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At least 1,711 records · Page 95Linked to original sources
PubMed1991

Construction, arraying, and high-density screening of large insert libraries of human chromosomes X and 21: their potential use as reference libraries.

We have constructed cosmid libraries from flow-sorted human chromosomes X and 21, each of which contains greater than 30 genome equivalents, and have developed systems allowing permanent storage of primary clones, easy screening of libraries in high-density filter formats, and the simultaneous generation of fingerprinting and mapping data on the same set of cosmid clones. Clones are picked into microtiter plate wells and stored at -70 degrees C. A semiautomatic robot system allows the generation of filter replicas containing up to 10,000 clones per membrane. Sets of membranes containing 15-20 chromosome equivalents of both chromosomes will be used for the construction of ordered clone libraries by hybridization fingerprinting protocols. In addition, multiple sets of two membranes containing 4 chromosome equivalents of the human X chromosome, and one membrane containing 3 chromosome equivalents of chromosome 21, have been distributed to other interested laboratories as part of a system of reference libraries. This system allows other groups easy access to the clones and offers an efficient protocol to combine results generated in different laboratories using these libraries. Here we describe the construction of the libraries and demonstrate the use of high-density screening filters in oligonucleotide probe hybridizations and the isolation of cosmids by hybridization with probes from the X chromosome.

Chromosomes, Human, Pair 21↗
PubMed2000

Use of restriction fragment length polymorphism analysis to differentiate strains of psychrophilic and psychrotropic clostridia associated with blown pack' spoilage of vacuum-packed meats.

Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP). Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing. DNA-RFLP resolved 19 of the 22 meat strains into 11 groups. Three meat strains were untypable using this method. All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes. With PCR-RFLP, the 22 meat strains produced eight distinct genotypes. 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence. PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used. Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species. Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms. The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir.

Clostridium↗
PubMed2006

Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells.

OBJECTIVE: To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA). METHODS: Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques. RESULTS: The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression. Furthermore, the expression of DR-3 protein was down-modulated in association with methylation of the promoter CpG island in RA synovial cells. CONCLUSION: The CpG island in the DR3 gene promoter was specifically methylated to down-modulate the expression of DR-3 protein in rheumatoid synovial cells, which may provide resistance to apoptosis in RA synovial cells.

Adult↗
PubMed2003

A two-dimensional proteome map of Shigella flexneri.

Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans. In this study, extracellular proteins from the culture medium and whole cell proteins in cellular extracts of S. flexneri 2a strain 2457T were examined by two-dimensional (2-D) gel electrophoresis using immobilized pH gradient (IPG) technology. Proteins were identified by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with Mascot search program. In total, among the 488 proteins spots processed, 388 proteins were identified. The identified proteins represented 169 genes. By comparing results of Mascot search against databases of Escherichia coli and genomes of S. flexneri 2a, one S. flexneri-specific protein was identified and one possible gap was found in 2457T genome sequences. Although this proteome map is still incomplete, it is already a useful reference for future studies involving pathogenicity, vaccine development, design of novel antibacterial drugs, etc. Proteome maps and a table of all identified proteins are available on the internet at www.proteomics.com.cn.

Bacterial Proteins↗
PubMed1989

Bovine leukemia virus trans-activator p38tax activates heterologous promoters with a common sequence known as a cAMP-responsive element or the binding site of a cellular transcription factor ATF.

The genome of bovine leukemia virus (BLV) encodes a transcriptional trans-activator p38tax (also referred to as pXBL-I) which amplifies the virus gene expression driven by its long terminal repeat (LTR). It was proposed that activation of cellular gene expression by p38tax might be involved in the mechanism of B-cell transformation caused in vivo by BLV infection. Here, we report that the U3 region of BLV LTR contains multiple regulatory elements responsive to p38tax. A core element composing the p38tax-inducible U3 structure is suggested to be a heptanucleotide motif of 5'TGACGTCA3', the consensus sequence proposed for a cAMP-responsive element (CRE) and for the binding sites of a cellular transcription factor (ATF). Adenovirus-5 E3 and E4, c-fos and somatostatin regulatory regions containing CRE/ATF-element exhibited responsiveness to p38tax in a chloramphenicol acetyltransferase transient expression assay. These suggest that in BLV-infected cells, cellular gene expression might be induced abnormally by the virus trans-activator through ATF or ATF-like factors.

Activating Transcription Factors↗
PubMed2006

Metal ions and carcinogenesis.

Metals are essential for the normal functioning of living organisms. Their uses in biological systems are varied, but are frequently associated with sites of critical protein function, such as zinc finger motifs and electron or oxygen carriers. These functions only require essential metals in minute amounts, hence they are termed trace metals. Other metals are, however, less beneficial, owing to their ability to promote a wide variety of deleterious health effects, including cancer. Metals such as arsenic, for example, can produce a variety of diseases ranging from keratosis of the palms and feet to cancers in multiple target organs. The nature and type of metal-induced pathologies appear to be dependent on the concentration, speciation, and length of exposure. Unfortunately, human contact with metals is an inescapable consequence of human life, with exposures occurring from both occupational and environmental sources. A uniform mechanism of action for all harmful metals is unlikely, if not implausible, given the diverse chemical properties of each metal. In this chapter we will review the mechanisms of carcinogenesis of arsenic, cadmium, chromium, and nickel, the four known carcinogenic metals that are best understood. The key areas of speciation, bioavailability, and mechanisms of action are discussed with particular reference to the role of metals in alteration of gene expression and maintenance of genomic integrity.

Animals↗
PubMed1993

Consequences of unique double-stranded breaks in yeast chromosomes: death or homozygosis.

We have developed a system in which a unique double-stranded break (DSB) can be introduced into a yeast chromosome during mitotic growth. The recognition site for the endonuclease I-SceI was inserted at different places in the yeast genome in haploid and diploid cells expressing this endonuclease. Induction of the break in haploids results in cell death if no intact copy of the cleaved region is present in the cell. If such a copy is provided on a plasmid, as an ectopic gene duplication, or on a homologous chromosome, the break can be repaired. Repair results in two identical copies in the genome of the locus which has been cut. We call this phenomenon homozygotization by reference to diploids heterozygous for the cut site in which repair leads to homozygosis at this site. We have compared the efficiencies of repair in the various topological situations examined, and conclude that some mechanism must search for regions of homology to both sides of the DSB and that repair is successful only if the homologies are provided by the same template molecule.

Chromosomes, Fungal↗
PubMed2005

Looking into flowering time in almond (Prunus dulcis (Mill) D. A. Webb): the candidate gene approach.

Blooming time is one of the most important agronomic traits in almond. Biochemical and molecular events underlying flowering regulation must be understood before methods to stimulate late flowering can be developed. Attempts to elucidate the genetic control of this process have led to the identification of a major gene (Lb) and quantitative trait loci (QTLs) linked to observed phenotypic differences, but although this gene and these QTLs have been placed on the Prunus reference genetic map, their sequences and specific functions remain unknown. The aim of our investigation was to associate these loci with known genes using a candidate gene approach. Two almond cDNAs and eight Prunus expressed sequence tags were selected as candidate genes (CGs) since their sequences were highly identical to those of flowering regulatory genes characterized in other species. The CGs were amplified from both parental lines of the mapping population using specific primers. Sequence comparison revealed DNA polymorphisms between the parental lines, mainly of the single nucleotide type. Polymorphisms were used to develop co-dominant cleaved amplified polymorphic sequence markers or length polymorphisms based on insertion/deletion events for mapping the candidate genes on the Prunus reference map. Ten candidate genes were assigned to six linkage groups in the Prunus genome. The positions of two of these were compatible with the regions where two QTLs for blooming time were detected. One additional candidate was localized close to the position of the Evergrowing gene, which determines a non-deciduous behaviour in peach.

Chromosome Mapping↗
PubMed2004

Mitochondria in health and disease: perspectives on a new mitochondrial biology.

The integrity of mitochondrial function is fundamental to cell life. It follows that disturbances of mitochondrial function will lead to disruption of cell function, expressed as disease or even death. In this review, I consider recent developments in our knowledge of basic aspects of mitochondrial biology as an essential step in developing our understanding of the contributions of mitochondria to disease. The identification of novel mechanisms that govern mitochondrial biogenesis and replication, and the delicately poised signalling pathways that coordinate the mitochondrial and nuclear genomes are discussed. As fluorescence imaging has made the study of mitochondrial function within cells accessible, the application of that technology to the exploration of mitochondrial bioenergetics is reviewed. Mitochondrial calcium uptake plays a major role in influencing cell signalling and in the regulation of mitochondrial function, while excessive mitochondrial calcium accumulation has been extensively implicated in disease. Mitochondria are major producers of free radical species, possibly also of nitric oxide, and are also major targets of oxidative damage. Mechanisms of mitochondrial radical generation, targets of oxidative injury and the potential role of uncoupling proteins as regulators of radical generation are discussed. The role of mitochondria in apoptotic and necrotic cell death is seminal and is briefly reviewed. This background leads to a discussion of ways in which these processes combine to cause illness in the neurodegenerative diseases and in cardiac reperfusion injury. The demands of mitochondria and their complex integration into cell biology extends far beyond the provision of ATP, prompting a radical change in our perception of mitochondria and placing these organelles centre stage in many aspects of cell biology and medicine.

Animals↗
PubMed2005

Recombinant expression, purification, and functional analysis of two novel cystatins from sugarcane (Saccharum officinarum).

Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins.

Chromatography, Affinity↗
PubMed2002

Beta-catenin mutations correlate with over expression of C-myc and cyclin D1 Genes in bladder cancer.

PURPOSE: We hypothesized that over expression of c-myc and cyclin D1 genes is transcriptionally activated by beta-catenin mutation independent of gene amplification in bladder cancer. To test this hypothesis we investigated the relationship of beta-catenin mutation to c-myc and cyclin D1 mRNA with special reference to the changes in copy number of the 2 genes. MATERIALS AND METHODS: Genomic DNA and total RNA were extracted from 59 bladder cancer specimens and from 31 histologically normal specimens of bladder mucosa. We performed beta-catenin deletion screening by polymerase chain reaction (PCR) using primers spanning exons 3 (including the glycogen synthase kinase-3beta consensus motif), 5 and 6. Mutational changes in beta-catenin in exons 3, 5 and 6 were detected by each PCR-single strand conformational polymorphism analysis followed by direct DNA sequencing. mRNA expression and copy numbers of c-myc and cyclin D1 were determined by semiquantitative reverse transcriptase-PCR and competitive genomic PCR. RESULTS: Missense mutations of beta-catenin found in grade 3 bladder cancer were involved in the consensus motif of glycogen synthase kinase-3beta in exon 3. These cancers showed strong intracellular accumulation of beta-catenin and intense expression of c-myc and cyclin D1 mRNA compared with samples lacking the beta-catenin mutation. When grade 3 cancers were compared, expression levels of c-myc and cyclin D1 mRNA were still higher in those with versus without the beta-catenin mutation. In bladder cancers with beta-catenin mutations copy numbers of the c-myc and cyclin D1 genes did not amplify. CONCLUSIONS: Bladder cancer harboring a beta-catenin mutation may represent aggressive biological behavior with enhanced proliferating activity. These findings are important for understanding the role of beta-catenin mutation in the pathogenesis of bladder cancer.

Carcinoma, Transitional Cell↗
PubMed2001

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle.

The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5' domain. This creates the binding site for the Alu RNA 3' domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3' domain folding back by 180 degrees onto the 5' domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived from Alu elements in genomic DNA.

Alu Elements↗
PubMed2005

Validation of a tomato-specific gene, LAT52, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic tomatoes.

Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR) detection methods of transgenic tomatoes, one tomato (Lycopersicon esculentum) species specific gene, LAT52, was selected and validated as suitable for using as an endogenous reference gene in transgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with 16 different tomato varieties, and identical amplified products or fluorescent signals were obtained with all of them. No amplified products and fluorescent signals were observed when DNA samples from 20 different plants such as soybean, maize, rapeseed, rice, and Arabidopsis thaliana were used as templates. These results demonstrated that the amplified LAT52 DNA sequence was specific for tomato. Furthermore, results of Southern blot showed that the LAT52 gene was a single-copy gene in the different tested tomato cultivars. In qualitative and quantitative PCR analysis, the detection sensitivities were 0.05 and 0.005 ng of tomato genomic DNA, respectively. In addition, two real-time assays employing this gene as an endogenous reference gene were established, one for the quantification of processed food samples derived from nontransgenic tomatoes that contained degraded target DNA and the other for the quantification of the junction region of CaMV35s promoter and the anti-sense ethylene-forming enzyme (EFE) gene in transgenic tomato Huafan No. 1 samples. All of these results indicated that the LAT52 gene could be successfully used as a tomato endogenous reference gene in practical qualitative and quantitative detection of transgenic tomatoes, even for some processed foods derived from transgenic and nontransgenic tomatoes.

DNA, Plant↗
PubMed2004

Development and comparison of four real-time polymerase chain reaction systems for specific detection and quantification of Zea mays L.

Four real-time polymerase chain reaction systems aiming at the specific detection and quantification of maize DNA are described. They have been developed in four independent laboratories targeting different maize sequences, i.e., alcohol dehydrogenase (Adh1), high mobility group protein (hmga), invertase A (ivr1), and zein, respectively. They were all fully specific, showing a very similar quantification accuracy along a number of distantly related maize cultivars and being either single or low copy number genes. They were highly sensitive and exhibited limits of quantification below 100 maize genomic copies. In consequence, they are considered suitable for use as maize specific endogenous reference genes in DNA analyses, including GMO quantitative tests.

Alcohol Dehydrogenase↗
PubMed2006

The impact of phage lambda: from restriction to recombineering.

Experiments using phage lambda provided early insights into important molecular mechanisms, including genetic recombination and the control of gene expression. Before recombinant DNA technology, the use of lambda, most particularly lambda transducing phages, illustrated the importance of cloning bacterial genes, already providing some insight into how to use cloned genes to advantage. Subsequently, lambda made significant contributions to recombinant DNA technology, including the early generation of genomic and cDNA libraries. More recently, lambda genes associated with recombination have enabled techniques referred to as 'recombineering' to be developed. These techniques permit the refined manipulation, including mutation, of foreign genes in Escherichia coli and their subsequent return to the donor organism.

Bacteriophage lambda↗
PubMed2002

Mapping microsatellite markers identified in porcine EST sequences.

A sequence search of swine expressed sequence tags (EST) data in GenBank identified over 100 sequence files which contained a microsatellite repeat or simple sequence repeat (SSR). Most of these repeat motifs were dinucleotide (CA/GT) repeats; however, a number of tri-, tetra-, penta- and hexa-nucleotide repeats were also detected. An initial assessment of six dinucleotide and 14 higher-order repeat markers indicated that only dinucleotide markers yielded a sufficient number of informative markers (100% vs. 14% for dinucleotide and higher order repeats, respectively). Primers were designed for an additional 50 di- and one tri-nucleotide SSRs. Overall, 42 markers were polymorphic in the US Meat Animal Research Center (MARC) reference population, 17 markers were uninformative and 12 primer pairs failed to satisfactorily amplify genomic DNA. A comparison of di-nucleotide repeat vs. markers with repeat motifs of three to six bases demonstrated that 72% of dinucleotide markers were informative relative to only 7% of other repeat motifs. The difference was the result of a much higher percentage of monomorphic markers in the three to six base repeat motif markers than in the dinucleotide markers (64% vs. 14%). Either higher order repeat motifs are less polymorphic in the porcine genome or our selection criteria for repeat length of more than 17 contiguous bases was too low. The mapped microsatellite markers add to the porcine genetic map and provide valuable links between the porcine and human genome.

Alleles↗
PubMed2000

Disruption of cell cycle control by human papillomaviruses with special reference to cervical carcinoma.

Human papillomaviruses (HPVs) play a major role in neoplastic transformation of squamous epithelial cells. The viral genome is small in size and only encodes a limited number of proteins, so one of the major functions of the viral proteins is to modulate the function of key cellular proteins involved in cell cycle control and DNA replication. During this process important host cell cycle checkpoints are lost which may lead to the accumulation of genetic abnormalities and eventual malignant transformation. This review briefly describes the normal cell cycle and also the mechanisms by which HPVs interfere with cell cycle control both as part of their productive life cycle and in the process of neoplastic transformation.

Journal Article↗
PubMed2004

Genetic dissection of human stature in a large sample of multiplex pedigrees.

Recently, we reported a whole genome scan on a sample of 630 Caucasian subjects from 53 human pedigrees. Several genomic regions were suggested to be linked to height. In an attempt to confirm the identified genomic regions, as well as to identify new genomic regions linked to height, we conducted a whole genome linkage study on an extended sample of 1,816 subjects from 79 pedigrees, which includes the 53 pedigrees containing the original 630 subjects from our previous whole genome study and an additional 128 new subjects, and 26 further pedigrees containing 1,058 subjects. Several regions achieved suggestive linkage signals, such as 9q22.32 [MLS (multipoint LOD score) = 2.74], 9q34.3 [MLS = 2.66], Xq24 [two-point LOD score = 2.64 at the marker DXS8067], and 7p14.2 [MLS = 2.05]. The importance of the above regions is supported either by other whole genome studies or by candidate genes within these regions relevant to linear growth or pathogenesis of short stature. In addition, this study has tentatively confirmed the Xq24 region's linkage to height, as this region was also detected in the previous whole genome study. To date, our study has achieved the largest sample size in the field of genetic linkage studies of human height. Together with the findings of other studies, the current study has further delineated the genetic basis of human stature.

Adult↗