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S Knudsen

Publications and source records attributed to S Knudsen.

29 records · Page 2Linked to original sources
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↗
PubMed1992

G+C-rich tract in 5' end of human introns.

Analysis of an artificial neural network trained to classify DNA as coding or non-coding revealed compositional differences between sequence parts translated into protein and those that were not. The 5' end of human introns was found to have a base composition that was non-random to an extent matching the non-randomness in the 3' end that contains the polypyrimidine tract. The prevailing nucleotides in the initial 50 nucleotides of human introns are guanine and cytosine, the trinucleotide GGG was found to occur almost four times as frequently as it would in sequences with a uniform distribution of the nucleotides. The initial part of terminal exons and their associated terminal introns were shown to have a very special base composition deviating strongly from the normal picture in other exons and introns.

Base Composition↗
PubMed1992

Prediction of gene structure.

We have developed a hierarchical rule base system for identifying genes in DNA sequences. Atomic sites (such as initiation codons, stop codons, acceptor sites and donor sites) are identified by a number of different methods and evaluated by a set of filters and rules chosen to maximize sensitivity; these are combined into higher-order gene elements (such as exons), evaluated, filtered and combined as equivalence classes into probable genes, which are evaluated and ranked. The system has been tested on an extensive collection of vertebrate genes smaller than 15,000 bases. Results obtained show that, on average, 88% of the predicted coding region for a transcription unit is actually coding, and 80% of the actual coding is correctly predicted. This will, in most applications, be sufficient for a search against protein sequence databases for the identification of probable gene function. In addition, the system provides a general test platform for both gene atomic site identification and the rules for their evaluation and assembly.

Algorithms↗
PubMed1991

Prediction of human mRNA donor and acceptor sites from the DNA sequence.

Artificial neural networks have been applied to the prediction of splice site location in human pre-mRNA. A joint prediction scheme where prediction of transition regions between introns and exons regulates a cutoff level for splice site assignment was able to predict splice site locations with confidence levels far better than previously reported in the literature. The problem of predicting donor and acceptor sites in human genes is hampered by the presence of numerous amounts of false positives: here, the distribution of these false splice sites is examined and linked to a possible scenario for the splicing mechanism in vivo. When the presented method detects 95% of the true donor and acceptor sites, it makes less than 0.1% false donor site assignments and less than 0.4% false acceptor site assignments. For the large data set used in this study, this means that on average there are one and a half false donor sites per true donor site and six false acceptor sites per true acceptor site. With the joint assignment method, more than a fifth of the true donor sites and around one fourth of the true acceptor sites could be detected without accompaniment of any false positive predictions. Highly confident splice sites could not be isolated with a widely used weight matrix method or by separate splice site networks. A complementary relation between the confidence levels of the coding/non-coding and the separate splice site networks was observed, with many weak splice sites having sharp transitions in the coding/non-coding signal and many stronger splice sites having more ill-defined transitions between coding and non-coding.

Base Sequence↗
PubMed1991

Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley.

A 1420 bp genomic fragment (lambda-hor1-17) encompassing a Hor-1 gene encoding a C-hordein polypeptide is presented. The deduced amino acid sequence is 261 residues long. It comprises a 20 amino acid signal peptide, unique NH2- and COOH-terminal regions and a coding region comprised of pentapeptide (PQQPY) and octapeptide (PQQPFPQQ) repeat motifs. The 431 bp of 5' non-coding region contains a 'TATA box' at -105, a 'CACA box' (-181 to -201) and a -300 prolamin element. In the 3' non-coding region there are two putative polyadenylation signals located 88 and 142 bp downstream of the stop codon. The structure of lambda-hor1-17 is compared with that of another gene (lambda-hor1-14) encoding a C-hordein polypeptide, which contains an amber codon interrupting the ORF. A functional assay in which the 5' non-coding regions of the two genes were fused to the beta-glucuronidase (GUS) gene demonstrated that both genes were transcriptionally active and that circa 430 bp of the C-hordein promoters were sufficient to drive the expression of the GUS gene in developing barley endosperms. It also demonstrated that both promoters had transcriptional efficiencies comparable with that of the 35S CaMV promoter. The in vitro translation of the coding region of lambda-hor1-14 in the wheat germ system showed that the premature stop codon could be partially suppressed. The suppression was also demonstrated in a transient expression assay in vivo using isolated barley endosperms.

Amino Acid Sequence↗
PubMed1990

Neural network detects errors in the assignment of mRNA splice sites.

The use of databanks in genetic research assumes reliability of the information they contain. Currently, error-detection in the manually or electronically entered data contained in the nucleotide sequence databanks at EMBL, Heidelberg and GenBank at Los Alamos is limited. We have used a subset of sequences from these databanks to train neural networks to recognize pre-mRNA splicing signals in human genes. During the training on 33 human genes from the EMBL databank seven genes appeared to disturb the learning process. Subsequent investigation revealed discrepancies from the original published papers, for three genes. In four genes, we found wrongly assigned splicing frames of introns. We believe this to be a reflection of the fact that splicing frames cannot always be unambiguously assigned on the basis of experimental data. Thus incorrect assignment appear both due to mere typographical misprints as well as erroneous interpretation of experiments. Training on 241 human sequences from GenBank revealed nine new errors. We propose that such errors could be detected by computer algorithms designed to check the consistency of data prior to their incorporation in databanks.

Algorithms↗