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PubMed1995

A comparison of the threshold detail detectability of a screen-film combination and computed radiology under conditions relevant to high-kVp chest radiography.

There is conflicting evidence regarding the viability of photostimulable phosphor computed radiology (CR) when used with high x-ray beam energies. In this study a standard screen-film combination was compared with CR using identical exposure conditions, which simulated those of a high-energy chest examination. The comparison was made using a psychophysical technique in which the threshold contrast detail detectability is assessed by human observers. Threshold contrast determination is an efficient way with which to assess the overall image quality. The CR images were processed and displayed using two independent image processing algorithms and hard-copy formats. The results showed, at the 5% level of statistical significance, that there was no difference in the overall image quality between any of the modalities being compared. It may be concluded therefore that CR is a potentially viable option for use in high-energy radiography of the chest.

Biophysical Phenomena↗
PubMed1992

Intracellular selection, conversion bias, and the expected substitution rate of organelle genes.

A key step in the substitution of a new organelle mutant throughout a population is the generation of germ-line cells homoplasmic for that mutant. Given that each cell typically contains multiple copies of organelles, each of which in turn contains multiple copies of the organelle genome, processes akin to drift and selection in a population are responsible for producing homoplasmic cells. This paper examines the expected substitution rate of new mutants by obtaining the probability that a new mutant is fixed throughout a cell, allowing for arbitrary rates of genome turnover within an organelle and organelle turnover within the cell, as well as (possibly biased) gene conversion and genetic differences in genome and/or organelle replication rates. Analysis is based on a variation of Moran's model for drift in a haploid population. One interesting result is that if the rate of unbiased conversion is sufficiently strong, it creates enough intracellular drift to overcome even strong differences in the replication rates of wild-type and mutant genomes. Thus, organelles with very high conversion rates are more resistant to intracellular selection based on differences in genome replication and/or degradation rates. It is found that the amount of genetic exchange between organelles within the cell greatly influences the probability of fixation. In the absence of exchange, biased gene conversion and/or differences in genome replication rates do not influence the probability of fixation beyond the initial fixation within a single organelle. With exchange, both these processes influence the probability of fixation throughout the entire cell. Generally speaking, exchange between organelles accentuates the effects of directional intracellular forces.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Replication↗
PubMed1994

Directed replacement of mt A by mt a-1 effects a mating type switch in Neurospora crassa.

To test the functions of a mating type genes, we developed an efficient strategy to select transformants of Neurospora crassa in which resident A mating type DNA was replaced by cloned DNA from the mt a idiomorph. Cloned a idiomorphic DNA could specify all functions, including fertility, of a mating type, but only when it replaced A DNA at the mating type locus. Only the mt a-1 region of the a idiomorph was necessary in order to specify a mating type. Gene replacement events involved the homologous sequences flanking the unique mating type idiomorphic DNA, resulting in apparently isogenic a and A strains. These isogenic strains were fertile when crossed with one another, indicating that no determinants outside the transforming DNA are necessary for fertility as a and that no host sequences of A strains interfere with fertility as a. One a replacement strain bore a duplication of the transforming mt a-1 and hph DNA. The duplication strain had unexpected properties. Although mating type segregated 1:1 in crosses of this strain to A, the duplicated regions were efficiently altered during the sexual process to generate a single copy in the progeny. No progeny were recovered that had undergone RIP (repeat induced point mutation) sufficient to inactivate the mt a-1 gene. We infer that the mt a-1 gene is necessary and sufficient to specify a mating type identity in all vegetative and sexual activities. Mt a-1 may also play an essential role in ascosporogenesis after fertilization.

Cloning, Molecular↗
PubMed2006

Chromosome XII context is important for rDNA function in yeast.

The rDNA cluster in Saccharomyces cerevisiae is located 450 kb from the left end and 610 kb from the right end of chromosome XII and consists of approximately 150 tandemly repeated copies of a 9.1 kb rDNA unit. To explore the biological significance of this specific chromosomal context, chromosome XII was split at both sides of the rDNA cluster and strains harboring deleted variants of chromosome XII consisting of 450 kb, 1500 kb (rDNA cluster only) and 610 kb were created. In the strain harboring the 1500 kb variant of chromosome XII consisting solely of rDNA, the size of the rDNA cluster was found to decrease as a result of a decrease in rDNA copy number. The frequency of silencing of URA3 inserted within the rDNA locus was found to be greater than in a wild-type strain. The localization and morphology of the nucleolus was also affected such that a single and occasionally (6-12% frequency) two foci for Nop1p and a rounded nucleolus were observed, whereas a typical crescent-shaped nucleolar structure was seen in the wild-type strain. Notably, strains harboring the 450 kb chromosome XII variant and/or the 1500 kb variant consisting solely of rDNA had shorter life spans than wild type and also accumulated extrachromosomal rDNA circles. These observations suggest that the context of chromosome XII plays an important role in maintaining a constant rDNA copy number and in physiological processes related to rDNA function in S.cerevisiae.

Cell Nucleolus↗
PubMed2005

Objective assessment of image quality in conventional and digital mammography taking into account dynamic range.

The goal of this work is to develop a method to objectively compare the performance of a digital and a screen-film mammography system in terms of image quality. The method takes into account the dynamic range of the image detector, the detection of high and low contrast structures, the visualisation of the images and the observer response. A test object, designed to represent a compressed breast, was constructed from various tissue equivalent materials ranging from purely adipose to purely glandular composition. Different areas within the test object permitted the evaluation of low and high contrast detection, spatial resolution and image noise. All the images (digital and conventional) were captured using a CCD camera to include the visualisation process in the image quality assessment. A mathematical model observer (non-prewhitening matched filter), that calculates the detectability of high and low contrast structures using spatial resolution, noise and contrast, was used to compare the two technologies. Our results show that for a given patient dose, the detection of high and low contrast structures is significantly better for the digital system than for the conventional screen-film system studied. The method of using a test object with a large tissue composition range combined with a camera to compare conventional and digital imaging modalities can be applied to other radiological imaging techniques. In particular it could be used to optimise the process of radiographic reading of soft copy images.

Breast↗
PubMed2003

Herpes simplex virus and Meniere's disease.

OBJECTIVE/HYPOTHESIS: This study was designed to investigate the hypothesis that Meniere's disease is associated with herpes simplex virus (HSV) reactivation in the vestibular ganglion. STUDY DESIGN: Case control study. METHODS: Vestibular ganglia were obtained from archival surgical pathology specimens from patients undergoing vestibular neurectomy for vertigo caused by Meniere's disease. All patients met criteria for classification as definite Meniere's disease according to American Academy of Otolaryngology/Head and Neck Surgery (AAO-HNS) criteria. Control specimens were obtained from willed body donors. Sections from each ganglion were studied for prevalence of viral DNA using a nested polymerase chain reaction designed to amplify the HSV DNA polymerase gene. Quantitative analysis determined the number of viral copies per standard unit of ganglionic DNA. RESULTS: HSV DNA was more prevalent in paraffin embedded ganglia from patients with Meniere's disease (100%) than in fresh-frozen control ganglia (81%) (P =.02). Fixation and paraffin embedding substantially reduced recovery of HSV virus in selected control specimens. Quantitative analysis found no correlation between viral copy number in control ganglia processed frozen versus formalin fixed and paraffin embedded. CONCLUSIONS: HSV is more commonly isolated from vestibular ganglia of patients with Meniere's disease than the general population. The routine histologic preparation of formalin fixation and paraffin embedding significantly altered the quantity of virus detected though not in a predictable manner. The study provides supportive evidence for a viral etiology in Meniere's disease.

Adult↗
PubMed1986

Intramolecular recombination between transfected repeated sequences in mammalian cells is nonconservative.

When plasmids carrying a fragmented gene with segments present as direct repeats are introduced into mammalian cells, recombination or gene conversion between the repeated sequences can reconstruct the gene. Intramolecular recombination leads to the deletion of the intervening sequences and the loss of one copy of the repeat. This process is known to be stimulated by double-strand breaks. Two current models for recombination in eucaryotic cells propose that the reaction is initiated by double-strand breaks, but differ in their predictions as to the fate of the intervening sequences. One model suggests that these sequences are always lost, while the other indicates that the reaction will be conservative as a function of the position of the double-strand break. We have constructed a plasmid in which two overlapping portions of the simian virus 40 early region, which contains the origin and T-antigen gene, are present as direct repeats separated by sequences containing a plasmid with a simian virus 40 origin of replication. Recombination across the repeated segments could produce a plasmid with an origin of replication and/or a plasmid with a gene for a functional T-antigen which would drive the replication of both. Introduction of this construction into African green monkey kidney cells, without coinfection, establishes a condition in which the products of the recombination or gene conversion can be interpreted unambiguously. We find that the majority of the reconstruction reactions are nonconservative.

Animals↗
PubMed1996

Mutations in the Dictyostelium heterotrimeric G protein alpha subunit G alpha5 alter the kinetics of tip morphogenesis.

Tip morphogenesis during the Dictyostelium developmental life cycle is a process by which prestalk cells sort to form the anterior region of the multicellular organism. We show that the temporal regulation of this morphological process is dependent on the copy number of the Dictyostelium G alpha5 gene. Tip formation is delayed in aggregates of g alpha5 null mutant cells and accelerated in aggregates overexpressing the G alpha5 gene compared to tip formation in wild-type cells. The onset of cell-type-specific gene expression associated with mound formation and tip morphogenesis is also temporally altered in G alpha5 mutants. Tip morphogenesis in chimeric organisms of G alpha5 mutants and wild-type cells is dependent on the copy number of the G alpha5 gene, indicating that G alpha5 function plays an integral role in the intercellular signaling of this stage of development. The G alpha5 gene encodes a G alpha subunit that has 51% identity to the Dictyostelium G alpha4 subunit. Like the G alpha4 gene, the G alpha5 gene is expressed in a subset of cells distributed throughout the multicellular organism, with a distribution that is similar to the anterior-like cell population. Amino acid substitutions in the G alpha5 subunit analogous to substitutions altering guanine nucleotide binding and hydrolysis in other G alpha subunits had no apparent effect on the rate of tip formation when a single copy of the mutant gene was used to replace the wild-type gene. Overexpression of these mutant G alpha5 genes by increased gene dosage resulted in cell death, suggesting that high levels of the altered subunits have detrimental effects during vegetative growth.

Amino Acid Sequence↗
PubMed1992

Multiple amplicons of discrete sizes encompassing N-myc in neuroblastoma cells evolve through differential recombination from a large precursor DNA.

Human neuroblastoma cells often carry cytogenetic abnormalities signaling amplification of the gene N-myc. In most cell lines amplified N-myc is localized in homogeneously staining regions (HSRs). Varying proportions of the amplified DNA consist of multiple tandem arrays of DNA segments encompassing N-myc. Here we report the cloning and sequencing of a DNA breakpoint which represents the joint of the tandem repeats of a 280-kb amplicon of neuroblastoma line NMB. The breakpoint is located in the first intron of the N-myc gene and leads to the deletion of the 5' region of N-myc in this 20-copy amplicon. The representation of DNA derived from the non-N-myc part of the novel joint in the different amplicons suggests that an increase in N-myc copy numbers involves a multistep process proceeding from large 'precursors' to smaller multicopy amplicons.

Base Sequence↗
PubMed1991

Structure of the chicken insulin-like growth factor I gene reveals conserved promoter elements.

Insulin-like growth factor I plays a major role in cellular growth by mediating many of the actions of growth hormone. In mammals, IGF-I gene expression is complicated, as the single-copy gene is transcribed and processed into multiple mRNAs that encode at least two peptide precursors. Recent cDNA cloning studies have suggested that in chickens the organization of the gene might be simpler, and that comparative mapping might define essential features of IGF-I that are common to vertebrates. To achieve this goal, we have cloned and characterized the chicken IGF-I gene. The chicken gene is more compact than its mammalian homologues. It is composed of 4 exons which are distributed over 50 kilobases (kb) of chromosomal DNA and are transcribed and processed into mRNAs of 1.9 and 2.6 kb. As in the rat and human genes, the mature 70-amino acid IGF-I molecule is encoded within exons 2 and 3, while exons 1 and 4 contain NH2- and COOH-terminal extension peptides, respectively, and untranslated sequences. Both ends of the chicken gene are structurally heterogeneous. IGF-I mRNAs were found to have two regions of polyadenylation which mapped to exon 4 and were separated by approximately 0.7 kb. At the 5' end, several transcription initiation sites were identified within a 74-nucleotide portion of exon 1 that was found to be highly conserved between chicken and mammalian IGF-I genes. To test promotor function, chimeric genes were constructed linking fragments of chicken IGF-I 5'-flanking DNA to a promoterless reporter plasmid. Hybrid genes containing at least 0.6 kb of IGF-I enhanced expression of luciferase after transfection into SK-N-MC cells, a human line that synthesizes IGF-I mRNA. These observations represent the first thorough characterization of an IGF-I gene and provide an impetus for discerning the mechanisms by which IGF-I expression is regulated during growth, development, and differentiation.

Amino Acid Sequence↗
PubMed1993

Amplification of bacteriophage Mu DNA by rolling circle DNA replication in vitro.

When mini-Mu DNA was allowed to transpose and replicate in vitro over a prolonged period, the products consisted not only of simple inserts and cointegrates but also high molecular weight DNA many times the unit length of mini-Mu. This high molecular weight product contained predominantly full-length mini-Mu DNA and relatively little non-Mu DNA (the vector harboring the mini-Mu element and the target for transposition in the reaction system). It arose from rolling circle DNA replication of templates created by intramolecular strand transfer, which is catalyzed by Mu transposition proteins. A donor substrate, which is a supercoiled plasmid bearing a mini-Mu element, gave rise to large amounts of the high molecular weight product provided that the vector segment outside the mini-Mu element was 2 kilobase pairs or more. When a donor substrate had a vector segment of only 600 base pairs, the mini-Mu element first had to transpose to a larger circular target before giving rise to the high molecular weight product. These results suggest a mechanism by which Mu DNA can be amplified for lytic development without transposing multiple times. By establishing a circular template, multiple copies of Mu can be processively generated from a single initiation event.

Bacteriophage mu↗
PubMed1996

Seventeen copies of the human 37 kDa laminin receptor precursor/p40 ribosome-associated protein gene are processed pseudogenes arisen from retropositional events.

A cDNA coding for a 37 kDa polypeptide has been identified in several species as both the potential precursor of the 67 kDa laminin receptor (37LRP) and a putative ribosome-associated protein (p40). Interestingly, increased expression of this polypeptide (37LRP/p40) is consistently observed in invasive and metastatic cancer cells and is associated with poor prognosis. Southern-blot analysis of human genomic DNA predicted multiple copies of the 37LRP/p40 gene. In this study, we report that the number of copies of this sequence in the human genome is 26 +/- 2. We have sequenced and analyzed 19 genomic clones corresponding to the 37LRP/p40 gene and found that they were all processed pseudogenes. They all lack intronic sequences and show multiple genetic alterations leading in some cases to the appearance of stop codons. Moreover, they all bear characteristic features of retroposons as the presence of a poly(A)-tail at their 3' end and short direct repeated flanking DNA sequences. None of the pseudogenes analyzed present cis-elements in their 5' flanking region such as TATA or GC boxes. Our date reveal that over 50% of the 37LRP/p40 gene copies are pseudogenes most probably generated by retropositional events. The finding of multiple pseudogenes for the 37LRP/p40 suggests that the accumulation of several copies of this gene might have given a survival advantage to the cell in the course of evolution.

Base Sequence↗
PubMed2003

Development of a fed-batch fermentation process to overproduce phosphoenolpyruvate carboxykinase using an expression vector with promoter and plasmid copy number controllable by heat.

To effectively achieve tight regulation and high-level expression of cloned genes, a novel expression plasmid has been developed to contain the promoter and allow the plasmid copy number to be controlled by heat. The feasibility of the plasmid was tested by overproducing the pck gene product (Pck), a protein responsible for cell growth on gluconeogenic carbons and with potential toxicity. By fusing the pck gene with the promoter on the plasmid, the Escherichia coli strain harboring the composite vector was shown to produce various amounts of Pck in response to different degrees of heat shock. With the use of a 30 degrees -->41 degrees C stepwise upshift, the shake-flask culture of recombinant cells enabled production of maximal Pck in soluble form accounting for 20% of total cell protein. In sharp contrast, Pck production was undetectable in the uninduced cell, and this was further confirmed by the failed growth of strain JCL1305, defective in the essential genes for gluconeogenesis, carrying the composite vector on succinate at 30 degrees C. By exploiting the fed-batch fermentation approach, the recombinant cell batch initially kept at 30 degrees C in a lab-scale fermentor was exposed to 41 degrees C for 2 h at the batch fermentation stage, followed by a reduction in temperature to 37 degrees C throughout the remainder of the culturing process. Consequently, this resulted in Pck production equivalent to 15% of total cell protein. The total Pck yield thus calculated was amplified 1880-fold over that obtained at the shake-flask scale. Overall, there is great promise for this expression system due to its tight control, high production, simple thermomodulation, and feasible scale-up of recombinant proteins.

Bacteriophage T7↗
PubMed2002

Stabilizing plasmid copy number to improve recombinant protein production.

The key objective for recombinant protein production in bacteria is the maximum exploitation of the cell factory's potential, whereby often strong expression vectors are used to increase product yield. If the metabolic load caused by recombinant expression exceeds the host's capacity, the system exhausts itself, resulting in a loss of protein yield. Excessive plasmid replication is observed after inducing recombinant gene expression, which greatly contributes to metabolic overload of the host cell. The transcriptional and translational machineries are extremely overstrained. By abolishing sequence homology between ColE1 RNA I/RNA II and tRNAs, we were able to restore the plasmid's replication control mechanisms and to keep the plasmid copy number constant throughout the culture process, thereby prolonging metabolic activity and productivity of the bacterial expression system. Because the bacterial host cell is not being exploited beyond its tolerable potential with this method, the constancy of the plasmid copy number level throughout the whole period of the bioprocess provides novel strategies for bioprocess optimization.

Bacterial Proteins↗
PubMed1999

Effects of bilateral stimulation and stimulus redundancy on interhemispheric interaction.

Recent visual laterality studies have included trials in which critical stimulus information is presented simultaneously in both visual half-fields and, thereby, simultaneously to both cerebral hemispheres. To investigate interhemispheric interaction, researchers compare performance on bilateral redundant trials with performance on unilateral trials in which a single copy of the target is presented to one hemisphere or the other. The authors used the identification of nonword letter trigrams to examine the relationship between unilateral and bilateral performance when the 2 types of trials were equated for the number of locations stimulated (Experiment 1) and the number of redundant copies of the target (Experiment 2). Results suggest that when the number of stimulated locations is held constant, each of 2 copies of a target stimulus can be processed with the same efficiency and the same strategy as it would have been had it been the only copy.

Adult↗
PubMed1992

Automatic control during hand reaching at undetected two-dimensional target displacements.

1. The aim of this study was to demonstrate that goal-directed pointing movements, executed at normal speed to a small visual target, but without vision of the movement, do not rely on preprogrammed commands (open-loop process); by contrast these responses are under the control of a feedback loop, which compares the ongoing response and the goal (or its internal representation). When the location of this goal is changed at the onset of the movement, an automatic correction of the path occurs. Modification of the goal was obtained by presenting a target in the peripheral visual field that the subject had to look at and point at as quickly and accurately as possible. When the orienting ocular saccade reached its peak velocity, statistically corresponding to the hand movement onset, the target was suddenly shifted 10 degrees in a random direction. This perturbation was undetected by the subject because of the absence of perception during the saccade. For the compensation to occur, the initial orientation of the movement and also its extent had to be modified. The results revealed 1) a nearly complete compensation of the movement path and a 66- to 80-ms duration lengthening; 2) relatively short reaction times to the perturbations (from 145 to 174 ms, with effective reaction times even 40 ms shorter); 3) nearly identical spatiotemporal movement characteristics to the perturbations, regardless of whether vision of the hand was allowed, suggesting that corrections were subserved by the same mechanisms. 2. The spatiotemporal characteristics of these unconscious corrections were similar to those observed in the classical double-step experiments investigating the intentional modifications of ongoing movements and suggest that they might share some common low-level mechanisms. That is, they could rely on visuokinesthetic feedback loops, which compare the updated information provided by the eye at the end of the saccade and the proprioceptive information of the end point effector (the fingertip here); they could also rely on feed-forward processes detecting the discrepancy between an efference copy of the movement and the new goal; or they could rely on a combination of those two main processes.

Acceleration↗
PubMed1991

Copying strategies and memory on the Complex Figure Test in psychiatric patients.

We assessed three copying strategies on the Rey-Osterrieth Complex Figure Test among 50 psychiatric patients. The strategies were featural (detail-focused), contextual (framework-focused), and mixed. Reliable classification of each patient's copying strategy showed 7 patients used a featural and 7 patients used a contextual strategy. The remaining 36 used a mixed strategy involving both elements. Analysis indicated that patients who met DSM-III-R criteria for schizophrenia tended to use a mixed strategy. Nonschizophrenic patients also favoured the mixed approach. Moreover, when subjects were divided into groups based on their strategies, there were no differences in copying accuracy. However, the groups differed on immediate and delayed recall of the Complex Figure Test. Patients who adopted a featural strategy on the copy trial had the lowest recall scores. "Process" variables may be important in understanding neurocognitive functioning in schizophrenia; however, there is little evidence that schizophrenic patients use copying strategies consistent with lateralized impairment of brain function.

Adult↗
PubMed1995

The Xenopus intron-encoded U17 snoRNA is produced by exonucleolytic processing of its precursor in oocytes.

U17 is a small nucleolar RNA encoded in the introns of the Xenopus laevis gene for ribosomal protein S7 (formerly S8, see Note). To study the mechanisms involved in its in vivo processing from S7 transcripts, various in vitro synthesized RNAs embedding a U17 sequence have been microinjected into the germinal vesicle of Xenopus oocytes and their processing analysed. In particular, the Xenopus U17 gene copies a and f and a U17 gene copy from the pufferfish Fugu rubripes have been used. Information about the nature of the processing activities involved in U17 RNA maturation have been sought by injecting transcripts protected from exonucleolytic attack at their 5'-end by capping and/or lengthened at their 3'-end by polyadenylation. The results obtained indicate that U17 RNA processing is a splicing-independent event and that it is mostly or entirely due to exonucleolytic degradation at both the 5'- and 3'-ends of the precursor molecules. Moreover, it is concluded that the enzymes involved are of the processive type. It is suggested that the apparatus for U17 RNA processing is that responsible for the degradation of all excised and debranched introns. Protection from exonucleolytic attack, due to the tight structure and/or to the binding of specific proteins, would be the mechanism by which U17 RNA is produced.

Animals↗