Gene identification with sequenced T‐DNA tags generated by transformation of <i>Arabidopsis</i> cell suspension

Centromere protein C (CENP-C) is a component of the kinetochore essential for correct segregation of sister chromatids in mammals. In Arabidopsis thaliana , a single-copy gene encoding a protein homologous to CENP-C has been found by homology in the whole-genome sequence. To investigate the CENP-C homolog (AtCENP-C), we cloned cDNAs by RT-PCR and determined its full-length coding sequence. Antibodies against the synthetic peptide for the C-terminal residues of AtCENP-C detected a polypeptide in Arabidopsis cell extracts on western blots. Immunofluorescence labeling with the antibodies and fluorescence in situ hybridization demonstrated clearly that AtCENP-C is present at the centromeric regions throughout the cell cycle.

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“…E-mail: yogura@rib.okayama-u.ac.jp ecotype Columbia was maintained as described (Mathur et al 1998). …”

Section: Methodsmentioning

confidence: 99%

Characterization of a CENP-C homolog in Arabidopsis thaliana

et al. 2004

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“…DNA fragments flanking the T-DNA insertions were amplified from purified plant DNA samples using either long-range inverse PCR or TAIL-PCR as described (Mathur et al, 1998;Szabados et al, 2002). Long-range inverse PCR was performed with DNA templates that were previously digested by EcoRI and self-ligated.…”

Section: Sequencing Of T-dna Insert Junctionsmentioning

confidence: 99%

Gene Trapping with Firefly Luciferase in Arabidopsis. Tagging of Stress-Responsive Genes

et al. 2004

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To monitor the expression of T-DNA-tagged plant genes in vivo, a collection of 20,261 transgenic lines of Arabidopsis (Columbia-0) were generated with the promoter trap vector pTluc, which carries a promoterless firefly luc (luciferase) reporter gene linked to the right T-DNA border. By detection of bioluminescence in 3-week-old seedlings, 753 lines were identified showing constitutive, organ-specific, and stress-responsive luciferase expression patterns. To facilitate the identification of well-defined luciferase expression patterns, a pooled seed stock was established. Several lines showed sugar, salt, and abscisic acid (ABA)-inducible luciferase activity. Segregation analysis of 215 promoter trap lines indicated that about 50% of plants contained single insertions, whereas 40% carried two and 10% carried three or more T-DNA tags. Sequencing the T-DNA insert junctions isolated from 17 luciferase-expressing lines identified T-DNA tags in 5Ј-and 3Ј-transcribed domains and translational gene fusions generated by T-DNA insertions in exons and introns of Arabidopsis genes. Tissue specific expression of eight wild-type Arabidopsis genes was confirmed to be similar to the luminescence patterns observed in the corresponding luciferase-tagged lines. Here, we describe the characterization of a transcriptional luc reporter gene fusion with the WBC-type ABC transporter gene At1g17840. Expression of wild-type and luciferase-tagged At1g17840 alleles revealed similar induction by salt, glucose, and ABA treatments and gibberellin-mediated down-regulation of ABA-induced expression. These results illustrate that luciferase gene traps are well suited for monitoring the expression of stress-responsive Arabidopsis genes in vivo.

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“…Suspension cultures of Arabidopsis (ecotype Columbia) or diminuto (dim) were initiated by adding root sections to liquid medium, as described in Mathur et al (1998). RNA was isolated using RNA Matrix (Bio-101 Inc., Richmond, CA) according to the manufacturer's instructions.…”

Section: Differential Displaymentioning

confidence: 99%

The Arabidopsis DIMINUTO/DWARF1 Gene Encodes a Protein Involved in Steroid Synthesis

et al. 1998

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We have identified the function of the Arabidopsis DIMINUTO / DWARF1 (DIM / DWF1) gene by analyzing the dim mutant, a severe dwarf with greatly reduced fertility. Both the mutant phenotype and gene expression could be rescued by the addition of exogenous brassinolide. Analysis of endogenous sterols demonstrated that dim accumulates 24-methylenecholesterol but is deficient in campesterol, an early precursor of brassinolide. In addition, we show that dim is deficient in brassinosteroids as well. Feeding experiments using deuterium-labeled 24-methylenecholesterol and 24-methyldesmosterol confirmed that DIM/DWF1 is involved in both the isomerization and reduction of the ⌬ 24(28) bond. This conversion is not required in cholesterol biosynthesis in animals but is a key step in the biosynthesis of plant sterols. Transient expression of a green fluorescent protein-DIM/DWF1 fusion protein and biochemical experiments showed that DIM/DWF1 is an integral membrane protein that most probably is associated with the endoplasmic reticulum.

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Gene identification with sequenced T‐DNA tags generated by transformation of Arabidopsis cell suspension

Cited by 119 publications

References 44 publications

Characterization of a CENP-C homolog in Arabidopsis thaliana

Characterization of a CENP-C homolog in Arabidopsis thaliana

Gene Trapping with Firefly Luciferase in Arabidopsis. Tagging of Stress-Responsive Genes

The Arabidopsis DIMINUTO/DWARF1 Gene Encodes a Protein Involved in Steroid Synthesis

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