Biomedical subjects

Derek J Lydiate

Publications and source records attributed to Derek J Lydiate.

8 recordsLinked to original sources

PubMed2006

Characterization of a beta-carotene hydroxylase of Adonis aestivalis and its expression in Arabidopsis thaliana.

Carotenoids are plant secondary metabolites that comprise two main groups: carotenes and xanthophylls. The latter group includes zeaxanthin which is synthesized by beta-carotene hydroxylase catalyzing the hydroxylation of the beta-rings of beta-carotene molecules. To develop tools to alter carotenoid biosynthesis in plants, we isolated a cDNA clone encoding a candidate beta-carotene hydroxylase, CrtH1, from the flower petals of Adonis aestivalis. CrtH1 protein has homology to beta-carotene hydroxylases from other organisms, and possesses the four histidine motifs conserved in this family of enzymes. Sequence analysis predicted the presence of a putative plastid transit peptide at the amino terminus and four transmembrane helical regions. Southern-blot analysis showed CrtH1 to be encoded by a multicopy gene family with at least three members in A. aestivalis. Analysis of CrtH1 transcript abundance by Northern blotting indicates it is highly expressed in flower petals, roots and stems, with relatively low expression in leaves and developing seeds. CrtH1 was able to catalyze the formation of zeaxanthin and its intermediate precursor beta-cryptoxanthin from beta-carotene in functional assays conducted in E. coli. Expression of CrtH1 in Arabidopsis thaliana wild type and a mutant deficient for endogenous beta-carotene hydroxylases enhanced the biosynthesis of violaxanthin in the seeds.

Adonis↗

PubMed2006

Isolation and characterization of a GCN5-interacting protein from Arabidopsis thaliana.

An Arabidopsis protein, AtEML, was isolated based on its interaction with the histone acetyltransferase AtGCN5 in a yeast two-hybrid screen. RNA blot and RT-PCR analysis showed that AtEML is expressed in flowers, leaves, stems and siliques. The promoter region of AtEML has several cis-acting elements associated with response to biotic and abiotic stress conditions, and the accumulation of the AtEML transcript was found to be regulated by cold and salt treatments. In vitro and in vivo protein-protein interaction assays indicated that AtEML interacts with AtGCN5 through the N-terminal region. Furthermore, AtEML was shown to activate expression of the lacZ reporter gene in yeast through recruitment of AtGCN5. Such recruitment was accompanied by an increase in histone H3 acetylation at the promoter driving lacZ expression, as determined by chromatin immunoprecipitation. A higher level of AtEML gene expression was detected in the Arabidopsis gcn5 knockout mutant as compared to wild type Arabidopsis, indicating that AtEML expression is regulated by AtGCN5. These results suggest that AtEML may be a transcription factor that co-ordinates the expression of target stress regulated genes through involvement in recruiting AtGCN5 to their promoters.

Amino Acid Sequence↗

PubMed2006

New evidence from Sinapis alba L. for ancestral triplication in a crucifer genome.

We present clear evidence of ancestral genome triplication in Sinapis alba, a close relative of the cultivated Brassica species. Exceptionally high levels of heterozygosity in the parents of an F1 intercross permitted the mapping of an estimated 87% of all detected restriction fragment length polymorphism (RFLP) loci, with each RFLP probe typically detecting 2 or 3 loci. These duplicated loci were arranged in 8 triplicated homologous linkage blocks and 2 small, duplicated, homologous linkage blocks covering the majority of the S. alba genome. Several large-scale inversions and translocations appear to have rearranged the order of loci within homologous blocks. The role of successive polyploidization events on the evolution of crucifer species is discussed.

Brassicaceae↗

PubMed2005

Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana.

Over 1000 genetically linked RFLP loci in Brassica napus were mapped to homologous positions in the Arabidopsis genome on the basis of sequence similarity. Blocks of genetically linked loci in B. napus frequently corresponded to physically linked markers in Arabidopsis. This comparative analysis allowed the identification of a minimum of 21 conserved genomic units within the Arabidopsis genome, which can be duplicated and rearranged to generate the present-day B. napus genome. The conserved regions extended over lengths as great as 50 cM in the B. napus genetic map, equivalent to approximately 9 Mb of contiguous sequence in the Arabidopsis genome. There was also evidence for conservation of chromosome landmarks, particularly centromeric regions, between the two species. The observed segmental structure of the Brassica genome strongly suggests that the extant Brassica diploid species evolved from a hexaploid ancestor. The comparative map assists in exploiting the Arabidopsis genomic sequence for marker and candidate gene identification within the larger, intractable genomes of the Brassica polyploids.

Arabidopsis↗

PubMed2005

Genome-wide analysis of the frequency and distribution of crossovers at male and female meiosis in Sinapis alba L. (white mustard).

We present the first genetic linkage maps of Sinapis alba (white mustard) and a rigorous analysis of sex effects on the frequency and distribution of crossovers at meiosis in this species. Sex-averaged maps representing recombination in two highly heterozygous parents were aligned to give a consensus map consisting of 382 loci defined by restriction fragment length polymorphisms and arranged in 12 linkage groups with no unlinked markers. The loci were distributed in a near-random manner across the genome, and there was little evidence of segregation distortion. From these dense maps, a subset of spaced informative markers was used to establish recombination frequencies assayed separately in male and female gametes and derived from two distinct genetic backgrounds. Analyses of 746 gametes indicated that recombination frequencies were greater in male gametes, with the greatest differences near the ends of linkage groups. Genetic background had a lesser effect on recombination frequencies, with no discernible pattern in the distribution of such differences. The possible causes of sex differences in recombination frequency and the implications for plant breeding are discussed.

Chromosome Mapping↗

PubMed2003

A novel protein from Brassica napus has a putative KID domain and responds to low temperature.

To identify factors that interact with histone deacetylase (HDAC) in Brassica napus, a yeast two-hybrid library was screened using the Arabidopsis HDA19 as bait. A novel protein, bnKCP1, containing a putative kinase-inducible domain (KID) was found to interact with HDA19. Southern blot analysis indicated that the bnKCP1 gene belongs to a small gene family of at least three members. Northern blot analysis showed bnKCP1 to be strongly expressed in stems, flowers, roots, and immature siliques, but not in leaf blades of seedlings. The accumulation of bnKCP1 transcript in the leaf blades was induced significantly within 4 h of exposure of B. napus seedlings to cold stress, whereas treatment of leaf blades with inomycin, an ionophore of Ca2+, caused a rapid (30 min) but transient induction of bnKCP1 expression. In contrast to that observed in leaf blades, expression of bnKCP1 in the stems was repressed upon cold treatment. In vitro and in vivo protein-binding assays showed that bnKCP1 interacts with HDA19 via the KID domain, and that S188 is critical for bnKCP1-HDA19 interaction. BnKCP1 also exerted modest transactivation of the lacZ reporter gene in yeast through its N-terminal region. These assays suggest that bnKCP1 may function as a transcription factor, which regulates gene expression through interaction with HDA19.

Amino Acid Sequence↗

PubMed2003

Detection and effects of a homeologous reciprocal transposition in Brassica napus.

A reciprocal chromosomal transposition was identified in several annual oilseed Brassica napus genotypes used as parents in crosses to biennial genotypes for genetic mapping studies. The transposition involved an exchange of interstitial homeologous regions on linkage groups N7 and N16, and its detection was made possible by the use of segregating populations of doubled haploid lines and codominant RFLP markers. RFLP probes detected pairs of homeologous loci on N7 and N16 for which the annual and biennial parents had identical alleles in regions expected to be homeologous. The existence of an interstitial reciprocal transposition was confirmed by cytological analysis of synaptonemal complexes of annual x biennial F1 hybrids. Although it included approximately one-third of the physical length of the N7 and N16 chromosomes, few recombination events within the region were recovered in the progenies of the hybrids. Significantly higher seed yields were associated with the parental configurations of the rearrangement in segregating progenies. These progenies contained complete complements of homeologous chromosomes from the diploid progenitors of B. napus, and thus their higher seed yields provide evidence for the selective advantage of allopolyploidy through the fixation of intergenomic heterozygosity.

Brassica↗

PubMed2003

Genetic analysis and genome mapping in Raphanus.

The first genetic map of the Raphanus genome was developed based on meiosis in a hybrid between Raphanus sativus (cultivated radish) and Raphanus raphanistrum (wild radish). This hybrid was used to produce a BC1 population of 54 individuals and an F2 population of 85 individuals. A total of 236 marker loci were assayed in these populations using a set of 144 informative Brassica RFLP probes previously used for genetic mapping in other crucifer species. The genetic maps derived from the BC1 and F2 populations were perfectly collinear and were integrated to produce a robust Raphanus map. Cytological observations demonstrated strict bivalent pairing in the R. sativus x R. raphanistrum hybrids. Productive pairing along the length of each chromosome was confirmed by the identification of nine extensive linkage groups and the lack of clustering of marker loci. Indeed, the distributions of both marker loci and crossovers was more random than those reported for other crop species. The genetic markers and the reference map of Raphanus will be of considerable value for future trait mapping and marker-assisted breeding in this crop, as well as in the intergenomic transfer of Raphanus genes into Brassica crops. The future benefits of comparative mapping with Arabidopsis and Brassica species are also discussed.

Chromosome Mapping↗