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PubMed2005

Identification and characterization of tandem repeats in exon III of dopamine receptor D4 (DRD4) genes from different mammalian species.

In this study we have identified and characterized dopamine receptor D4 (DRD4) exon III tandem repeats in 33 public available nucleotide sequences from different mammalian species. We found that the tandem repeat in canids could be described in a novel and simple way, namely, as a structure composed of 15- and 12- bp modules. Tandem repeats composed of 18-bp modules were found in sequences from the horse, zebra, onager, and donkey, Asiatic bear, polar bear, common raccoon, dolphin, harbor porpoise, and domestic cat. Several of these sequences have been analyzed previously without a tandem repeat being found. In the domestic cow and gray seal we identified tandem repeats composed of 36-bp modules, each consisting of two closely related 18-bp basic units. A tandem repeat consisting of 9-bp modules was identified in sequences from mink and ferret. In the European otter we detected an 18-bp tandem repeat, while a tandem repeat consisting of 27-bp modules was identified in a sequence from European badger. Both these tandem repeats were composed of 9-bp basic units, which were closely related with the 9-bp repeat modules identified in the mink and ferret. Tandem repeats could not be identified in sequences from rodents. All tandem repeats possessed a high GC content with a strong bias for C. On phylogenetic analysis of the tandem repeats evolutionary related species were clustered into the same groups. The degree of conservation of the tandem repeats varied significantly between species. The deduced amino acid sequences of most of the tandem repeats exhibited a high propensity for disorder. This was also the case with an amino acid sequence of the human DRD4 exon III tandem repeat, which was included in the study for comparative purposes. We identified proline-containing motifs for SH3 and WW domain binding proteins, potential phosphorylation sites, PDZ domain binding motifs, and FHA domain binding motifs in the amino acid sequences of the tandem repeats. The numbers of potential functional sites varied pronouncedly between species. Our observations provide a platform for future studies of the architecture and evolution of the DRD4 exon III tandem repeat, and they suggest that differences in the structure of this tandem repeat contribute to specialization and generation of diversity in receptor function.

Amino Acid Motifs↗

PubMed1998

DIVCLUS: an automatic method in the GEANFAMMER package that finds homologous domains in single- and multi-domain proteins.

MOTIVATION: Large-scale determination of relationships between the proteins produced by genome sequences is now common. All protein sequences are matched and those that have high match scores are clustered into families. In cases where the proteins are built of several domains or duplication modules, this can lead to misleading results. Consider the very simple example of three proteins: 1, formed by duplication modules A and B; 2, formed by duplication modules B' and C; and 3, formed by duplication modules C' and D. Duplication modules B and B' are homologous, as are C and C'. Matching the sequences of 1, 2 and 3 followed by simple single-linkage clustering would put all three in the same family, even though proteins 1 and 3 are not related. This is because the different parts of 2 match 1 and 3. This paper describes a procedure, DIVCLUS, that divides such complex clusters of partially related sequences into simple clusters that contain only related duplication modules. In the example just given, it would produce two groups of sequences: the first with domains B of sequence 1 and B of sequence 2, and the second with domain C of sequence 2 and C of sequence 3. DIVCLUS is part of a package called GEANFAMMER, for GEnome ANalysis and protein FAMily MakER. The package automates the detection of families of duplication modules from a protein sequence database. RESULTS: DIVCLUS has been applied to the division of single-linkage clusters generated from the protein sequences of six completely sequenced bacterial genomes. Out of 12 013 genes in these six genomes, 4563 single- and multi-domain sequences formed 1071 complex clusters. Application of the DIVCLUS program resolved these clusters into 2113 clusters corresponding to single duplication modules. AVAILABILITY: The perl5 program and its documentation are available at the following address: http://www.mrc-lmb.cam.ac.uk/genomes/ and by anonymous ftp at ftp.mrc-lmb.cam.ac.uk in the directory /pub/genomes/Software/. CONTACT: sat@mrc-lmb.cam.ac.uk; jong@mrc-lmb. cam.ac.uk

Algorithms↗

PubMed2000

Phoma glomerata as a mycoparasite of powdery mildew.

Ampelomyces and Phoma species are frequently confused with each other. Isolates previously attributed to the genus Ampelomyces were shown to be Phoma isolates through studies of their morphology and life cycle and ribosomal DNA internal transcribed spacer region 1 sequence analysis. Phoma glomerata can colonize and suppress development of powdery mildew on oak and may have utility as a mycoparasitic agent.

Ascomycota↗

PubMed2006

Comparative genomics of fungal allergens and epitopes shows widespread distribution of closely related allergen and epitope orthologues.

BACKGROUND: Allergy is a common debilitating and occasionally life threatening condition. The fungal kingdom contains a number of species that produce a wide range of well defined protein allergens although the vast majority of fungal species have unknown allergenic potential. The recent genome sequencing of a variety of fungi provides the opportunity to assess the occurrence of allergen orthologues across the fungal kingdom. Here we use comparative genomics to survey the occurrence of allergen orthologues in fungi. RESULTS: A database of 82 allergen sequences was compiled and used to search 22 fungal genomes. Additionally we were able to model allergen structure for representative members of several highly homologous allergen orthologue classes. We found that some allergen orthologue classes that had predicted structural congruence to allergens and allergen epitopes were ubiquitous in all fungi. Other allergen orthologues classes were less well conserved and may not possess conserved allergen epitope orthologues in all fungi. A final group of allergen orthologues, including the major allergens Asp f 1 and Alt a 1, appear to be present in only a limited number of species. CONCLUSION: These results imply that most fungi may possess proteins that have potential to be allergens or to cross react with allergens. This, together with the observation that important allergens such as Asp f 1 are limited to genera or species, has significant implications for understating fungal sensitization, and interpreting diagnosis and management of fungal allergy.

Allergens↗

PubMed2005

[Application to allergic diseases].

The increasing prevalence of allergic diseases in developed countries is considered to be caused, at least in part, by rapid improvement of human hygiene. In human beings, the immune system developed as an ingenious device for defending against frequent attacks by microbes. Therefore, our immune system seems to have become deranged in our recent, unprecedentedly hygienic environment. It is now necessary to understand the total functional elements comprising the immune system, not just a single molecule present in an immunocyte working in our immune system. Microarray analysis is now becoming capable of detecting the whole transcripts present in a cell. It is anticipated that we can understand the deranged human immunity using the system biology. It is also expected to predict previously unexpected drug-related adverse events caused by interaction of a drug with responsible molecules present in vital organs.

Animals↗

PubMed2001

Advances in the analysis of DNA sequence variations using oligonucleotide microchip technology.

The analysis of DNA variation (polymorphisms and mutations) on a genome-wide scale is becoming both increasingly important and technically challenging. An integration of a growing number of molecular biological methods of DNA-sequence analysis with the high-throughput feature of oligonucleotide microarray-based technologies is one of the most promising current directions of research and development.

Base Sequence↗

PubMed2006

Gene expression profiles of vitrified in vivo derived 8-cell stage mouse embryos detected by high density oligonucleotide microarrays.

Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to compare the gene expression patterns, and identify those most affected. For this, 8-cell stage embryos were collected from ICR mice and vitrified with solid surface vitrification technique, while maintaining equal numbers of embryos as control. Total RNAs were extracted and two rounds of amplification were employed. Finally three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22 K oligonucleotide slides and the results were analyzed with subsequent verification by independent real-time PCR analyses. The two rounds of amplification with 5 ng tRNA input have yielded 15-16 microg of cRNA. The analyses of repeated hybridizations showed 20,183 genes/ESTs as common signatures, and unsupervised analysis identified 628 differentially expressed (P < 0.01) genes. However, with at least 1.5-fold change considerations, 183 genes were differentially expressed (P < 0.01) out of which 107 were upregulated. The independent analysis with real-time PCR and unamplified samples fully confirmed the results of microarray, indicating the linearity of amplification. Furthermore, this novel gene expression study for vitrified embryos identified many new candidate genes with overrepresentation in some important biological processes. Thus, it is possible to conclude that the expression pattern reflected a broad spectrum of consequences of vitrification on embryos, with most effects on metabolism, regulatory role and stress response genes and allowed the identification of new candidate marker genes for cryosurvival.

Animals↗

PubMed2026

META-DIFF: a k-mer-based pipeline that detects differentially abundant sequences in metagenomics whole genome sequencing.

Traditional case-control metagenomic studies are constrained by their dependence on taxonomic and functional databases. Because annotation occurs before differential analysis, they are limited to known elements and keep function and taxonomy separate. Although binning strategies have emerged to reconstruct genomes and mitigate this issue, they still require an assembly step, preventing the use of all available sequencing data. Here, we introduce META-DIFF, a pipeline based on differentially abundant k-mers independently of any prior annotation. From those k-mers, it reconstructs longer sequences and provides biological context, as well as the best set of unitigs to discriminate between conditions. Across both taxonomy-centric and functionally-centric benchmarks, it showed robust performance and displayed great reproducibility. It also behaved more conservatively than did other univariate methodologies, i.e. it maintained a high precision at the expense of recall, particularly in conditions of low fold-change and limited sequencing depth. The efficacy of META-DIFF was further validated through its application to a real-world colorectal cancer dataset, which produced both confirmatory and novel results compared with those of previous publications. The pipeline is able to exploit all reads and identify differentially abundant elements, including unknown DNA, prior to annotation. With the guidelines provided, META-DIFF provides users with great exploratory power to unravel microbiome changes.

Metagenomics↗

PubMed2005

Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins.

The Western corn rootworm is the major pest of corn in the USA and has recently become the target for insect-resistant transgenic crops. Transgenic crops have switched the focus for identifying insecticide targets from the insect nervous system to the midgut. Here we describe a collection of 691 sequences from the Western corn rootworm midgut, 27% of which predict proteins with no matches in current databases. Of the remaining sequences, most predict proteins with either catalytic (62%) or binding (19%) functions, as expected for proteins expressed in the insect midgut. The utility of this approach for the identification of targets for novel toxins is demonstrated by analysis of the first coleopteran cadherin gene, a putative Bt receptor, and a large class of cysteine-proteases, the cathepsins.

Amino Acid Sequence↗

PubMed1982

Molecular cloning, genomic analysis, and biological properties of rat leukemia virus and the onc sequences of Rasheed rat sarcoma virus.

Rasheed rat sarcoma virus (RaSV) has been shown to code for a protein of 29,000 Mr not present in replication-competent rat type C helper virus (RaLV)-infected cells. This protein is a fused gene product consisting of a portion of the RaLV p15 gag protein and the transformation-specific 21,000 Mr (p21) ras protein, which is also found in Harvey murine sarcoma virus. We now report the molecular cloning of both the SD-1 (Sprague-Dawley) strain of RaLV and the transforming ras sequences of RaSV. Heteroduplex analysis of these cloned DNAs demonstrated that the RaSV ras gene (v-Ra-ras) was inserted into the rat type C viral genome with a small deletion of RaLV genetic information in the 5' region of the gag gene and that the v-Ra-ras gene (0.72 kilobase pair) is homologous to and colinear with the p21 ras gene of Harvey murine sarcoma virus (v-Ha-ras). Restriction enzyme mapping confirmed the homology demonstrated by heteroduplex mapping, showing strong site conservation of restriction endonucleases known to cleave v-Ha-ras. Cloned v-Ra-ras DNA transformed NIH 3T3 cells, inducing the synthesis of the p29 RaSVgag-ras protein.

Base Sequence↗

PubMed1994

Complete nucleotide sequence of the RNA 3 from subgroup II of cucumber mosaic virus (CMV) strain: Trk7.

The complete nucleotide sequence (2209 nucleotides) of the RNA 3 of the cucumber mosaic virus strain Trk7 was determined. Trk7-CMV possesses two open reading frames which encode the 3a protein (279 amino acids) and the coat protein (218 amino acids). Sequence analysis proved the earlier biological and serological classification of the RNA 3 molecule of Trk7-CMV in subgroup II. A high degree of homology was found in the strains Q and Kin of CMV, while the average similarity between the two different subgroups is much lower.

Amino Acid Sequence↗

PubMed2001

Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies.

A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-IZ solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-IIZ solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASHII Vector and GigapackIII XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h.

Cloning, Molecular↗

PubMed2005

SplitTester: software to identify domains responsible for functional divergence in protein family.

BACKGROUND: Many protein families have undergone functional divergence after gene duplications such that current subgroups of the family carry out overlapping but distinct biological roles. For the protein families with known functional subtypes (a functional split), we developed the software, SplitTester, to identify potential regions that are responsible for the observed distinct functional subtypes within the same protein family. RESULTS: Our software, SplitTester, takes a multiple protein sequences alignment as input, generated from protein members of two subgroups with known functional divergence. SplitTester was designed to construct the neighbor joining tree (a split cluster) from variable-sized sliding windows across the alignment in a process called split-clustering. SplitTester identifies the regions, whose split cluster is consistent with the functional split, but may be inconsistent with the phylogeny of the protein family. We hypothesize that at least some number of these identified regions, which are not following a random mutation process, are responsible for the observed functional split. To test our method, we used reverse transcriptase from a group of Pseudoviridae retrotransposons: to identify residues specific for diverged primer recognition. Candidate regions were then mapped onto the three dimensional structures of reverse transcriptase. The locations of these amino acids within the enzyme are consistent with their biological roles. CONCLUSION: SplitTester aims to identify specific domain sequences responsible for functional divergence of subgroups within a protein family. From the analysis of retroelements reverse transcriptase family, we successfully identified the regions splitting this family according to the primer specificity, implying their functions in the specific primer selection.

Algorithms↗

PubMed1998

[Variability of Trichophyton cerrucosum isolates from vaccinated herds with cattle ringworm].

Twenty-seven strains of Trichophyton verrucosum from 14 cattle herds in the Federal States of Thuringia and Mecklenburg-Vorpommern were examined by culture morphological and molecular biological (PCR fingerprinting, AFLP analysis sequencing of ITS region) methods. Six reference strains of the same species, among them the so-called album and ochraceum varieties, were also included. Despite great variability in terms of culture morphology, which suggested their possible classification into 4 different colony types, all T. verrucosum isolates were genotypically almost identical. Even the 2 field isolates growing with yellow pigment, which could possibly be regarded as belonging to the ochraceum variety, could not be differentiated using molecular biological methods. The results do not provide indications of a separate taxonomic position of the 3 T. verrucosum varieties. Furthermore, there is no evidence confirming the suspected infection of cattle herds with ochraceum strains as the cause of the failure of immune prophylaxis using various T. verrucosum vaccines. The frequent occurrence of animals not responding to vaccination could not be explained either. It should be assumed that the main factors responsible for this situation include poor handling of the vaccine strains and errors in application, especially the absence of continuous and systematic immune prophylaxis in the herds.

Animals↗

PubMed2004

The curious adventures of Trichophyton equinum in the realm of molecular biology: a modern fairy tale.

Sequence analysis of the highly variable internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA (rDNA) of the nuclear DNA has been used to explore the phylogeny of the dermatophytes. Results have led some investigators to recommend that a number of dermatophyte species be reduced to synonymy with other established species. One such recommendation is that both varieties of the zoophilic dermatophyte Trichophyton equinum be reduced to synonymy with the anthropophilic species T. tonsurans. The morphologies of both species are reviewed, as are their roles in human infection, their physiological characteristics and their respective ecologies. Close examination of these attributes shows clear differences between the varieties of T. equinum and T. tonsurans. The significance of the homogeneity of the ITS sequences of these two dermatophytes is discussed in the context of more recent appraisals of the results of ITS sequencing in other fungi. It is concluded that the results of ITS analysis for both varieties of T. equinum and T. tonsurans are indicative only of common ancestry. Similar arguments could be put forward for rescinding other changes in dermatophyte nomenclature that have been published in recent years.

Animals↗

PubMed2000

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa.

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21-carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.

Animals↗

PubMed2001

[Analysis of the most frequent mutations in girls with Rett syndrome].

BACKGROUND: Rett syndrome is an X-linked dominant neurodevelopmental disorder affecting 1 from 10,000 to 15,000 females worldwide. The responsible gene, encoding methyl-CpG binding protein 2 was recently identified. Methyl-CpG binding protein 2 is thought to act as a global transcriptional repressor. In the methyl-CpG binding protein 2 gene are known 5 prevalent mutations that cause Rett syndrome. Four of them are detectable by restriction analysis. In this study we present the results of the molecular study of four prevalent mutations in the gene for methyl-CpG binding protein 2 in Czech and Slovak patients with Rett syndrome. METHODS AND RESULTS: 22 females with Rett syndrome were investigated by methods of molecular biology. Restriction analysis and direct sequencing of PCR products revealed in methyl-CpG binding protein 2 gene 3 different mutations (T158M, R168X, R270X) in six unrelated patients with Rett syndrome. Mutation R306C, frequent in Great Britain and Sweden, was not detected in our group of patients with Rett syndrome. CONCLUSIONS: The diagnosis of Rett syndrome and genetic counselling in affected families should go out from the close cooperation of the pediatric, neurologic, and genetic departments with the specialized laboratories dealing with the molecular biological diagnosis.

Adolescent↗

PubMed1990

Receptor mechanisms. Structure and molecular biology of transmitter receptors.

Details of receptor structure and function that were unavailable as recently as two years ago are now readily obtainable through the application of molecular biological techniques. Cloning and sequence analysis of neurotransmitter receptor genes have provided information on the primary structure of these proteins, revealing the relationship between pharmacologically diverse families of receptors. Knowledge of the primary structure of receptors has allowed for prediction of secondary structure and the construction of three-dimensional models. Permanent expression of cloned neurotransmitter receptor genes in cultured cells is providing unlimited sources of pure receptor, which allows for pharmacological and biochemical studies on single receptor subtypes. The use of site-directed mutagenesis to elucidate the relationship between protein structure and function has provided considerable information on the role of certain conserved amino acids in receptor function and has suggested possible molecular mechanisms of signal transduction across membranes. The article will review some of these recent developments in the area of neurotransmitter receptors and point out the utility of molecular biology in these endeavors.

Amino Acid Sequence↗