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PubMed1982

Diverse mechanisms in the generation of human beta-tubulin pseudogenes.

The sequence of two human beta-tubulin pseudogenes is described. One contains an intervening sequence but lacks sequences encoding the 55 N-terminal amino acids of the polypeptide chain. A second has no introns but has a polyadenylate signal and an oligoadenylate tract at its 3' end, and it is flanked by a short direct repeat. These sequences have arisen by different mechanisms, including one that probably involves reverse transcription of a processed messenger RNA and reintegration of the complementary DNA copy into the genome.

Base Sequence↗
PubMed2003

Transferable vanB2 Tn5382-containing elements in fecal streptococcal strains from veal calves.

Three vancomycin-resistant veal calf fecal streptococci, identified as Streptococcus gallolyticus (n = 2) and Streptococcus lutetiensis, were shown to harbor vanB2 Tn5382-like elements earlier described in enterococci. One S. gallolyticus strain had a 1,495-bp IS256-related element inserted in vanS(B). The vanB2 Tn5382 element present in the plasmid-free S. lutetiensis strain was transferable to Enterococcus faecium BM4105-RF, Enterococcus faecalis JH2-2, and its recombination-deficient derivative, UV202. The transfer frequencies were comparable between recipient strains (from 1 x 10(-7) to 7 x 10(-6)). All transconjugants acquired a vanB-containing chromosomal insert of approximately 100 kb, apparently by site-specific integration. Secondary transconjugants were not observed in intraspecies retransfer experiments. These observations are consistent with a conjugative, selftransmissible, integrative element that might be involved in the interspecies spread of vanB2 resistance determinants. Two JH2-2-derived transconjugants had also gained additional copies of large vanB-containing chromosomal fragments, a process that involves unexplained mechanisms that seems to require functional host cell-dependent recombination mechanisms.

Animals↗
PubMed1981

Efficiency of processing of viral RNA during the early and late phases of productive infection by polyoma virus.

The efficiency of processing of polyoma viral RNA and of its export from nucleus to cytoplasm was measured in primary mouse kidney cells by comparing the initial rates of incorporation of [3H]uridine into cytoplasmic and nuclear viral RNA. Appropriate methods of cell fractionation were chosen to maximize yields of cytoplasmic RNA and to minimize leakage of nuclear RNA. Incorporation of [3H]uridine into cellular 4S RNA in the cytoplasm was followed to monitor pool equilibration and maintenance of an excess of radioactive precursor throughout the experimental period. During the early phase of infection (9 to 11 h, in the presence of 5-fluorodeoxyuridine), viral RNA was rapidly and efficiently exported from nucleus to cytoplasm. Viral RNA appeared in the cytoplasm within 6 min of its synthesis, greater than half of the viral RNA synthesized in the nucleus was exported to the cytoplasm. In contrast, during the late phase of infection (28 to 30 h), viral RNA was exported more slowly, appearing in the cytoplasm 12 to 20 min after its synthesis, and much less efficiently-only 5% of late nuclear transcripts was exported. The poor efficiency of processing of late viral RNA may be, in part, a result of (i) the presence in nuclear transcripts of non-mRNA sequences which are removed during processing; (ii) the presence in nuclear transcripts of multiple copies of mRNA sequences, only one of which is incorporated into mature mRNA; and (iii) inefficient polyadenylation of viral nuclear RNA.

Animals↗
PubMed1990

Spacer promoters are orientation-dependent activators of pre-rRNA transcription in Drosophila melanogaster.

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggest that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.

Animals↗
PubMed1996

Circular oligonucleotides: new concepts in oligonucleotide design.

Recent progress in the synthesis and properties of circular oligonucleotides as ligands for DNA and RNA and as templates for polymerase enzymes is described. Small synthetic circular DNAs, RNAs, and chimeric analogues ranging from 28 to 74 nucleotides in size have been synthesized with the use of nonenzymatic ligation strategy. Some of these were designed to undergo triplex formation with single-stranded DNA and RNA targets, and many bind with affinities and sequence selectivities considerably greater than those seen for linear oligonucleotides. Design strategies and modes of binding are discussed in the light of possible use of such molecules as hybridization probes, molecular diagnostics, and sequence-specific inhibitors of gene expression. Small circular oligonucleotides have also been shown to act as unusually efficient templates for DNA and RNA polymerases, which produce long, repeating copies of the circular sequence by a rolling circle process.

DNA↗
PubMed2007

Digital superresolution and the generalized sampling theorem.

The technique of reconstructing a higher-resolution (HR) image of size MLxML by digitally processing LxL subpixel-shifted lower-resolution (LR) copies of it, each of size MxM, has now become well established. This particular digital superresolution problem is analyzed from the standpoint of the generalized sampling theorem. It is shown both theoretically and by computer simulation that the choice of regularly spaced subpixel shifts for the LR images tends to maximize the robustness and minimize the error of reconstruction of the HR image. In practice, since subpixel-level control of LR image shifts may be nearly impossible to achieve, however, a more likely scenario, which is also discussed, is one involving random subpixel shifts. It is shown that without reasonably tight bounds on the range of random shifts, the reconstruction is likely to fail in the presence of even small amounts of noise unless either reliable prior information or additional data are available.

Journal Article↗
PubMed2000

Comparing the performance of mammographic enhancement algorithms: a preference study.

OBJECTIVE: The objective of this study was to compare the performance of four image enhancement algorithms on secondarily digitized (i.e., digitized from film) mammograms containing masses and microcalcifications of known pathology in a clinical soft-copy display setting. MATERIALS AND METHODS: Four different image processing algorithms (adaptive unsharp masking, contrast-limited adaptive histogram equalization, adaptive neighborhood contrast enhancement, and wavelet-based enhancement) were applied to one image of secondarily digitized mammograms of forty cases (10 each of benign and malignant masses and 10 each of benign and malignant microcalcifications). The four enhanced images and the one unenhanced image were displayed randomly across three high-resolution monitors. Four expert mammographers ranked the unenhanced and the four enhanced images from 1 (best) to 5 (worst). RESULTS: For microcalcifications, the adaptive neighborhood contrast enhancement algorithm was the most preferred in 49% of the interpretations, the wavelet-based enhancement in 28%, and the unenhanced image in 13%. For masses, the unenhanced image was the most preferred in 58% of cases, followed by the unsharp masking algorithm (28%). CONCLUSION: Appropriate image enhancement improves the visibility of microcalcifications. Among the different algorithms, the adaptive neighborhood contrast enhancement algorithm was preferred most often. For masses, no significant improvement was observed with any of these image processing approaches compared with the unenhanced image. Different image processing approaches may need to be used, depending on the type of lesion. This study has implications for the practice of digital mammography.

Aged↗
PubMed2001

The detection, cloning, and characterisation of WIS 2-1A retrotransposon-like sequences in Triticum aestivum L. and xTriticosecale Wittmack and an examination of their evolution in related Triticeae.

Retrotransposons and other mobile elements are major components of the repeated DNA fraction in higher-plant genomes. They have undoubtedly played an important role in higher plant genome evolution. The present work details the detection and characterisation of a WIS 2-1A related sequence in direct wheat relatives, and discusses the prevalence and evolution of its copy number in their genomes. An increase in copy number is detected when following the natural hybridisation processes that gave rise to bread and durum wheats. However, the opposite is observed in the development of triticale, a synthetic hybrid.

Base Sequence↗
PubMed1992

Rapid high resolution western blotting: from gel to image in a single day.

A streamlined protocol is described that allows high sensitivity antigen detection by Western blotting in a single day. The choice of membrane blotting matrix, as well as blocking reagents, has been optimized in order to allow rapid development of the blot with chemiluminescent reagents. The entire process, from gel to blot to a permanent, hard copy image on x-ray film, can be accomplished within six hours.

Animals↗
PubMed2005

Parallelisation of the blast algorithm.

Retrieving homologous DNA and protein sequences from existing databases is a fundamental routine in bioinformatics research. Programs of the NCBI BLAST family are widely used for this purpose. We evaluated paraBLAST, a parallelised version of the NCBI BLAST algorithm, using a Message Passing Interface (MPI) on a multi-node compute cluster. Here, we propose static and dynamic database-partitioning schemes based on the availability of the cluster. We evaluated the application of the algorithm in querying nucleotide sequences against a large-scale sequence database with different numbers of database partitions, and hence, different numbers of CPUs. Since the program's tasks are performed independently of each other, each available CPU can run its own copy of BLAST queries, resulting in reduced interference between processes and leading to a highly scalable solution.

Algorithms↗
PubMed1990

Two precursors of thyrotropin-releasing hormone from skin of Xenopus laevis. Each contains seven copies of the end product.

From the skin of Xenopus laevis, the cloned cDNAs of two precursors of thyrotropin-releasing hormone have been isolated and sequenced. These encode almost identical precursor polypeptides each containing seven copies of the end product flanked by typical prohormones processing signals. Northern blot analysis has corroborated the existence of two thyrotropin-releasing hormone precursor mRNAs of similar size as the cloned cDNA and demonstrated the existence of a third smaller species as well. Two or more mRNAs for this precursor are also present in the brain and eyes of X. laevis adults and tadpoles.

Amino Acid Sequence↗
PubMed1989

Production of human cells expressing individual transferred HLA-A,-B,-C genes using an HLA-A,-B,-C null human cell line.

We detail in this report the characterization of a human B-lymphoblastoid cell line, .221, that does not express endogenous HLA-A, HLA-B, or HLA-C class I Ag due to gamma-ray-induced mutations in the HLA complex. Mutant .221 is characterized by: 1) complete absence of HLA-A,-B,-C mRNA transcripts and alpha-chains, and 2) intracellular expression of two non-A,-B,-C class I alpha-chains with an abundance less than or equal to 1% of normal HLA-A,-B,-C expression on similar cells. However, transferred HLA-A, HLA-B, and HLA-C genomic genes are expressed as cell surface Ag in amounts similar to expression of the same endogenous genes in human B-lymphoblastoid cells. The amount of class I transcript produced from transferred class I genes is roughly proportional to the number of gene copies but, in every case studied, post-transcriptional processes limited cell surface Ag expressions to amounts approximately normal for the cell type. The ability of mutant .221 to express quantitatively normal amounts of transferred class I genes suggests that: 1) it can serve as a recipient for, and then express, any cloned HLA-A,-B, or -C gene that would normally be expressible in human B-lymphoblastoid cells; 2) the absence of a background of HLA-A,-B,-C Ag permits its use for studying the expression of normal non-A,-B,-C class I genes and of class I genes that have mutations; 3) mutant .221 can be used to create human cells that express on their surfaces just one defined class I Ag encoded by a transferred class I gene.

B-Lymphocytes↗
PubMedDate not supplied

New trends in blood purification.

Considering the different methods of blood purification, designed and developed during the last ten years, some common trends can be pointed out (Table 4). The uniformity of treatment during the first 30 years has been substituted by a variety of new methods, allowing a specific attribution to different underlying diseases. Whereas haemodialysis, haemofiltration and intermittent peritoneal dialysis exhibit a relatively broad spectrum of indications, CAPD, haemoperfusion and plasmapheresis are restricted to a reduced number of underlying diseases. Certain diseases, until a few years ago regarded as a contra-indication for blood purification treatment, are today considered to be specific indications for newly developed methods of treatment, for example diabetic nephropathy for CAPD and vascular instability for haemofiltration or its modifications. Apart from this individualization an important trend of modern artificial kidney design has been the attempt to miniaturize the devices in order to facilitate their application and to encourage self-treatment. This trend could result in a concentration of the patients on self-care and a substantial reduction of treatment costs. Moreover, a miniaturized artificial organ could reduce many immunological problems, which today result from transplantation of natural kidney. Imitation of the natural kidney has been an important goal of many investigators during the last years. Promising approaches have been attempted by the development of haemofiltration, imitating the process of glomerular filtration, or continuous ambulatory peritoneal dialysis, copying the continuously excreting natural kidney. In his assay 'De Urina', Homer Smith says, 'This liquid, which commonly inspires men only with contempt and disgust, which is generally ranked amongst vile and repulsive matters has become, in the hands of the chemists, a source of important discoveries'. Today the inspiration of the chemists has been replaced by the unceasing endeavours of technicians to create an artificial organ which produces a urine of similar qualities.

Blood↗
PubMed1995

Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase.

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.

Animals↗
PubMed1987

Transcription of multiple copies of the yeast GAL7 gene is limited by specific factors in addition to GAL4.

High levels of the GAL7 gene in the yeast cell appear to titrate regulatory factors and to impair transcription of related sequences. To investigate the role that the GAL regulatory factors GAL4 and GAL80 have in this process we have compared the accumulation of mRNA transcribed from single-copy (plasmid-borne GAL7 and chromosomal GAL10) and high-copy (plasmid-borne GAL7) genes in several GAL regulatory mutants. Our results show that functional GAL4 gene product is required for induction of transcription from the single- and high-copy genes. In a strain containing the GAL4 gene fused to the high expression ADH1 promoter, glucose can replace galactose to induce high levels of transcription of GAL7 and GAL10 genes, although the kinetics of accumulation induced by the two sugars are distinctly different. In the presence of high levels of GAL4, maximum accumulation of mRNA from single and high copy genes is elevated two-fold; disruption of the gal80 gene in combination with high levels of GAL4 results in a further two-fold increase in transcription. In this genetic background, galactose-induced transcription of the high copy GAL7 gene results in a greater than 50-fold increase in the levels of GAL7 mRNA, representing 30%-50% of the total cellular mRNA. Our results are consistent with a cooperative effect of saturation of multiple GAL4 DNA binding sites and with a limiting factor, in addition to GAL4, that is required for transcription of the GAL genes.

Alcohol Dehydrogenase↗
PubMed2000

Differential localisation of an Eimeria tenella aspartyl proteinase during the infection process.

Aspartyl proteinases are essential for the survival of many pathogens. A single copy gene in species of Eimeria encodes an aspartyl proteinase, which we propose should be called eimepsin to conform to the commonly used names of this family of proteinases. An epitope map, constructed using BIAcore technology, confirmed the specificity of 14 mAbs for eimepsin and defined four antigenic domains, which were conserved between native and recombinant forms of eimepsin. In resting sporozoites, mAb defining antigenic domains I and II stained the refractile body organelles, whereas those defining antigenic domains III and IV stained cytoplasmic granules. During host cell invasion, the staining patterns of mAb defining antigenic domains I, III and IV changed dramatically with the apical tips of invading sporozoites becoming strongly stained. In contrast, mAb defining antigenic domain II continued to stain only the refractile bodies. During early schizogony, mAb to all four domains stained the single fused refractile body, but when schizonts matured, mAb to antigenic domains I, III and IV stained the apical tip of merozoites whereas those to antigenic domain II continued to follow the developmental redistribution of the refractile body. Irrespective of localisation, mAb to three antigenic domains recognised a polypeptide of 49 kDa, which from N-terminal sequencing corresponds to a mature form of eimepsin. Staining with fluorescent pepstatin localised a mature, active form of eimepsin to the refractile bodies of the sporozoite, schizont and first generation merozoite. It remains to be determined whether eimepsin has a catalytic function within the refractile body or whether the activated enzyme is stored in the refractile body so that it can be rapidly redistributed to the apical tip during parasite invasion.

Animals↗
PubMed2006

Detection and quantitation of colored deposit-forming Meiothermus spp. in paper industry processes and end products.

Colored biofilms cause problems in paper industry. In this work we used real-time PCR to detect and to quantitate members of the genus Meiothermus from the process samples and end products from 24 machines manufacturing pulp, paper and board in four countries. The results obtained from 200 samples showed the importance of members of the genus Meiothermus as ubiquitous biofoulers in paper machines. This genus was the dominant biofouler in some mills. From < or =10(4) to 10(11) copies of Meiothermus 16S rRNA genes were found per gram of process deposit (wet weight). Meiothermus spp. were found in paper and board products with colored defects and connection between deposit-forming microbes and end-product spots was shown. 16S rRNA gene sequences of 29 biofilm producing bacterial isolates from different mills were determined. Based on sequence data, 25 of the isolates were assigned to the genus Meiothermus, with Meiothermus silvanus and M. ruber as the most frequent species.

DNA Primers↗
PubMed1991

High-level secretion of biologically active aprotinin from the yeast Pichia pastoris.

A synthetic gene encoding aprotinin (bovine pancreatic trypsin inhibitor) was fused to the Saccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site. Pichia pastoris strains were developed to express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion. P. pastoris containing a single copy of the vector secreted approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipotent with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.

Amino Acid Sequence↗