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The transcriptome of the salivary glands of the female western black-legged tick Ixodes pacificus (Acari: Ixodidae).

Sequencing of an Ixodes pacificus salivary gland cDNA library yielded 1068 sequences with an average undetermined nucleotide of 1.9% and an average length of 487 base pairs. Assembly of the expressed sequence tags yielded 557 contigs, 138 of which appear to code for secreted peptides or proteins based on translation of a putative signal peptide. Based on the BLASTX similarity of these contigs to 66 matches of Ixodes scapularis peptide sequences, only 58% sequence identity was found, indicating a rapid divergence of salivary proteins as observed previously for mosquito and triatomine bug salivary proteins. Here we report 106 mostly full-length sequences that clustered in 16 different families: Basic-tail proteins rich in lysine in the carboxy-terminal, Kunitz-containing proteins (monolaris, ixolaris and penthalaris families), proline-rich peptides, 5-, 9.4- and 18.7-kDa proteins of unknown functions, in addition to metalloproteases (class PIII-like) similar to reprolysins. We also have found a family of disintegrins, named ixodegrins that display homology to variabilin, a GPIIb/IIIa antagonist from the tick Dermacentor variabilis. In addition, we describe peptides (here named ixostatins) that display remarkable similarities to the cysteine-rich domain of ADAMST-4 (aggrecanase). Many molecules were assigned in the lipocalin family (histamine-binding proteins); others appear to be involved in oxidant metabolism, and still others were similar to ixodid proteins such as the anticomplement ISAC. We also identified for the first time a neuropeptide-like protein (nlp-31) with GGY repeats that may have antimicrobial activity. In addition, 16 novel proteins without significant similarities to other tick proteins and 37 housekeeping proteins that may be useful for phylogenetic studies are described. Some of these proteins may be useful for studying vascular biology or the immune system, for vaccine development, or as immunoreagents to detect prior exposure to ticks. Electronic version of the manuscript can be found at.

Amino Acid Sequence↗

Effect of the applied organic load rate on biodegradable polymer production by mixed microbial cultures in a sequencing batch reactor.

This article studies the operation of a new process for the production of biopolymers (polyhydroxyalkanoates, PHAs) at different applied organic load rates (OLRs). The process is based on the aerobic enrichment of activated sludge to obtain mixed cultures able to store PHAs at high rates and yields. A mixture of acetic, lactic, and propionic acids at different concentrations (in the range 8.5-31.25 gCOD/L) was fed every 2 h in a sequencing batch reactor (SBR). The resulting applied OLR was in the range 8.5-31.25 gCOD/L/day. Even though, as expected, the increase in the OLR caused an increase in biomass concentration (up to about 8.7 g COD/L), it also caused a relevant decrease of maximal polymer production rate. This decrease in polymer production rate was related to the different extent of "feast and famine" conditions, as function of the applied OLR and of the start-up conditions. As a consequence the best performance of the process was obtained at an intermediate OLR (20 gCOD/L/day) where both biomass productivity and PHA storage were high enough. However, at this high OLR the process was unstable and sudden decrease of performance was also observed. The sludge characterized by the highest PHA storage response was investigated by 16S rDNA clone library. The clone library contained sequences mostly from PHA producers (e.g., Alcaligenes and Comamonas genera); however many genera and among them, one of the dominant (Thauera), were never described before in relation to PHA storage response.

Acids, Acyclic↗

A new class of transcription initiation factors, intermediate between TATA box-binding proteins (TBPs) and TBP-like factors (TLFs), is present in the marine unicellular organism, the dinoflagellate Crypthecodinium cohnii.

Dinoflagellates are marine unicellular eukaryotes that exhibit unique features including a very low level of basic proteins bound to the chromatin and the complete absence of histones and nucleosomal structure. A cDNA encoding a protein with a strong homology to the TATA box-binding proteins (TBP) has been isolated from an expressed sequence tag library of the dinoflagellate Crypthecodinium cohnii. The typical TBP repeat signature and the amino acid motives involved in TFIIA and TFIIB interactions were conserved in this new TBP-like protein. However, the four phenylalanines known to interact with the TATA box were substituted with hydrophilic residues (His(77), Arg(94), Tyr(171), Thr(188)) as has been described for TBP-like factors (TLF)/TBP-related proteins (TRP). A phylogenetic analysis showed that cTBP is intermediate between TBP and TLF/TRP protein families, and the structural similarity of cTBP with TLF was confirmed by low affinity binding to a consensus' TATA box in an equivalent manner to that usually observed for TLFs. Six 5'-upstream gene regions of dinoflagellate genes have been analyzed and neither a TATA box nor a consensus-promoting element could be found within these different sequences. Our results showed that cTBP could bind stronger to a TTTT box sequence than to the canonical TATA box, especially at high salt concentration. Same binding results were obtained with a mutated cTBP (mcTBP), in which the four phenylalanines were restored. To our knowledge, this is the first description of a TBP-like protein in a unicellular organism, which also appears as the major form of TBP present in C. cohnii.

Amino Acid Sequence↗

Examination of fungal stress response genes using Saccharomyces cerevisiae as a model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link.

Saccharomyces cerevisiae served as a model fungal system to examine functional genomics of oxidative stress responses and reactions to test antioxidant compounds. Twenty-two strains of S. cerevisiae, including a broad spectrum of singular gene deletion mutants, were exposed to hydrogen peroxide (H2O2) to examine phenotypic response to oxidative stress. Responses of particular mutants treated with gallic, tannic or caffeic acids, or methyl gallate, during H2O2 exposure, indicated that these compounds alleviated oxidative stress. These compounds are also potent inhibitors of aflatoxin biosynthesis in Aspergillus flavus. To gain further insights into a potential link between oxidative stress and aflatoxin biosynthesis, 43 orthologs of S. cerevisiae genes involved in gene regulation, signal transduction (e.g., SHO1, HOG1, etc.) and antioxidation (e.g., CTT1, CTA1, etc.) were identified in an A. flavus expressed sequence tag library. A successful exemplary functional complementation of an antioxidative stress gene from A. flavus, mitochondrial superoxide dismutase (sodA), in a sod2Delta yeast mutant further supported the potential of S. cerevisiae deletion mutants to serve as a model system to study A. flavus. Use of this system to further examine functional genomics of oxidative stress in aflatoxigenesis and reduction of aflatoxin biosynthesis by antioxidants is discussed.

Aflatoxins↗

Adaptor-mediated amplification of minute amounts of severely fragmented ancient nucleic acids.

It has been repeatedly shown that high copy number mitochondrial DNA sequences can be recovered from ancient samples. A significant increase in the volume of information available to researchers will be observed when the amplification of nuclear DNA becomes commonplace and reproducible. To this end we established a modification of the Rapid Amplification of cDNA Ends (RACE) procedure normally used for the generation of cDNA ends from adaptor-ligated expressed sequence tag libraries. The modifications were designed to specifically address the problems associated with the highly damaged nucleic acids extracted from palaeontological specimens. For this study we used 6 human samples dating to 450 AD and approximately 6.500 BP that were refractory to reliable amplification of single copy loci by PCR. Racemate contents (ratio of D/L enantiomers) of aspartic acid, alanine, and leucine also indicated that no amplifiable DNA is present in 5 of the 6 samples. The proposed technique allowed us (i) to amplify four X-chromosomal loci from 5 human specimens, and (ii) to correct allelic drop-out phenomena at the amelogenin locus in one individual; thus showing that the threshold of 80 x 10-3 for D/Lasp as a borderline for the presence/absence of amplifiable aDNA requires reassessment. Reliability of the proposed technique (i.e. amplification of DNA sequences endogenous to the find) was validated by the application of "ancient RACE" (aRACE) to prehistoric animal samples.

Journal Article↗

Identification of genes up-regulated in urothelial tumors: the 67-kd laminin receptor and tumor-associated trypsin inhibitor.

Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified approximately 100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades.

Carcinoma↗

Nonrandom distribution of genes with sex-biased expression in the chicken genome.

Evolutionary theory predicts that sexually antagonistic genes should show a nonrandom genomic distribution with sex chromosomes usually being enriched for such genes. However, empirical observations from model organisms (Drosophila melanogaster, Caenorhabditis elegans, mammals) on the genomic location of genes with sex-biased expression have provided conflicting data and are not easily explained by a unified framework based on standard models of the evolution of sexually antagonistic genes. Previous studies have been confined to organisms with male heterogamety, meaning that effects related to homo- or heterozygosity of sex chromosomes cannot be separated from effects related to sex-specific characteristics. We therefore studied the genomic distribution of genes with sex-biased expression in the chicken, that is, in an organism with female heterogamety (males ZZ, females ZW). From the abundance of transcripts in expressed sequence tag libraries, we found an underrepresentation of female-specific genes (germ line and somatic tissue) and an overrepresentation of male-specific genes (somatic) on the Z chromosome. This is consistent with theoretical predictions only if mutations beneficial to one sex generally tend to be at least partly dominant (h > 0.5). We also note that sexual selection for a male-biased trait is facilitated by Z-linkage, because sons in organisms with female heterogamety will always inherit a Z chromosome from their fathers.

Animals↗

Identification of cDNAs encoding pterocarpan reductase involved in isoflavan phytoalexin biosynthesis in Lotus japonicus by EST mining.

Isoflavans and pterocarpans are the major biosynthetically connected phytoalexins in legumes. A search of the expressed sequence tag library of a model legume Lotus japonicus, which produces an (-)-isoflavan, for homologs of phenylcoumaran benzylic ether reductase catalyzing the reductive cleavage of dihydrofurans, yielded seven full-length cDNAs, and the encoded proteins were analyzed in vitro. Four of them cleaved the dihydrofuran of a pterocarpan medicarpin to yield an isoflavan (-)-vestitol and were designated pterocarpan reductase (PTR). Two PTRs displayed enantiospecificity to (-)-medicarpin, representing genuine L. japonicus PTRs, while the other two lacked enantiospecificity and were presumed to be evolutionarily primitive types.

DNA, Complementary↗

Strengths and weaknesses of EST-based prediction of tissue-specific alternative splicing.

BACKGROUND: Alternative splicing contributes significantly to the complexity of the human transcriptome and proteome. Computational prediction of alternative splice isoforms are usually based on EST sequences that also allow to approximate the expression pattern of the related transcripts. However, the limited number of tissues represented in the EST data as well as the different cDNA construction protocols may influence the predictive capacity of ESTs to unravel tissue-specifically expressed transcripts. METHODS: We predict tissue and tumor specific splice isoforms based on the genomic mapping (SpliceNest) of the EST consensus sequences and library annotation provided in the GeneNest database. We further ascertain the potentially rare tissue specific transcripts as the ones represented only by ESTs derived from normalized libraries. A subset of the predicted tissue and tumor specific isoforms are then validated via RT-PCR experiments over a spectrum of 40 tissue types. RESULTS: Our strategy revealed 427 genes with at least one tissue specific transcript as well as 1120 genes showing tumor specific isoforms. While our experimental evaluation of computationally predicted tissue-specific isoforms revealed a high success rate in confirming the expression of these isoforms in the respective tissue, the strategy frequently failed to detect the expected restricted expression pattern. The analysis of putative lowly expressed transcripts using normalized cDNA libraries suggests that our ability to detect tissue-specific isoforms strongly depends on the expression level of the respective transcript as well as on the sensitivity of the experimental methods. Especially splice isoforms predicted to be disease-specific tend to represent transcripts that are expressed in a set of healthy tissues rather than novel isoforms. CONCLUSIONS: We propose to combine the computational prediction of alternative splice isoforms with experimental validation for efficient delineation of an accurate set of tissue-specific transcripts.

Alternative Splicing↗

Identifying components of the NF-kappaB pathway in the beneficial Euprymna scolopes-Vibrio fischeri light organ symbiosis.

The Toll/NF-kappaB pathway is a common, evolutionarily conserved innate immune pathway that modulates the responses of animal cells to microbe-associated molecular patterns (MAMPs). Because MAMPs have been implicated as critical elements in the signaling of symbiont-induced development, an expressed sequence tag library from the juvenile light organ of Euprymna scolopes was used to identify members of the Toll/NF-kappaB pathway. Full-length transcripts were identified by using 5' and 3' RACE PCR. Seven transcripts critical for MAMP-induced triggering of the Toll/NF-kappaB phosphorylation cascade have been identified, including receptors, signal transducers, and a transcription factor. Further investigations should elucidate the role of the Toll/NF-kappaB pathway in the initiation of the beneficial symbiosis between E. scolopes and Vibrio fischeri.

Aliivibrio fischeri↗

Carbon monoxide binding by de novo heme proteins derived from designed combinatorial libraries.

Carbon monoxide binding was studied in a collection of de novo heme proteins derived from combinatorial libraries of sequences designed to fold into 4-helix bundles. The design of the de novo sequences was based on the previously reported "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains are varied extensively.(1) The combinatorial mixture of amino acids included histidine and methionine, which ligate heme iron in natural proteins. However, no attempt was made to explicitly design a heme binding site. Nonetheless, as reported previously, approximately half of the binary code proteins bind heme.(2) This collection of novel heme proteins provides a unique opportunity for an unbiased assessment of the functional potentialities of heme proteins that have not been prejudiced either by explicit design or by evolutionary selection. To assess the capabilities of the de novo heme proteins to bind diatomic ligands, we measured the affinity for CO, the kinetics of CO binding and release, and the resonance Raman spectra of the CO complexes for eight de novo heme proteins from two combinatorial libraries. The CO binding affinities for all eight proteins were similar to that of myoglobin, with dissociation constants (K(d)) in the low nanomolar range. The CO association kinetics (k(on)) revealed that the heme environment in all eight of the de novo proteins is partially buried, and the resonance Raman studies indicated that the local environment around the bound CO is devoid of hydrogen-bonding groups. Overall, the CO binding properties of the de novo heme proteins span a narrow range of values near the center of the range observed for diverse families of natural heme proteins. The measured properties of the de novo heme proteins can be considered as a "default" range for CO binding in alpha-helical proteins that have neither been designed to bind heme or CO, nor subjected to genetic selections for heme or CO binding.

Carbon Monoxide↗

Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower.

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.

DNA, Complementary↗

Unbiased selection of localization elements reveals cis-acting determinants of mRNA bud localization in Saccharomyces cerevisiae.

Cytoplasmic mRNA localization is a mechanism used by many organisms to generate asymmetry and sequester protein activity. In the yeast Saccharomyces cerevisiae, mRNA transport to bud tips of dividing cells is mediated by the binding of She2p, She3p, and Myo4p to coding regions of the RNA. To date, 24 bud-localized mRNAs have been identified, yet the RNA determinants that mediate localization remain poorly understood. Here, we used nonhomologous random recombination to generate libraries of sequences that could be selected for their ability to bind She-complex proteins, thereby providing an unbiased approach for minimizing and mapping localization elements in several transported RNAs. Analysis of the derived sequences and predicted secondary structures revealed short sequence motifs that mediate binding to the She complex and RNA localization to the bud tip in vivo. A predicted single-stranded core CG dinucleotide appears to be an important component of the RNA-protein interface, although other nucleotides contribute in a context-dependent manner. Our findings further our understanding of RNA recognition by the She complex, and the methods used here should be applicable for elucidating minimal RNA motifs involved in many other types of interactions.

Base Pairing↗

HuR mRNA ligands expressed after seizure.

HuR is a ubiquitously expressed AU-rich element (ARE)-binding protein that interacts with and stabilizes selective early response gene (ERG) mRNAs after cell activation or stress. To date, approximately 20 mRNAs have been unambiguously defined as HuR ligands. Given the discordance between the large number of ERG mRNAs and those few defined as ligands, we applied in vitro selection to isolate a broad range of HuR mRNA ligands using postseizure mouse hippocampal tissue. Selected mRNAs were converted into cDNA libraries and sequenced. Using this approach, we have identified over 600 novel, putative HuR mRNA ligands. These genes code for a variety of proteins, including transcription factors, signaling molecules, and kinases, but many have unknown function. Consistent with the means of their selection, several of these HuR ligands are differentially expressed in hippocampus after seizure. These results demonstrate a biochemical approach to identify and characterize the diverse repertoire of ligands for HuR and other regulatory mRNA-binding proteins.

Animals↗

Narbonin, a novel 2S protein from Vicia narbonensis L. seeds: cDNA, gene structure and developmentally regulated formation.

cDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced. The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (M(r) ca. 33 100) and an initiation methionine. The deduced amino acid sequence lacks a transient N-terminal signal peptide. The genomic clones do not contain any intron. No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries. The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins. However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein. Southern analysis suggests the existence of a small narbonin gene family. Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max. In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds.

Amino Acid Sequence↗

Assembly of the 68- and 72-kD proteins of signal recognition particle with 7S RNA.

Signal recognition particle (SRP), the cytoplasmic ribonucleoprotein particle that mediates the targeting of proteins to the ER, consists of a 7S RNA and six different proteins. The 68- (SRP68) and 72- (SRP72) kD proteins of SRP are bound to the 7S RNA of SRP as a heterodimeric complex (SRP68/72). Here we describe the primary structure of SRP72 and the assembly of SRP68, SRP72 and 7S RNA into a ribonucleoprotein particle. The amino acid sequence deduced from the cDNA of SRP72 reveals a basic protein of 671 amino acids which shares no sequence similarity with any protein in the sequence data libraries. Assembly of SRP72 into a ribonucleoprotein particle required the presence of 7S RNA and SRP68. In contrast, SRP68 alone specifically bound to 7S RNA. SRP68 contacts the 7S RNA via its NH2-terminal half while COOH-terminal portions of SRP68 and SRP72 are in contact with each other in SRP. SRP68 thus serves as a link between 7S RNA and SRP72. As a large NH2-terminal domain of SRP72 is exposed on SRP it may be a site of contact to other molecules involved in the SRP cycle between the ribosome and the ER membrane.

Amino Acid Sequence↗

[Isolation of maize genes related to aluminum tolerance].

To investigate gene expression profile in response to aluminum stress and to cloning the key genes related to aluminum tolerance, are crucial to genetic improvement of plant aluminum tolerance. In this study, suppression subtractive hybridization method was adopted to construct SSH-cDNA libraries at seedling stage of two maize inbred lines (Fig. 1), of which Mo17 is sensitive to aluminum toxicity and TL94B is tolerant. As a result, a forward SSH-cDNA library including 762 clones and a reverse SSH-cDNA library including 382 clones were constructed for Mo17. In the same way, a forward SSH-cDNA library including 760 clones and a reverse SSH-cDNA library including 380 clones were constructed for TL94B. Identification of these SSH-cDNA libraries shows that the length of inserted fragments ranges from 250 bp to 1.0 kb (Fig. 2), of which nearly 18% are positive clones. Through differential hybridization screening (Fig. 3), 124 and 47 positive clones were screened from forward and reverse SSH-cDNA libraries of Mo17 respectively; 103 and 64 positive clones from forward and reverse SSH-cDNA libraries of TL94B respectively. Total 338 positive clones from four SSH-cDNA libraries were sequenced, and all of the sequences of inserted fragments were analyzed using bioinformatical methods. A total of 232 kinds of EST sequences were obtained. Among these ESTs, 70.2% had significant homology with known genes, and the remaining 29.8% were function-unknown including 21 kinds of newly found ESTs (Table 1). An aluminum tolerant gene, GDP dissociation inhibitor gene, was detected its expression character by Northern hybridization (Fig. 4). These results indicate that the responses of maize to aluminum stress involve the interactions among different signal/metabolism pathways, such as signal transduction of stress-related factors, transcription and regulation of responsive genes, synthesis and transport of substances, changes in cell structures and functions.

Adaptation, Physiological↗

Analysis of the quality and utility of random shotgun sequencing at low redundancies.

The currently favored approach for sequencing the human genome involves selecting representative large-insert clones (100-200 kb), randomly shearing this DNA to construct shotgun libraries, and then sequencing many different isolates from the library. This method, entitled directed random shotgun sequencing, requires highly redundant sequencing to obtain a complete and accurate finished consensus sequence. Recently it has been suggested that a rapidly generated lower redundancy sequence might be of use to the scientific community. Low-redundancy sequencing has been examined previously using simulated data sets. Here we utilize trace data from a number of projects submitted to GenBank to perform reconstruction experiments that mimic low-redundancy sequencing. These low-redundancy sequences have been examined for the completeness and quality of the consensus product, information content, and usefulness for interspecies comparisons. The data presented here suggest three different sequencing strategies, each with different utilities. (1) Nearly complete sequence data can be obtained by sequencing a random shotgun library at sixfold redundancy. This may therefore represent a good point to switch from a random to directed approach. (2) Sequencing can be performed with as little as twofold redundancy to find most of the information about exons, EST hits, and putative exon similarity matches. (3) To obtain contiguity of coding regions, sequencing at three- to fourfold redundancy would be appropriate. From these results, we suggest that a useful intermediate product for genome sequencing might be obtained by three- to fourfold redundancy. Such a product would allow a large amount of biologically useful data to be extracted while postponing the majority of work involved in producing a high quality consensus sequence.

Animals↗