Changing images of the gene.

During the twentieth century the gene emerged as the major driving force of biology. Initially, even the nature and behavior of gene vehicles, the chromosomes, were subjected to doubts. The basic or standard gene concept, as a unit of function, mutation, and recombination, had to be revised.

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes.

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection.

Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome.

Induction of knockout mutations by T-DNA insertion mutagenesis is widely used in studies of plant gene functions. To assess the efficiency of this genetic approach, we have sequenced PCR amplified junctions of 1000 T-DNA insertions and analysed their distribution in the Arabidopsis genome. Map positions of 973 tags could be determined unequivocally, indicating that the majority of T-DNA insertions landed in chromosomal domains of high gene density.

Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis.

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation.

Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis.

The cpd mutation localized by T-DNA tagging on Arabidopsis chromosome 5-14.3 inhibits cell elongation controlled by the ecdysone-like brassinosteroid hormone brassinolide. The cpd mutant displays de-etiolation and derepression of light-induced genes in the dark, as well as dwarfism, male sterility, and activation of stress-regulated genes in the light.

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides.

Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana.

A recessive pale mutation, designated as cs, was identified by transferred-DNA (T-DNA)-mediated insertional mutagenesis in Arabidopsis thaliana. The pale mutation, cosegregating with the hygromycin resistance marker of the T-DNA, was mapped to the position of the ch-42 (chlorata) locus on chromosome 4. Lack of genetic complementation between cs and ch-42 mutants indicated allelism.

High-frequency T-DNA-mediated gene tagging in plants.

An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')II (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')II enzyme activity in diverse tissues of transgenic plants.

Aneuploidy detection with a short-term hexaploid wheat assay.

A new type of assay for the identification of agents causing aneuploidy is described. This assay takes advantage of allohexaploid wheat in which monosomic and nullisomic cell lineages can be genetically detected. The wheat strain used (Neatby's virescens) was homozygous for a pair of recessive alleles (v1) which in homozygous condition interfere with normal pigmentation of the leaves at low temperature whereas at higher temperature nearly normal green color formation is permitted.

Mutagen assay with Arabidopsis. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The small crucifer Arabidopsis is very economical for short-term yet complete assay for chemical mutagens. Thousands of gene loci can be simultaneously monitored for alterations within one culture by screening for segregation among the embryos (M2) developing on the M1 plants. This species is quite sensitive to a wide range of compounds and can activate promutagens to mutagens.

Gene locus specificity of the glucose effect in the thiamine pathway of the angiosperm, Arabidopsis.

All mutants at 3 loci in Arabidopsis thaliana (L.) Heynh., a higher plant, that are associated with the synthesis or coupling of the thiazole moiety of thiamine are susceptible to reversible glucose inhibition.

Direct evidence for models of heterosis provided by mutants of Arabidopsis blocked in the thiamine pathway.

Auxotrophic mutants genetically blocked at different steps of the thiamine pathway dramatically demonstrate the biochemical mechanism of hybrid vigor due to simple and perfect dominance at two unlinked loci. Heteroallelic hybrids of mutants requiring the pyrimidine moiety of thiamine display allelic complementation and thus furnish clear biochemical and genetic evidence for the superdominance hypothesis. Hybrids of low- and high-temperature-requiring leaky mutants demonstrate that heterozygosity at a single gene locus may confer developmental homeostasis on the heteroallelic combinations superior to that of the homoallelic parents.

Acceleration of flowering of the long-day plant Arabidopsis by 8-azaadenine.

The incorporation of 8-azaadenine (2×10(-5)M) to the aseptic culture medium reduced the time required for developing macroscopically visible flower primordia and the number of leaves appearing before the first flower buds to about half under 8 hrs daily illuumination in both wild type and a late monogenic mutant. In the presence of equimolar amount of adenine the flowering was not accelerated by the analog.

Early flowering in Arabidopsis induced by DNA base analogs.

Various isogenic lines of Arabidopsis differing in a single factor controlling flower initiation were cultured aseptically on media containing bromodeoxycytidine and bromodeoxyuridine (10(-5) M). The wild type under short-day illumination (8 hours daily) and the late mutant gi (2), in both continuous light and under short day, responded with dramatically earlier flower initiation. Another late mutant (ld) failed to respond.