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The chemokine information source: identification and characterization of novel chemokines using the WorldWideWeb and expressed sequence tag databases.

The chemokine superfamily is a large group of more than 30 small proteins. Many of these were originally identified because of their role in the selective recruitment and activation of leukocytes during inflammation. More recently, some of the chemokine receptors and ligands have been implicated in the mechanism of viral infection for primate lentiviruses such as HIV-1. From the original identification of interleukin-8 (IL-8; the most studied member of the superfamily), the number of new family members has mushroomed over the last few years. Two events have dramatically altered the speed at which sequence information concerning novel chemokines has become available to the scientific community. First, many groups have been obtaining large amounts of sequence information from cDNA libraries by sequencing the clones at random, generating expressed sequence tags (ESTs). Although these ESTs are relatively short, typically less than 500 bases, this amount of sequence is usually sufficient to obtain the entire open reading frame for chemokines. Second, there has been a rapid growth in the use of the WorldWideWeb by bioinformatics groups. The Web was originally set up by the European Centre for Particle Physics (CERN) in Geneva as a method of transferring data between collaborating groups throughout the world. It has enabled biologists throughout the world to have almost instantaneous access both to the databases containing the EST sequences and to the automated tools that are required for searching such databases. With such methods we have been able to rapidly identify more than 10 new human chemokines from public domain databases. In addition to the known categories of chemokines, which are named C, CC, and CXC based on the spacings of N-terminal cysteine residues, we have been able to identify the first member of a novel chemokine subfamily, with a novel CXXXC cysteine spacing. Furthermore, we can subdivide the CC chemokines into monocyte chemotactic protein and macrophage inflammatory protein families based on their sequence identity levels, but also their clustering on the human genome, as identified on other Web sites. The rapid availability of all this data has reduced the amount of time spent on conventional gene identification, enabling us to move quickly on to trying to understand the biology and physiological relevance of these molecules. The novel chemokine sequences obtained and alignments with existing members of the superfamily are now contained within a Chemokine Information Source on an open access server, allowing further searching of chemokine sequences and increasing the availability of such data to the scientific community.

Amino Acid Sequence↗

Assembly and functional expression of murine glutathione reductase cDNA: a sequence missing in expressed sequence tag libraries.

Glutathione reductase (GR) is a chemotherapeutic target. Murine GRcDNA, which contains 85% GC in the 38 codons following the start codon, was assembled from the PCR-amplified exon 1 and a downstream cDNA prior to expression in Escherichia coli as a His(6)-tagged protein. Recombinant GR, an FAD-containing homodimer, corresponds in its enzymic and spectral properties to GR isolated from murine Ehrlich ascites tumor cells. Another cDNA, representing GR with a mitochondrial targeting sequence, yielded two distinct enzymically active expression products.

Animals↗

Gene-sequence-tag expression analyses of 1,800 genes related to chloroplast functions.

Quantification of the expression levels of nuclear genes encoding plastid proteins under different genetic or environmental conditions can contribute to the genetic dissection of plastid functions. To facilitate such measurements, a set of 1,827 Arabidopsis thaliana genes coding for plastid proteins was PCR-amplified from genomic DNA and spotted on nylon membranes to generate an array of chloroplast-specific gene-sequence-tags (GSTs). The sensitivity and reliability of the experimental system was evaluated and a procedure was developed for detecting differential gene expression. The GST array was found to serve as a reliable monitor of changes in gene expression induced by environmental and genetic alteration of chloroplast functions. Based on comparisons of dark- versus light-grown seedlings, and wild-type versus prpl11-1 plants, lists of differentially expressed genes are provided which include 193/7 and 25/42 up/down-regulated genes, respectively. The cut-off values for differential expression were 2.5-times (up) and 0.40 (down). Additional up-regulated genes with relatively low expression ratios (from 1.5- to 2.5-times) or down-regulated with relatively high ratios (0.4-0.67) can be accessed at the website: http://www.mpiz-koeln.mpg.de/~richly/GST-array.html. A sample of genes analysed by quantitative reverse transcription PCR confirmed the expression profiles monitored by the GST array. Differential hybridisation experiments with the prpl11-1 mutant revealed the existence of regulatory networks sensing the protein state of the chloroplast and transmitting the signal to the nucleus.

Arabidopsis↗

Microarray analysis of expressed sequence tags from haustoria of the rust fungus Uromyces fabae.

Rust fungi are plant parasites which colonise host tissue with an intercellular mycelium that forms haustoria within living plant cells. To identify genes expressed during biotrophic growth, EST sequencing was performed with a haustorium-specific cDNA library from Uromyces fabae. One thousand seventeen ESTs were generated, which assembled into 530 contigs. Several of the most frequently represented sequences in the EST database were identical to the in planta induced genes (PIGs) identified previously (Hahn, M., Mendgen, K., 1997. Characterisation of in planta-induced rust genes isolated from a haustorium-specific cDNA library, Mol. Plant-Microbe Interact. 10, 427-437). Virus-encoded sequences were identified, providing evidence for two novel RNA mycoviruses in U. fabae. Microarray hybridisation revealed many cDNAs that were significantly activated in rust-infected leaves compared to germinated uredospores. Very strong in planta expression was found for two PIGs encoding putative metallothioneins. Furthermore, several genes involved in ribosome biogenesis and translation, glycolysis, amino acid metabolism, stress response, and detoxification showed an increased expression in the parasitic mycelium. These data indicate a strong shift in gene expression in rust fungi between germination and the biotrophic stage of development.

Basidiomycota↗

Identification and initial characterization of 6,000 expressed sequenced tags (ESTs) from rat normal-growing cartilage and swarm rat chondrosarcoma cDNA libraries.

Chondrosarcoma is the second most common type of skeletal malignancy with a survival rate at five years for histological grade III of only 29 percent. The development of a reliable chondrosarcoma animal model could enable the study of tumor growth and progression, the effect of the host on tumor behavior, and the effectiveness of various therapeutic modalities. The Swarm rat chondrosarcoma is a tumor tissue line that has been maintained through the years by serial subcutaneous injections, and the histochemical characteristics of the tumor have remained essentially similar in all transplants over the years. This study was designed to initiate the characterization of the Swarm rat chondrosarcoma model by gene expression profiling as compared to normal-growing rat cartilage. Analysis of the gene expression from both libraries revealed a complex pattern of gene expression, including many genes not yet reported to be expressed by chondrocytes. It suggests that the biochemical characterization of growing cartilage and chondrosarcoma reported to date has only begun to describe the complexity of these tissues.

Animals↗

Recently evolved genes identified from Drosophila yakuba and D. erecta accessory gland expressed sequence tags.

The fraction of the genome associated with male reproduction in Drosophila may be unusually dynamic. For example, male reproduction-related genes show higher-than-average rates of protein divergence and gene expression evolution compared to most Drosophila genes. Drosophila male reproduction may also be enriched for novel genetic functions. Our earlier work, based on accessory gland protein genes (Acp's) in D. simulans and D. melanogaster, suggested that the melanogaster subgroup Acp's may be lost and/or gained on a relatively rapid timescale. Here we investigate this possibility more thoroughly through description of the accessory gland transcriptome in two melanogaster subgroup species, D. yakuba and D. erecta. A genomic analysis of previously unknown genes isolated from cDNA libraries of these species revealed several cases of genes present in one or both species, yet absent from ingroup and outgroup species. We found no evidence that these novel genes are attributable primarily to duplication and divergence, which suggests the possibility that Acp's or other genes coding for small proteins may originate from ancestrally noncoding DNA.

Animals↗

Nuclear expressed sequence Tag (NEST) analysis: a novel means to study transcription through amplification of nuclear RNA.

We describe a novel concept and corresponding methods for the analysis of transcription in higher plant cells. The concept is that an examination of the presence of different polyadenylated transcripts within isolated nuclei reflects the state of gene expression at a given moment more precisely than do conventional techniques using total cellular mRNA. The methods involve isolation of polyadenylated nuclear transcripts from flow-sorted nuclei, reverse transcription, amplification using the polymerase chain reaction, and analysis of the products through gel electrophoresis and sequencing. By using specific primers, we have demonstrated detection of selected gene products in nuclei from transgenic plants. We also employed a technique for analysis of individual transcripts based on the length polymorphisms of restriction fragments derived from their 3' ends. Because the technique does not require a priori knowledge of the analyzed sequences, it is suitable for displaying the complete spectra of RNA transcripts present in nuclei at the moment of their isolation. These fragments can be easily isolated and sequenced and the sequence information used for assignment of putative function of corresponding genes. These techniques have been used to identify leaf-, root-, and cell cycle-specific transcripts. In principle, they should be applicable to the tissues of any eukaryotic species that contain transcriptionally active nuclei.

Base Sequence↗

Generation and characterization of cDNA clones from Sarcoptes scabiei var. hominis for an expressed sequence tag library: identification of homologues of house dust mite allergens.

Molecular studies on scabies, a disease of considerable human and veterinary significance, have been limited because of the difficulty of obtaining the causative organism Sarcoptes scabiei, the "itch mite." We have used skin from the bedding of crusted scabies patients as a source of mites for the construction of libraries of cDNAs from S. scabiei var. hominis in the bacteriophage lambda vector lambdaZAP express. Sequences of 145 clones established that the libraries predominantly contain sequences from S. scabiei, enabling a major sequencing program to begin. Among those sequenced to date, cDNAs encoding S. scabiei homologues of 3 house dust mite allergens-the M-177 apolipoprotein, glutathione S-transferase, and paramyosin--were identified. The availability of cDNA libraries from S. scabiei var. hominis and S. scabiei var. vulpes and the emerging public sequence databases from both opens up new possibilities in scabies research.

Allergens↗

Expressed sequence tags (ESTs) and single nucleotide polymorphisms (SNPs): what large-scale sequencing projects can tell us about ADME.

To date over 800 complete genomes have been sequenced, with many more partially complete. Coupled with the large amount of mRNA transcript sequence data being produced from expression studies, there is now a daunting amount of information available to the research scientist. This review examines how this information may be best used, focusing on examples from sequences encoding absorption, distribution, metabolism and excretion (ADME)-related proteins in particular. Through the use of phylogenetic, splice variant and single nucleotide polymorphism (SNP) analysis, the review examines not only how insights into species-specific responses to drug exposure may be gained, but also how best to utilize this information to predict both individual human responses and the impact of population variance in response.

Alternative Splicing↗

Analysis of expressed sequence tags from Gibberella zeae (anamorph Fusarium graminearum).

Gibberella zeae is a broad host range pathogen that infects many crop plants, including wheat and barley, and causes head blight and rot diseases throughout the world. To better understand fungal development and pathogenicity, we have generated 7996 ESTs from three cDNA libraries. Two libraries were generated from carbon-(C-) and nitrogen- (N-) starved mycelia and one library was generated from cultures of maturing perithecia (P). In other fungal pathogens, starvation conditions have been shown to act as cues to induce infection-related gene expression. To assign putative function to cDNAs, sequences were initially assembled using StackPack. The estimated total number of genes identified from the three EST databases was 2110: 1088 contigs and 1022 singleton sequences. These 2110 sequences were compared to a yeast protein sequence reference set and to the GenBank nonredundant database using BLASTX. Based on presumptive gene function identified by this process, we found that the two starved cultures had similar, but not identical, patterns of gene expression, whereas the developmental cultures were distinct in their pattern of expression. Of the three libraries, the perithecium library had the greatest percentage (46%) of ESTS falling into the "unclassified" category. Homologues of some known fungal virulence or pathogenicity factors were found primarily in the N- and C-libraries. Comparisons also were made with ESTs from the related fungi, Neurospora crassa and Magnaporthe grisea and the genomic sequence of N. crassa.

Expressed Sequence Tags↗