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PubMed2005

CR-EST: a resource for crop ESTs.

The crop expressed sequence tag database, CR-EST (http://pgrc.ipk-gatersleben.de/cr-est/), is a publicly available online resource providing access to sequence, classification, clustering and annotation data of crop EST projects. CR-EST currently holds more than 200,000 sequences derived from 41 cDNA libraries of four species: barley, wheat, pea and potato. The barley section comprises approximately one-third of all publicly available ESTs. CR-EST deploys an automatic EST preparation pipeline that includes the identification of chimeric clones in order to transparently display the data quality. Sequences are clustered in species-specific projects to currently generate a non-redundant set of approximately 22,600 consensus sequences and approximately 17,200 singletons, which form the basis of the provided set of unigenes. A web application allows the user to compute BLAST alignments of query sequences against the CR-EST database, query data from Gene Ontology and metabolic pathway annotations and query sequence similarities from stored BLAST results. CR-EST also features interactive JAVA-based tools, allowing the visualization of open reading frames and the explorative analysis of Gene Ontology mappings applied to ESTs.

Crops, Agricultural↗
PubMed2001

Comparing vertebrate whole-genome shotgun reads to the human genome.

Multi-species sequence comparisons are a very efficient way to reveal conserved genes. Because sequence finishing is expensive and time consuming, many genome sequences are likely to stay incomplete. A challenge is to use these fragmented data for understanding the human genome. Methods for using cross-species whole-genome shotgun sequence (WGS) for genome annotation are described in this paper. About one-half million high-quality rat WGS reads (covering 7.5% of the rat genome) generated at the Baylor College of Medicine Human Genome Sequencing Center were compared with the human genome. Using computer-generated random reads as a negative control, a set of parameters was determined for reliable interpretation of BLAST search results. About 10% of the rat reads contain regions that are conserved in the human genomic sequence and about one-third of these include known gene-coding regions. Mapping the conserved regions to human chromosomes showed a 23-fold enrichment for coding regions compared with noncoding regions. This approach can also be applied to other mammalian genomes for gene finding. These data predicted approximately 42,500 genes in the human, slightly more than reported previously.

Animals↗
PubMed1995

Locus-specific amplification of HLA class I genes from genomic DNA: locus-specific sequences in the first and third introns of HLA-A, -B, and -C alleles.

We have identified locus-specific sequences in the first and third introns flanking the polymorphic second and third exons of HLA class I genes. PCR primers derived from these conserved sequences produced DNA fragments of the expected sizes for each of the HLA-A, -B, and -C loci in the amplification of genomic DNA. PCR products generated using each of the locus-specific sets of primers displayed exquisite locus specificity, as assessed by hybridization with oligonucleotide probes specific for ten classical and non-classical HLA class I genes. Amplification with these primer sets was effective and specific for the HLA alleles tested under the given PCR conditions. When hybridized with oligonucleotides derived from shared polymorphic sequence motifs, reaction patterns of PCR products from each locus were precisely as expected from published or database sequences. Chemiluminescent signals generated from digoxygenin-ddUTP-labeled probes were even for all samples and as strong as those obtained in MHC class II typing. These locus-specific primer sets derived from intron sequences provide an effective means to amplify genomic DNA which will facilitate PCR-based HLA class I typing methods. This will also allow HLA class I typing to be conducted with greater precision, at lower cost, and faster than previously described class I typing methodologies.

Alleles↗
PubMed2006

Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 beta-lactamase.

A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.

Drug Resistance, Multiple, Bacterial↗
PubMed1985

Analysis of mobilization elements in plasmids from Shigella flexneri.

The mobilization properties of three plasmids were examined after cotransfer from Shigella flexneri to Escherichia coli. The largest plasmid, pCN1, was shown to be a conjugative R factor that could promote its own transfer and allow cotransfer of a 4.1-kilobase plasmid, pCN3; mobilization of the third plasmid, pCN2 (6.3 kilobases), required the presence of both pCN1 and pCN3. Sequences from pCN2 and pCN3 homologous to the bom (basis of mobilization) sites of ColE1 and pBR322 were localized by analysis of site-specific deletion derivatives generated in vivo during the transfer of composite plasmids and were characterized by DNA sequencing.

Base Sequence↗
PubMed1997

Molecular characterization of patients with 18q23 deletions.

The 18q- syndrome is a deletion syndrome that is characterized by mental retardation, hearing loss, midfacial hypoplasia, growth deficiency, and limb anomalies. Most patients with this syndrome have deletions from 18q21-qter. We report on three patients with deletions of 18q23. A mother and daughter with identical deletions of 18q23 have many of the typical features of the 18q- syndrome, including midfacial hypoplasia and hearing loss. In contrast, the third patient has few of the symptoms of the 18q- syndrome. A contig of the 18q23 region was generated to aid in the mapping of the breakpoints. FISH was used to map both breakpoints to the same YAC clone. Furthermore, somatic-cell hybrids from the daughter and the third patient were isolated. The mapping results of sequence-tagged sites relative to the two breakpoints were identical, suggesting that the two deletion breakpoints map very close to one another. The analyses of these patients demonstrate that the critical region for the 18q- syndrome maps to 18q23 but that a deletion of 18q23 does not always lead to the clinical features associated with the syndrome. These patients demonstrate the wide phenotypic variability associated with deletions of 18q.

Abnormalities, Multiple↗
PubMed1976

Size differences among immunoglobulin heavy chains from phosphorylcholine-binding proteins.

The entire sequences of the heavy chain variable regions of M167 and TEPC 15 (phosphorylcholine-binding myeloma proteins of BALB/c origin) have been determined. These sequences are compared with the phosphorylcholine-binding protein M603. T15 differs from M603 at four positions, all of which are located in antigen-binding complementarity regions. M167, in addition to having differences in the complementarity regions, also has five substitutions in the conserved framework portion of the variable region when compared to T15 and M603. Each of the three proteins has a different length in the third complementarity region. It is unlikely that complementarity regions of different lengths associated with similar framework regions could be generated by proposed mechanisms of somatic mutation which are generally limited to point mutations. It appears more likely that these products are directly encoded by different structural germ line genes.

Amino Acid Sequence↗
PubMed2026

Efficacy of the NMIC-150 system in identifying extended-spectrum beta-lactamases in clinical isolates.

Extended-spectrum beta-lactamases (ESBLs) are significant contributors to the growing global crisis of antimicrobial resistance. This study evaluated the performance of the NMIC-150 System for susceptibility testing of third-generation cephalosporins (3GCs) and assessed whether ceftazidime-avibactam and aztreonam-avibactam could identify ESBL-producing carbapenem-resistant Enterobacterales (CREs). A total of 278 non-duplicate clinical isolates (Klebsiella pneumoniae, E. coli, and Proteus mirabilis) were analyzed. Antimicrobial susceptibility was determined using reference broth microdilution (BMD) and the NMIC-150 System. ESBL production was defined as an ≥eight-fold reduction in the minimum inhibitory concentration (MIC) of 3GCs in the presence of clavulanic acid, according to CLSI criteria. Whole-genome sequencing was performed to characterize ESBL and carbapenemase genes among 3GC-resistant isolates. A Random Forest model was used to predict ESBL-producing isolates based on MIC values. The NMIC-150 System demonstrated over 90% categorical and essential agreement with BMD for ceftazidime and ceftriaxone, along with robust predictive performance via Random Forest analysis. These findings suggest that the NMIC-150 System is a reliable platform for 3GC susceptibility testing and that an ≥eight-fold MIC reduction with ceftazidime-avibactam or aztreonam-avibactam may serve as a phenotypic indicator of ESBL production in CRE isolates. In conclusion, the NMIC-150 System shows potential for routine antimicrobial resistance surveillance and may facilitate the rapid identification of ESBL-producing CREs in clinical settings.

Microbial Sensitivity Tests↗
PubMed1992

Identification of clusters of biallelic polymorphic sequence-tagged sites (pSTSs) that generate highly informative and automatable markers for genetic linkage mapping.

Using a combination of denaturing gradient gel electrophoresis and direct DNA sequencing, we have found that multiple (4 to 7) biallelic sequence polymorphisms can be located within short DNA segments, 300 to 2400 bp. Here, we report on the identification of three clusters of DNA polymorphisms, one in each of the constant regions of the human T cell receptor alpha and beta gene complexes on human chromosomes 14 and 7, respectively, and a third among the human t-RNA genes on human chromosome 14. The frequency of these polymorphisms and the extent of linkage disequilibrium between individual polymorphisms have been determined using a semiautomated DNA typing system combining DNA target amplification by the polymerase chain reaction with the analysis of internal sequence polymorphisms by a colorimetric oligonucleotide ligation assay. We have found that individual biallelic polymorphisms in each cluster are often in partial linkage disequilibrium with one another. This partial linkage disequilibrium permits the combined use of three to four markers in a cluster to generate a haplotype with high levels of heterozygosity, 71 to 88%. Therefore, clusters of physically linked biallelic polymorphisms provide an automatable and highly informative type of genetic marker for general linkage analysis as well as an attractive alternative marker system for fine-point mapping of disease-causing genes and phenotypic traits relative to their framework locations in the genome.

Alleles↗
PubMed1993

Detection of Borrelia burgdorferi by nested polymerase chain reaction in cerebrospinal fluid and urine of children with neuroborreliosis.

Diagnosis of neuroborreliosis is often difficult since history and clinical presentation may be non-specific and serological tests may initially be negative. We therefore tested the polymerase chain reaction (PCR) for the detection of borrelial sequences in CSF and urine samples of consecutive children with neuroborreliosis seen in a single summer season. Four of eight children were negative in serum for antibodies to Borrelia burgdorferi. Two of eight children were PCR-positive in CSF and one other child was positive in urine. In two out of four children PCR was the only laboratory test confirming the clinical diagnosis. All children recovered after treatment with third generation cephalosporins. When seven of eight children were re-examined 6 months later all were healthy and antibodies to B. burgdorferi were detected in their serum. PCR may assist the paediatrician in establishing a diagnosis of neuroborreliosis; however, a negative result does not rule out neuroborreliosis. PCR is an adjunct, but no substitute for clinical judgement and serology.

Adolescent↗
PubMed2006

A new mechanism for generating TCR diversity: A TCR Jalpha-like gene that inserts partial nucleotide sequences in a D-gene manner.

delta-TCR genes of two gammadelta-T cell hybridomas were found to contain an identical 19-nt sequence in their non-germline N-regions. To determine if this sequence represented a third murine TCR Ddelta gene, genomic PCR was performed by using it as a primer together with primers for interspersed repetitive elements (IRE). Sequencing revealed that the 19-nt segment is part of a 61-nt gene with flanking 5' and 3' recombination signaling sequences (RSS). Southern blot analysis confirmed the presence of this 61-nt gene in the genome of several mouse strains. The gene is unusual in that the distal 24 nucleotides of its 3' RSS region are contributed by the 5' portion of a B2 IRE sequence that includes an apparently non-functional RNA splice site within the 3' nonamer sequence. It has sequence similarities with the Ddelta1 gene (81%) at its 5' end and with Jalpha genes (73%) overall. Tyramide-FISH analysis identified the gene to exist within or adjacent to the TCR alpha/delta locus on chromosome 14. Surveys of available TCR sequences reveal possible partial insertions of the 61-nt gene in other delta-TCR and in alpha-TCR gene sequences. Thus, the unique 61-nt gene is Jalpha gene-like in structure but D gene-like in function.

Amino Acid Sequence↗
PubMed2003

Three novel PAX6 mutations in patients with aniridia.

AIMS: To describe mutations in the PAX6 gene in five patients with aniridia from three unrelated families. METHODS: The PAX6 gene was analysed using single stranded conformational polymorphism analysis and direct sequencing. RESULTS: In one family, three individuals from two generations had aniridia, whereas in each of the other families only one member was affected. The first patient had the heterozygous Q221X (1023C --> T) nonsense mutation in exon 8. The same mutation was found in his mother and sister. Another patient had a heterozygous Q297X (1252C --> T) mutation in exon 10. The third patient carried a heterozygous IVS5+2T --> C mutation leading to aberrant splicing of mRNA. CONCLUSIONS: These findings provide further examples of haploinsufficiency of PAX6 in aniridia.

Aniridia↗
PubMed2003

Timing of network synchronization by refractory mechanisms.

Even without active pacemaker mechanisms, temporally patterned synchronization of neural network activity can emerge spontaneously and is involved in neural development and information processing. Generation of spontaneous synchronization is thought to arise as an alternating sequence between a state of elevated excitation followed by a period of quiescence associated with neuronal and/or synaptic refractoriness. However, the cellular factors controlling recruitment and timing of synchronized events have remained difficult to specify, although the specific temporal pattern of spontaneous rhythmogenesis determines its impact on developmental processes. We studied spontaneous synchronization in a model of 600-1,000 integrate-and-fire neurons interconnected with a probability of 5-30%. One-third of neurons generated spontaneous discharges and provided a background of intrinsic activity to the network. The heterogeneity and random coupling of these neurons maintained this background activity asynchronous. Refractoriness was modeled either by use-dependent synaptic depression or by cellular afterhyperpolarization. In both cases, the recruitment of neurons into spontaneous synchronized discharges was determined by the interplay of refractory mechanisms with stochastic fluctuations in background activity. Subgroups of easily recruitable neurons served as amplifiers of these fluctuations, thereby initiating a cascade-like recruitment of neurons ("avalanche effect"). In contrast, timing depended on the precise implementation of neuronal refractoriness and synaptic connectivity. With synaptic depression, neuronal synchronization always occurred stochastically, whereas with cellular afterhyperpolarization, stochastic turned into periodic behavior with increasing synaptic strength. These results associate the type of refractory mechanism with the temporal statistics and the mechanism of synchronization, thereby providing a framework for differentiating between cellular mechanisms of spontaneous rhythmogenesis.

Action Potentials↗
PubMed2000

A cDNA encoding a nuclear hormone receptor of the steroid/thyroid hormone-receptor superfamily from the human parasitic nematode Strongyloides stercoralis.

A cDNA encoding a nuclear hormone receptor of the steroid/thyroid receptor superfamily was obtained from third-stage larvae(L3) of the parasitic roundworm Strongyloides stercoralis. A recombinant clone was isolated via screening of an S. stercoralis cDNA library with a polymerase chain reaction (PCR)-generated probe. The insert of 2,583 bp contained the complete coding sequence of the receptor homologue. The conceptually translated amino acid sequence of this open reading frame encodes a 753-amino-acid-residue protein with an apparent molecular weight of 83.6 kDa and a predicted isoelectric point (pI) of 8.52. The coding sequence is 69% AT and the noncoding sequence is 72% AT, reflecting a characteristic A/T codon bias of S. stercoralis. In this report the amino acid sequence of the S. stercoralis nuclear hormone receptor of the steroid/thyroid receptor superfamily is compared with that of nuclear hormones of Caenorhabditis elegans, human orphan nuclear receptors, and insect ecdysone receptors. The potential role of steroids in the induction of hyperinfection syndrome is also discussed.

Amino Acid Sequence↗
PubMed1995

A single amino-acid change between the antigenically different extracellular serine proteases V2 and B2 from Dichelobacter nodosus.

Dichelobacter nodosus (Dn), the causative organism of ovine footrot, secrets three distinct types of extracellular serine proteases which have been implicated in virulence. Southern analyses have shown that the proteases are encoded by three separate genes, and the genes encoding an acidic protease V5 and a basic protease have already been characterised from virulent Dn strain 198. The gene encoding the third protease type, as represented by acidic protease V2, was isolated from an EcoRI-BamHI library of strain 198 genomic DNA by probing with a polymerase chain reaction (PCR) fragment generated with oligodeoxyribonucleotides based on protease V2 amino acid (aa) sequences. A further clone from an RsaI library was isolated to complete the 5' region of the gene to yield an ORF of 1803 bp encoding a protein precursor of 601 aa. The acidic protease V2 gene, aprV2, shows the same precursor structure as the bprV and aprV5 genes with 72% and 69% similarity at the nucleotide (nt) level and with 73% and 69% similarity at the aa level, respectively. As monoclonal antibodies consistently distinguish the virulent (V) and benign (B) forms of this protease, the gene encoding the acidic protease B2 from benign Dn strain 305 was isolated using the PCR and characterized to investigate the molecular basis for this difference in antigenicity. A 2-bp substitution in a single codon was identified which appeared to be responsible for a change of epitope.

Amino Acid Sequence↗
PubMed2001

Properties of multidrug-resistant, ESBL-producing Proteus mirabilis isolates and possible role of beta-lactam/beta-lactamase inhibitor combinations.

At our institution, isolation rates of clinical strains of ESBL-producing Proteus mirabilis increased to 8.8% of all P. mirabilis isolates during the period 1997-1999. To evaluate the susceptibility of ESBL-producing P. mirabilis strains against commonly used drugs, we studied 50 non-duplicated isolates selected on the basis of synergy between clavulanate and beta-lactams (ceftazidime, aztreonam, cefotaxime, and ceftriaxone). The presence of ESBL-coding genes was confirmed by colony hybridization with bla(TEM-1) and bla(SHV-1) probes. Minimum inhibitory concentrations of several antimicrobial agents for each isolate were obtained using the Etest method. All strains were encoding for TEM-derived enzymes. Gene sequencing showed that at least three different genes (TEM-15, TEM-20, and TEM-52) were present. These enzymes have not been previously reported in P. mirabilis. Isolates were characterized by: (a) reduced susceptibility or resistance to third- and fourth-generation cephalosporins (MIC > or = 2 mg/l), (b) resistance to piperacillin that was abolished by tazobactam (MIC > or = 256 vs. < or = 2 mg/l, respectively), (c) multiple antibiotic resistance that included gentamicin, fluoroquinolones and co-trimoxazole. Therapeutic failure and lack of eradication of ESBL-positive P. mirabilis by third-generation cephalosporins has been repeatedly observed both at our Institution and elsewhere. Piperacillin-tazobactam, as well as amikacin and meropenem appear to be important therapeutic options for infections due to multidrug-resistant, ESBL-producing P. mirabilis isolates.

Anti-Bacterial Agents↗
PubMed1999

Shared early autoantibody recognition events in the development of anti-Sm B/B' in human lupus.

Many aspects of the immune maturation are uncharted. For ordinary human autoimmune systems there are no complete descriptions of the progression from an initial antigenic epitope to a maximally complex immune response. In this study we have exploited a large serial collection of human sera to investigate the development of the anti-Sm autoimmune response in systemic lupus erythematosus (SLE). The results suggest a similar, if not virtually identical, stepwise progression in the early humoral immune maturation of anti-Sm. The amino acid sequence PPPGMRPP comprises the first epitope in the anti-Sm B/B'response and its close relative, PPPGMRGP, the second. Epitopes are subsequently enlarged by the incorporation of neighbouring amino acids. The third and fourth epitopes are also recognised by an antibody in a nearly identical sequence in different lupus patients. A column absorption with PPPGMRPP demonstrates that the epitope spreading among the first four early epitopes appears to occur by the sequential generation of cross-reactive antibodies. Unexpectedly, epitope spreading in this system occurs in a predictable fashion by involving essentially the same sequence of antigenic structures from person to person. In addition, these data support the lupus anti-Sm antibodies originating against a single antigenic structure and, hence, strongly support a unifying mechanism in the generation of these autoantibodies.

Amino Acid Sequence↗
PubMed2004

The bipartite 3'-cis-acting signal for replication is required for formation of a ribonucleoprotein complex in vivo between the viral genome and its RNA polymerase in yeast 23 S RNA virus.

23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.9 kb) encodes only its RNA-dependent RNA polymerase, p104, and forms a ribonucleoprotein complex with p104 in vivo. Previously we succeeded in generating 23 S RNA virus in yeast from an expression vector containing the entire viral cDNA sequence. Using this system, we have recently identified a bipartite 3' cis-acting signal for replication. The signal consists of a stretch of four cytidines (Cs) at the 3' end and a mismatched pair of purines in a stem-loop structure that partially overlaps the terminal four Cs. Although the 3' terminal and penultimate Cs are not essential for virus launching, the generated viruses efficiently recovered these terminal nucleotides. In this work, we expressed RNA transcripts containing the entire 23 S RNA genome but incapable of generating the virus because of the presence of non-viral extra sequences at the 3' ends. These transcripts could form complexes with p104 in vivo, and a detailed analysis indicated that the mismatched pair of purines as well as the third and fourth Cs from the viral 3' end was essential for this complex-forming activity. Given that 23 S RNA virus does not have genes for capsid proteins, the binding of p104 to the viral 3' end, in addition to the efficient 3' terminal repair, may play a crucial role in virus persistence by protecting and maintaining the correct viral 3' end in vivo.

Base Sequence↗