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David J Cove

Publications and source records attributed to David J Cove.

5 recordsLinked to original sources
PubMed2006

The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration.

The model bryophyte Physcomitrella patens exhibits high frequencies of gene targeting when transformed with DNA constructs containing sequences homologous with genomic loci. 'Targeted gene replacement' (TGR) resulting from homologous recombination (HR) between each end of a targeting construct and the targeted locus occurs when either single or multiple targeting vectors are delivered. In the latter instance simultaneous, multiple, independent integration of different transgenes occurs at the targeted loci. In both single gene and 'batch' transformations, DNA can also be found to undergo 'targeted insertion' (TI), integrating at one end of the targeted locus by HR with one flanking sequence of the vector accompanied by an apparent non-homologous end-joining (NHEJ) event at the other. Untargeted integration at nonhomologous sites also occurs, but at a lower frequency. Molecular analysis of TI at a single locus shows that this occurs as a consequence of concatenation of the transforming DNA, in planta, prior to integration, followed by HR between a single site in the genomic target and two of its repeated homologues in the concatenated vector. This reinforces the view that HR is the major pathway by which transforming DNA is integrated in Physcomitrella.

Alleles↗
PubMed2006

Identification and functional characterization of the moss Physcomitrella patens delta5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis.

The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a delta5-desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known delta5-desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b5-like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the delta5-containing fatty acid arachidonic acid in cells that were provided di-homo-gamma-linolenic acid. In addition, the expressed enzyme generated delta5-desaturation products with the C20 substrates omega-6 eicosadienoic and omega-3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and alpha-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-gamma-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the delta5-fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis.

Arachidonic Acid↗
PubMed2006

Agravitropic mutants of the moss Ceratodon purpureus do not complement mutants having a reversed gravitropic response.

New mutants of the moss Ceratodon purpureus have been isolated, which showed abnormal gravitropic responses. The apical cells of protonemal filaments of wild-type strains respond to gravity by growing upwards and are well aligned to the gravity vector. This response only occurs in darkness. Mutants show a range of phenotypes. Some are insensitive to gravity, showing symmetrical growth, while others align to the gravity vector but orient growth downwards. A further class grows in darkness as though it were in light, showing insensitivity to gravity and continued chlorophyll synthesis. Somatic hybrids between mutants and wild-type strains and between pairs of mutants have been selected using transgenic antibiotic resistance as selective markers. Hybrids between wild-type strains and all of the mutants have a wild-type phenotype, and so all mutants therefore have recessive phenotypes. Mutants comprise three complementation groups. One group has a single member, while another has three members. The third has at least 16 members and shows a complex pattern of complementation consistent with a single gene product functioning in both orientation and alignment to gravity, as well as contributing more than one subunit to the mature product.

Bryopsida↗
PubMed2005

Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens.

In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates predominantly by homologous recombination with its genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology length and targeting frequency for replacement vectors (a selectable marker flanked by homologous DNA). Overall homology of only 1 kb is sufficient to achieve a 50% yield of targeted transformants. Targeting may occur through homologous recombination in one arm, accompanied by non-homologous end-joining by the other arm of the vector, or by allele replacement following two homologous recombination events. Allele replacement frequency depends on the symmetry of the targeting vector, being proportional to the length of the shorter arm. Allele replacement may involve insertion of multiple copies of the transforming DNA, accompanied by ectopic insertions at non-homologous sites. Single-copy and single insertions at targeted loci (targeted gene replacements, 'TGR') occur with a frequency of 7-20% of all transformants when the minimum requirements for allele replacement are met. Homologous recombination in Physcomitrella is substantially more efficient than in any multicellular eukaryote, recommending it as the outstanding model for the study of homologous recombination in plants.

Bryopsida↗