Classification and identification of photocopiers: a progress report.
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Copying Processes↗
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Preferential cis-syn thymine dimer bypass by DNA polymerase eta occurs with biased fidelity.
Human DNA polymerase eta (Pol eta) modulates susceptibility to skin cancer by promoting DNA synthesis past sunlight-induced cyclobutane pyrimidine dimers that escape nucleotide excision repair (NER). Here we have determined the efficiency and fidelity of dimer bypass. We show that Pol eta copies thymine dimers and the flanking bases with higher processivity than it copies undamaged DNA, and then switches to less processive synthesis. This ability of Pol eta to sense the dimer location as synthesis proceeds may facilitate polymerase switching before and after lesion bypass. Pol eta bypasses a dimer with low fidelity and with higher error rates at the 3' thymine than at the 5' thymine. A similar bias is seen with Sulfolobus solfataricus DNA polymerase 4, which forms a Watson-Crick base pair at the 3' thymine of a dimer but a Hoogsteen base pair at the 5' thymine (ref. 3). Ultraviolet-induced mutagenesis is also higher at the 3' base of dipyrimidine sequences. Thus, in normal people and particularly in individuals with NER-defective xeroderma pigmentosum who accumulate dimers, errors made by Pol eta during dimer bypass could contribute to mutagenesis and skin cancer.
Base Pairing↗
[Optimized image processing with modified preprocessing of image data sets of a transparent imaging plate by way of the lateral view of the cervical spine].
PURPOSE: To improve the diagnostic quality of lateral radiographs of the cervical spine by pre-processing the image data sets produced by a transparent imaging plate with both-side reading and to evaluate any possible impact on minimizing the number of additional radiographs and supplementary investigations. MATERIAL AND METHODS: One hundred lateral digital radiographs of the cervical spine were processed with two different methods: processing of each data set using the system-imminent parameters and using the manual mode. The difference between the two types of processing is the level of the latitude value. Hard copies of the processed images were judged by five radiologists and three neurosurgeons. The evaluation applied the image criteria score (ICS) without conventional reference images. RESULTS: In 99 % of the lateral radiographs of the cervical spine, all vertebral bodies could be completed delineated using the manual mode, but only 76 % oft the images processed by the system-imminent parameters showed all vertebral bodies. Thus, the manual mode enabled the evaluation of up to two additional more caudal vertebral bodies. The manual mode processing was significantly better concerning object size and processing artifacts. This optimized image processing and the resultant minimization of supplementary investigations was calculated to correspond to a theoretical dose reduction of about 50 %. CONCLUSION: The introduction of optimized organ programs for the upper and lower cervical spine based on the 12-bit data of the images should improve the evaluation of the lateral radiograph of the cervical spine without reducing the latitude value.
Cervical Vertebrae↗
Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage.
Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage.
Amino Acid Sequence↗
Digital chest radiography with a solid-state flat-panel x-ray detector: contrast-detail evaluation with processed images printed on film hard copy.
PURPOSE: To evaluate and compare human observer performance in a contrast-detail test by using postprocessed hard-copy images from a digital chest radiography system and conventional screen-film radiographs. MATERIALS AND METHODS: The digital radiography system is based on a large-area flat-panel x-ray detector with a structured cesium iodide scintillator layer and an amorphous silicon thin-film transistor array for image readout. Images of a contrast-detail phantom were acquired at two exposure levels by using two standard thoracic screen-film systems and the digital system at matched dose. By using images of the phantom processed with standard chest image postprocessing techniques, a four-alternative forced-choice observer perception study was performed, and the number of detectable test signals (disk-shaped objects 0.3-4.0 mm in diameter) was determined for each image type. RESULTS: On average, observers detected more test signals on digital images than on screen-film radiographs at all diameters up to 2.0 mm and an equivalent number at larger diameters. Test signals with lower inherent subject contrast were detected more readily on digital images than on screen-film images, even when x-ray exposure levels for the digital system were reduced by 20%. CONCLUSION: Observer performance in a contrast-detail detection task can be improved by using images acquired with the flat-panel digital chest radiography system as compared with those acquired with state-of-the-art screen-film combinations.
Humans↗
Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.
The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.
Animals↗
Molecular cloning of a preprohormone from Hydra magnipapillata containing multiple copies of Hydra-L Wamide (Leu-Trp-NH2) neuropeptides: evidence for processing at Ser and Asn residues.
The simple, freshwater polyp Hydra is often used as a model to study development in cnidarians. Recently, a neuropeptide, < Glu-Gln-Pro-Gly-Leu-Trp-NH2, has been isolated from sea anemones that induces metamorphosis in a hydroid planula larva to become a polyp. Here, we have cloned a preprohormone from Hydra magnipapillata containing 11 (eight different) immature neuropeptide sequences that are structurally related to the metamorphosis-inducing neuropeptide from sea anermones. During the final phase of our cloning experiments, another research team independently isolated and sequenced five of the neuropeptides originally found on the preprohormone. Comparison of these mature neuropeptide structures with the immature neuropeptide sequences on the preprohormone shows that most immature neuropeptide sequences are preceded by Ser or Asn residues, indicating that these residues must be novel processing sites. Thus, the structure of the Hydra preprohormone confirms our earlier findings that cnidarian preprohormones contain unusual or novel processing sites. Nearly all neuropeptide copies located on the Hydra preprohormone will give rise to mature neuropeptides with a C-terminal Gly-Leu-Trp-NH2 sequence (the most frequent one being Gly-Pro-Pro-Pro-Gly-Leu-Trp-NH2; Hydra-LWamide l; three copies). Based on their structural similarities with the metamorphosis-inducing neuropeptide from sea anemones, the mature peptides derived from the Hydra-LWamide preprohormone are potential candidates for being developmentally active neurohormones in Hydra.
Amino Acid Sequence↗
cDNA copies of the testis-specific lactate dehydrogenase (LDH-C) mRNA are present in spermatogenic cells in mice, but processed pseudogenes are not derived from mRNAs that are expressed in haploid and late meiotic spermatogenic cells.
Retroposons are a class of genes created by reverse transcribing a processed mRNA and inserting the DNA copy into genomic DNA in germ-line cells. The present study concerns the question: Are retroposons created in meiotic and haploid spermatogenic cells? We demonstrate that polymerase chain reaction amplifies cytoplasmic DNAs with the expected intronless-structure of endogenous reverse transcriptase copies of the processed lactate dehydrogenase C mRNA encoding the testis-specific isoform of lactate dehydrogenase. Quantification of cytoplasmic LDH-C mRNA and endogenous cDNA by competitive RT-PCR and PCR, respectively, indicates that the level of LDH-C cDNA is lower by a factor of about 10(7) than the level of LDH-C mRNA in the cytoplasmic nucleic acids extracted from the testes of 14-day-old mice, and that about 1 in 10(5) meiotic cells contains an endogenous cDNA copy of LDH-C mRNA. A review of the literature reveals that a large number of genes including the LDH-C gene, whose expression is restricted to spermatogenic cells, are always single copy. Collectively, these observations suggest that reverse transcriptase cDNA copies of mRNAs are present in meiotic and haploid spermatogenic cells, but these cDNAs are not integrated into genomic DNA.
Animals↗
Gene transfer experiments imply instructive role of major histocompatibility complex class I molecules in cellular peptide processing.
DBA/2-derived mouse tumor cells were transfected with the H-2 Kb gene. Naturally processed minor histocompatibility (H) peptides were extracted from both transfected and non-transfected cells by acid elution, and were separated by high-performance liquid chromatography. Kb-restricted minor H epitopes corresponding to H-4b and mapki, both encoded by non-major histocompatibility complex genes of DBA/2, were readily detected by the respective cytotoxic T lymphocyte in peptides extracted from Kb-transfected, but not from non-transfected or Db-transfected cells. Titration experiments indicated at least 3000-fold less copies of correctly processed Kb-restricted epitopes in cells without Kb as compared to cells with Kb. Since we estimate the copy number of Kb-restricted H-4b epitopes in Kb-expressing transfectants to be less than 1000 per cell, the pool size of H-4b epitopes correctly processed in the absence of Kb should be less than 1/3 copy per cell.
Animals↗
Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments.
Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and phi 29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations >or= 10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples.
Bacillus Phages↗
Cloning and characterization of two processed p53 pseudogenes from the rat genome.
We have cloned, by the polymerase chain reaction (PCR), two rat genomic fragments of 1.3 and 1.2 kb, both of which hybridize to a human p53 cDNA probe. Nucleotide sequencing revealed that they are two intronless rat p53 pseudogenes (designated as psi R53-1 and psi R53-2, respectively), representing a start-to-stop-codon-length copy of the processed transcript of the rat p53 gene. Further PCR analysis of DNA from rat organs (skin, kidney, etc.) of two different strains demonstrated that psi R53-1 and psi R53-2 were formed in the germ-line. As psi R53-1 and psi R53-2 share 85 and 83% homology with the rat p53 cDNA, it is thus estimated that psi R53-1 was created approx. 10 million years (Myr) ago and psi R53-2 arose 12 Myr ago. Moreover, GenBank scanning indicated that a 95-bp insert in psi R53-1, as compared with the cDNA, was 90% homologous with a sequence of mouse alternatively spliced p53 mRNA, where the spliced p53 mRNA contains an additional 96 nt derived from intron 10. Although the rat alternatively spliced p53 mRNA has so far not been described, our data suggest that these two processed pseudogenes may have been generated by integration of different mRNA intermediates into germ-line DNA.
Animals↗
Mild hearing impairment can reduce verbal memory performance in a healthy adult population.
We studied to what extent immediate and delayed recall in an auditory verbal learning paradigm was affected by basic information processing speed (digit copying) and hearing acuity (average hearing acuity at 1, 2 and 4 KHz at the better ear). A group of 453 individuals in the age between 23 and 82 years with no overt hearing pathology was recruited from a larger study of cognitive aging (Maastricht Aging Study, MAAS). After controlling for age, sex, educational level, and processing speed it was found that a mild to moderate hearing loss predicted lower verbal memory performance. Auditory administered verbal memory tests can underestimate true memory performance, particularly in older individuals with unknown hearing status.
Adult↗
Retroposition of processed pseudogenes: the impact of RNA stability and translational control.
Human processed pseudogenes are copies of cellular RNAs reverse transcribed and inserted into the nuclear genome by the enzymatic machinery of L1 (LINE1) non-LTR retrotransposons. Although it is generally accepted that germline expression is crucial for the heritable retroposition of cellular mRNAs, little is known about the influences of RNA stability, mRNA quality control and compartmentalization of translation on the retroposition of processed pseudogenes. We found that frequently retroposed human mRNAs are derived from stable transcripts with translation-competent functional reading frames that are resistant to nonsense-mediated RNA decay. They are preferentially translated on free cytoplasmic ribosomes and encode soluble proteins. Our results indicate that interactions between mRNAs and L1 proteins seem to occur at free cytoplasmic ribosomes.
Animals↗
What is a linear process?
We argue that given even an infinitely long data sequence, it is impossible (with any test statistic) to distinguish perfectly between linear and nonlinear processes (including slightly noisy chaotic processes). Our approach is to consider the set of moving-average (linear) processes and study its closure under a suitable metric. We give the precise characterization of this closure, which is unexpectedly large, containing nonergodic processes, which are Poisson sums of independent and identically distributed copies of a stationary process. Proofs of these results will appear elsewhere.
Linear Models↗
APOBEC3G cytidine deaminase inhibits retrotransposition of endogenous retroviruses.
Endogenous retroviruses are multicopy retroelements accounting for nearly 10% of murine or human genomes. These retroelements spread into our ancestral genome millions of years ago and have acted as a driving force for genome evolution. Endogenous retroviruses may also be deleterious for their host, and have been implicated in cancers and autoimmune diseases. Most retroelements have lost replication competence because of the accumulation of inactivating mutations, but several, including some murine intracisternal A-particle (IAP) and MusD sequences, are still mobile. These elements encode a reverse transcriptase activity and move by retrotransposition, an intracellular copy-and-paste process involving an RNA intermediate. The host has developed mechanisms to silence their expression, mainly cosuppression and gene methylation. Here we identify another level of antiviral control, mediated by APOBEC3G, a member of the cytidine deaminase family that was previously shown to block HIV replication. We show that APOBEC3G markedly inhibits retrotransposition of IAP and MusD elements, and induces G-to-A hypermutations in their DNA copies. APOBEC3G, by editing viral genetic material, provides an ancestral wide cellular defence against endogenous and exogenous invaders.
APOBEC-3G Deaminase↗
Multiple simultaneous stimulus presentation facilitates lexical processing.
Bilateral presentation of two copies of the same word leads to faster lexical decisions compared to unilateral presentation alone (bilateral gain). This has implications for theories of interhemispheric interaction, because it suggests that, under certain conditions, both hemispheres cooperate rather than inhibit each other or act independently. Experiment 1 confirmed that the bilateral gain is word-specific and does not occur for pseudowords. Whereas the bilateral gain proved to be present for words of different word frequencies, results of Experiment 2 suggest that it is slightly stronger for high-frequency words compared to words of lower frequencies. Experiment 3 revealed that higher numbers of stimuli (two versus four copies of the same word/pseudoword) presented at the same time lead to an additional improvement of word processing. These results support a neurobiological model of word representation assuming that words are cortically represented in widely distributed interhemispheric cell assemblies. Summation of activity in such assemblies leads to faster and more reliable ignition of the network.
Adult↗
Reverse transcriptase: mediator of genomic plasticity.
Reverse transcription has been an important mediator of genomic change. This influence dates back more than three billion years, when the RNA genome was converted into the DNA genome. While the current cellular role(s) of reverse transcriptase are not yet completely understood, it has become clear over the last few years that this enzyme is still responsible for generating significant genomic change and that its activities are one of the driving forces of evolution. Reverse transcriptase generates, for example, extra gene copies (retrogenes), using as a template mature messenger RNAs. Such retrogenes do not always end up as nonfunctional pseudogenes but form, after reinsertion into the genome, new unions with resident promoter elements that may alter the gene's temporal and/or spatial expression levels. More frequently, reverse transcriptase produces copies of nonmessenger RNAs, such as small nuclear or cytoplasmic RNAs. Extremely high copy numbers can be generated by this process. The resulting reinserted DNA copies are therefore referred to as short interspersed repetitive elements (SINEs). SINEs have long been considered selfish DNA, littering the genome via exponential propagation but not contributing to the host's fitness. Many SINEs, however, can give rise to novel genes encoding small RNAs, and are the migrant carriers of numerous control elements and sequence motifs that can equip resident genes with novel regulatory elements [Brosius J. and Gould S.J., Proc Natl Acad Sci USA 89, 10706-10710, 1992]. Retrosequences, such as SINEs and portions of retroelements (e.g., long terminal repeats, LTRs), are capable of donating sequence motifs for nucleosome positioning, DNA methylation, transcriptional enhancers and silencers, poly(A) addition sequences, determinants of RNA stability or transport, splice sites, and even amino acid codons for incorporation into open reading frames as novel protein domains. Retroposition can therefore be considered as a major pacemaker for evolution (including speciation). Retroposons, with their unique properties and actions, form the molecular basis of important evolutionary concepts, such as exaptation [Gould S.J. and Vrba E., Paleobiology 8, 4-15, 1982] and punctuated equilibrium [Elredge N. and Gould S.J. in Schopf T.J.M. (ed). Models in Paleobiology. Freeman, Cooper, San Francisco, 1972, pp. 82-115].
Animals↗
Exploratory data analysis groupware for qualitative and quantitative electrophoretic gel analysis over the Internet-WebGel.
Many scientists use quantitative measurements to compare the presence and amount, of various proteins and nucleotides among series of one- and two-dimensional (1-D and 2-D) electrophoretic gels. These gels are often scanned into digital image files. Gel spots are then quantified using stand-alone analysis software. However, as more research collaborations take place over the Internet, it has become useful to share intermediate quantitative data between researchers. This allows research group members to investigate their data and share their work in progress. We developed a World Wide Web group-accessible software system, WebGel, for interactively exploring qualitative and quantitative differences between electrophoretic gels. Such Internet databases are useful for publishing quantitative data and allow other researchers to explore the data with respect to their own research. Because intermediate results of one user may be shared with their collaborators using WebGel, this form of active data-sharing constitutes a groupware method for enhancing collaborative research. Quantitative and image gel data from a stand-alone gel image processing system are copied to a database accessible on the WebGel Web server. These data are then available for analysis by the WebGel database program residing on that server. Visualization is critical for better understanding of the data. WebGel helps organize labeled gel images into montages of corresponding spots as seen in these different gels. Various views of multiple gel images, including sets of spots, normalization spots, labeled spots, segmented gels, etc. may also be displayed. These displays are active and may be used for performing database operations directly on individual protein spots by simply clicking on them. Corresponding regions between sets of gels may be visually analyzed using Flicker-comparison (Electrophoresis 1997, 18, 122-140) as one of the WebGel methods for qualitative analysis. Quantitative exploratory data analysis can be performed by comparing protein concentration values between corresponding spots for multiple samples run in separate gels. These data are then used to generate reports on statistical differences between sets of gels (e.g., between different disease states such as benign or metastatic cancers, etc.). Using combined visual and quantitative methods, WebGel can help bridge the analysis of dissimilar gels which are difficult to analyze with stand-alone systems and can serve as a collaborative Internet tool in a groupware setting.
Electrophoresis↗