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PubMed1987

Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters.

Specific interactions between T7 RNA polymerase and its promoter have been studied by a simple steady-state kinetic assay using synthetic oligonucleotide promoters that produce a short five-base message. A series of promoters with upstream lengths extending to promoter positions -19, -17, -14, and -12 show that promoters extending to -19 and -17 produce very specific transcripts with initiation rate constant Kcat = 50 min-1 and a Michaelis constant Km = 0.02 microM, indicating that the consensus sequence to position -17 is sufficient for maximum promoter usage. Shortening the upstream region of the promoter to -14 substantially increases Km (0.3 microM) but does not significantly reduce the maximum velocity (kcat = 30 min-1). Finally, truncation of the promoter at position -12 results in extremely low levels of specific transcription. The coding and noncoding strands appear to make different contributions to promoter recognition. Although the double-stranded promoter of upstream length -12 is very poor as a transcription template, extension of only the noncoding strand to -17 very significantly improves both Kcat and Km. In contrast, extension of only the coding strand results in no significant improvement. Substitution of an AT base pair at position -10 by CG (as found in T3 RNA polymerase promoters) produces a 10-fold increase in Km, with little effect on Kcat. Comparison of two promoters containing a base pair mismatch at this site (AG or CT) demonstrates that promoter recognition is very sensitive to the nature of the base on the noncoding strand and is only slightly affected by the presence of a mismatch created by a wrong base in the coding strands.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed2004

The effect of colour congruency on shape discriminations of novel objects.

Although visual object recognition is primarily shape driven, colour assists the recognition of some objects. It is unclear, however, just how colour information is coded with respect to shape in long-term memory and how the availability of colour in the visual image facilitates object recognition. We examined the role of colour in the recognition of novel, 3-D objects by manipulating the congruency of object colour across the study and test phases, using an old/new shape-identification task. In experiment 1, we found that participants were faster at correctly identifying old objects on the basis of shape information when these objects were presented in their original colour, rather than in a different colour. In experiments 2 and 3, we found that participants were faster at correctly identifying old objects on the basis of shape information when these objects were presented with their original part-colour conjunctions, rather than in different or in reversed part-colour conjunctions. In experiment 4, we found that participants were quite poor at the verbal recall of part-colour conjunctions for correctly identified old objects, presented as grey-scale images at test. In experiment 5, we found that participants were significantly slower at correctly identifying old objects when object colour was incongruent across study and test, than when background colour was incongruent across study and test. The results of these experiments suggest that both shape and colour information are stored as part of the long-term representation of these novel objects. Results are discussed in terms of how colour might be coded with respect to shape in stored object representations.

Adult↗

PubMed1994

Evidence for adenine methylation within the mouse myogenic gene Myo-D1.

Previous studies have indicated that there may be uncleavable TaqI sites (TCGA) within the mouse myogenic gene, Myo-D1. Fragments of DNA bearing most of the presumed insensitive TaqI sites have been reproduced using PCR. The presence of each of the originally uncleavable TaqI sites has been confirmed and each TaqI site has been shown to be sensitive to TaqI hydrolysis in PCR-synthesized genomic DNA. Since TaqI is inhibited by methylation of the adenine residue within its recognition sequence (but not by cytosine methylation), it is suggested that specific adenine bases are methylated in the coding region of Myo-DI and maintained throughout cell division. The same TaqI recognition sequences are insensitive to digestion in genomic DNA isolated from various mouse tissues including fetus, regenerating skeletal muscle and a myogenic cell line, all of which express Myo-D1. Thus, adenine methylation is not a modification of DNA following gametic fusion nor does it appear to play a major role in regulation of Myo-D1 expression.

Adenine↗

PubMed2000

Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development.

RNA binding proteins play key roles in the post-transcriptional regulation of gene expression. Here we present the molecular cloning and spatio-temporal expression of Xseb-4, which codes for a putative RNA binding protein containing a single RNA recognition motif (RRM). XSEB-4 shares 60-65% identity with the mammalian SEB-4 proteins. Xseb-4 is strongly expressed maternally. Zygotic transcription is initiated in the early gastrula embryo in paraxial mesoderm that is fated to give rise to somites. During the course of gastrulation and neurulation Xseb-4 expression in somitic paraxial mesoderm is centered within the XmyoD expression domain. As development proceeds Xseb-4 expression is in addition initiated in the cardiac primordium and the lens vesicle. In the heart expression is confined to the myocardium. Thus, the RRM-containing putative RNA binding protein XSEB-4 is differentially expressed during embryonic development in Xenopus.

Amino Acid Motifs↗

PubMed1997

Recognition of dengue virus NS1-NS2a proteins by human CD4+ cytotoxic T lymphocyte clones.

We examined nine dengue virus-specific human CD4+ CD8- cytotoxic T lymphocyte (CTL) clones for protein recognition, using recombinant vaccinia viruses which contain genes coding for dengue virus proteins. These clones were established from peripheral blood mononuclear cells of a donor previously immunized with a live-attenuated experimental dengue-1 vaccine. Of nine CD4+ T cell clones, seven were dengue-1-specific and two were dengue-1-dengue-3 cross-reactive. Four dengue-1-specific clones and one dengue-1-dengue-3 cross-reactive clone recognized epitopes within the NS1 or NS2a proteins. Analysis of human leukocyte antigen (HLA) restriction revealed that three dengue-1-specific clones are HLA-DR1-restricted and one dengue-1-dengue-3 cross-reactive clone is HLA-DPw3-restricted. These results indicate that NS1 and NS2a proteins as well as C, E, and NS3 proteins reported earlier contain one or more epitopes recognized by dengue virus-specific human CD4+ T lymphocytes.

Antigens, Viral↗

PubMed1979

Computer-aided analysis of chromosomal aberrations occurring in an abnormal human karyotype.

A semi-automatic method for recognition of chromosomal changes is presented: from a coded description of an abnormal chromosome (formulated with the aid of an R-banding technique), a computer program makes simulations to reconstitute this chromosomal image from a group of normal chromosomes. Nine types of aberrations (see text) can thus be recognized and the aggregate structure of a group of abnormal chromosomes occurring in a human karyotype can also be rediscovered.

Chromosome Aberrations↗

PubMed1996

Gip-2 codon 179 oncogene mutations: absent in adrenal cortical tumors.

The role of G protein mutations in the pathogenesis of adrenal cortex neoplasms is controversial. Two published studies disagree on the existence of a cysteine or histidine for arginine substitution at position 179 (R179C/H) of the GTP binding region of the alpha chain of an inhibitory G protein (Gi2alpha) in these tumors. Prior studies using detection by mutation-specific oligonucleotide hybridization showed either 3 of 11 or 0 of 56 tumors harbored mutations. To resolve this discrepancy and ascertain the importance of the R179C/H Gi2alpha mutation in the development of adrenal cortex tumors, we screened tumors from 29 patients (24 with adenoma, 5 with carcinoma) using a more sensitive assay employing polymerase chain reaction (PCR) and examination for restriction fragment length polymorphisms (RFLP). Detection of the potential R179C/H mutation by this technique was possible because the wild-type coding sequence includes the BSTU-1 restriction endonuclease recognition site CGCG, whereas the mutated gene does not. Results showed complete digestion of the amplified DNA samples from all 29 patients and the negative control DNA by BSTU-1, indicating that all tumor samples exhibited only the wild-type sequence. Direct sequencing of PCR product from four tumor samples confirmed the presence of only the wild-type sequence. The 0 of 29 rate of R179C/H mutations we found in Gi2alpha is different than the 3 of 11 positive rate (p < 0.05, Fishers' exact) previously reported but agrees with the report showing 0 of 56 mutations. We conclude a mutation at position 179 of Gi2alpha is not important in the pathogenesis of most adrenal cortical tumors.

Adenoma↗

PubMed2003

A comprehensive search for HNF-3alpha-regulated genes in mouse hepatoma cells by 60K cDNA microarray and chromatin immunoprecipitation/PCR analysis.

To characterize the regulatory pattern by a specific transcription regulatory factor, we used a combination of expression analysis with the mouse cDNA microarray composed of 60,000 cDNA clones and cross-linking/chromatin immunoprecipitation (X-ChIP) followed by comparative PCR. Overexpression of mouse hepatocyte nuclear factor-3alpha (HNF-3alpha) in a mouse hepatoma cell line resulted in accompanied perturbed expression of more than 1500 genes. Search for HNF-3alpha consensus recognition sequences in the upstream regions of their coding sequences, which were mapped on the mouse genome, enabled us to mine 300 genes as the potential HNF-3alpha-regulated genes and classify 135 annotated ones into several functional categories. Further X-ChIP/PCR analysis demonstrated in vivo binding of HNF-3alpha to the 5(')-flanking sequences of 25 members selected out of these genes. Besides known HNF-3alpha-regulated genes such as albumin and alpha-fetoprotein genes, the genes newly identified as the HNF-3alpha-regulated ones include three encoding CDP-diacylglycerol-inositol 3-phosphatidyltransferase, phosphatidylserine decarboxylase, and phospholipase A2, which are located en suite in the lipid metabolic pathway in liver. The potential usefulness of the present approach to extensive characterization of gene expression framework directed by a specific transcription regulatory factor is discussed.

5' Flanking Region↗

PubMed2004

Method for producing coded micro-carrier and test method by using a novel type biochip.

This paper provides a method for producing a novel type coded micro-carrier. A simple and cost effective solution for bio-molecule applications was developed. Application relevant items such as manufacture process, biospecific interaction, and analysis method are discussed. For low cost fabrication, the use of LIGA-like process is suggested. LIGA-like process is used as a dry patterning process in which an intense beam of light from an excimer laser is used to pattern a material directly. This process has found extensive application in the microelectronics industry for patterning of polymer materials. The use of LIGA-like techniques offers two attractive features: first, we can cut the polymer into many tiny micro-carriers with micrometer precision. Second, LIGA-like process allows to encode with high precision spatial information onto the micro-carrier that can be used in the identification of the bio-molecule. This paper gives a description of the basic idea, describes the fabrication of the novel micro-carrier that we called "coded micro-carrier," and of the image processing algorithms used for the analysis of bio-molecules. This study also provides a test method for identifying a bio-molecule, which includes mixing several coded micro-carriers with the hybridized unknown bio-molecules; and identifying the codes on the micro-carrier via image recognition system. The numbers and types of the known micro-carrier can be flexibly adjusted according to the number of tested bio-molecules.

Animals↗

PubMed2005

Simultaneous production of two foreign proteins from a polyvirus-based vector.

With the aim of developing a biotechnological tool for the production of foreign proteins in plants, we first engineered an infectious turnip mosaic virus (TuMV) cDNA that contained the jellyfish green fluorescent protein (GFP) gene or the bacterial beta-glucuronidase (GUS) gene (uidA). Two insertion sites were assessed, either between P1 and HCPro cistrons or Pol and CP cistrons. In each construct, the junctions flanking the inserted gene coded for P1 and/or VPg-Pro cleavage recognition site sequences, to produce free GUS or GFP. After transfection by particle bombardment on Brassica perviridis, characteristic symptoms for TuMV infection appeared and Western blot analyses showed that GFP and GUS had been excised from the viral polyprotein. No significant differences in expression level were noticed between the two insertion sites. By RT-PCR, gfp was found to be stable over 30 days post-transfection (dpt) while uidA was gradually lost at 15 dpt. We also created two constructs containing either gene at each insertion sites on the same molecule. Attenuated systemic symptoms were observed after particle bombardment on B. perviridis and Western blot analyses showed that both foreign proteins were produced. Also, the same stability/instability as for the single-gene constructs were observed. These results indicate that it is possible to produce at least two foreign proteins simultaneously in a TuMV-based vector.

Amino Acid Sequence↗

PubMed2004

Processing concrete words: fMRI evidence against a specific right-hemisphere involvement.

Behavioral, patient, and electrophysiological studies have been taken as support for the assumption that processing of abstract words is confined to the left hemisphere, whereas concrete words are processed also by right-hemispheric brain areas. These are thought to provide additional information from an imaginal representational system, as postulated in the dual-coding theory of memory and cognition. Here we report new event-related fMRI data on the processing of concrete and abstract words in a lexical decision task. While abstract words activated a subregion of the left inferior frontal gyrus (BA 45) more strongly than concrete words, specific activity for concrete words was observed in the left basal temporal cortex. These data as well as data from other neuroimaging studies reviewed here are not compatible with the assumption of a specific right-hemispheric involvement for concrete words. The combined findings rather suggest a revised view of the neuroanatomical bases of the imaginal representational system assumed in the dual-coding theory, at least with respect to word recognition.

Adult↗

PubMed2002

Comparison of speech perception benefits with SPEAK and ACE coding strategies in pediatric Nucleus CI24M cochlear implant recipients.

Nine congenitally deaf children who received a Nucleus CI24M cochlear implant and who were fitted with the SPrint speech processor participated in this study. All subjects were initially programmed with the SPEAK coding strategy and then converted to the ACE strategy. Speech perception was evaluated before and after conversion to the new coding strategy using word and Common Phrase speech recognition tests in both the presence and absence of noise. In quiet conditions, the mean percent correct scores for words were 68.8% with SPEAK and 91% with ACE; for phrases the percentage was 66.6% with SPEAK and 85.5% with ACE. In the presence of noise (at +10 dB signal-to-noise ratio), the mean percent correct scores for words were 43.3% with SPEAK compared to 84.4% with ACE; for phrases the percentage was 41.1% with SPEAK and 82.2% with ACE. Statistical analysis revealed significant improvement in open-set speech recognition with ACE compared to SPEAK. Preliminary data suggest that converting children from SPEAK to the ACE strategy improves their performance. Subjects showed significant improvements for open-set word and sentence recognition in quiet as well as in noise when ACE was used in comparison with SPEAK. The greatest improvements were obtained when tests were presented in the presence of noise.

Acoustic Stimulation↗

PubMed2002

Real-time RT-PCR for quantitation of hepatitis C virus RNA.

A newly developed real-time RT-polymerase chain reaction assay for quantitation of hepatitis C virus (HCV) RNA in human plasma and serum was applied. A pair of primers and a probe (molecular beacon) were designed that are specific for the recognition of a highly conservative 5'-non-coding region (5'-NCR) in HCV genome. HCV real-time RT-PCR assay had a sensitivity of 1000 RNA copies per reaction, with a dynamic range of detection between 10(3) and 10(7) RNA copies. The coefficient variation of threshold cycle (Ct) values in intra- and inter-runs were less than 1.37 and 4.66%, respectively. The real-time RT-PCR assay on the HCV sero-positive samples yielded reproducible data, with less than 2.09% of the inter-assay variation. In order to determine its potential for clinical diagnosis, real-time RT-PCR was used to examine the HCV RNA levels in plasma from sero-positive and negative subjects, showing that the assay is highly sensitive and has specificity of 100%. It was demonstrated that the real-time RT-PCR was able to amplify HCV RNA in reference sera with seven genotypes (1A, 1B, 2B, 3A, 4, 5A and 6A) that include six major HCV genotypes circulated in the world. Since HCV is a major pathogen of post-transfusion and community-transmitted non-A, non-B hepatitis, this assay has a broad application for basic and clinical investigations.

5' Untranslated Regions↗

PubMed2000

The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis.

We have previously used Tn917 for the identification and characterization of regulated promoters from Lactococcus lactis [Israelsen et al., Appl. Environ. Microbiol. 61 (1995) 2540-2547]. We describe here the construction of a new Tn917-transposon derivative, termed TnNuc, which includes the Staphylococcus aureus nuclease gene (nuc) as a reporter for secretion. Transposition of TnNuc into the L. lactis chromosome allows the generation of fusions in-frame with the nuc gene. TnNuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e. clones with Lac+ phenotype result from transposition of TnNuc into a functional gene on the L. lactis chromosome. The presence of a functional signal sequence at the upstream flanking region of the left repeat of the transposed element results in the detection of nuclease activity using a sensitive plate assay. TnNuc was used for the identification of novel secretion signals from L. lactis. The sequences identified included known and unknown lactococcal-secreted proteins containing either a signal peptidase-I or -II recognition sequence. In one case, the gene identified codes for a transmembrane protein. The sequences identified were used to study functionality when located in a plasmid under the control of the pH and growth phase-dependent promoter P170 [Madsen et al., Mol. Microbiol. 32 (1999) 75-87]. In all cases, concurrent secretion of nuclease was observed during induction of P170 in a fermentor.

Bacterial Proteins↗

PubMed2006

Additive genetic variation of transcriptional regulation: metallothionein expression in the soil insect Orchesella cincta.

Field-selected metal tolerance in Orchesella cincta is correlated with overexpression of the single copy cadmium (Cd) inducible metallothionein (mt). Previously, we have demonstrated large phenotypic variation in mt gene expression, and a higher frequency of high-expression phenotypes in a tolerant population. Here, we describe midparent-offspring regression analysis of mt gene expression in a laboratory culture originating from a noncontaminated natural population. Families were either not exposed (n=47) or exposed to 0.5 micromol Cd per gram dry food (n=46). Mean mt gene expressions normalized to 28S rRNA and beta-actin RNA were generated using real-time RT-PCR applied to parents and offspring RNA and subjected to regression analysis. A significant heritability (h2) for mt gene expression was estimated between 0.36 (beta-actin normalized) and 0.46 (28S normalized) in Cd exposed families. Nontreated families did not yield a significant h2 value. Restriction Fragment Length Polymorphism analysis of the metallothionein promoter sequence revealed eight promoter alleles that show structural variation. Three alleles show increased frequencies in families with high mt expression. Another gene, croquemort (isolated from a differential screening for 1 micromole Cd treatment) showed no h2 of gene expression in response to 0.5 micromol Cd. This gene codes for a receptor-protein involved in recognition of apoptotic cells and may participate in the general stress response. The present data suggest that evolution of metal tolerance in O. cincta can occur in the field by selection for high mt expression due to structural changes in mt cis-regulation.

Adaptation, Physiological↗

PubMed2002

A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator Zac-1.

p53 exerts its tumor suppressor effects by activating genes involved in cell growth arrest and programmed cell death. The p53 target genes inducing growth arrest are well defined whereas those inducing apoptosis are not fully characterized. Proapoptotic activity of p53 was shown to involve several genes like Bax, Noxa and Puma, which may function in the release of cytochrome c from the mitochondria. Cytochrome c associates with Apaf-1 and caspase 9 to form the apoptosome. Genetic and cellular data indicate that Apaf-1 deficiency abrogates the apoptotic effect of p53 and substitutes for p53 loss in promoting tumor formation. Here we show that Apaf-1, the mammalian homologue of C. elegans CED4, is a direct target of p53 as demonstrated by gel shift analysis of the target site sequence in the presence of p53 and by Apaf-1 promoter-luciferase assays. We also show that the p53 activation of the Apaf-1 luciferase construct can be enhanced by the putative tumor suppressor gene product, Zac-1, a transcription factor that has previously been shown to inhibit cell proliferation. Furthermore, we demonstrate that Zac-1 is a possible direct target of p53 since the sequence upstream to the first coding exon of Zac-1 contains a p53 recognition site and the luciferase construct containing this region is activated by p53. These results suggests the existence of a tightly controlled self amplifying mechanism of transcriptional activation leading to apoptosis by p53.

Animals↗

PubMed2001

Intragenic promoter adaptation and facilitated RNA polymerase III recycling in the transcription of SCR1, the 7SL RNA gene of Saccharomyces cerevisiae.

The SCR1 gene, coding for the 7SL RNA of the signal recognition particle, is the last known class III gene of Saccharomyces cerevisiae that remains to be characterized with respect to its mode of transcription and promoter organization. We show here that SCR1 represents a unique case of a non-tRNA class III gene in which intragenic promoter elements (the TFIIIC-binding A- and B-blocks), corresponding to the D and TpsiC arms of mature tRNAs, have been adapted to a structurally different small RNA without losing their transcriptional function. In fact, despite the presence of an upstream canonical TATA box, SCR1 transcription strictly depends on the presence of functional, albeit quite unusual, A- and B-blocks and requires all the basal components of the RNA polymerase III transcription apparatus, including TFIIIC. Accordingly, TFIIIC was found to protect from DNase I digestion an 80-bp region comprising the A- and B-blocks. B-block inactivation completely compromised TFIIIC binding and transcription capacity in vitro and in vivo. An inactivating mutation in the A-block selectively affected TFIIIC binding to this promoter element but resulted in much more dramatic impairment of in vivo than in vitro transcription. Transcriptional competition and nucleosome disruption experiments showed that this stronger in vivo defect is due to a reduced ability of A-block-mutated SCR1 to compete with other genes for TFIIIC binding and to counteract the assembly of repressive chromatin structures through TFIIIC recruitment. A kinetic analysis further revealed that facilitated RNA polymerase III recycling, far from being restricted to typical small sized class III templates, also takes place on the 522-bp-long SCR1 gene, the longest known class III transcriptional unit.

Amino Acid Sequence↗

PubMed2004

Leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus recognizes minihelices.

Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix(Leu) is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (alpha beta-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA(Leu) is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix(Leu) charging by alpha beta-LeuRS, indicating possible domain-domain communication within alpha beta-LeuRS. The leucine-specific domain of alpha beta-LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA(Leu), minihelix(Leu) is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix(Leu) cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of alpha beta-LeuRS.

Adenosine Triphosphate↗