1,720,965 research outputs found
Structure of dihydrofolate reductase-thymidylate synthase gene (Accession No. AJ003139) from Daucus carota. (PGR98-055)
No full textA reliable amplification technique with single-sided specificity for the isolation of 5' gene regulating regions.
No full textA simple and efficient method is described for the isolation of extension fragments of known DNA sequences by polymerase chain reaction (PCR) using a single specific primer. With this method, size-selected genomic DNA fragments are ligated to a plasmid Vector (pGEM-4Z) which contains sequencing primers and the population of chimeric plasmids is used for transforming Escherichia coli. DNA is extracted from an aliquot of the resulting mini-library and PCR performed using a sequence-specific primer and either of the standard sequencing primers of the plasmid vector. This method appears to be more Versatile than inverse PCR (IPCR), since: ii) the DNA sequence needed as the specific primer can be as short as about 20 nucleotides (nt); (ii) the DNA templates to be used in PCR are available in high amount, thus facilitating all manipulations; and (iii) if relinearization of the DNA by restriction enzyme digestion is desired before the PCR reaction, many restriction sites can be chosen from the vector polylinker. Using this method, we have isolated the genomic 5' region of the carrot bifunctional dihydrofolate reductase-thymidylate synthase-encoding gene dhfr-ts using a 21-nt sequence of the 5' region of the dhfr-ts cDNA clone as the specific primer
Amplification of Gene-Regulating Regions with Single-Sided Specificity.
No full textFRom molecular cloning to Genetic engineerin
Analysis of the structure of the 5' end of the gene coding for carrot dihydrofolate reductase-thymidylate synthase.
No full textCarrot cells contain two transcript types for the dhfr-ts gene and evidence was obtained for a plastidial localization of the product of the longer transcripts. This indication contrasted with previous circumstantial evidence which suggested a cytosolic Iodization. The conflicting evidence was found to bt due to the presence of a large intron in the region corresponding to the 5' end of the mRNA. In this paper we report the structure of the genomic region corresponding to the 5' end of the carrot DHFR-TS-encoding gene and the cloning by inverse polymerase chain reaction of the promoter region which has been partially sequenced showing the presence of putative TATA boxes
and AT-rich regions. It is also observed that the two dhfr-ts gene transcripts contain TC repeats and a TC-rich stretch, respectively, whose possible role in the regulation of gene expression is discussed
Cloning and characterisation of a carrot cDNA coding for a WD repeat protein homologous to Drosophila fizzy and CDC20-like proteins
No full textThe present study describes the isolation of a cDNA coding for a carrot protein of 450 amino acids that contains WD repeats (DcWD1) and is homologous to Drosophila melanogaster fizzy protein, mammalian p55CDC and yeast Cdc20p. As for the known related proteins, sequence conservation concerned the majority of the polypeptide except the far N-terminus. Results of Southern blot analysis with genomic DNA under high stringency conditions showed the occurrence of a single gene. Northern blot analyses revealed the accumulation of DcWD1 mRNA in all tested tissues (leaves, petioles and hypocotyls, apical meristems, roots and suspension cultured cells), though at a different extent. Lack of induction of relevant transcripts in proliferating auxin-stimulated hypocotyls suggests a mode of expression not strictly related to the cell proliferation
Molecular cloning and analysis of a cDNA coding for the bifunctional dihydrofolate reductase-thymidylate synthase of Daucus carota.
Full text linkMolecular cloning of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota was
achieved by immunoscreening of a cDNA library obtaining a 2 kbp clone which contains an open reading
frame of 1528 bp. Comparison of the deduced amino acid sequence with those from other sources revealed the presence of motifs typical of DHFR and TS thus confirming the bifunctional nature of the carrot protein. As in other organisms, a higher degree of conservation was observed in the TS domain. Analysis of the dhfr-ts gene content in carrot revealed the presence of several copies per diploid genome
Multiple transcription start sites of the carrot dihydrofolate reductase-thymidylate synthase and sub-cellular localisation of the bifunctional protein
No full textThe analysis of clones obtained by rapid amplification of the 5' end and by primer extension of the mRNA for carrot bifunctional dihydrofolate reductase-thymidylate synthase showed transcripts of differing lengths that belonged to two sub-populations. The longer transcripts were found to contain a translation start site 147 nt upstream of, and in frame with, the one which is present in the shorter transcripts. The ORF that begins at this ATG codes for a protein of 64 714 Da, which is much larger than mature DHFR-TS subunit. The N-terminus region of this polypeptide shows features typical of plant transit peptides. Immunogold labelling studies and immunorecognition of the plastid-containing sub-cellular fraction suggested a plastidial localisation of the bifunctional protein. Although plant cells were shown to contain folate pools in plastids, in mitochondria and in the cytosol, few enzymes of the folate pathway have been associated with any sub-cellular compartment. Thus, this is the first indication for the presence of an enzyme of the folate biosynthetic pathway in plastids. The longer transcripts revealed the presence of a TC microsatellite at the 5'-untranslated end
Cloning and characterisation of a carrot cDNA coding for a WD repeat protein homologous to Drosophila fizzy and CDC20-like proteins
No full textThe present study describes the isolation of a cDNA coding for a carrot protein of 450 amino acids that contains WD repeats (DcWD1) and is homologous to Drosophila melanogaster fizzy protein, mammalian p55CDC and yeast Cdc20p. As for the known related proteins, sequence conservation concerned the majority of the polypeptide except the far N-terminus. Results of Southern blot analysis with genomic DNA under high stringency conditions showed the occurrence of a single gene. Northern blot analyses revealed the accumulation of DcWD1 mRNA in all tested tissues (leaves, petioles and hypocotyls, apical meristems, roots and suspension cultured cells), though at a different extent. Lack of induction of relevant transcripts in proliferating auxin-stimulated hypocotyls suggests a mode of expression not strictly related to the cell proliferation
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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