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At least 127 records · Page 7Linked to original sources

PubMed2006

Prediction of protein subcellular localization.

Because the protein's function is usually related to its subcellular localization, the ability to predict subcellular localization directly from protein sequences will be useful for inferring protein functions. Recent years have seen a surging interest in the development of novel computational tools to predict subcellular localization. At present, these approaches, based on a wide range of algorithms, have achieved varying degrees of success for specific organisms and for certain localization categories. A number of authors have noticed that sequence similarity is useful in predicting subcellular localization. For example, Nair and Rost (Protein Sci 2002;11:2836-2847) have carried out extensive analysis of the relation between sequence similarity and identity in subcellular localization, and have found a close relationship between them above a certain similarity threshold. However, many existing benchmark data sets used for the prediction accuracy assessment contain highly homologous sequences-some data sets comprising sequences up to 80-90% sequence identity. Using these benchmark test data will surely lead to overestimation of the performance of the methods considered. Here, we develop an approach based on a two-level support vector machine (SVM) system: the first level comprises a number of SVM classifiers, each based on a specific type of feature vectors derived from sequences; the second level SVM classifier functions as the jury machine to generate the probability distribution of decisions for possible localizations. We compare our approach with a global sequence alignment approach and other existing approaches for two benchmark data sets-one comprising prokaryotic sequences and the other eukaryotic sequences. Furthermore, we carried out all-against-all sequence alignment for several data sets to investigate the relationship between sequence homology and subcellular localization. Our results, which are consistent with previous studies, indicate that the homology search approach performs well down to 30% sequence identity, although its performance deteriorates considerably for sequences sharing lower sequence identity. A data set of high homology levels will undoubtedly lead to biased assessment of the performances of the predictive approaches-especially those relying on homology search or sequence annotations. Our two-level classification system based on SVM does not rely on homology search; therefore, its performance remains relatively unaffected by sequence homology. When compared with other approaches, our approach performed significantly better. Furthermore, we also develop a practical hybrid method, which combines the two-level SVM classifier and the homology search method, as a general tool for the sequence annotation of subcellular localization.

Algorithms↗

PubMed2026

Clinical Utility of Next-Generation Sequencing in Tumors Diagnosed as Lung Squamous Cell Carcinoma: Real-World Data of Diagnostic and Therapeutic Implications.

Lung squamous cell carcinoma (LUSC) is the second most common subtype of non-small cell lung carcinoma (NSCLC), typically associated with a poor prognosis. Unlike lung adenocarcinoma, the application of next-generation sequencing (NGS) in LUSC has lagged because of the long-standing perception of low therapeutic yield, primarily based on highly selected, resected cohorts. We sought to determine the real-world clinical utility of NGS in LUSC. We analyzed an institutional cohort of 576 tumors initially diagnosed as LUSC that underwent NGS profiling. We defined "clinical yield" as either diagnostic reclassification or the identification of a targetable mitogenic alteration. Twenty cases (3.5%) were reclassified, including rediagnosis to cutaneous squamous cell carcinoma, transformed adenocarcinoma (post targeted therapy), and rare entities such as nuclear protein of the testis-rearranged carcinoma and lymphoepithelial carcinoma. Primary mitogenic drivers were identified in 83 cases (14.4% of the total cohort), of which 43 (7.5% of the total cohort) harbored alterations with currently Food and Drug Administration-approved therapies for NSCLC (including KRAS, EGFR, MET, ALK, and ROS1). Overall clinical yield-defined as the sum of diagnostic reclassifications and identification of NSCLC-specific targetable alterations-was 11.0% (63/576). Univariate and multivariate analysis demonstrated that never or light smoking history was the strongest independent predictor of clinical yield, with 57.3% of tumors in this subset being reclassified or harboring a strong driver. Our findings demonstrate that NGS provides significant diagnostic and therapeutic value in a real-world LUSC cohort, challenging the historical premise of low yield. Although clinicodemographic features can help prioritize testing in resource-limited settings, the identification of targetable drivers across all smoking groups supports the universal application of comprehensive NGS for all patients diagnosed with LUSC.

Humans↗

PubMed1990

Influence of cellular sequences on instability of plasmid integration sites in human cells.

To learn more about mechanisms of genome instability in human cells, I investigated DNA sequences that promote high rates of recombination by analyzing rare unstable plasmid integration sites in simian virus 40-transformed human fibroblasts. Previous studies had hypothesized that rearrangement or loss of integrated sequences could be attributed to adjacent cellular DNA. Consistent with this interpretation, a cloned fragment containing both the integrated plasmid and 2.0 kb of adjacent cell DNA from one such unstable integration site in the cell line LM205 demonstrated a much higher incidence of rearrangements when integrated into other chromosome locations than did the original plasmid. To further test this hypothesis, portions of cellular DNA from this region were integrated in duplicate in other locations to determine their ability to promote restriction-fragment-length polymorphism, an indicator of high rates of homologous recombination. Although two types of instability were observed, neither could be attributed solely to the cell sequences being tested in the plasmid. The first type of instability was a transient deletion or amplification of the plasmid DNA soon after integration, which appeared to be a general phenomenon often associated with any type of newly integrated sequence. A second type of instability continued indefinitely for many cell generations, as did that observed in cell line LM205. Because this was rare (one of 78 clones tested), it could not be attributed solely to cell sequences contained within the plasmid. However, the rearrangements in this cell clone occurred exclusively within the cell DNA adjacent to the integration site, again suggesting a role for cis-acting cell sequences in this process. The inability to identify specific cell sequences responsible for instability may therefore indicate that a complex combination of sequences is involved, possibly within both the plasmid and cell DNA.

Base Sequence↗

PubMed2003

Effect of prolonged vegetative reproduction of olive tree cultivars (Olea europaea L.) in mitochondrial homoplasmy and heteroplasmy.

The inheritance of mitochondrial and chloroplast genomes does not follow Mendelian laws, but proceeds by vegetative segregation. Most organisms show organelle homoplasmy, which is probably produced and maintained during sexual reproduction. We have tested the effect of prolonged vegetative multiplication in the maintenance of mitochondrial homoplasmy and the generation of heteroplasmy in cultivated olive trees, Olea europaea L. Seven trees, each representing a different variety of olive, were analysed by the study of an intergenic spacer region of the mitochondrial genome. A very high level of heteroplasmy was detected in all cases. We found multiple genome variants of the sequence analysed. The frequency of genomes with no changes in the spacer region was 11.5%. This means that 88.5% of genomes contain at least one change. The same spacer mitochondrial region was sequenced in several clones from four olive trees of a second generation of sexually reproduced trees. In these trees, many clones were identical and had no changes, which represents a clear reduction of the heteroplasmy (p < 0.001). Therefore, this work supports the relevance of the role of sexual reproduction in the maintenance of mitochondrial homoplasmy and also shows that mutations accumulate in a non-coding sequence of the mitochondrial genome when vegetative propagation is maintained for a long period of time.

Base Sequence↗

PubMed2000

Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death.

Medieval Black Death is believed to have killed up to one-third of the Western European population during the 14th century. It was identified as plague at this time, but recently the causative organism was debated because no definitive evidence has been obtained to confirm the role of Yersinia pestis as the agent of plague. We obtained the teeth of a child and two adults from a 14th century grave in France, disrupted them to obtain the pulp, and applied the new "suicide PCR" protocol in which the primers are used only once. There were no positive controls: Neither Yersinia nor Yersinia DNA were introduced in the laboratory. A negative result is followed by a new test using other primers; a positive result is followed by sequencing. The second and third primer pair used, coding for a part of the pla gene, generated amplicons whose sequence confirmed that it was Y. pestis in 1 tooth from the child and 19/19 teeth from the adults. Negative controls were negative. Attempts to detect the putative alternative etiologic agents Bacillus anthracis and Rickettsia prowazekii failed. Suicide PCR avoids any risk of contamination as it uses a single-shot primer-its specificity is absolute. We believe that we can end the controversy: Medieval Black Death was plague.

Adult↗

PubMed2001

Evidence for the contribution of point mutations to vlsE variation and for apparent constraints on the net accumulation of sequence changes in vlsE during infection with Lyme disease spirochetes.

In the Lyme disease spirochetes, both the ospE and vlsE gene families have been demonstrated to undergo sequence variation during infection. To further investigate the mechanisms associated with the generation of vls variation, single-nucleotide polymorphism and subsequent DNA sequence analyses were performed on the vlsE gene and its paralog, BBJ51, a related gene with a frameshift mutation. These analyses focused on a series of postinfection clonal populations obtained from mice infected with Borrelia burgdorferi B31MIpc or its clonal derivative, B31MIc53. vlsE, but not BBJ51, was found to undergo sequence changes during infection. Consistent with that reported previously (J.-R. Zhang et al., Cell 89:275-285, 1997) many of the sequence changes appear to have arisen through gene conversion events and to be localized to the variable regions of vlsE. However, analysis of the vlsE nucleotide sequences revealed that some sequence changes were the result of point mutations, as these changes did not have potential contributing sources in the vls cassettes. To determine if sequence changes accumulate in vlsE over long-term infection, the vlsE genes of clonal populations recovered after 7 months of infection in mice were analyzed. While new sequence changes developed, a significant number of these changes resulted in the restoration of the vlsE sequence of the original infecting clone. In addition, we noted that some positions within the variable regions (VR) are stable even though the cassettes possess residues that could contribute to sequence variation through gene conversion. These analyses suggest that the total number of amino acid sequence changes that can be maintained by VlsE levels off during infection. In summary, in this report we demonstrate that the development of point mutations serves as a second mechanism by which vlsE sequence variation can be generated and that the capacity for vlsE variation, while still significant, is less than previously postulated.

Amino Acid Sequence↗

PubMed2002

Transovarial transmission of sugarcane white leaf phytoplasma in the insect vector Matsumuratettix hiroglyphicus (Matsumura).

White leaf is a serious disease of sugarcane caused by phytoplasma. The disease is transmitted to the plant by the leafhopper Matsumuratettix hiroglyphicus (Matsumura). The reservoir of phytoplasma was suspected to be weeds that grow in sugarcane farming areas because they can be infected with phytoplasma and show symptoms similar to sugarcane white leaf. However in previous work we have demonstrated by RFLP and sequencing that this is not the case. Here we have reared M. hiroglyphicus through two generations by feeding them phytoplasma free sugarcane grown from tissue culture. By nested-PCR followed by sequencing, we demonstrated the presence of the phytoplasma in eggs, nymphs and adults of the first and second generations thereby showing transovarial transmission. We have also shown by in situ PCR that phytoplasmas were widely distributed throughout the body of the insect. RFLP and sequencing showed that the same phytoplasma was present in the vector and in the plant. Together, these data point to the leafhopper M. hiroglyphicus as the reservoir of phytoplasma that cause sugarcane white leaf disease.

Animals↗

PubMed2001

Advanced integrated mouse YAC map including BAC framework.

Functional characterization of the mouse genome requires the availability of a comprehensive physical map to obtain molecular access to chromosomal regions of interest. Positional cloning remains a crucial way of linking phenotype with particular genes. A key step and frequent stumbling block in positional cloning is making a contig of a genetically defined candidate region. The most efficient first step is isolating YAC (Yeast Artificial Chromosome) clones. A robust, detailed YAC contig map is thus an important tool. Employing Interspersed Repetitive Sequence (IRS)-PCR genomics, we have generated an advanced second-generation YAC contig map of the mouse genome that doubles both the depth of clones and the density of markers available. In addition to the primarily YAC-based map, we located 1942 BAC (Bacterial Artificial Chromosome) clones. This allows us to present for the first time a dense framework of BACs spanning the genome of the mouse, which, for instance, can serve as a nucleus for genomic sequencing. Four large-insert mouse YAC libraries from three different strains are included in our data, and our analysis incorporates the data of Hunter et al. and Nusbaum et al. There is a total of 20,205 markers on the final map, 12,033 from our own data, and a total of 56,093 YACs, of which 44,401 are positive for more than one marker.

Algorithms↗

PubMed2006

Lessons from Nature: microRNA-based shRNA libraries.

Loss-of-function genetics has proven essential for interrogating the functions of genes and for probing their roles within the complex circuitry of biological pathways. In many systems, technologies allowing the use of such approaches were lacking before the discovery of RNA interference (RNAi). We have constructed first-generation short hairpin RNA (shRNA) libraries modeled after precursor microRNAs (miRNAs) and second-generation libraries modeled after primary miRNA transcripts (the Hannon-Elledge libraries). These libraries were arrayed, sequence-verified, and cover a substantial portion of all known and predicted genes in the human and mouse genomes. Comparison of first- and second-generation libraries indicates that RNAi triggers that enter the RNAi pathway through a more natural route yield more effective silencing. These large-scale resources are functionally versatile, as they can be used in transient and stable studies, and for constitutive or inducible silencing. Library cassettes can be easily shuttled into vectors that contain different promoters and/or that provide different modes of viral delivery.

Animals↗

PubMed2004

"Deoxyribo nanonucleic acid"; antiparallel, parallel, and unparalleled.

The crystal structure of a single-stranded DNA oligonucleotide has revealed formation of a unique three-dimensional array by continuous antiparallel and parallel pairing between monomers. The array is based on tertiary interactions and represents a second-generation nanotechnological system.

Base Sequence↗

PubMed1995

Expression and site-directed mutagenesis of mouse prostaglandin E2 receptor EP3 subtype in insect cells.

A cDNA encoding for mouse prostaglandin E2 (PGE2) receptor EP3 subtype was cloned from a mouse kidney cDNA library by PCR using terminal primers derived from the known sequence of mouse lung EP3 receptor cDNA. The cloned cDNA was confirmed by sequencing and was expressed in Trichoplusia ni (MG1) insect cells using a baculovirus expression system. A specific protein of 60 kDa was detected by immunoblot with antibodies generated against a unique decapeptide sequence present in the second extracellular loop of the EP3 receptor. Specific binding of [3H]PGE2 with a Kd of 3 nM was also found in the membrane fraction of the insect cells. Ligand binding of the receptor was further studied by site-directed mutagenesis. Arg-309 of the receptor was separately mutated to lysine, glutamate and valine. cDNAs of the wild-type and mutant EP3 receptors were respectively expressed and studied in MG1 insect cells. Binding studies indicated that both glutamate and valine mutant EP3 receptors had no binding of [3H]PGE2. On the contrary, the lysine mutant receptor exhibited an even tighter binding (Kd = 1.3 nM) than the wild-type EP3 receptor. Immunoblot studies indicated that these receptors were expressed in a comparable amount in MG1 insect cells. These results suggest that Arg-309 of EP3 receptor may be essential in ligand binding through ionic interaction.

Amino Acid Sequence↗

PubMed2002

Imprint control element-mediated secondary methylation imprints at the Igf2/H19 locus.

Understanding the molecular basis of monoallelic expression as observed at imprinted loci is helpful in understanding the mechanisms underlying epigenetic regulation. Genomic imprinting begins during gametogenesis with the establishment of epigenetic marks on the chromosomes such that paternal and maternal chromosomes are rendered distinct. During embryonic development, the primary imprint can lead to generation of secondary epigenetic modifications (secondary imprints) of the chromosomes. Eventually, either the primary imprints or the secondary imprints interfere with transcription, leading to parent-of-origin-dependent silencing of one of the two alleles. Here we investigated several aspects pertaining to the generation and functional necessity of secondary methylation imprints at the Igf2/H19 locus. At the H19 locus, these secondary imprints are, in fact, the signals mediating paternal chromosome-specific silencing of that gene. We first demonstrated that the H19 secondary methylation imprints are entirely stable through multiple cell divisions, even in the absence of the primary imprint. Second, we generated mouse mutations to determine which DNA sequences are important in mediating establishment and maintenance of the silent state of the paternal H19 allele. Finally, we analyzed the dependence of the methylation of Igf2DMR1 region on the primary methylation imprint about 90 kilobases away.

Animals↗

PubMed2001

Establishment of a high throughput EST sequencing system using poly(A) tail-removed cDNA libraries and determination of 36,000 bovine ESTs.

We determined 36,310 bovine expressed sequence tag (EST) sequences using 10 different cDNA libraries. For massive EST sequencing, we devised a new system with two major features. First, we constructed cDNA libraries in which the poly(A) tails were removed using nested deletion at the 3'-ends. This permitted high quality reading of sequences from the 3'-end of the cDNA, which is otherwise difficult to do. Second, we increased throughput by sequencing directly on templates generated by colony PCR. Using this system, we determined 600 cDNA sequences per day. The read-out length was >450 bases in >90% of the sequences. Furthermore, we established a data management system for analyses, storage and manipulation of the sequence data. Finally, 16,358 non-redundant ESTs were derived from approximately 6900 independent genes. These data will facilitate construction of a precise comparative map across mammalian species and isolate the functional genes that govern economic traits. This system is applicable to other organisms, including livestock, for which EST data are limited.

Animals↗

PubMed1990

Rearrangements in unintegrated retroviral DNA are complex and are the result of multiple genetic determinants.

We used a replication-competent retrovirus shuttle vector based on a DNA clone of the Schmidt-Ruppin A strain of Rous sarcoma virus to characterize rearrangements in circular viral DNA. In this system, circular molecules of viral DNA present after acute infection of cultured cells were cloned as plasmids directly into bacteria. The use of a replication-competent shuttle vector permitted convenient isolation of a large number of viral DNA clones; in this study, over 1,000 clones were analyzed. The circular DNA molecules could be placed into a limited number of categories. Approximately one-third of the rescued molecules had deletions in which one boundary was very near the edge of a long terminal repeat (LTR) unit. Subtle differences in the patterns of deletions in circular DNAs with one versus two copies of the LTR sequence were observed, and differences between deletions emanating from the right and left boundaries of the LTR were seen. A virus with a missense mutation in the region of the pol gene responsible for integration and exhibiting a temperature sensitivity phenotype for replication had a marked decrease in the number of rescued molecules with LTR-associated deletions when infection was performed at the nonpermissive temperature. This result suggests that determinants in the pol gene, possibly in the integration protein, play a role in the generation of LTR-associated deletions. Sequences in a second region of the genome, probably within the viral gag gene, were also found to affect the types of circular viral DNA molecules present after infection. Sequences in this region from different strains of avian sarcoma-leukosis viruses influenced the fraction of circular molecules with LTR-associated deletions, as well as the relative proportion of circular molecules with either one or two copies of the LTR. Thus, the profile of rearrangements in unintegrated viral DNA is complex and dependent upon the nature of sequences in the gag and pol regions.

Animals↗

PubMed1993

Cerebral perfusion in children: detection with dynamic contrast-enhanced T2*-weighted MR images.

Cerebral perfusion dynamics were assessed with dynamic contrast material-enhanced T2*-weighted magnetic resonance (MR) imaging in 33 subjects aged 3-20 years. Group A (n = 20) had sickle cell anemia without clinical evidence of cerebrovascular disease. Group B (n = 13) consisted of 12 patients with cerebrovascular disease and homozygous sickle cell anemia and one patient without that anemia. All subjects underwent conventional MR imaging and a dynamic study in which a spoiled gradient-echo pulse sequence was used to generate images (acquisition time, 2.5 seconds) during injection of a compact bolus of gadopentetate dimeglumine (0.1 mmol/kg). For qualitative analysis, the dynamic images were displayed in cine mode. Group A demonstrated symmetric sequential region patterns of loss of signal intensity within 10 seconds of injection. Group B exhibited signal loss asymmetries that corresponded to cerebrovascular lesions on conventional MR images. Quantitative analysis enabled estimation of hemodynamic parameters, including relative cerebral blood volume, relative cerebral blood flow, and mean transit time. This method of assessment of cerebral perfusion dynamics complements conventional MR imaging.

Adolescent↗

PubMed2001

PSMA specific antibodies and their diagnostic and therapeutic use.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glycoprotein highly restricted to prostatic epithelial cells. PSMA expression is increased in association with prostatic cancer, particularly in hormone refractory disease. Given its membrane-bound character, PSMA is an ideal sentinel molecule for use in targeting prostatic cancer cells. Monoclonal antibodies specific for PSMA are available, beginning with the antibody 7E11.C5 which originally defined PSMA and which has been developed for use in cancer detection via immunoscintiscanning in the ProstaScint test. Newer second generation antibodies specific for both linear amino acid sequence epitopes and protein conformational epitopes on the extracellular domain of PSMA have been reported. Although most of these are murine antibodies, both humanised and fully human examples have been developed. These antibodies are beginning to work their way into clinical applications for potential improved diagnostic and therapeutic uses. Results to date suggest that antibodies specific for extracellular epitopes are significantly better for clinical uses in vivo than the 7E11.C5 antibody that is specific for an intracellular epitope. Current knowledge relating to PSMA-specific antibodies and their clinical uses and potential is described and evaluated.

Animals↗

PubMed2026

Genetic Analysis of Genomic and Methylomic Variation and Identification of Multi-Trait Mutants in Rice Carried on Chang'e-5.

Global food security is facing challenges from population growth to diminishing arable land. Space mutation breeding holds promise for overcoming the variation limitations in conventional breeding; however, the mutagenic effects of the deep-space environment on rice and the transgenerational inheritance patterns of induced variations remain unclear. In this study, rice seeds carried by the Chang'e-5 spacecraft were used as materials. Whole-genome sequencing and whole-genome bisulfite sequencing were performed on the first (SP1) and second generations (SP2) of space-mutagenized plants after their return to Earth. The results showed that the number of genomic variants in the SP2 generation increased significantly compared with SP1, and SNPs, homozygous sites, and variants in coding regions were more heritable. The genome-wide methylation level was elevated in the SP2 generation, and among differentially methylated cytosines, those in the CG context exhibited the highest heritability. Furthermore, large-scale screening for nitrogen efficiency, tolerance to PEG-induced stress, and germination-stage cold resistant mutants was conducted in the SP2 generation, and phenotypic validation was performed in the third generation (SP3). By integrating multi-omics analyses of representative mutants to mine candidate genes, a number of heritable elite mutants were obtained, and seven candidate genes for key traits were identified. This study systematically elucidates the transgenerational inheritance patterns of deep-space-induced variation in rice. The multi-trait mutants obtained provide valuable germplasm resources for gene cloning and breeding applications in rice.

DNA methylation↗

PubMed1990

[Sex differences in memory performance for odors, tone sequences and colors].

Sense of olfaction would seem to be of little importance for human behavior. However, a closer look at this from the psychological point of view reveals many interesting aspects, such as sex differences in olfactory perception, that are of interest to differential psychology. The present study deals with sex differences in the memory for odors; we assume that women will do better here than men while other memory tasks involving acoustical and optical stimuli will show no such differences. Sixty women and 40 men were examined. On the first day, they had to retain 10 odors, 10 random-generated tone-sequences, and 10 colors. On the second day, 20 such stimuli for each memory task were presented, and the subjects had to remember and to tell which were known and which of them were unknown stimuli to them. A significant advantage in the olfaction memory task was found for women, while acoustical and optical memory scores showed no such differences. This expected finding is discussed in two ways. First, the female advantage might result from phylogenetic sources. Second, it might arise because women in general more often than men seem to deal with olfactory cues, so that they might simply have more experience and therefore the greater chance to score higher in an odor memory task.

Adult↗