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PubMed1987

Unusual structure, evolutionary conservation of non-coding sequences and numerous pseudogenes characterize the human H3.3 histone multigene family.

The genomic organization of the replication-independent, basally expressed, human H3.3 gene is atypical of traditional histone gene organization. The gene contains 3 introns totalling 7.8 kb and unusual direct repeats flank all three intron-exon splice junctions. The transcription initiation site was mapped by S1 nuclease protection analysis and confirms that cDNA clones previously reported were full length. Sequence similarities between regions at the 5' and 3' termini of this human gene and a chicken H3.3 gene lead us to propose that either the previous assignments of termini of the chicken gene are in error, or there are alternative transcription start and polyadenylation sites. The 85% base matching of human and chicken H3.3 3'UTR sequences for 520 bases is unprecedented among homolog 3'UTR segments, especially considering that these species are separated by over 250 Myr of evolution. We also present the sequence of three related processed human H3.3 pseudogenes and provide evidence demonstrating that most of the 20 to 30 copies of the H3.3 gene within the human genome are in fact processed pseudogenes.

Amino Acid Sequence↗
PubMed2003

Genetic adaptation controlled by methylations and acetylations at the nuclear and cytosolic levels: a hypothetical model.

Metabolic sensors related to the maturation of metabolism seem to control a process of generic adaptation involving the silencing of genes and the expression of their copies more adapted to environmental changes. Nuclear methylases and histone deacetylases control the gene silencing process. Nuclear methylases compete with cytosolic methylases for the same methyl donnors, this will favor the expression of unmethylated more adapted gene copies, when cytosotic methylases take over. Methylated cytosolic compounds may then represent an index of this adaptation. If a more adapted gene copy is mutated, the regulatory ligand of the gene product that does not find its target may induce a reexpression of the silenced gene. The hypothetical model proposed considers that gene silencing and expression of a more adequate copy involves a non-specific gene silencer switch that depends on the histone status; the silencer switch is counteracted by the ligand of the adapted gene copy product acting like an inducer.

Acetylation↗
PubMed1995

[Priming effects in picture problems: preliminary solutions].

Two experiments explored whether picture puzzles are an adequate instrument to investigate implicit memory for pictorial information. During the testing phase, the subjects had to identify hidden figures in picture puzzles. In a preceding learning phase, the priming conditions were varied systematically. In the first experiment, some subjects had to solve picture puzzles in the learning phase, whereas others made esthetic judgments (global processing) or estimated the number of triangles in the picture puzzles (local processing). In the second experiment, the subjects inspected copies of figures that were hidden at testing, modified versions of these figures, or their names in the learning phase. In the first experiment, the subjects of the different encoding conditions showed comparable priming effects. Picture puzzles that had already been processed or seen during learning were solved more often than new ones. Interview data revealed that subjects in the local or global processing conditions did not identify hidden figures at encoding. Furthermore, these subjects could not discriminate between old and new picture puzzles in a final explicit test of recognition. Thus, nonconscious storage of perceptual information that is not semantically interpreted may be sufficient to evoke priming effects. In the second experiment, the subjects in the different encoding conditions showed reliable priming effects, too. The presentation of the duplicates at encoding produced the greatest amount of priming. Effects of verbal priming, however, indicate that the solution of picture puzzles is not based solely on perceptual information. Depending on the priming conditions at learning, the solution of picture puzzles may be based primarily on data-driven processing or may be guided more heavily by conceptual information. It is concluded that perceptual, lexical, and/or conceptual information can contribute to the solution of picture puzzles.

Adult↗
PubMed2001

Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis.

Intraerythrocytic malaria parasites replicate by the process of schizogeny, during which time they copy their genetic material and package it into infective merozoites. These merozoites must then exit the host cell to invade new erythrocytes. To better characterize the events of merozoite escape, erythrocytes containing Plasmodium falciparum schizonts were cultured in the presence of the cysteine protease inhibitor, l-transepoxy-succinyl-leucylamido-(4-guanidino)butane (E64). This treatment resulted in the accumulation of extraerythrocytic merozoites locked within a thin, transparent membrane. Immunomicroscopy demonstrated that the single membrane surrounding the merozoites is not erythrocytic but rather is derived from the parasitophorous vacuolar membrane (PVM). Importantly, structures identical in appearance can be detected in untreated cultures at low frequency. Further studies revealed that (i) merozoites from the PVM-enclosed merozoite structures (PEMS) are invasive, viable, and capable of normal development; (ii) PEMS can be purified easily and efficiently; and (iii) when PEMS are added to uninfected red blood cells, released merozoites can establish a synchronous wave of infection. These observations suggest that l-transepoxy-succinyl-leucylamido-(4-guanidino)butane (E64) causes an accumulation of an intermediate normally present during the process of rupture. We propose a model for the process of rupture: merozoites enclosed within the PVM first exit from the host erythrocyte and then rapidly escape from the PVM by a proteolysis-dependent mechanism.

Animals↗
PubMed1991

Mutation pattern of immunoglobulin transgenes is compatible with a model of somatic hypermutation in which targeting of the mutator is linked to the direction of DNA replication.

We have previously demonstrated that B lymphocyte specific somatic mutations are introduced into the variable regions of immunoglobulin kappa transgenes in two independent transgenic mouse lines. The frequency, distribution and nature of these mutations strongly suggest that they arose as a result of the process of somatic hypermutation, which is responsible, in part, for affinity maturation during an immune response. Unexpectedly, in these multiple copy transgenic lines, many of the transgene copies showed no evidence of somatic mutation. This paradox was addressed by determining the sequence of each transgene copy in several B cell hybridomas derived from a mouse line carrying three copies of the kappa transgene. It was found that the somatic hypermutation process in different B cells from the same mouse preferentially targets one, but not the same, transgene copy. We present a model, based on the pattern of this targeting, which links somatic hypermutation to the orientation of the Ig gene relative to the direction of DNA replication.

Animals↗
PubMed1988

Human ubiquitin genes: one member of the UbB gene subfamily is a tetrameric non-processed pseudogene.

The human ubiquitin gene family consists of three subfamilies. One of these, the UbB subfamily, includes a functional gene coding for a polyubiquitin protein that contains three ubiquitin copies tandemly repeated, as well as three pseudogenes of the processed type. We have now isolated a fifth human UbB type gene, different from any of the previously identified ones. This newly isolated gene is a tetrameric pseudogene which has presumably arisen by unequal crossing-over of two ancestral trimeric alleles. Southern blotting data indicate that all members of the human UbB gene subfamily are now accounted for.

Adult↗
PubMed2004

Improved interpretation of digitized mammography with wavelet processing: a localization response operating characteristic study.

OBJECTIVE: Our objective was the implementation and evaluation of a novel enhancement technique for improved interpretation of high-resolution digitized mammograms from computer monitors. MATERIALS AND METHODS: A wavelet algorithm was designed to attenuate the image spectral characteristics responsible for the long-range image correlation that often interferes with digital display. The algorithm was evaluated with a localization response operating characteristic (LROC) experiment with 500 negative, benign, and cancer cases with masses and calcification clusters. Three observers reviewed the original and wavelet-enhanced images on a 5-Mpixel monitor using a custom-made workstation user interface. RESULTS: Performance indexes were estimated for four different case combinations, each observer, and each interpretation mode. Wavelet enhancement improved the performance of all observers in all case combinations. Detection accuracy ranged from 0.678 to 0.827 for the unprocessed original data and 0.709-0.871 for the enhanced cases. Localization accuracy ranged from 0.547 to 0.785 for the original images and 0.568-0.847 for the enhanced cases, yielding increases of 5-15%. The difference between enhanced and original performances was statistically significant at the 0.10 level and in a few combinations at the 0.05 level. CONCLUSION: Soft-copy digitized mammography could replace standard film mammography under appropriate display parameters and conditions. The optimization of the soft-copy quality is expected to require more advanced processing techniques than standard gray-scale adjustments. Wavelet-based algorithms, such as the one proposed here, offer better soft-copy quality than the originals and a better starting point for additional manual gray-scale adjustments or automated postprocessing.

Algorithms↗
PubMed1995

Regulated expression of the Alcaligenes eutrophus pha biosynthesis genes in Escherichia coli.

A novel poly-beta-hydroxybutyrate (PHB) production system in which the expression and gene dosage of the Alcaligenes eutrophus pha biosynthetic operon were effectively regulated by cultivation temperature was constructed in Escherichia coli. The pha operon was fused to the negatively regulated tac promoter and cloned into a vector in which the copy number is temperature dependent. A two-phase process was employed to produce PHB during fed-batch growth. In the growth phase, the culture was maintained at a low temperature. Under this condition, the plasmid copy number was depressed and the number of LacI proteins was sufficient to repress tacupha transcription. The production phase was initiated by temperature upshift. At the elevated temperature, the number of plasmids surpassed the number of LacI repressors, which resulted in rapid induction of tacupha transcription, synthesis of poly-beta-hydroxyalkanoate-specific proteins, and polymer synthesis. During the production phase, the PHB production rate was 1.07 g of PHB liter-1 h-1 under optimized conditions. This rate is comparable to that of bacteria which naturally produce this polymer.

Alcaligenes↗
PubMed2006

Use of chromogenic in situ hybridization to identify MYCN gene copy number in neuroblastoma using routine tissue sections.

Amplification of MYCN in neuroblastoma is associated with a poor prognosis. However, methods for estimating the number of MYCN genes based on pooled cells do not address copy number heterogeneity at the cell level and can underestimate or even miss amplification. MYCN copy number can be directly assessed by fluorescence in situ hybridization, but evaluation of tissue histology is next to impossible. We have used a chromogenic method for in situ hybridization (CISH) that enables determination of MYCN copy number using routine light microscopy on routinely processed paraffin sections. Of 41 cases studied, CISH identified 100% of the 18 cases that were determined to be amplified by other techniques and was more sensitive than Southern blotting or quantitative DNA polymerase chain reaction. Because the technique evaluates individual tumor cells, heterogeneity of MYCN copy number was apparent from cell to cell. When defined as 50% or greater variation in copy number between cells in amplified tumors, almost 30% of cases were scored as heterogeneous. Heterogeneity reflects different tumor clones and its role has likely been under-recognized and underestimated in neuroblastoma biology. CISH will provide a valuable tool to assess this phenomenon in conjunction with other morphologic parameters in neuroblastoma specimens, to further our understanding of the biology of this childhood tumor.

Blotting, Southern↗
PubMed1989

A direct selection procedure for isolating yeast mutants with an impaired segregation of artificial minichromosomes.

The nondisjunction of artificial yeast minichromosomes (2:0 segregation events) during mitosis is accompanied by the appearance of cells containing more than one copy of the minichromosome. A mathematical simulation of this process has demonstrated that under certain conditions, a nondisjunction of the minichromosomes may result in their accumulation in a considerable portion of the cell population. An increase in the copy number of artificial minichromosomes as a result of impaired segregation has been used to develop a new experimental procedure for directly selecting yeast mutants showing an impaired segregation of artificial minichromosomes during mitosis. Four new genes, AMC1, AMC2, AMC3, and AMC4, which control the segregation of artificial minichromosomes in mitosis, have been identified (AMC3 and AMC4 are mapped to chromosome IV and VII, respectively). Mutations in the genes AMC1-AMC4 also affect the mitotic transmission of natural chromosomes. We suggest that the genes AMC1, AMC2, AMC3, and AMC4 control the segregation of natural chromosomes in yeast.

Chromosomes↗
PubMed2002

Gene content and function of the ancestral chromosome fusion site in human chromosome 2q13-2q14.1 and paralogous regions.

Various portions of the region surrounding the site where two ancestral chromosomes fused to form human chromosome 2 are duplicated elsewhere in the human genome, primarily in subtelomeric and pericentromeric locations. At least 24 potentially functional genes and 16 pseudogenes reside in the 614-kb of sequence surrounding the fusion site and paralogous segments on other chromosomes. By comparing the sequences of genomic copies and transcripts, we show that at least 18 of the genes in these paralogous regions are transcriptionally active. Among these genes are new members of the cobalamin synthetase W domain (CBWD) and forkhead domain FOXD4 gene families. Copies of RPL23A and SNRPA1 on chromosome 2 are retrotransposed-processed pseudogenes that were included in segmental duplications; we find 53 RPL23A pseudogenes in the human genome and map the functional copy of SNRPA1 to 15qter. The draft sequence of the human genome also provides new information on the location and intron-exon structure of functional copies of other 2q-fusion genes (PGM5, retina-specific F379, helicase CHLR1, and acrosin). This study illustrates that the duplication and rearrangement of subtelomeric and pericentromeric regions have functional relevance to human biology; these processes can change gene dosage and/or generate genes with new functions.

Amino Acid Sequence↗
PubMed2001

Chromosomal distribution of the transposable elements Osvaldo and blanco in original and colonizer populations of Drosophila buzzatii.

Chromosomal distribution of transposable elements (TEs) Osvaldo and blanco in D. buzzatii was studied in three original natural populations from Argentina (Berna, Puerto Tirol and La Nostalgia) and a colonizer population from the Iberian Peninsula (Carboneras). The Spanish population showed significant differences for Osvaldo and blanco copy numbers when we compared the X chromosome and the autosomes; but it is mainly the accumulation of copies in chromosome 2, where most sites with high insertion frequency were located, that causes the discrepancy with the negative selection model. We found no significant differences in TE frequency between chromosomal regions with different exchange rates, and no evident accumulation of TE was detected within chromosomal inversions where recombination rate is reduced. The Carboneras population shows euchromatic sites of Osvaldo and blanco with high occupancy and others with low copy number. On the contrary, Argentinian populations show only a generalized low occupancy per insertion site. Moreover, the mean copy number of both elements is higher in Spain than in Argentina. All these results suggest an important role of the colonization process in the distribution of TEs. The increase in the copy number of the TEs analysed and their elevated frequency in some chromosomal sites in Carboneras is, most probably, a sequel of the founder event and drift that took place at the time of the colonization of the Old World by D. buzzatii from the New World some 300 years ago.

Animals↗
PubMed2005

Event-related potentials show configural specificity of global form processing.

Glass patterns are a type of moiré created when a random-dot field is overlaid with a rotated, translated or dilated copy. The overall form of the moiré cannot be detected using local processing mechanisms, and because of this, Glass patterns are useful probes of global form processing. Here, we use event-related potentials to show that certain global organizations (concentric structure created by rotation and radial structure produced by dilation) produce much larger brain responses than others (linear structure created by translation). The results are consistent with the existence of specialized form processing mechanisms in the extrastriate cortex.

Evoked Potentials, Visual↗
PubMed1987

Evolution of nuclear gene families in primates. Copy-number variation in the argininosuccinate synthetase (ASS) pseudogene family and the anonymous DNA sequence, D1S1.

Changes in the copy number of nuclear genes provide the raw material for the creation of new gene functions. To better understand the mechanisms for such events, and their physiologic and evolutionary consequences, it is valuable to study a well characterized and closely related group of species such as primates. Fortuitously, most of the powerful molecular techniques and DNA probes developed for research in humans are equally applicable to non-human primates. We review what is known of copy number variation in primates and describe two informative DNA probes: pAS-1, a cDNA probe to the human urea cycle enzyme argininosuccinate synthetase (ASS), and an anonymous DNA probe, D1S1. In addition to the ASS structural locus on human chromosome 9, pAS-1 detects at least 14 dispersed, processed pseudogenes in humans. The number of pseudogene copies appears to be approximately the same in humans, chimpanzees, gorillas, orangutans and baboons; less in marmosets; and least in some rodents. Chimpanzees and gorillas appear to have all of the human pseudogenes though an Xp copy may be missing from gorillas. The Y pseudogene is apparently absent from orangutans and baboons, and, finally, a comparison of humans and chimpanzees revealed that the number of nucleotide substitutions in the Y chromosome pseudogenes is approximately 1 per 100. D1S1 maps to human chromosome 3 but also detects a high homology copy on chromosome 1. Chimpanzees, gorillas and orangutans all appear to have only the chromosome 3 homolog suggesting that this is the ancestral sequence and that the duplication occurred after separation of humans and the great apes. Both the ASS pseudogene family and the D1S1 system provide valuable information on the evolution of nuclear gene families in primates.

Animals↗
PubMed2025

FRET-FLIM for the Study of Protein-Protein Interactions Underpinning Mitosis Checkpoints.

Cell division is a key cellular process that ensures the continuation of life on Earth. In order to protect the genetic integrity of organisms, cell division must happen accurately, ensuring each daughter cell receives a complete copy of the original genome. The accuracy of this process is, in part, preserved by various cell cycle checkpoints. These checkpoints rely on the physical interactions of their components to ensure proper function. The spindle assembly checkpoint (SAC), for example, produces an inhibitory complex of BUBR1-BUB3 and MAD2 bound to CDC20. Many of these cell cycle checkpoint components have been identified in plants, but it has not yet been established whether plants have a mitotic checkpoint architecture that is similar to mammalian cells. To understand the function of plant cell cycle homologues, it is imperative to characterize their interactions in vivo. FRET-FLIM (Förster resonance energy transfer-fluorescence lifetime imaging microscopy), is a rapidly expanding technique that can be used to rapidly and simply characterize protein-protein interactions.

Fluorescence Resonance Energy Transfer↗
PubMed2001

Factor structure and explanatory variables of the Mini-Mental State Examination (MMSE) for elderly persons in Taiwan.

BACKGROUND AND PURPOSE: The Mini-Mental State Examination (MMSE) is commonly used in studies and clinical assessment of cognitive functioning. The purpose of this study was to examine the factor structure and variables explaining the scores of the MMSE in elderly persons in Taiwan, in order to provide a reference for instrument selection and data collection for clinicians and researchers. METHODS: Secondary data from an epidemiologic study that included 1,442 randomly selected subjects aged 64 to 98 with an average age of 72.6 years were used. Males comprised 57.1% of the sample, and females 42.9%. Exploratory and confirmatory factor analysis and covariance structure modeling were used to analyze the data. RESULTS: In the cross validation of the covariance structure modeling, 50.1% of the variance in simple processing, 86.9% in complex processing, and 66.9% in memory/attention were explained. Better education background predicted better score in complex processing ability (path coefficient = .860). Greater independence in self-care ability predicted better scores in simple processing (path coefficient = -.716) and memory/attention (path coefficient = -.811). CONCLUSION: The results of this study indicate that items in the MMSE measuring complex processing ability including writing, reading and obeying, and copy design are seriously biased by educational background, and that the remaining items are better indicators of the subject's cognitive functioning for elderly persons in Taiwan. In our model, self-care ability was strongly related to cognitive functioning as measured by the MMSE, especially for items in the factors of simple processing (registration, naming, repetition, and commanding) and attention/memory (orientation to place and time, attention, and recall).

Activities of Daily Living↗
PubMed1998

Changes in epidermal growth factor receptor expression and competence to generate glia regulate timing and choice of differentiation in the retina.

Previous studies demonstrated that the level of epidermal growth factor receptors (EGF-Rs) expressed by progenitor cells in the newborn (P0) rat retina was limiting for the generation of Muller glial cells but not for proliferation. To determine whether EGF-R signaling biases cells to generate a specific cell type or regulates more general processes during progenitor cell development, we have introduced extra copies of the EGF-R into progenitor cells at earlier stages (E15 and E18), when different cell types are produced. We show that progenitor cells in early embryonic retina (E15) normally express lower levels of EGF-Rs than progenitor cells in later retina (E18 and P0). Whereas lower levels of stimulation of endogenous and virally transduced EGF-Rs enhanced proliferation, higher levels reduced proliferation, resulting in premature differentiation. At E15, very few EGF-R-Infected progenitor cells differentiated prematurely into Muller glial cells, unlike E18 and P0 cells, even when they were exposed to an older retinal environment. Higher levels of EGF-R-mediated signaling alone therefore do not specify a glial fate, indicating that competence to generate glia is temporally regulated by additional mechanisms. The differences in EGF-R expression observed among retinal progenitor cells at distinct developmental stages may instead help to define signaling thresholds which delay or accelerate their differentiation.

3T3 Cells↗
PubMed1981

Copy number control and incompatibility of plasmid R1: identification of a protein that seems to be involved in both processes.

Investigations into the genetic determinants for incompatibility of miniplasmids and hybrid replicons constructed from wide type and mutant R1 revealed the presence of an incompatibility function at the junction f two small PstI fragments. These two fragments were not distinguished in earlier experiments since they have the same mobility on agarose gels. This incompatibility function is distinct from other inc-determinants of R1 (Kollek and Goebel 1979; Molin and Nordström, 1980) and independent of R1-type replication. By means of specific deletions and subcloning of DNA fragments, the location of this new inc-determinant could be determined further. After deletion of this inc-determinant from inc-determinant from miniplasmids, a 5-fold increase in copy number was observed which could then be reduced to a copy number of about 1 plasmid per cell by complementation with hybrid plasmids having this function. Incompatibility of miniplasmids deleted in this determinant is not reduced, whereas analogous deletions introduced into recombinant plasmids nearly abolished their incompatibility. This determinant seems to exert strong incompatibility only when cloned on pBR322. Therefore, its main function is plasmid R1 is probably restricted to copy control. The appearance of low copy numbers of of miniplasmids carrying this determinant and of trans-acting copy control and strong incompatibility exerted by hybrid plasmids is consistently correlated with the presence of a protein of 11,000 molecular weight, synthesized in relatively large amounts in Escherichia coli minicells.

Bacterial Proteins↗