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PubMed1996

Common core sequences are found in skeletal muscle slow- and fast-fiber-type-specific regulatory elements.

The molecular mechanisms generating muscle diversity during development are unknown. The phenotypic properties of slow- and fast-twitch myofibers are determined by the selective transcription of genes coding for contractile proteins and metabolic enzymes in these muscles, properties that fail to develop in cultured muscle. Using transgenic mice, we have identified regulatory elements in the evolutionarily related troponin slow (TnIs) and fast (TnIf) genes that confer specific transcription in either slow or fast muscles. Analysis of serial deletions of the rat TnIs upstream region revealed that sequences between kb -0.95 and -0.5 are necessary to confer slow-fiber-specific transcription; the -0.5-kb fragment containing the basal promoter was inactive in five transgenic mouse lines tested. We identified a 128-bp regulatory element residing at kb -0.8 that, when linked to the -0.5-kb TnIs promoter, specifically confers transcription to slow-twitch muscles. To identify sequences directing fast-fiber-specific transcription, we generated transgenic mice harboring a construct containing the TnIs kb -0.5 promoter fused to a 144-bp enhancer derived from the quail TnIf gene. Mice harboring the TnIf/TnIs chimera construct expressed the transgene in fast but not in slow muscles, indicating that these regulatory elements are sufficient to confer fiber-type-specific transcription. Alignment of rat TnIs and quail TnIf regulatory sequences indicates that there is a conserved spatial organization of core elements, namely, an E box, a CCAC box, a MEF-2-like sequence, and a previously uncharacterized motif. The core elements were shown to bind their cognate factors by electrophoretic mobility shift assays, and their mutation demonstrated that the TnIs CCAC and E boxes are necessary for transgene expression. Our results suggest that the interaction of closely related transcriptional protein-DNA complexes is utilized to specify fiber type diversity.

Animals↗

PubMed2001

Interaction of poliovirus-encoded 2C/2BC polypeptides with the 3' terminus negative-strand cloverleaf requires an intact stem-loop b.

The poliovirus-encoded, membrane-associated polypeptide 2C and its precursor, 2BC, is believed to be required for initiation and elongation of viral RNA synthesis. Previous studies have shown that the 2C polypeptide specifically interacts with the 3'-terminal sequence of poliovirus negative-strand RNA. This interaction is facilitated by the presence of the conserved sequence UGUUUU in the "stem a" within the 3'-terminal cloverleaf structure. We show that the 2C precursor, 2BC, also interacts with the 3'-terminal cloverleaf of the negative-strand RNA. We also demonstrate here that interaction of 2C/2BC with the negative strand 3'-terminal sequence not only depends on an intact "stem a" containing the UGUUUU sequence but is equally influenced by the presence of an intact "stem b" within the negative-strand cloverleaf. The results presented here suggest that the spatial configuration of stem a UGUUUU sequence with respect to an intact stem-b is crucial for 2C/2BC interaction with the 3'-terminal negative-strand cloverleaf structure.

Base Sequence↗

PubMed1997

[Turbo STIR sequence: optimization and comparison with conventional STIR sequence in bone diseases].

INTRODUCTION: The most common fat-suppressed sequence used to study skeletal conditions is the STIR sequence which has shown high sensitivity in the detection of skeletal lesions and whose main drawback is its long acquisition time. Currently, Turbo-STIR (T-STIR) sequences can shorten the acquisition time. The purpose of this study was therefore to compare the conventional STIR sequence with the new T-STIR sequence in the study of skeletal conditions to compare their diagnostic yield. MATERIAL AND METHODS: Twenty patients with different types of skeletal lesions were examined. MR examinations were performed with a Philips Gyroscan S15/ACS II unit (1.5 T). All the patients underwent a STIR sequence (TR/TE = 1500/20, TI = 180 ms, matrix = 204 x 256, NEX = 2, slice thickness = 5 mm, acquisition time = 9 min 24 s) and a T-STIR sequence (TR/TE = 1500/20, TI = 180 ms, matrix = 204 x 256, NEX = 2, slice thickness = 5 mm, TFL = 3, acquisition time = 3 min 33 s). The images were evaluated by measuring both quantitative parameters--percent contrast (%C), contrast to noise ratio (C/N), signal to noise ratio (S/N)--and qualitative parameters--lesion conspicuity, margins and extension, motion artifacts, image quality. RESULTS: The only statistically significant difference between the two sequences was image quality, which was superior in the conventional STIR sequence (p < .05). No statistically significant difference was demonstrated with the quantitative evaluation. DISCUSSION: In this study, T-STIR sequences were performed with low-high acquisition profile to acquire an actual echo time of 20 ms which permits to obtain optimal S/N with good spatial resolution. Therefore, T-STIR sequences with low-high acquisition profile provides better results than T-STIR sequences with linear acquisition profile which permits to obtain an actual echo time of 40 ms. CONCLUSION: This work shows that T-STIR sequences can replace conventional STIR sequences in the study of skeletal conditions reducing the acquisition time by 60%. This result can be obtained only by an accurate optimization of acquisition parameters.

Adolescent↗

PubMed2003

Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints.

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.

Algorithms↗

PubMed1988

Chromatin structure of altered yeast centromeres.

We have investigated the chromatin structure of wild-type and mutationally altered centromere sequences in the yeast Saccharomyces cerevisiae by using an indirect end-labeling mapping strategy. Wild-type centromere DNA from chromosome III (CEN3) exhibits a nuclease-resistant chromatin structure 220-250 base pairs long, centered around the conserved centromere DNA element (CDE) III. A point mutation in CDE III that changes a central cytidine to a thymidine and completely disrupts centromere function has lost the chromatin conformation typically associated with the wild-type centromere. A second conserved DNA element, CDE I, is spatially separated from CDE III by 78-86 A + T-rich base pairs, which is termed CDE II. The sequence and spatial requirements for CDE II are less stringent; alterations in CDE II length and sequence can be tolerated to a limited extent. Nuclease-resistant cores are altered in dimension in two CDE II CEN3 mutations. Two CDE I deletion mutations that retain partial centromere function also show nuclease-resistant regions of reduced size and intensity. The results from a number of such altered centromeres indicate a correlation between the presence of a protected core and centromere function.

Base Sequence↗

PubMed1999

High resolution FISH in plants - techniques and applications.

Fluorescence in situ hybridization (FISH) is an effective and accurate cytogenetic tool for mapping single copy and repetitive DNA sequences on chromosomes. Attempts to increase the detection sensitivity of very small chromosomal targets, and to improve the spatial resolution of signals derived from flanking sequences, have led to the development of a variety of novel techniques: it is now possible to perform in situ hybridizations on interphase nuclei, meiotic pachytene chromosomes and isolated chromatin (DNA fibres). The recent application of these techniques has indicated that a spatial resolution of 1 kb between adjacent targets and a sensitivity of targets smaller than 1 kb is now feasible. Here, we describe the benefits of these novel chromosome analysis techniques and discuss their relevance for the study of plant genomes.

Journal Article↗

PubMed1996

Alternative splicing of the Endo16 transcript produces differentially expressed mRNAs during sea urchin gastrulation.

The Endo16 gene codes for an RGD-containing calcium-binding protein that is found on the basal surfaces and in the extracellular matrix of cells of the invaginating archenteron during sea urchin gastrulation. Previously, we have shown that Endo16 is a single copy gene and we have determined the coding sequence and analyzed the temporal and spatial expression of a 6.6-kb mRNA. In this report we demonstrate that two additional longer Endo16 mRNAs are produced by differential splicing rather than alternative promoter usage. cDNA clones for two 8.5-kb mRNAs have been isolated and analyzed. The two 8.5-kb mRNAs are identical to each other in the coding region and differ only in their 3' UTRs. The extended open reading frame of the 8.5-kb mRNAs code for domains already identified in the 6.6-kb mRNA, including two different types of calcium-binding motifs and a region with a highly conserved cysteine pattern similar to that found in Ecm1 in the mouse. The 6.6- and 8.5-kb mRNAs show overlapping but distinct temporal as well as spatial expression patterns during gastrulation.

Alternative Splicing↗

PubMed1981

Familiarity, redundancy, and the spatial control of visual attention.

Highly familiar letter sequences (English words) in noncued portions of a tachistoscopic display were shown to substantially reduce accuracy of partial report. These findings suggest that in addition to facilitating character scanning, familiarity may operate in automatically directing attentional resources to a particular spatial region of a display. Such attentional capture may be disruptive if the material to be reported is presented at another location.

Attention↗

PubMed2004

Identification of binding epitope of a monoclonal antibody (Z12) against human TNF-alpha using computer modeling and deletion mutant technique.

The genes of the heavy and light chain variable region (VH, VL) of Z12 antibody against hTNF-alpha were cloned, and according to the translated sequence of amino acids, the spatial structures of VH and VL domains were modeled by using homology-based modeling method, followed by constructing the whole three-dimensional structure of Fv fragment. The complex model of Fv interacting with hTNF-alpha was gained with computer-guided molecular docking method, based on which, it was predicted that the epitope recognized by Z12 was from 141 to 146 of hTNF-alpha. hTNF-alpha molecule was divided into two fragments of N-terminal region from 1 to 91 and C-terminal region from 92 to 157 with prokaryotic expression. The measured results suggested that the antigenic epitope recognized by Z12 antibody was located in the C-terminal region 92-157 of hTNF-alpha, proving the predicted result reliable preliminarily. Further experimental results showed that after hTNF-a 141-146 residues were deleted, Z12 antibody almost lost the ability to recognize the mutant, suggesting that the amino acid residues from 141 to 146 of hTNF-alpha were specially recognized by Z12 antibody.

Animals↗

PubMed2026

Understanding tumor adaptations and resistance to MET inhibitors in MET-altered non-small cell lung cancer.

AIM: Type Ib MET inhibitors are clinically active in selected MET-altered non-small cell lung cancer, particularly tumors with MET exon 14 skipping or MET amplification, but acquired resistance remains incompletely understood. Here, we investigated resistance across biologically distinct MET-altered contexts, including MET exon 14 skipping, MET amplification, and MET overexpression. METHODS: Paired baseline and progression samples from seven patients treated with tepotinib or capmatinib were analyzed using spatial transcriptomics, whole-exome sequencing, RNA sequencing, CRISPR screening, and drug-combination assays. Patient-derived cultures and resistant cell-line models were used to explore resistance-associated changes. RESULTS: MET inhibitor resistance was heterogeneous, with persistence of the initial MET alteration in most evaluable cases and emergence of patient-specific genomic events. Three main resistance-associated, often overlapping, routes were identified: on-target MET evolution through kinase-domain alterations; extracellular matrix and tumor-microenvironment remodeling, including collagen and fibronectin upregulation, complement-related signaling, and partial EMT-associated programs; and bypass signaling involving EGFR/HER, MAPK, and PI3K/Akt pathways. In vitro models reproduced several tumor-cell-intrinsic features but only partially captured microenvironment-associated changes. CONCLUSIONS: MET inhibitor resistance in this cohort involved overlapping, context-dependent genomic, phenotypic, and signaling adaptations, supporting combination strategies for MET-altered lung cancer.

CRISPR screen↗

PubMed2001

Identification, sequence and expression of a crustacean cardioactive peptide (CCAP) gene in the moth Manduca sexta.

The crustacean cardioactive peptide (CCAP) gene was isolated from the tobacco hawkmoth Manduca sexta. The gene has an open reading frame of 125 amino acid residues containing a single, complete copy of CCAP. Analysis of the gene structure revealed three introns interrupting the coding region. A comparison of the M. sexta CCAP gene with the Drosophila melanogaster genome database reveals significant similarities in sequence and gene structure. The spatial and temporal expression patterns of the CCAP gene in the M. sexta central nervous system were determined in all major post-embryonic stages using in situ hybridization techniques. The CCAP gene is expressed in a total of 116 neurons in the post-embryonic M. sexta central nervous system. Nine pairs of cells are observed in the brain, 4.5 pairs in the subesophageal ganglion, three pairs in each thoracic ganglion (T1-T3), three pairs in the first abdominal ganglion (A1), five pairs each in the second to sixth abdominal ganglia (A2-A6) and 7.5 pairs in the terminal ganglion. The CCAP gene is expressed in every ganglion in each post-embryonic stage, except in the thoracic ganglia of first- and second-instar larvae. The number of cells expressing the CCAP gene varies during post-embryonic life, starting at 52 cells in the first instar and reaching a maximum of 116 shortly after pupation. One set of thoracic neurons expressing CCAP mRNA shows unusual variability in expression levels immediately prior to larval ecdysis. Using previously published CCAP immunocytochemical data, it was determined that 91 of 95 CCAP-immunopositive neurons in the M. sexta central nervous system also express the M. sexta CCAP gene, indicating that there is likely to be only a single CCAP gene in M. sexta.

Amino Acid Sequence↗

PubMedDate not supplied

Comparison of conventional single echo and multi-echo sequences with a fast spin-echo sequence for quantitative T2 mapping: application to the prostate.

The accuracy of water T2 maps generated from a fast spin-echo (FSE) sequence was compared with data obtained by conventional single and multi-echo spin-echo pulse sequences using a commercial gel phantom. Spatially localized stimulated echo acquisition mode (STEAM) proton spectroscopy was also used to confirm the reported water T2 values of the gels contained in the phantom. The FSE sequence was shown to be superior in accuracy to both the single and multi-echo spin echo sequences and comparable to STEAM, producing results that were within 10% of known values. The effectiveness of the FSE sequence was further demonstrated by generating T2 maps of the normal and diseased prostate in clinically acceptable imaging times, resulting in comparable T2 values to those obtained using STEAM. Accurate quantitative T2 maps can be produced with the FSE sequence.

Carcinoma↗

PubMed1998

Model of cortical-basal ganglionic processing: encoding the serial order of sensory events.

Several lines of evidence suggest that the prefrontal (PF) cortex and basal ganglia are important in cognitive aspects of serial order in behavior. We present a modular neural network model of these areas that encodes the serial order of events into spatial patterns of PF activity. The model is based on the topographically specific circuits linking the PF with the basal ganglia. Each module traces a pathway from the PF, through the basal ganglia and thalamus, and back to the PF. The complete model consists of an array of modules interacting through recurrent corticostriatal projections and collateral inhibition between striatal spiny units. The model's architecture positions spiny units for the classification of cortical contexts and events and provides bistable cortical-thalamic loops for sustaining a representation of these contextual events in working memory activations. The model was tested with a simulated version of a delayed-sequencing task. In single-unit studies, the task begins with the presentation of a sequence of target lights. After a short delay, the monkey must touch the targets in the order in which they were presented. When instantiated with randomly distributed corticostriatal weights, the model produces different patterns of PF activation in response to different target sequences. These patterns represent an unambiguous and spatially distributed encoding of the sequence. Parameter studies of these random networks were used to compare the computational consequences of collateral and feed-forward inhibition within the striatum. In addition, we studied the receptive fields of 20,640 model units and uncovered an interesting set of cue-, rank- and sequence-related responses that qualitatively resemble responses reported in single unit studies of the PF. The majority of units respond to more than one sequence of stimuli. A method for analyzing serial receptive fields is presented and utilized for comparing the model units to single-unit data.

Algorithms↗

PubMed2000

Farey sequences of spatiotemporal patterns in video feedback.

In this paper we present an experimental and theoretical description of the dynamic of spatial patterns obtained in a video feedback loop. A video camera monitors the screen to which it is connected and can turn around its optical axis at an angle alpha. Under certain conditions of brightness and magnification, this optoelectronic system produces spatiotemporal patterns in the form of spots located on a circle on the screen. These patterns are very similar to the spatial transverse modes obtained in other optical devices such as lasers or photorefractive media. It is possible to generate stationary patterns of n-fold symmetries for angles alpha=2pi/n. When the angle alpha varies around 2pi/n, the pattern rotates with a certain frequency proportional to the difference between 2pi/n and alpha. We discover more general patterns at angles 2pi/(p/k) with p-fold symmetry, following the hierarchy of the Farey algorithm which theoretically can produce stationary patterns at any angle alpha. Very accurate experiments were performed to observe these patterns up to the level k=6. This is the first time a Farey tree has been observed as a sequence of spatial patterns to our knowledge. Previous observations of this hierarchy were made only in the temporal domain.

Animals↗

PubMed2006

Biogeography and landscape-scale diversity of the dominant Crenarchaeota of soil.

We surveyed the diversity of soil Archaea across a large scale elevational gradient of ecosystem types, from foothills forest to alpine tundra in the Front Range of the Rocky Mountains. We used a dilution technique to sequence the single most abundant archaeal 16S rDNA sequence in each of the 40 soil cores distributed across the gradient to compare our results to those of typical 16S clone library studies. We found a greater diversity of sequences than has typically been found in clone library studies from a single site or core, identifying sequences both from the Terrestrial Group and the FFSB Group at several sites. We did not observe any significant environmental correlates with the dominant sequence type, nor was there any relationship between the spatial distance between samples and the phylogenetic similarity of the dominant sequence types. Despite using a very different methodology, our collective results are in remarkably good agreement with other studies of soil Crenarchaeota in terms of the diversity and relative abundance of sequence types identified. We are able to identify two instances of very tightly clustered sequences which we suggest are the results of global selective sweeps-one closely related to SCA1145, an abundant globally distributed group within the Terrestrial Group of Crenarchaeota, and another nested within the more basal FFSB group of sequences. We replicated our sequence results at two levels: first, by repeating the dilution and PCR processes from the same soil core DNA extraction, and second, by performing a replicate DNA extraction from the same homogenized soil core sample. Pairs of sequences produced by the dilution replicates were significantly more similar than the pairs of sequences produced by the extraction replicates, suggesting that soil Crenarchaeota exists in highly localized and discrete clonal populations.

Base Sequence↗

PubMed1993

Drosophila tyrosine hydroxylase is encoded by the pale locus.

We have reintroduced an 8 kb genomic fragment from the Drosophila tyrosine hydroxylase (DTH) locus into the genome of mutant pale (ple) flies. ple was first recovered as a recessive embryonic lethal by Jurgens et al. (1984) and maps to the same chromosomal region as DTH (65A-E). Mutant ple alleles affect pigmentation of the cuticle (L-DOPA, the product of the reaction catalyzed by TH, is an intermediate in the cuticular sclerotization and pigmentation pathways) and catecholamine biosynthesis. In this report we demonstrate that ple does encode the structural gene for TH, since the reintroduced sequences rescue ple flies from lethality to viable adults. Morphological, immunocytochemical, and behavioral characterization of three transformant lines suggests that the reintroduced sequences contain the necessary elements for correct temporal and spatial expression of the gene, but may not contain all the sequences essential for quantitative expression.

Animals↗

PubMed1999

Visualization of a specific sequence on a single large DNA molecule using fluorescence microscopy based on a new DNA-stretching method.

A method for analyzing large DNA which makes it possible to obtain spatial information on the positions of specific sequences along a DNA molecule has been developed. Making use of the fact that large DNA molecules are stably elongated under an alternating-current field in a concentrated linear polymer solution, the direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. The spatial positions of the fluorescent spots of the probe (fluorescence-labeled restriction endonuclease EcoRI) on DNA molecules were determined by image analysis. As expected, fluorescent spots of EcoRI were observed at certain positions on lambda DNA, where sequences to which EcoRI binds are located. Finally, the potential application of single large DNA molecule analysis using this DNA-stretching method is discussed.

Bacteriophage lambda↗

PubMed1989

Evolution of the temporal contraction sequence after acute experimental myocardial infarction.

The effect of infarct maturation on the temporal sequence of contraction within infarct zones has not previously been described. Accordingly, the time-varying pattern of contraction within ischemic/infarct zones was studied with use of cross-sectional echocardiography in 17 dogs at 10 min to 6 weeks after acute experimental myocardial infarction. Left ventricular short-axis images were digitized from end-diastole to end-systole and endocardial fractional radial change along 36 evenly spaced rays was calculated. The circumferential extent of dyskinesia and the number of rays that exhibited maximal dyskinesia were determined for each decile of the normalized contraction sequence. Between 10 min and 1 week after infarction, the greatest circumferential extent of dyskinesia occurred between the 3rd and 4th deciles of the normalized contraction sequence. However, as the infarct matured, the greatest spatial expanse of dyskinesia was noted to occur progressively earlier in the contraction sequence (second decile at 6 weeks), and the extent of mid- to late-systolic dyskinesia decreased markedly. Whereas end-systolic dyskinesia was present in 30% to 50% of ischemic/infarct zone rays from 10 min to 48 h, end-systolic dyskinesia was no longer observed at 6 weeks. Similarly, the maximal amplitude of dyskinesia was most commonly observed during midsystole from 10 min to 48 h, but occurred progressively earlier as the infarct matured, falling during the first decile at 6 weeks after infarction. These data suggest that maximal circumferential extent and amplitude of dyskinesia occur progressively earlier in the systolic contraction sequence as the infarct matures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗