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At least 1,459 records · Page 81Linked to original sources
PubMed2003

HIV-1 p55Gag encoded in the lysosome-associated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class II compartment, and elicits enhanced immune responses.

Several genetic vaccines encoding antigen chimeras containing the lysosome-associated membrane protein (LAMP) translocon, transmembrane, and cytoplasmic domain sequences have elicited strong mouse antigen-specific immune responses. The increased immune response is attributed to trafficking of the antigen chimera to the major histocompatibility class II (MHC II) compartment where LAMP is colocalized with MHC II. In this report, we describe a new form of an HIV-1 p55gag DNA vaccine, with the gag sequence incorporated into the complete LAMP cDNA sequence. Gag encoded with the translocon, transmembrane and cytoplasmic lysosomal membrane targeting sequences of LAMP, without the luminal domain, was poorly expressed, did not traffic to lysosomes or MHC II compartments of transfected cells, and elicited a limited immune response from DNA immunized mice. In contrast, addition of the LAMP luminal domain sequence to the construct resulted in a high level of expression of the LAMP/Gag protein chimera in transfected cells that was further increased by including the inverted terminal repeat sequences of the adeno-associated virus to the plasmid vector. This LAMP/Gag chimera with the complete LAMP protein colocalized with endogenous MHC II of transfected cells and elicited strong cellular and humoral immune responses of immunized mice as compared with the response to DNA-encoding native Gag, with a 10-fold increase in CD4+ responses, a 4- to 5-fold increase in CD8+ T-cell responses, and antibody titers of >100,000. These results reveal novel roles of the LAMP luminal domain as a determinant of Gag protein expression, lysosomal trafficking, and possibly of the immune response to Gag.

3T3 Cells↗
PubMed2005

Cloning, expression, and characterization of the human mitochondrial beta-ketoacyl synthase. Complementation of the yeast CEM1 knock-out strain.

A human beta-ketoacyl synthase implicated in a mitochondrial pathway for fatty acid synthesis has been identified, cloned, expressed, and characterized. Sequence analysis indicates that the protein is more closely related to freestanding counterparts found in prokaryotes and chloroplasts than it is to the beta-ketoacyl synthase domain of the human cytosolic fatty acid synthase. The full-length nuclear-encoded 459-residue protein includes an N-terminal sequence element of approximately 38 residues that functions as a mitochondrial targeting sequence. The enzyme can elongate acyl-chains containing 2-14 carbon atoms with malonyl moieties attached in thioester linkage to the human mitochondrial acyl carrier protein and is able to restore growth to the respiratory-deficient yeast mutant cem1 that lacks the endogenous mitochondrial beta-ketoacyl synthase and exhibits lowered lipoic acid levels. To date, four components of a putative type II mitochondrial fatty acid synthase pathway have been identified in humans: acyl carrier protein, malonyl transferase, beta-ketoacyl synthase, and enoyl reductase. The substrate specificity and complementation data for the beta-ketoacyl synthase suggest that, as in plants and fungi, in humans this pathway may play an important role in the generation of octanoyl-acyl carrier protein, the lipoic acid precursor, as well as longer chain fatty acids that are required for optimal mitochondrial function.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2005

Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation.

Sumoylation has recently been identified as an important mechanism that regulates protein interactions and localization in essential cellular functions, such as gene transcription, subnuclear structure formation, viral infection, and cell cycle progression. A SUMO binding amino acid sequence motif (SBM), which recognizes the SUMO moiety of modified proteins in sumoylation-dependent cellular functions, has been consistently identified by several recent studies. To understand the mechanism of SUMO recognition by the SBM, we have solved the solution structure of SUMO-1 in complex with a peptide containing the SBM derived from the protein PIASX (KVDVIDLTIESSSDEEEDPPAKR). Surprisingly, the structure reveals that the bound orientation of the SBM can reverse depending on the sequence context. The structure also reveals a novel mechanism of recognizing target sequences by a ubiquitin-like module. Unlike ubiquitin binding motifs, which all form helices and bind to the main beta-sheet of ubiquitin, the SBM forms an extended structure that binds between the alpha-helix and a beta-strand of SUMO-1. This study provides a clear mechanism of the SBM sequence variations and its recognition of the SUMO moiety in sumoylated proteins.

Amino Acid Motifs↗
PubMedDate not supplied

Assessment of DNA damage produced by 125I-triplex-forming oligonucleotides in cells.

PURPOSE: Triplex-forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger-electron-emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two 125I-labeled TFOs, one with a single target in the genome and another with multiple targets. MATERIALS AND METHODS: Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone gamma-H2AX foci formed at the sites of DNA double strand breaks (DSBs). RESULTS: The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more gamma-H2AX foci per cell than the TFO with a single target. CONCLUSION: Since the two methods gave comparable results, measuring the number of gamma-H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.

Cell Line↗
PubMed1998

Hidden Markov models for detecting remote protein homologies.

MOTIVATION: A new hidden Markov model method (SAM-T98) for finding remote homologs of protein sequences is described and evaluated. The method begins with a single target sequence and iteratively builds a hidden Markov model (HMM) from the sequence and homologs found using the HMM for database search. SAM-T98 is also used to construct model libraries automatically from sequences in structural databases. METHODS: We evaluate the SAM-T98 method with four datasets. Three of the test sets are fold-recognition tests, where the correct answers are determined by structural similarity. The fourth uses a curated database. The method is compared against WU-BLASTP and against DOUBLE-BLAST, a two-step method similar to ISS, but using BLAST instead of FASTA. RESULTS: SAM-T98 had the fewest errors in all tests-dramatically so for the fold-recognition tests. At the minimum-error point on the SCOP (Structural Classification of Proteins)-domains test, SAM-T98 got 880 true positives and 68 false positives, DOUBLE-BLAST got 533 true positives with 71 false positives, and WU-BLASTP got 353 true positives with 24 false positives. The method is optimized to recognize superfamilies, and would require parameter adjustment to be used to find family or fold relationships. One key to the performance of the HMM method is a new score-normalization technique that compares the score to the score with a reversed model rather than to a uniform null model. AVAILABILITY: A World Wide Web server, as well as information on obtaining the Sequence Alignment and Modeling (SAM) software suite, can be found at http://www.cse.ucsc.edu/research/compbi o/ CONTACT: karplus@cse.ucsc.edu; http://www.cse.ucsc.edu/karplus

Algorithms↗
PubMed1987

Hybrid dysgenesis in Drosophila melanogaster: evidence from sterility and southern hybridization tests that P cytotype is not maintained in the absence of chromosomal P factors.

A two-generation crossing program was used to replace the entire chromosome complement of P strains by M strain chromosomes, the maternal contribution being from the P strain. The cytotype of chromosomally substituted females was indistinguishable from M strain cytotype, judged by the sterility of offspring from the cross of such females to P strain males. In addition, following replacement of the chromosomes, the level of DNA homologous to the P factor was sufficiently low to be explicable by low levels of P factor transposition. These results are consistent with immediate chromosomal control for the switching from P to M cytotype. However, the reverse chromosome substitution, replacing all chromosomes of an M strain with P chromosomes, did not usually lead to immediate change of cytotype properties, showing that there is a true maternal effect in the M to P direction. The absence of true maternal inheritance for P cytotype argues against models of P factor repression which depend on autonomous replication of a nonchromosomal element. The repression could still be explained by nonchromosomal copies of the P factor, provided that these are replenished from chromosomal P factors. A model is put forward in which P cytotype is due to the presence of circular P factors carrying a P factor target sequence, leading to preferential transposition of chromosomal P factors to nonchromosomal target sites.

Animals↗
PubMed2003

Parallel gene analysis with allele-specific padlock probes and tag microarrays.

Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes.

Adenosine Triphosphatases↗
PubMed1990

Identification, purification, and cloning of a polypeptide (PRTF/GRM) that binds to mating-specific promoter elements in yeast.

In yeast the alpha-specific regulators, alpha 1 and alpha 2 have been proposed to be DNA-binding proteins, both of which have to interact with an additional factor called PRTF or GRM, respectively, to exert their biological functions. Although the cis-acting sequence requirements for alpha 1 and alpha 2 are different, their target sequences share a common motif. PRTF or GRM is thought to act via this common DNA sequence; therefore, it has been suggested that they represent the same factor. I purified a protein that binds to this common promoter element by DNA affinity chromatography. The purified protein is able to recruit the alpha-specific activator alpha 1 to its binding sites, suggesting that it is indeed PRTF. Further evidence is presented to show that PRTF and GRM are the same protein and that PRTF plays a role in the activation of a-specific genes. Specific antibodies to the purified protein were obtained. They identify the protein as a component of DNA-protein complexes that formed with cell-type-specific promoter sequences. Using these antibodies, the gene encoding the protein was cloned from a yeast lambda gt11 expression library. The DNA sequence established that the gene encoding PRTF/GRM is identical with a previously described gene, FUN80 (essential factor of unknown function) or MCM1 (minichromosome maintenance). Sequence comparison showed further that PRTF/GRM shares similarities with a repressor from yeast, ARGRI, and the mammalian transcription factor SRF.

Amino Acid Sequence↗
PubMed1994

A phylogenetic tree of 16S rRNA sequences from sulfate-reducing bacteria in a sandy marine sediment.

The divergence of 16S rDNA sequences in marine sediment was investigated. Twenty unique partial sequences were found among 33 cloned following PCR. Thirteen shared 82 to 91% similarity with sequences of delta subclass sulfate-reducing bacteria. Three contained the target sequence for a sulfate-reducing bacterium-specific oligonucleotide probe designed from pure-culture studies.

Bacteria↗
PubMed1999

Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1.

Pseudomonas fluorescens, a gram-negative psychrotrophic bacterium, secretes a thermostable lipase into the extracellular medium. In our previous study, the lipase of P. fluorescens SIK W1 was cloned and expressed in Escherichia coli, but it accumulated as inactive inclusion bodies. Amino acid sequence analysis of the lipase revealed a potential C-terminal targeting sequence recognized by the ATP-binding cassette (ABC) transporter. The genetic loci around the lipase gene were searched, and a secretory gene was identified. Nucleotide sequencing of an 8.5-kb DNA fragment revealed three components of the ABC transporter, tliD, tliE, and tliF, upstream of the lipase gene, tliA. In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. tliDEF showed high similarity to ABC transporters of Pseudomonas aeruginosa alkaline protease, Erwinia chrysanthemi protease, Serratia marcescens lipase, and Pseudomonas fluorescens CY091 protease. tliDEF and the lipase structural gene in a single operon were sufficient for E. coli cells to secrete the lipase. In addition, E. coli harboring the lipase gene secreted the lipase by complementation of tliDEF in a different plasmid. The ABC transporter of P. fluorescens was optimally functional at 20 and 25 degrees C, while the ABC transporter, aprD, aprE, and aprF, of P. aeruginosa secreted the lipase irrespective of temperature between 20 and 37 degrees C. These results demonstrated that the lipase is secreted by the P. fluorescens SIK W1 ABC transporter, which is organized as an operon with tliA, and that its secretory function is temperature dependent.

ATP-Binding Cassette Transporters↗
PubMed1991

Rapid identification of dengue virus serotypes by using polymerase chain reaction.

Four primer pairs were selected on the basis of the published sequence data of four dengue virus serotypes so that each unique target sequence size could be amplified for each serotype by polymerase chain reaction. The procedure consists of (i) RNA preparation, (ii) reverse transcription, and (iii) polymerase chain reaction, all of which could be completed within 2 h in a single tube for each specimen. The amplified sequence size revealed by ethidium bromide-stained agarose gel electrophoresis was unique for each serotype, using infected culture fluid of isolates from dengue fever or dengue hemorrhagic fever patients in Thailand, Indonesia, and the Philippines as well as from prototype viruses, thus facilitating simultaneous identification and typing.

Animals↗
PubMed1991

vRel is an inactive member of the Rel family of transcriptional activating proteins.

The vRel oncoprotein is member of a family of related proteins that also includes cRel, NF-kappa B, and Dorsal. We investigated the transcriptional regulatory properties of several Rel proteins in cotransfection assays with chicken embryo fibroblasts (CEF). Retroviral vectors expressing hybrid proteins that contain the DNA-binding domain of LexA fused to portions of the viral oncoprotein vRel or chicken, mouse, human, or Drosophila melanogaster (Dorsal) cRel proteins were cotransfected with a reporter plasmid that contains the DNA sequence recognized by LexA, a promoter, and the assayable gene for chloramphenicol acetyltransferase. In transient assays, a LexA-vRel protein did not activate transcription in CEF. Full-length chicken cRel, mouse cRel, and Dorsal fusion proteins all activated transcription weakly; however, deletion of N-terminal Rel sequences from each of these proto-oncogene encoded proteins resulted in strong activation by LexA fusion proteins containing only C-terminal sequences. Inhibition of the C-terminal chicken cRel gene activation domain by N-terminal sequences was seen in CEF and mouse and monkey fibroblasts. These results show that cRel proteins from different species have the same general organization: an N-terminal inhibitory domain and a C-terminal activation domain. Sequence comparison suggests that the inhibitory domain is conserved but the activation domain is species specific. In contrast, vRel lacks a strong C-terminal gene activation function, since a LexA fusion protein containing C-terminal vRel sequences alone only weakly activated transcription. In addition, the wild-type vRel protein (lacking LexA sequences) repressed transcription from reporter plasmids containing NF-kappa B target sequences; nontransforming vRel mutants did not repress transcription from these plasmids. Our results suggest that vRel transforms cells by interfering with transcriptional activation by cellular Rel proteins.

Animals↗
PubMed1994

Transcriptional suppression of the human T-cell leukemia virus type I long terminal repeat occurs by an unconventional interaction of a CREB factor with the R region.

To analyze regulation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR), cell lines were generated from LTR-tax x LTR-beta-galactosidase (beta-Gal) doubly transgenic mouse fibroblastic tumors. The HTLV-I LTR directs expression of both the tax and lacZ genes, and Tax up-modulates both promoters in primary cells. However, once cells were transformed by tax, beta-Gal but not tax expression was suppressed. Supertransformation of these cells with v-src suppressed both beta-Gal and tax expression. This suppression was reversed by treatment with the tyrosine kinase inhibitor herbimycin A or protein kinase A inhibitor H8. Electrophoretic mobility shift assays demonstrated augmented binding in the R but not U3 region. This binding was competitively inhibited by a high-affinity CREB oligodeoxynucleotide and super-shifted with a specific CREB antibody. Treatment of cells with the cyclic AMP analog dibutyryl cyclic AMP also transiently increased the R region binding dramatically. In vitro DNase I footprint analysis identified a protein-binding sequence in the R region which corresponded with suppression. However, this target sequence lacked a conventional CREB-binding site. A 70.5-kDa DNA-binding protein was partially purified by affinity chromatography, along with a 49-kDa protein which reacted with CREB-specific sera. These data demonstrate that HTLV-I LTR suppression is associated with CREB factor binding in the R region, probably by direct interaction with a 70.5-kDa protein, and provide a novel mechanism for maintenance of viral latency.

Animals↗
PubMed2000

Postcleavage sequence specificity in V(D)J recombination.

Unintended DNA rearrangements in a differentiating lymphocyte can have severe, oncogenic consequences, but the mechanisms for avoiding pathogenic outcomes in V(D)J recombination are not well understood. The first level at which fidelity is instituted is in discrimination by the recombination proteins between authentic and inauthentic recombination signal sequences. Nevertheless, this discrimination is not absolute and cannot fully eliminate targeting errors. To learn more about the basis of specificity during V(D)J recombination, we have investigated whether it is possible for the recombination machinery to detect an inaccurately targeted sequence subsequent to cleavage. These studies indicate that even postcleavage steps in V(D)J recombination are sequence specific and that noncanonical sequences will not efficiently support the resolution of recombination intermediates in vivo. Accordingly, interventions after a mistargeting event conceivably occur at a late stage in the joining process and the likelihood may well be crucial to enforcing fidelity during antigen receptor gene rearrangement.

Animals↗
PubMed2002

The quality of DNA recovered from the archival tissues of atomic bomb survivors is good enough for the single nucleotide polymorphism analysis in spite of the decade-long preservation in formalin.

It is well known that the yield of DNA recovered form tissues preserved in formalin is inversely proportional to the stored duration. How is the quality? We tested the quality of DNA from archival tissues of atomic-bomb survivors stored in formalin for decades with the parameters of gene amplification efficiency by a polymerase chain reaction. All of the DNA extracted from the tissues preserved in formalin for 30 years amplified the 54- and 61-base pairs of the DNA fragments successfully. The direct sequencing of the PCR products confirmed the accurate amplification of the target sequence. A further trial to amplify the longer sequence of 111 base pairs succeeded in 20% of the samples tested. From these results, we propose a new utility of archival samples for the analysis of single nucleotide sequence polymorphism of genes, no matter how long the samples have been preserved in formalin.

Animals↗
PubMed1991

An improved technique for the in situ detection of DNA after polymerase chain reaction amplification.

In situ detection of polymerase chain reaction (PCR)-amplified DNA in cell and tissue preparations previously required 5 to 7 primer pairs designed to generate a long (greater than 1,000 base pair) product. The authors describe a nonisotopic PCR in situ technique, employing a single primer pair and target sequences as short as 115 base pairs, that can detect one target molecule per cell. The essential procedural change is to withhold the DNA polymerase or primers until the reaction temperature approaches 80 degrees C. The Hot-Start method greatly increased amplification specificity which, more than product size, appears to determine success of in situ PCR. The marked improvement in specificity may permit target detection by direct incorporation of labeled nucleotides.

DNA, Viral↗
PubMed1990

Properties of the maize transposable element Activator in transgenic tobacco plants: a versatile inter-species genetic tool.

The maize controlling element Activator (Ac) transposes autonomously from an integrated T-DNA vector to new sites in the genomes of tobacco and other heterologous plant species. Here we demonstrate that critical functions required for transposition of Ac in maize are conserved in tobacco and that Ac transposes at high frequency for at least five generations. Ac structure and terminal sequences are conserved upon transposition and a characteristic 8-bp duplication of target sequences is generated upon integration. Ac remains unmethylated, transcriptionally active, and capable to trans-activate transposition of the nonautonomous Dissociation (Ds) element throughout several generations. In tobacco, as in maize, Ac transposes adjacent to low copy or unique DNA, and transcriptional analysis of unique target DNA provides evidence that an Ac element transposed into a gene. In maize, increasing copies of Ac delay the timing and reduce the frequency of early transposition of Ac and Ds. In tobacco, increasing copies of Ac correlate with an increased frequency of Ds trans-activation. These data firmly establish that the Ac and Ds transposable elements are versatile genetic tools well suited for use as insertional mutagens and demonstrate that thorough investigation of mechanism and regulation of transposition is facilitated in heterologous settings.

Base Sequence↗
PubMed1999

BUNDLE SHEATH DEFECTIVE2, a novel protein required for post-translational regulation of the rbcL gene of maize.

The Bundle sheath defective2 (Bsd2) gene is required for ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Using a Mutator transposable element as a molecular probe, we identified a tightly linked restriction fragment length polymorphism that cosegregated with the bsd2-conferred phenotype. This fragment was cloned, and sequences flanking the Mutator insertion were used to screen a maize leaf cDNA library. Using a full-length cDNA clone isolated in this screen, we show that an abundant 0.6-kb transcript could be detected in wild-type plants but not in bsd2-m1 plants. This 0.6-kb transcript accumulated to low levels in plants carrying an allele derived from bsd2-m1 that conditions a less severe mutant phenotype. Taken together, these data strongly suggest that we have cloned the Bsd2 gene. Sequence analysis of the full-length cDNA clone revealed a chloroplast targeting sequence and a region of homology shared between BSD2 and the DnaJ class of molecular chaperones. This region of homology is limited to a cysteine-rich Zn binding domain in DnaJ believed to play a role in protein-protein interactions. We show that BSD2 is targeted to the chloroplast but is not involved in general photosynthetic complex assembly or protein import. In bsd2 mutants, we could not detect the Rubisco protein, but the chloroplast-encoded Rubisco large subunit transcript (rbcL) was abundant and associated with polysomes in both bundle sheath and mesophyll cells. By characterizing Bsd2 expression patterns and analyzing the bsd2-conferred phenotype, we propose a model for BSD2 in the post-translational regulation of rbcL in maize.

Amino Acid Sequence↗