Abstract: Lyme disease is a tick-borne infection that can lead to chronic, debilitating problems if not recognized or treated appropriately. Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature by a complex enzootic cycle involving Ixodes ticks and mammalian hosts. Many previous studies support the notion that B. burgdorferi differentially expresses numerous genes and proteins to help it adapt to growth in the mammalian host. In this regard, several studies have utilized a dialysis membrane chamber (DMC) cultivation system to generate “mammalian host-adapted” spirochetes for the identification of genes selectively expressed during mammalian infection. Here, we have exploited the DMC cultivation system in conjunction with microarray technology to examine the global changes in gene expression that occur in the mammalian host. To identify genes regulated by only mammal-specific signals and not by temperature, borrelial microarrays were hybridized with cDNA generated either from organisms temperature shifted in vitro from 23°C to 37°C or from organisms cultivated by using the DMC model system. Statistical analyses of the combined data sets revealed that 125 genes were expressed at significantly different levels in the mammalian host, with almost equivalent numbers of genes being up- or down-regulated by B. burgdorferi within DMCs compared to those undergoing temperature shift. Interestingly, during DMC cultivation, the vast majority of genes identified on the plasmids were down-regulated (79%), while the differentially expressed chromosomal genes were almost entirely upregulated (93%). Global analysis of the upstream promoter regions of differentially expressed genes revealed that several share a common motif that may be important in transcriptional regulation during mammalian infection. Among genes with known or putative functions, the cell envelope category, which includes outer membrane proteins, was found to contain the most differentially expressed genes. The combined findings have generated a subset of genes that can now be further characterized to help define their role or roles with regard to B. burgdorferi virulence and Lyme disease pathogenesis.

Full-length plant cDNA and uses thereof

Abstract: Full-length cDNAs of plants and their uses are provided. Source plants are preferably monocot plants, more preferably poaceous plants, and most preferably rice. Vectors carrying said cDNAs and transformants containing said cDNAs or said vectors, transgenic plants containing said transformants, polypeptides encoded by said cDNAs are also provided. The full-length cDNA clones play important roles in the annotation of correct gene coding region, determination of exons and introns, comprehensive expression analysis on the transcription level and proteome analysis. Furthermore, full-length cDNA clones are industrially useful in producing plants having different properties from those of the wild type due to the inhibition of expression and functional suppression in plant bodies.

Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain

Abstract: Escherichia coli is a diverse bacterial species that comprises commensal nonpathogenic strains such as E. coli K-12 and pathogenic strains that cause a variety of diseases in different host species. Avian pathogenic E. coli strain χ7122 (O78:K80:H9) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-E. coli K-12) transcripts, pathogen-specific cDNAs were identified. Pathogen-specific transcripts corresponded to putative adhesins, lipopolysaccharide core synthesis, iron-responsive, plasmid- and phage-encoded genes, and genes of unknown function. Specific deletion of the aerobactin siderophore system and E. coli iro locus, which were identified by selective capture of transcribed sequences, demonstrated that these pathogen-specific systems contribute to the virulence of strain χ7122. Consecutive blocking to enrich for selection of pathogen-specific genes did not completely eliminate the presence of transcripts that corresponded to sequences also present in E. coli K-12. These E. coli conserved genes are likely to be highly expressed in vivo and contribute to growth or virulence. Overall, the approach we have used simultaneously provided a means to identify novel pathogen-specific genes expressed in vivo and insight regarding the global gene expression and physiology of a pathogenic E. coli strain in a natural animal host during the infectious process.

Characterizing the stress/defense transcriptome of Arabidopsis

Abstract: Background: To understand the gene networks that underlie plant stress and defense responses, it is necessary to identify and characterize the genes that respond both initially and as the physiological response to the stress or pathogen develops. We used PCR-based suppression subtractive hybridization to identify Arabidopsis genes that are differentially expressed in response to ozone, bacterial and oomycete pathogens and the signaling molecules salicylic acid (SA) and jasmonic acid. Results: We identified a total of 1,058 differentially expressed genes from eight stress cDNA libraries. Digital northern analysis revealed that 55% of the stress-inducible genes are rarely transcribed in unstressed plants and 17% of them were not previously represented in Arabidopsis expressed sequence tag databases. More than two-thirds of the genes in the stress cDNA collection have not been identified in previous studies as stress/defense response genes. Several stress-responsive cis-elements showed a statistically significant over-representation in the promoters of the genes in the stress cDNA collection. These include W- and G-boxes, the SAinducible element, the abscisic acid response element and the TGA motif. Conclusions: The stress cDNA collection comprises a broad repertoire of stress-responsive genes encoding proteins that are involved in both the initial and subsequent stages of the physiological response to abiotic stress and pathogens. This set of stress-, pathogen- and hormone-modulated genes is an important resource for understanding the genetic interactions underlying stress signaling and responses and may contribute to the characterization of the stress transcriptome through the construction of standardized specialized arrays.

A profile of putative parasitism genes expressed in the esophageal gland cells of the root-knot nematode Meloidogyne incognita.

Abstract: Identifying parasitism genes encoding proteins secreted from a nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Meloidogyne incognita parasitism genes were cloned by microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. Of 2,452 cDNA clones sequenced, deduced protein sequences of 185 cDNAs had a signal peptide for secretion and, thus, could have a role in root-knot nematode parasitism of plants. High-throughput in situ hybridization with cDNA clones encoding signal peptides resulted in probes of 37 unique clones specifically hybridizing to transcripts accumulating within the subventral (13 clones) or dorsal (24 clones) esophageal gland cells of M. incognita. In BLASTP analyses, 73% of the predicted proteins were novel proteins. Those with similarities to known proteins included a pectate lyase, acid phosphatase, and hypothetical proteins from other organisms. Our cell-specific analysis of genes encoding secretory proteins provided, for the first time, a profile of putative parasitism genes expressed in the M. incognita esophageal gland cells throughout the parasitic cycle.

Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference.

Abstract: Purpose: The purpose of this study was to profile gene expression changes in colorectal tumors to identify new targets and strategies for the management of this disease. Experimental Design: cDNA microarray analysis was used to detect differences in gene expression between normal tissue and colon tumors and polyps isolated from 20 patients. To identify genes that are important in regulating the growth properties of colorectal cancer, RNA interference (RNAi) was used to disrupt expression of several of the overexpressed genes in a colon tumor cell line, HCT116, which showed similar patterns of gene expression as many of the patient tumors. Results: Expression changes of ≥2-fold in approximately one-third of the patients were consistently observed for 2632 of a total of 9592 genes (574 up-regulated genes and 2058 down-regulated genes). Subsequent analysis of 13 genes by quantitative real-time PCR confirmed the reliability of this analysis. RNAi-mediated disruption of the expression of one of these genes, survivin, a potent inhibitor of apoptosis, severely reduced tumor growth both in vitro and in an in vivo xenograft model. Conclusions: The combined use of microarray analysis and RNAi provides an excellent system to define the role of specific genes that are up-regulated in cancer lead to the increased in vitro and in vivo growth of colon tumors.

A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication.

Abstract: Yellow fever virus (YF) is the prototype member of the Flavivirus genus. Here, we report the successful construction of a full-length infectious cDNA clone of the vaccine strain YF-17D. YF cDNA was cloned into a low-copy-number plasmid backbone and stably maintained in several E. coli strains. Transcribed RNAs had a specific infectivity of 105–106 p.f.u. (μg RNA)−1, and the resulting virus exhibited growth kinetics, plaque morphology and proteolytic processing similar to the parental virus in cell culture. This clone was used to analyse the importance of conserved flavivirus RNA sequences and the 3′ stem–loop structure in virus replication. The conserved sequences 5′CS and CS1, as well as the 3′ stem–loop structure, were found to be essential for virus replication in cell culture, whereas the conserved sequence CS2 and the region containing YF-specific repeated sequences were dispensable. This infectious clone will aid future studies on YF replication and pathogenesis, as well as facilitate the development of YF-17D-based recombinant vaccines.

DNA detection and signal amplification via an engineered allosteric enzyme.

Abstract: Rapid, sensitive, and sequence-specific DNA detection can be achieved in one step using an engineered intrasterically regulated enzyme. The semi-synthetic inhibitor-DNA-enzyme (IDE) construct (left) rests in the inactive state but upon exposure to a complementary DNA sequence undergoes a DNA hybridization-triggered allosteric enzyme activation (right). The ensuing rapid substrate turnover provides the built-in signal amplification mechanism for detecting approximately 10 fmol DNA in less than 3 min under physiological conditions.

A lipase isolated from the silkworm Bombyx mori shows antiviral activity against nucleopolyhedrovirus.

Abstract: A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. A homology search of the deduced amino acid sequence of the protein cDNA revealed 56% homology with Drosophila melanogaster lipase and 21% homology with human lipase. As lipase activity of the protein was confirmed in vitro, this protein was designated Bmlipase-1. Northern blot analysis showed that the Bmlipase-1 gene is expressed in the midgut but not in other tissues, nor is it activated by BmNPV infection. In addition, the Bmlipase-1 gene was shown not to be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that an insect digestive enzyme has potential as a physiological barrier against BmNPV at the initial site of viral infection.