Open access to sequence data 'will boost hunt for breast cancer gene'.
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BRCA2 Protein↗
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Regiospecific inhibition of DNA duplication by antisense phosphate-methylated oligodeoxynucleotides.
A new synthesis route for long phosphate-methylated oligodeoxynucleotides is described, which were used as antisense inhibitors of the DNA replication. Phosphate-methylated oligomers hybridize more strongly with natural DNA than their natural analogues, due to the absence of electrostatic interstrand repulsions. Compared with phosphate-ethylated and methyl phosphonate systems, phosphate-methylated systems are preferable as antisense DNA, which was concluded from the high Tm values and sharp melting transitions of duplexes of phosphate-methylated and natural DNA. By using the Sanger dideoxy technique, it was shown that a complementary phosphate-methylated 18-mer can effectively and site-specifically block the DNA replication process at room temperature.
Base Sequence↗
Equilibrium point control of a monkey arm simulator by a fast learning tree structured artificial neural network.
A planar 17 muscle model of the monkey's arm based on realistic biomechanical measurements was simulated on a Symbolics Lisp Machine. The simulator implements the equilibrium point hypothesis for the control of arm movements. Given initial and final desired positions, it generates a minimum-jerk desired trajectory of the hand and uses the backdriving algorithm to determine an appropriate sequence of motor commands to the muscles (Flash 1987; Mussa-Ivaldi et al. 1991; Dornay 1991b). These motor commands specify a temporal sequence of stable (attractive) equilibrium positions which lead to the desired hand movement. A strong disadvantage of the simulator is that it has no memory of previous computations. Determining the desired trajectory using the minimum-jerk model is instantaneous, but the laborious backdriving algorithm is slow, and can take up to one hour for some trajectories. The complexity of the required computations makes it a poor model for biological motor control. We propose a computationally simpler and more biologically plausible method for control which achieves the benefits of the backdriving algorithm. A fast learning, tree-structured network (Sanger 1991c) was trained to remember the knowledge obtained by the backdriving algorithm. The neural network learned the nonlinear mapping from a 2-dimensional cartesian planar hand position (x,y) to a 17-dimensional motor command space (u1, . . ., u17). Learning 20 training trajectories, each composed of 26 sample points [[x,y], [u1, . . ., u17] took only 20 min on a Sun-4 Sparc workstation. After the learning stage, new, untrained test trajectories as well as the original trajectories of the hand were given to the neural network as input. The network calculated the required motor commands for these movements. The resulting movements were close to the desired ones for both the training and test cases.
Algorithms↗
Unique gene expression and clinical characteristics are associated with the 11q23 deletion in chronic lymphocytic leukaemia.
Chromosome abnormalities influence prognosis and tumour progression in B-cell Chronic Lymphocytic Leukaemia (CLL). This study sought to determine whether these different disease subgroups were associated with unique gene expression patterns. Thirty-four cases of CLL were screened for the 11q23, 13q14, 17p13 deletions, and trisomy 12 by fluorescence in situ hybridization (FISH). Expression of 205 cell signalling and apoptosis genes were compared by cDNA array among cases with different chromosome abnormalities. A majority of the statistically differentially expressed genes were present in the 11q23 deletion group by hierarchical clustering. CDC2, a serine/threonine kinase, was overexpressed in the 11q23 deletion group (P = 0.0004) and confirmed by Taqman real-time polymerase chain reaction. Several other genes associated with cell signalling were overexpressed in the 11q23 deletion group. A strong overall correlation existed between the presence of different chromosome abnormalities and a number of prognostic factors including immunoglobulin heavy chain variable region mutation status (P = 0.011), time to treatment (P = 0.025) and lymphocyte doubling time (P = 0.034). This study confirmed the prognostic impact of chromosome abnormalities identified by FISH in CLL, particularly the 11q23 deletion and trisomy 12. In addition, the 11q23 deletion group was associated with a unique gene expression pattern involving cell signalling and apoptosis genes.
Adult↗
Genomic abnormalities in chronic lymphocytic leukemia influence gene expression by a gene dosage effect.
This work describes the identification and impact of somatic genomic abnormalities in human chronic lymphocytic leukemia (CLL). Using molecular cytogenetics (FISH) and G-banding cytogenetic analysis, chromosome abnormalities were detected in 37 of 46 (80.4%) CLL patients. 13q14 deletion was the most common finding followed by trisomy 12 and 11q22.3 deletion. 17p13 deletion was also detected as were several less frequent chromosome abnormalities. The presence of these abnormalities significantly influenced the period of treatment-free survival as well as other clinical characteristics. In particular, CLL samples with trisomy 12 and 11q22.3 deletion were associated with shorter treatment-free survival. In order to identify the under-lying molecular differences among CLL subgroups with different chromosome abnormalities, gene expression profiling was performed on a custom DNA microarray consisting of 10,000 human gene-specific oligonucleotides. A gene dosage effect was observed where the expression of genes at the genetic loci of the sites of the somatic genomic abnormality was altered in a fashion according to the type of genomic change. This phenomenon was particularly evident in CLL samples with trisomy 12 and 17p13 deletion. Thus, this study demonstrates that genomic abnormalities influence gene expression in CLL by a dosage effect.
Adult↗
An ASIC-chip for stereoscopic depth analysis in video-real-time based on visual cortical cell behavior.
In a stereoscopic system both eyes or cameras have a slightly different view. As a consequence small variations between the projected images exist ("disparities") which are spatially evaluated in order to retrieve depth information. We will show that two related algorithmic versions can be designed which recover disparity. Both approaches are based on the comparison of filter outputs from filtering the left and the right image. The difference of the phase components between left and right filter responses encodes the disparity. One approach uses regular Gabor filters and computes the spatial phase differences in a conventional way as described already in 1988 by Sanger. Novel to this approach, however, is that we formulate it in a way which is fully compatible with neural operations in the visual cortex. The second approach uses the apparently paradoxical similarity between the analysis of visual disparities and the determination of the azimuth of a sound source. Animals determine the direction of the sound from the temporal delay between the left and right ear signals. Similarly, in our second approach we transpose the spatially defined problem of disparity analysis into the temporal domain and utilize two resonators implemented in the form of causal (electronic) filters to determine the disparity as local temporal phase differences between the left and right filter responses. This approach permits video real-time analysis of stereo image sequences (see movies at http://www.neurop.ruhr-uni-bochum.de/Real- Time-Stereo) and a FPGA-based PC-board has been developed which performs stereo-analysis at full PAL resolution in video real-time. An ASIC chip will be available in March 2000.
Animals↗
An assessment technique for children with auditory-language processing problems.
The purpose of this study was to develop a new multilayer clinical assessment technique to evaluate auditory-language processing abilities in children. Following a 90-min in-service workshop on auditory-language processing problems, 46 nonhandicapped first-, second-, and third-grade students were referred by their classroom teachers for an evaluation of auditory-language processing abilities. Twelve "normally" achieving first-, second-, and third-grade students were randomly selected as controls. Standardized and nonstandardized measures included a pure tone and impedance test, selected subtests of the Clinical Evaluation of Language Functions (Linguistic Concepts, Relationships and Ambiguities, Oral Directions, Spoken Paragraphs, Word Associations, and Model Sentences), the Goldman-Fristoe-Woodcock (GFW) Memory for Sequence Test, Sound Mimicry Test, Sound-Symbol Association Test, and the GFW Test of Auditory Discrimination. Nonstandardized measures included an Observational Profile of Classroom Communication and an informal language sample. Results indicated 87% of 46 (n = 40) children were identified as having auditory-language processing problems. In-service training was an effective means to heighten teachers' awareness for referring subjects. Additionally, the Observational Profile of Classroom Communication was an effective procedure for teachers to systematically observe and document communication behaviors in the context of the classroom.
Auditory Perceptual Disorders↗
The otter annotation system.
With the completion of the human genome sequence and genome sequence available for other vertebrate genomes, the task of manual annotation at the large genome scale has become a priority. Possibly even more important, is the requirement to curate and improve this annotation in the light of future data. For this to be possible, there is a need for tools to access and manage the annotation. Ensembl provides an excellent means for storing gene structures, genome features, and sequence, but it does not support the extra textual data necessary for manual annotation. We have extended Ensembl to create the Otter manual annotation system. This comprises a relational database schema for storing the manual annotation data, an application-programming interface (API) to access it, an extensible markup language (XML) format to allow transfer of the data, and a server to allow multiuser/multimachine access to the data. We have also written a data-adaptor plugin for the Apollo Browser/Editor to enable it to utilize an Otter server. The otter database is currently used by the Vertebrate Genome Annotation (VEGA) site (http://vega.sanger.ac.uk), which provides access to manually curated human chromosomes. Support is also being developed for using the AceDB annotation editor, FMap, via a perl wrapper called Lace. The Human and Vertebrate Annotation (HAVANA) group annotators at the Sanger center are using this to annotate human chromosomes 1 and 20.
Computational Biology↗
The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.
BACKGROUND: The survival of patients with diffuse large-B-cell lymphoma after chemotherapy is influenced by molecular features of the tumors. We used the gene-expression profiles of these lymphomas to develop a molecular predictor of survival. METHODS: Biopsy samples of diffuse large-B-cell lymphoma from 240 patients were examined for gene expression with the use of DNA microarrays and analyzed for genomic abnormalities. Subgroups with distinctive gene-expression profiles were defined on the basis of hierarchical clustering. A molecular predictor of risk was constructed with the use of genes with expression patterns that were associated with survival in a preliminary group of 160 patients and was then tested in a validation group of 80 patients. The accuracy of this predictor was compared with that of the international prognostic index. RESULTS: Three gene-expression subgroups--germinal-center B-cell-like, activated B-cell-like, and type 3 diffuse large-B-cell lymphoma--were identified. Two common oncogenic events in diffuse large-B-cell lymphoma, bcl-2 translocation and c-rel amplification, were detected only in the germinal-center B-cell-like subgroup. Patients in this subgroup had the highest five-year survival rate. To identify other molecular determinants of outcome, we searched for individual genes with expression patterns that correlated with survival in the preliminary group of patients. Most of these genes fell within four gene-expression signatures characteristic of germinal-center B cells, proliferating cells, reactive stromal and immune cells in the lymph node, or major-histocompatibility-complex class II complex. We used 17 genes to construct a predictor of overall survival after chemotherapy. This gene-based predictor and the international prognostic index were independent prognostic indicators. CONCLUSIONS: DNA microarrays can be used to formulate a molecular predictor of survival after chemotherapy for diffuse large-B-cell lymphoma.
Antibiotics, Antineoplastic↗
Effects of d-amphetamine on the behavior of pigeons maintained by a second-order schedule of reinforcement.
The effects of various doses of d-amphetamine were studied on the responding of two pigeons exposed to a second-order schedule of reinforcement. With this schedule, food was presented following the completion of a sequence of three 2-minute fixed-interval components. A visual stimulus was presented at the completion of each fixed interval, including the one which was terminated with food. The pigeons' behavior was characterized by a pause immediately after each stimulus presentation followed by a gradual increase in response rate as the interval progressed. d-Amphetamine was found to increase the low rates of responding which occurred early in each interval and to decrease the high rates of responding at the end of each interval. These effects occurred whether responding preceded the presentation of food or the brief stimulus alone.
Animals↗
Molecular cloning, expression and characterization of the first three genes in the mevalonate-independent isoprenoid pathway in Streptomyces coelicolor.
The mevalonate-independent biosynthetic pathway to isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors to the isoprenoids, operates in eubacteria, including Escherichia coli, in algae, and in the plastids of higher plants. A search of the Sanger Centre Streptomyces coelicolor genome database revealed open reading frames with ca. 40--50% identity at the deduced amino acid level to the first three E. coli enzymes of this pathway, corresponding to deoxyxylulose phosphate synthase, deoxyxylulose phosphate reductoisomerase and 2-C-methyl erythritol 4-phosphate cytidylyltransferase. The S. coelicolor genes have been cloned and expressed in E. coli, and the recombinant proteins characterized physically and kinetically. The presence of the corresponding enzyme activities in extracts of S. coelicolor CH999 further supports the operation of the mevalonate-independent pathway in this organism.
Aldose-Ketose Isomerases↗
Lymphedema in the replanted limb of the rat: scintigraphic evaluation.
The sequence of scintigraphic patterns in resolving lymphedema in the replanted hind limb of the rat is reported. Compared to simple observation of the limb, scintigraphy using 99mTc-antimony trisulfide colloid (99mTc SbSC) provides multiple qualitative parameters for assessing lymphedema, ie, diffuse uptake in the implanted limb, focal uptake at the site of surgical repair, accumulation in the popliteal node, and activity in the liver. Quantitative analysis in this model was not helpful. Scintigraphic criteria may be utilized to study the effect of alteration in the animal model described. It is hoped that this model can be used to clarify the clinical factors influencing the resolution of lymphedema.
Animals↗
Molecular diagnosis of Burkitt's lymphoma.
BACKGROUND: The distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is crucial because these two types of lymphoma require different treatments. We examined whether gene-expression profiling could reliably distinguish Burkitt's lymphoma from diffuse large-B-cell lymphoma. METHODS: Tumor-biopsy specimens from 303 patients with aggressive lymphomas were profiled for gene expression and were also classified according to morphology, immunohistochemistry, and detection of the t(8;14) c-myc translocation. RESULTS: A classifier based on gene expression correctly identified all 25 pathologically verified cases of classic Burkitt's lymphoma. Burkitt's lymphoma was readily distinguished from diffuse large-B-cell lymphoma by the high level of expression of c-myc target genes, the expression of a subgroup of germinal-center B-cell genes, and the low level of expression of major-histocompatibility-complex class I genes and nuclear factor-kappaB target genes. Eight specimens with a pathological diagnosis of diffuse large-B-cell lymphoma had the typical gene-expression profile of Burkitt's lymphoma, suggesting they represent cases of Burkitt's lymphoma that are difficult to diagnose by current methods. Among 28 of the patients with a molecular diagnosis of Burkitt's lymphoma, the overall survival was superior among those who had received intensive chemotherapy regimens instead of lower-dose regimens. CONCLUSIONS: Gene-expression profiling is an accurate, quantitative method for distinguishing Burkitt's lymphoma from diffuse large-B-cell lymphoma.
Adolescent↗
Studies on the structure and mechanism of an exo-(1 yields 3)-beta-D-glucanase from Basidiomycete QM806.
A method for the large-scale production of a (1 yields 3)-beta-D-glucan glucohydrolase (EC 3.2.1.58) from the culture filtrate of Basidiomycete QM806 is described. The final preparation is homogeneous by disc electrophoresis under non-dissociating and denaturing conditions, by ultracentrifugation, and by isoelectric focusing. Various physical and chemical characteristics of the enzyme have been determined, including terminal amino acid residues, extinction coefficient, and stability to pH extremes. The N-terminal amino acids are leucine and serine (Sanger's method) and the C-terminal amino acids are alanine, serine, and glycine (hydrazinolysis). pH profile studies show that no group titrating in the region 2.5-8 is directly involved with substrate binding and that a single group having a pKa of 6.5 is involved in the catalysis. Photooxidation of the enzyme caused rapid inactivation. The pH-dependence of this photooxidation, and amino acid analysis of the photooxidized enzyme, indicate that decomposition of histidine is probably responsible for the loss of activity. Other chemical modifications performed were: treatment with hydrogen peroxide under acidic conditions, esterification with diphenyldiazomethane, and oxidation with N-bromosuccinimide. Oxidation with N-bromosuccinimide indicated that a tryptophan side-chain is involved in, but not necessary for, the catalytic activity.
Amino Acid Sequence↗
Myofibrillogenesis in skeletal muscle cells.
How are myofibrils assembled in skeletal muscles? The current authors present evidence that myofibrils assemble through a three-step model: premyofibrils to nascent myofibrils to mature myofibrils. This three-step sequence was based initially on studies of living and fixed cultured cells from cardiac muscle. Data from avian primary muscle cells and from a transgenic skeletal mouse cell line indicate that a premyofibril model for myofibrillogenesis also holds for skeletal muscle cells. Premyofibrils are characterized by minisarcomeres bounded by Z-bodies composed of the muscle isoform of alpha-actinin. Actin filaments are connected to these Z-bodies and to the mini-A-bands composed of nonmuscle myosin II filaments. Nascent myofibrils are formed when premyofibrils align and are modified by the addition of titin and muscle myosin II filaments. Mature myofibrils result when nonmuscle myosin II is eliminated from the myofibrils and the alpha-actinin rich Z-bodies fuse as the distance between them increases from 0.5 microm in premyofibrils to 2 to 2.5 microm in the mature myofibrils.
Actins↗
Bi-directional dideoxy fingerprinting (Bi-ddF): a rapid method for quantitative detection of mutations in genomic regions of 300-600 bp.
There is a great need for rapid screening methods that detect essentially all mutations. Dideoxy fingerprinting (ddF) is a highly sensitive screening method that is performed by electrophoresing one lane of a Sanger dideoxy termination reaction through a nondenaturing gel. Mutations may produce an extra segment or eliminate a segment from the termination products (informative dideoxy component). In addition, mutations can be detected by the altered mobility of one or more termination segments (informative SSCP component). To screen larger segments with virtually 100% sensitivity, bi-directional ddF (Bi-ddF) was developed. Bi-ddF is a 'second generation ddF' in which the dideoxy termination reaction is performed simultaneously with two opposing primers. Bi-ddF has two important advantages over ddF: (i) the dideoxy component can detect 10 of the 12 types of possible single-base substitutions; and (ii) the SSCP component is enhanced because alterations of mobility can be detected in either the downstream or upstream direction. As a result, Bi-ddF can screen larger regions of genomic DNA with virtually 100% sensitivity. Bi-ddF detected 100% of 28 single-base substitutions in a 494 bp segment containing exons B and C of the human factor IX gene and 100% of 42 single-base substitutions and one microdeletion present in a 577 bp region containing exon H. In a blinded analysis in which 39 wildtype samples were randomly mixed with 51 mutant samples, all mutations were detected with no false positives. Bi-ddF requires essentially the same effort as ddF, yet twofold more DNA sequence can be screened reliably per unit effort.
DNA↗
BCL2 translocation defines a unique tumor subset within the germinal center B-cell-like diffuse large B-cell lymphoma.
Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed prognostically important subgroups: germinal center B-cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal large B-cell lymphoma. The t(14;18)(q32;q21) has been reported previously to define a unique subset within the GCB-DLBCL. We evaluated for the translocation in 141 cases of DLBCL that were successfully gene expression profiled. Using a dual-probe fluorescence in situ hybridization assay, we detected the t(14;18) in 17% of DLBCLs and in 34% of the GCB subgroup which contained the vast majority of positive cases. In addition, 12 t(14;18)-positive cases detected by polymerase chain reaction assays on additional samples were added to the fluorescence in situ hybridization-positive cases for subsequent analysis. Immunohistochemical data indicated that BCL2, BCL6, and CD10 protein were preferentially expressed in the t(14;18)-positive cases as compared to t(14;18)-negative cases. Within the GCB subgroup, the expression of BCL2 and CD10, but not BCL6, differed significantly between cases with or without the t(14;18): 88% versus 24% for BCL2 and 72% versus 32% for CD10, respectively. In the GCB-DLBCL subgroup, a heterogeneous group of genes is overexpressed in the t(14;18)-positive subset, among which BCL2 is a significant discriminator. Interestingly, the t(14;18)-negative subset is dominated by overexpression of cell cycle-associated genes, indicating that these tumors are significantly more proliferative, suggesting distinctive pathogenetic mechanisms. However, despite this higher proliferative activity, there was no significant difference in overall or failure-free survival between the t(14;18)-positive and -negative subsets within the GCB subgroup.
Apoptosis Regulatory Proteins↗
Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin.
Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.
Actinin↗