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At least 469 records · Page 26Linked to original sources

PubMed1996

A novel expression vector for transfection of bovine MHC class I genes.

A vector is described for the expression of genomic or cDNA copies of bovine major histocompatibility complex (MHC) class I genes in transfected mouse Ltk- cells. Class I gene fragments are amplified by the polymerase chain reaction, using primers in conserved parts of exon 2 and the 3'-untranslated region of the gene. Amplified class I gene fragments can then be subcloned into the expression vector, pBoLA-21, which contains the necessary 5'- and 3'-sequences for correct expression. The vector was tested by subcloning and expressing genomic and cDNA clones.

Animals↗

PubMed2004

A simple mathematical formula for stoichiometry quantification of viral and nanobiological assemblage using slopes of log/log plot curves.

In nanotechnology, biomolecular assemblies serve not only as model systems for the construction of nanodevices, but they can also be used directly as templates for the formation of nanostructures. Biological nano-building blocks can either be isolated as complete functional units from living cells or viruses (biological "Top down" approach) or formed by biomolecular assembly from recombinant or synthetic components ("Bottom up" approach). In both cases, rational design of nanostructures requires knowledge of the stoichiometry of the biological structures, which frequently occur as multimers, i.e., the morphological complex is composed of multiple copies of one or more macromolecules. In this paper, a method is described for the stoichiometric quantification of molecules in bio-nanostructures. The method is based on using dilution factors and relative concentrations rather than absolute quantities, which are often difficult to determine, especially in short-lived assembly intermediates. The approach exploits the fact that the larger the stoichiometry of the component is, the more dramatic is the influence of the dilution factor (decrease in concentration) on the reaction. We established and used the method to determine the stoichiometry of components of bacterial virus phi29. The log of dilution factors was plotted against the log of reaction yield. The stoichiometry Z was determined with the equation Z=-1.58+2.4193T-0.001746T(2) [T in (0,1000), or 90 degree angle alpha in (0 degrees, 89.9 degrees )], where T is the slope of the curve (tangent of 90 degree angle alpha, which is the angle between the x-axis and the concentration dependent curve). Z can also be determined from a standard table given in this report. With the bacteriophage phi29 in vitro assembly system, up to 5x10(8) infectious virions per ml can be assembled from 11 purified components, giving our method a sensitivity of nine orders of magnitude. We confirmed the stoichiometries of phi29 components that were determined previously with microscopic approaches. The described method also responded to programmed stoichiometry changes, which were generated by assembling the phi29 DNA packaging motor from modified pRNA (DNA-packaging RNA) molecules forming a trimer of dimers or a dimer of trimers, instead of the wild-type hexamer.

Bacillus Phages↗

PubMed2003

Glycoproteins expression on platelet membrane in inherited macrothrombocytopenias.

INTRODUCTION: Inherited giant platelet disorders (IGPD) are a heterogeneous group of rare diseases characterized by thrombocytopenia, large platelets and variable bleeding symptoms. Glycoprotein (GP) expression on platelet surface in these conditions is poorly characterized. We have investigated the expression of constitutively expressed platelet membrane GP and the response to TRAP activation in a group of patients with different forms of inherited macrothrombocytopenias. MATERIALS AND METHODS: Two patients diagnosed Epstein syndrome (ES), two with Bernard-Soulier syndrome (BSS) and eight with Mediterranean macrothrombocytopenia (MM) were studied using flow cytometry combined to monoclonal antibodies (MoAbs). Mean platelet volume was also measured. RESULTS: In general, there was an increase in the binding of MoAbs directed against platelet membrane GPs in the patients studied (except, obviously, in BSS patients). The observed increase ranged between 0.9 and 3.36 times the value of the control for GPIbalpha, between 1.36 and 7.2 times for GPIIb-IIIa and 0.94 and 8.07 times for GPIV. However, when the observed value was divided by the measured platelet volume, the estimated density was similar to controls in the case of GPIbalpha but were increased for GPIIb-IIIa and GPIV. TRAP activation provoked significant increments in the number of copies of GPIIb-IIIa complexes present on platelet surface in controls (91%), MM patients (49%), but almost no changes in BSS platelets (6%). CONCLUSION: In the group of macrothrombocytopenia studied, an increase in the expression of platelet membrane GP was observed, although a wide variability exists even in patients with the same disorder.

Adolescent↗

PubMed2006

The implications of DNA transposons in the evolution of P elements in zebrafish (Danio rerio).

We have carried out detailed analyses of Drosophila P-transposon-homologous Pdre elements in zebrafish (Danio rerio) and present a model to explain the evolutionary origin of the complete and terminally truncated copies. Analysis of the most complex terminally truncated element led to the identification of novel DNA transposons, referred to as demio, hexi, and lady elements. We also show that the existence of a P-homologous stable gene can be explained in two ways: it is a P-transposon source gene, and insertion into or recombination with another transposon can lead to mobilization by recruitment of structures necessary for transposition (known as the source gene hypothesis), or it is the result of a domestication event involving a P transposon (domestication hypothesis). Our results indicate that both scenarios are possible and that they reflect different stages of the P-element life cycle.

Animals↗

PubMed2000

Polysomy 13 with concomitant deletion of 13q13-14 involving the retinoblastoma gene and the D13S25 locus in a case of acute myeloid leukemia.

We herein describe a case of acute myeloblastic leukemia (AML), FAB subtype M4, with an unfavorable clinical course and a complex karyotype, including 4-9 copies of chromosome 13. Polysomy 13 was a result of clonal evolution. Fluorescence in situ hybridization (FISH) revealed a cytogenetically unrecognizable deletion within 13q13-14 that included the retinoblastoma gene (RB) and the D13S25 locus in all but one copy of chromosome 13. The only chromosome 13 that did not show a deletion affecting the q13-14 region was translocated to chromosome 7, resulting in a dic(7;13)(q21;p11). In this case, the coexistence of polysomy and a partial deletion within the same chromosome point toward a possible formation of a fusion product with oncogenic potential and its consecutive amplification as a critical alteration in this case.

Aged↗

PubMed1998

Identification and characterisation of a functional peroxidoxin from Leishmania major.

Leishmania spp. encounter damaging oxygen metabolites from endogenous metabolic processes as well as from exogenous sources, such as inside the gut of the sandfly vector and within host macrophages. The recently described peroxidoxin protein family form part of a novel pathway for metabolising hydrogen peroxide that, in trypanosomatids, links peroxide reduction to NADPH oxidation via trypanothione. Here we report the cloning and characterisation of the Leishmania major peroxidoxin gene, tryparedoxin peroxidase (TryP). TryP is a multi-copy gene arranged in a complex tandem array located on the size polymorphic homologues of chromosome 15. Northern analysis showed that TryP expresses a single 1.6 kb mRNA throughout promastigote development. TryP encodes a 22-kDa protein with two conserved cysteine-containing domains that defines it as a 2-Cys peroxidoxin. Purified recombinant TryP protein catabolised hydrogen peroxide in the presence of the tryparedoxin homologue from Crithidia fasciculata (Cf-TryX), trypanothione, trypanothione reductase and NADPH. The demonstration that L. major utilises a three-protein peroxidase system confirms that this is a mechanism of protection against oxidative damage in this parasite.

Amino Acid Sequence↗

PubMed2004

Visuoconstructive performance, implicit hemispatial inattention, and schizotypy.

The present study investigates visuoconstructive performance and hemispatial inattention in schizotypic college students and control subjects using the Rey-Osterrieth Complex Figure Test (CFT). Both copy and delay CFT accuracy, as well as line bisection scores on the CFT, were examined in right-handed males reporting positive schizotypy (deviantly high scorers on the Perceptual Aberration and Magical Ideation Scales; n=24) and those reporting negative schizotypy (deviantly high scorers on the revised Social Anhedonia Scale and Physical Anhedonia Scale; n=26), as well as controls (n=45). The negative schizotypy group displayed reduced visuoconstructive performance, as measured by performance on both the copy and delay trials of the CFT. We did not find a significant relationship between schizotypy and hemispatial inattention. These findings suggest that negative schizotypy may be associated with memory performance deficits, but do not support prior assertions of a relationship between hemispatial inattention and positive schizotypy.

Adolescent↗

PubMed2000

Learning to read is much more than learning to read: a neuropsychologically based reading program.

Departing from the observation that illiterates significantly underscore in some neuropsychological tests, a learning-to-read method named NEUROALFA was developed. NEUROALFA is directed to reinforce these underscored abilities during the learning-to-read process. It was administered to a sample of 21 adult illiterates in Colima (Mexico). Results were compared with 2 control groups using more traditional procedures in learning to read. The NEUROPSI neuropsychological test battery was administered to all the participants before and after completing the learning-to-read training program. All 3 groups presented some improvement in the test scores. Gains, however, were significantly higher in the experimental group in Orientation in Time, Digits Backward, Visual Detection, Verbal Memory, Copy of a Semi-Complex Figure, Language Comprehension, Phonological Verbal Fluency, Similarities, Calculation Abilities, Sequences, and all the recall subtests, excluding Recognition. Performance in standard reading tests was also significantly higher in the experimental group. Correlations between pretest NEUROPSI scores and reading ability were low. However, correlations between posttest NEUROPSI scores and reading scores were higher and significant for several subtests. Results are interpreting as supporting the assumption that reinforcement of those abilities in which illiterates significantly underscore results in a significant improvement in neuropsychological test scores and strongly facilitates the learning-to-read process. The NEUROALFA method of teaching reading to adult illiterates is beginning to be used extensively in Mexico. To our knowledge, this is the first attempt to apply neuropsychological principles to social problems.

Adolescent↗

PubMed2000

RNA location and modeling of a WD40 repeat domain within the vault.

The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.

Animals↗

PubMed2004

Identification and characterization of a novel form of the human L-dopa decarboxylase mRNA.

L-Dopa decarboxylase (DDC) has been cloned from several species and was shown to undergo alternative splicing within its 5'-untranslated and coding regions. In this report, we describe a novel splice variant of DDC mRNA in human tissue, lacking exons 10-15 of the full-length transcript but including an alternative exon 10. The isolated alternative human DDC cDNA (alt-DDC) was cloned from human placenta, and was found to be of the neuronal type. Northern blot analysis indicated that the alt-DDC transcript is expressed in high levels in human kidney. Our results demonstrate the detection of a new alternative splicing event within the coding region of the human DDC mRNA, further suggesting that the single copy human DDC gene undergoes complex processing leading to the formation of multiple mRNA isoforms.

Alternative Splicing↗

PubMed2002

Coordinate regulation of energy transduction modules in Halobacterium sp. analyzed by a global systems approach.

The extremely halophilic archaeon Halobacterium NRC-1 can switch from aerobic energy production (energy from organic compounds) to anaerobic phototrophy (energy from light) by induction of purple membrane biogenesis. The purple membrane is made up of multiple copies of a 1:1 complex of bacterioopsin (Bop) and retinal called bacteriorhodopsin that functions as a light-driven proton pump. A light- and redox-sensing transcription regulator, Bat, regulates critical genes encoding the biogenesis of the purple membrane. To better understand the regulatory network underlying this physiological state, we report a systems approach using global mRNA and protein analyses of four strains of Halobacterium sp.: the wild-type, NRC-1; and three genetically perturbed strains: S9 (bat+), a purple membrane overproducer, and two purple membrane deficient strains, SD23 (a bop knockout) and SD20 (a bat knockout). The integrated DNA microarray and proteomic data reveal the coordinated coregulation of several interconnected biochemical pathways for phototrophy: isoprenoid synthesis, carotenoid synthesis, and bacteriorhodopsin assembly. In phototrophy, the second major biomodule for ATP production, arginine fermentation, is repressed. The primary systems level insight provided by this study is that two major energy production pathways in Halobacterium sp., phototrophy and arginine fermentation, are inversely regulated, presumably to achieve a balance in ATP production under anaerobic conditions.

Adenosine Triphosphate↗

PubMed1979

Chromosome 1 contains the endogenous RAV-0 retrovirus sequences in chicken cells.

We have developed a structurally unique probe which can be used to determine the chromosomal location of nonreiterated genes in vertebrate organisms by the method of in situ hybridization. The probe consists of several specific RNA molecules attached by means of poly(A) . poly(BrdUrd) hybrids to 125I-labeled DNA of high molecular weight. The probe can be synthesized with a variety of RNA molecules, giving it versatility for detecting a variety of genes irrespective of gene size, copy frequency, and host genome complexity. Using this probe prepared with retrovirus genomic RNAs, we have physically mapped all three detectable endogenous genomes of Rous-associated virus type 0 (RAV-0) in Spafas gs- chf- (group-specific antigen negative, chicken helper factor negative) chicken fibroblasts to specific sites on chromosome 1. This finding suggests that these multiple nontranscribed RAV-0 genomes evolved through gene duplication of an original RAV-0 genome. The endogenous src gene coding for a 60,000-dalton protein also has been localized to one of the small macrochromosomes, 10, 11, or 12, in both chicken and Japanese quail cells. The results presented here are consistent with and greatly extend previously reported data obtained by using both chromosome fractionation and restriction endonuclease techniques and thus support the soundness of this hybridization approach.

Animals↗

PubMed1985

Mutations that impair a posttranscriptional step in expression of HLA-A and -B antigens.

Mutations can interfere with posttranscriptional expression of the HLA-A and -B genes. B-lymphoblastoid cells that contain one copy of the major histocompatibility complex (MHC) were subjected to mutagenesis and immunoselection for MHC antigen-loss mutants. Some mutations partially reduced surface expression of HLA-A and eliminated HLA-B expression concurrently, although the HLA-A and -B genes were present and transcribed. Antigen expression was fully restored in hybrids of these mutants with other B-lymphoblastoid cells. Therefore, normal cell surface expression of the HLA-A and -B antigens on B lymphoblasts requires (i) execution of at least one trans-active step in the production of the antigens after transcription of the HLA-A and -B genes or (ii) association of the class I antigens with other molecules. DNA analysis of one mutant suggests the possibility that a locus required for the normal expression of the HLA-A and -B antigens is located between the MHC complement genes and the HLA-DP alpha II locus.

Antibodies, Monoclonal↗

PubMed1993

The TFIIIB-assembling subunit of yeast transcription factor TFIIIC has both tetratricopeptide repeats and basic helix-loop-helix motifs.

The multisubunit yeast transcription factor IIIC (TFIIIC; also called tau) can undergo considerable conformational changes upon binding to the A and B blocks of tRNA genes. After binding to DNA encoding tRNA (tDNA), TFIIIC acts as an assembly factor to recruit an initiation factor, TFIIIB, via its tau 131 subunit. We have cloned the gene encoding the tau 131 subunit and named it TFC4. This gene is unique, essential for cell viability, and encodes a M(r) 120,153 protein. Epitope-tagging and mobility-shift assays indicated the presence of a single tau 131 subunit in TFIIIC-tDNA complexes. tau 131 contains two sequence motifs, accounting for nearly one-half of the protein mass, that may provide a molecular explanation for the properties of TFIIIC-tDNA complex. A series of 11 copies of the tetratricopeptide repeat motif may account for the flexibility and interaction properties of TFIIIC. A motif akin to the basic helix-loop-helix motif of MyoD suggests the direct involvement of tau 131 in promoting DNA binding of TFIIIB.

Amino Acid Sequence↗

PubMed2004

The aryl hydrocarbon (Ah) receptor transcriptional regulator hepatitis B virus X-associated protein 2 antagonizes p23 binding to Ah receptor-Hsp90 complexes and is dispensable for receptor function.

To further understand the role that the hepatitis B virus X-associated protein 2 (XAP2) plays in regulating aryl hydrocarbon receptor (AhR) function, a point mutation was introduced at tyrosine 408 of the AhR, changing the residue to an alanine or lysine. These mutations resulted in the loss of AhR binding to endogenous XAP2 in COS-1 cells and reduced binding of exogenously expressed XAP2. Cellular localization of the mutant AhR-yellow fluorescent protein fusion proteins remained nuclear when XAP2 was co-expressed, while the non-mutant receptor was redistributed to the cytoplasm. XAP2 expression caused an overall repression of constitutive and ligand-induced AhR transcriptional activity. However, increased expression of XAP2 had no effect on the AhRY408A mutant transcriptional activity. Additionally the XAP2 binding-deficient AhR mutants showed overall higher transcriptional activity when compared with the non-mutant receptor. Interestingly reduced incorporation of the Hsp90 associated co-chaperone p23 in the unliganded AhR complex was observed with increasing XAP2 expression. The displacement of p23 from Hsp90 did not occur when increasing levels of XAP2 were introduced in COS-1 cells in the absence of the AhR; thus this displacement event occurs specifically within an AhR complex. Finally XAP2 itself was capable of existing in multimeric complexes, and these complexes did not require Hsp90 or AhR to form. However, it is not yet clear whether XAP2 can exist within the AhR complex in more than one copy.

Animals↗

PubMed1999

Human cochlear expressed sequence tags provide insight into cochlear gene expression and identify candidate genes for deafness.

To identify candidate genes for human hearing disorders and to understand better human hearing at the molecular level, we constructed a human cochlear cDNA library. An aliquot of the unsubtracted cochlear library was contributed to the IMAGE Consortium at Lawrence Livermore National Laboratory for the generation of expressed sequence tags (ESTs) by the Merck/WashU EST project. Over 4000 ESTs were developed from the cochlear cDNA library and deposited in the GenBank EST database. Sequence clustering shows that the majority of clones are in low copy numbers, demonstrating the high complexity of the library. The sequences of 1388 cochlear ESTs (33%) match 517 known human genes. Among these are genes previously shown to cause both syndromic and non-syndromic hearing loss. A number of the cochlear ESTs show high homology to non-human genes, suggesting new gene family members or human homologs of animal genes. We also report the chromosomal map positions of 437 cochlear ESTs. These provide positional candidate genes for 18 different non-syndromic hearing disorders. A Human Cochlear EST Database web site (http://www.bwh.partners. org/pathology ) has been created to provide access to the cochlear clone data for gene discovery investigations.

Animals↗

PubMed1984

PLASMAP: an interactive computational tool for storage, retrieval and device-independent graphic display of conventional restriction maps.

We describe an interactive computational tool, PLASMAP, which allows the user to electronically store, retrieve, and display circular restriction maps. PLASMAP permits users to construct libraries of plasmid restriction maps as a set of files which may be edited in the laboratory at any time. The display feature of PLASMAP quickly generates device-independent, artist-quality, full-color or monochrome, hard copies or CRT screens of complex, conventional circular restriction maps.

Base Sequence↗

PubMed1985

Transposition of a long member of the L1 major interspersed DNA family into the mouse beta globin gene locus.

A long member of the highly repeated long interspersed DNA family L1Md (for L1 in Mus domesticus) has integrated by transposition into a target site which lies between the two adult beta globin genes of mouse. DNA hybridization and nucleotide sequence analysis show that this target site, which is part of the single copy DNA flanking the globin genes, is interrupted by the L1 element in one chromosome but is uninterrupted in both allelic and ancestral chromosomes. Other large DNA rearrangements of the region between the two adult beta globin genes are also associated with these allelic chromosomes, and include insertions or deletions of both single copy DNA and simple and complex repetitive DNA. This has caused extensive reorganization of this intergenic region. However, the distance between the two genes flanking this region remains conserved, suggesting that the spacing of the globin genes may be subject to conservative selection.

Alleles↗