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S Knudsen

Publications and source records attributed to S Knudsen.

At least 19 recordsLinked to original sources
PubMed2002

Prediction of human protein function from post-translational modifications and localization features.

We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects such as the length, isoelectric point and composition of the polypeptide chain.

Computational Biology↗
PubMed2001

Identification of gene expression patterns in superficial and invasive human bladder cancer.

Multiple transcriptional events take place when normal urothelium is transformed into tumor tissue. These can now be monitored simultaneously by the use of oligonucleotide arrays, and expression patterns of superficial and invasive tumors can be established. Single-cell suspensions were prepared from bladder biopsies (36 normal, 29 tumor). Pools of cells were made from normal urothelium and from pTa grade I and II and pT2 grade III and IV bladder tumors. From these suspensions, and from 10 single-tumor biopsies, labeled cRNA was hybridized to oligonucleotide arrays carrying probes for 6500 genes. The obtained expression data were sorted according to a weighting scheme and were subjected to hierarchical cluster analysis of tissues and genes. Northern blotting was used to verify the array data, and immunohistology was used to correlate between RNA and protein levels. Hierarchical clustering of samples correctly identified the stage using both 4076 genes and a subset of 400 genes covarying with the stages and grades of tumors. Hierarchical clustering of gene expression levels identified several stage-characteristic, functionally related clusters, encoding proteins that were related to cell proliferation, oncogenes and growth factors, cell adhesion, immunology, transcription, proteinases, and ribosomes. Northern blotting correlated well with array data. Immunohistology showed a good concordance between transcript level and protein staining. The study indicates that gene expression patterns may be identified in bladder cancer by combining oligonucleotide arrays and cluster analysis. These patterns give new biological insight and may form a basis for the construction of molecular classifiers and for developing new therapy for bladder cancer.

Algorithms↗
PubMed2000

The frequency of mutators in populations of Escherichia coli.

Owing to occasional spontaneous mutations in genes encoding DNA repair, any population of a reasonable size is expected to harbor a sub-population of genetic mutators. Using a genetically modified strain of Escherichia coli K-12, we have estimated the frequency of mutators to be about 3x10(-5). By and large, this corresponds to a mutation rate from non-mutators to mutators of 5x10(-6) per bacterium per generation. Using a mutS∷Tn10 derivative as representative for mutators, we estimated the increase in mutation rates in mutators to be 19- to 82-fold, depending on the test-mutation under consideration. The load associated with this increase in mutation rate resulted in a growth inhibition of 1%. From these data, we estimated that the rate of detrimental mutations in the non-mutators to be 2x10(-4)-8x10(-4). The situations where adaptive mutations may result in an increase in the frequency of mutators are discussed.

Adenosine Triphosphatases↗
PubMed2000

Transformation of barley by microinjection into isolated zygote protoplasts.

Barley zygote protoplasts were mechanically isolated, embedded in agarose droplets, and microinjected with a rice actin promoter Act1-gusA-nos gene construct. On average 62% of the cells survived the injection and of these 55% continued development into embryo-like structures and eventually to plants. PCR screening for the presence of a 307-bp fragment in the middle of the gusA gene showed that on average 21% of the derived structures contained this fragment. However, among the hundreds of injected zygotes, derived structures and regenerants we only found significant GUS expression in two cases (embryo-like structures nine days after injection). Two lines of green plants, derived from zygotes microinjected with linearized plasmid (line A147-1) or an isolated Act1-gusA-nos gene cassette (line A166-h) proved to be transgenic. Line A147-1 appeared to contain a single and intact copy of the expression cassette but a PCR based progeny analysis indicated the presence of additional shorter fragments of the cassette. Line A166-h appeared to contain a single fragment of the gusA gene that was transferred to the progeny as a single Mendelian trait. One additional fragment of the gusA gene was identified in this line. The present data show that transformation of barley by microinjection of DNA into isolated zygotes is feasible but also that gene expression rarely is achieved, possibly due to degradation of the introduced DNA.

Actins↗
PubMed2000

Automatic discovery of regulatory patterns in promoter regions based on whole cell expression data and functional annotation.

MOTIVATION: The whole genomes submitted to GenBank contain valuable information about the function of genes as well as the upstream sequences and whole cell expression provides valuable information on gene regulation. To utilize these large amounts of data for a biological understanding of the regulation of gene expression, new automatic methods for pattern finding are needed. RESULTS: Two word-analysis algorithms for automatic discovery of regulatory sequence elements have been developed. We show that sequence patterns correlated to whole cell expression data can be found using Kolmogorov-Smirnov tests on the raw data, thereby eliminating the need for clustering co-regulated genes. Regulatory elements have also been identified by systematic calculations of the significance of correlations between words found in the functional annotation of genes and DNA words occurring in their promoter regions. Application of these algorithms to the Saccharomyces cerevisiae genome and publicly available DNA array data sets revealed a highly conserved 9-mer occurring in the upstream regions of genes coding for proteasomal subunits. Several other putative and known regulatory elements were also found. AVAILABILITY: Upon request.

Algorithms↗
PubMed1999

Promoter2.0: for the recognition of PolII promoter sequences.

MOTIVATION: A new approach to the prediction of eukaryotic PolII promoters from DNA sequence takes advantage of a combination of elements similar to neural networks and genetic algorithms to recognize a set of discrete subpatterns with variable separation as one pattern: a promoter. The neural networks use as input a small window of DNA sequence, as well as the output of other neural networks. Through the use of genetic algorithms, the weights in the neural networks are optimized to discriminate maximally between promoters and non-promoters. RESULTS: After several thousand generations of optimization, the algorithm was able to discriminate between vertebrate promoter and non-promoter sequences in a test set with a correlation coefficient of 0.63. In addition, all five known transcription start sites on the plus strand of the complete adenovirus genome were within 161 bp of 35 predicted transcription start sites. On standardized test sets consisting of human genomic DNA, the performance of Promoter2.0 compares well with other software developed for the same purpose. AVAILABILITY: Promoter2.0 is available as a Web server at http://www.cbs.dtu. dk/services/promoter/

Algorithms↗
PubMed1998

The effect of the lacY gene on the induction of IPTG inducible promoters, studied in Escherichia coli and Pseudomonas fluorescens.

The role of the Escherichia coli lacY gene product (the lactose permease) in the induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible promoters was studied in E. coli and P. fluorescens. This was done by comparing strains containing a lacIPOZYA chromosomal insert with newly constructed strains containing inserts without the lacY gene (lacIPOZ). The lactose operon inserts were introduced as single-copy chromosomal inserts to eliminate differences in expression caused by differences in copy number. Comparison between the two types of inserts showed that the lactose permease was essential to allow growth on lactose by both bacteria and that the lactose permease plays an important role in transporting the inducer IPTG across the membrane of P. fluorescens. The use of a functional lactose permease allows expression of beta-galactosidase to increase more than fivefold from a wild-type lac promoter in P. fluorescens SS1001. We suggest that an increase in the rate of protein synthesis from lac-type promoters could be enhanced if an active lactose permease is present as well.

Escherichia coli↗
PubMed1998

Combined Epstein-Barr virus and human papillomavirus infection in nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) has been shown to be a likely etiologic agent in nasopharyngeal carcinogenesis. Human papillomaviruses (HPVs) have previously been identified in numerous upper aerodigestive tract carcinomas. This pilot study was undertaken to investigate the prevalence of combined EBV and HPV infection in 17 patients with nasopharyngeal carcinoma (NPCA) using polymerase chain reaction (PCR). The primary goal was to determine if the presence of HPV could be correlated with molecular, histologic, or clinical parameters. There were seven patients with undifferentiated NPCA (World Health Organization [WHO] type III) and 10 patients with squamous cell carcinoma (WHO type I). All 17 patients had stage IV disease at presentation. EBV was identified in 15 patients (88.2%), and HPV subtypes were identified in samples from nine patients (52.9%). All HPV-positive cases were also EBV positive. Western blot analysis of six samples showed a high level of expression of c-myc and cdc2 kinase and a low level of p53 protein in NPCAs that contained both HPV and EBV (n = 3). Increased expression of c-myc and cdc2 kinase was seen in the cases that contained EBV only, but to a lesser extent (n = 2). These findings indicate an effect of the virus on cellular proliferation and differentiation. Similarly, an elevated level of Rb protein was found only in the HPV-containing NPCAs. Moderate differentiation (keratinization) occurred in four of eight HPV-negative and none of the nine HPV-positive NPCAs. (All HPV-positive cases were poorly differentiated or undifferentiated.) This difference is statistically significant for this sample size (P < 0.03). There was a trend for the group that was HPV positive to have WHO III histology and for the HPV-negative group to have WHO I. The presence of HPV could not be correlated with any clinical parameters in this small group of patients with advanced disease; however, these data suggest that coexistence of EBV and HPV infection may be a factor in the pathogenesis of NPCA and may have an effect on regulation of cellular proliferation and differentiation.

Adolescent↗
PubMed1996

Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase.

In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T1 generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0-9.5-fold increase for DHPS. T1 seeds of DHPS transformants showed the same changes in free amino acids as observed in T0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.

Amino Acids↗
PubMed1995

Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei.

Particle gun acceleration appears to be a possible way to transform mycelium cells of obligate plant parasites growing on host surfaces. GUS expression was obtained in E. graminis f.sp. hordei cells after bombardment with the GUS gene under the control of the E. graminis f.sp. hordei &beta;-tubulin promoter. Three heterologous promoters, onefrom Aspergillus nidulans and two from Cochliobolus heterostrophus, gave very low or no expression of GUS.

Ascomycota↗
PubMed1995

Development and testing of improved suicide functions for biological containment of bacteria.

We have developed very efficient suicide functions for biological containment based on the lethal Escherichia coli relF gene. The suicide functions are placed in duplicate within a plasmid and arranged to prevent inactivation by deletion, recombination, and insertional inactivation. The efficiency of this concept was tested in a plasmid containment system that prevents transfer of plasmids to wild-type bacteria. Protection against plasmid transfer was assayed in test tubes and in rat intestine. Protection was efficient and refractory to inactivation by mutation and transposons. The efficiency of the suicide system was also tested in soil and seawater. We show that unprecedented suicide efficiency can be achieved in soil and seawater after suicide induction by IPTG (isopropyl-beta-D-thiogalactopyranoside). More than 7 orders of magnitude reduction in suicide bacteria was achieved.

Animals↗
PubMed1994

Identification of an enhancer/silencer sequence directing the aleurone-specific expression of a barley chitinase gene.

Chitinases are expressed in various plant tissues where they are thought to play a role in defense against chitin-containing pathogens. Transient gene expression assays have been used in tissues of barley to delineate promoter sequences involved in the regulation of an aleurone-specific chitinase gene (Chi26), and of a vegetatively expressed chitinase gene (Chi33). The assays measured the activities of transcriptional fusions between chitinase 5' upstream sequences and GUS reporter genes after DNA delivery by particle bombardment. Analysis of Chi26 5' and 3' promoter deletions indicated that sequences between -200 and -140 confer developmental and aleurone-specific expression. Deletions/replacements covering this part of the promoter indicated that sequences between -179 and -147 (E-region) direct expression in aleurone cells. The ability of the 33bp E-region of the Chi26 promoter to activate transcription specifically in aleurone was confirmed by constructing and testing two types of chimeric promoters. The first type, which contained two copies of the E-region fused to the CaMV 35S TATA box, conferred aleurone-specific expression of a GUS reporter gene. The second type, which contained a single copy of the E-region inserted into a deleted, inactive Chi33 promoter derivative, was also capable of directing transcription in aleurone but not in leaves. The pattern of expression of this and other Chi26/Chi33 chimeric promoters suggest that the E-region contains cis-acting sequences which activate transcription in aleurone and silence transcription in leaves. DNA sequence motifs implicated in the regulation of Chi26 and Chi33 are described.

Base Sequence↗
PubMed1993

The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box.

The 431 bp C-hordein promoter of lambda-1-17 exhibits a specific response to amino acids and NH4NO3 in developing barley (Hordeum vulgare L.) endosperms. With the aid of particle bombardment it is shown that the GCN4 motif ATGA(C/G)TCAT is the dominating cis-acting element in this response. But synergistic interaction with the neighbouring endosperm motif TGTAAAGT within the bifactorial prolamin element and cooperation with upstream sequences including a second prolamin-like element is an absolute requirement for a strong, positive regulation by an optimal nitrogen regime. Low nitrogen levels convert the GCN4 box into a negative motif. In contrast the endosperm box on its own exerted a silencing activity, independent of nitrogen nutrition. Sequence comparisons revealed that GCN4- and endosperm-like motifs are widely distributed among plant promoters. Their putative role in nitrogen regulation is discussed.

Base Sequence↗
PubMed1993

Molecular characterization of the gene for carrot cell wall beta-fructosidase.

Carrot cell wall beta-fructosidase, previously purified and cloned, is encoded by a single, wound- and pathogen-inducible gene. The developmental regulation of the gene was studied by determining the steady-state mRNA levels in different organs during carrot development: cell wall beta-fructosidase mRNA was detected in roots and leaves of young plants but not during tap root development. A genomic clone was isolated and characterized. The transcription start site was determined by primer extension analysis. Inspection of the promoter sequence (1488 bp) revealed the presence of sequences with high homology to cis-acting elements for the regulation of plant genes by wounding and infection. The 5'-regulatory sequence was fused to the reporter gene beta-glucuronidase (GUS) and tested in a transient expression assay with carrot suspension cells and wounded carrot root tissue (aged disks of carrot roots). The expression of the GUS gene in the transfected cells proved that the isolated promoter was functional. In transgenic tobacco plants containing the cell wall beta-fructosidase promoter fused to GUS, the reporter gene was predominantly expressed in the shoot and root meristems of young seedlings. No GUS expression was detected in mature tobacco plants, showing that the development-specific regulation of the cell wall beta-fructosidase promoter seen in carrot was maintained in tobacco plants. In contrast, expression of the GUS reporter gene in transgenic tobacco was not wound inducible. To analyze the functional organization of the cell wall beta-fructosidase promoter, a 5'-deletion series was generated and tested in a transient expression assay in protoplasts of Nicotiana plumbaginifolia. Two regions containing putative silencer elements were identified. A comparison of these regions with known silencer elements identified in both regions one copy of the negative dominant cis-acting element found in a chalcone synthase promoter of petunia.

Amino Acid Sequence↗
PubMed1993

Expression of the dihydroflavonol reductase gene in an anthocyanin-free barley mutant.

The barley gene encoding dihydroflavonol-4-reductase (DFR) was delivered by micoprojectile bombardment into leaf sheath tissue of the anthocyanin-free barley mutant ant 18-162, a mutant which lacks DFR activity-probably because of a missense mutation in the structural gene for DFR. The delivered gene complemented the mutation, as evidenced by the synthesis of anthocyanin in individual leaf sheath cells of the bombarded tissues. Pigment synthesis appeared two days after gene delivery and both the number of pigmented cells and the intensity of pigmentation increased over the following days. Depending on the physiological condition of the host plants, up to 15 pigmented cells per 10 tissue segments were detected. These results demonstrate that the Ant 18 gene of barley encodes dihydroflavonol-4-reductase. A series of gene constructs encoding DFR were expressed in the anthocyanin-free mutant tissue. The genomic clone complemented the mutation whereas an equivalent plasmid with all introns deleted did not. The highest number of pigmented cells was obtained using plasmids containing the DFR-coding sequence interrupted by intron 1 of the genomic clone, indicating that the presence of an intron stabilizes the DFR message.

Alcohol Oxidoreductases↗