Search

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. Formation of precise junctions at the right T‐DNA border, and DNA sequence homology between the left termini of T‐DNA segments and break‐points of target deletions were observed in those cases where full‐length canonical T‐DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T‐DNA segments showed no homology to break‐points of target sequence deletions. Homology between short segments within target sites and T‐DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T‐DNA integration results from illegitimate recombination. The data suggest that while the left T‐DNA terminus and both target termini participate in partial pairing and DNA repair, the right T‐DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

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. Plant boundaries of the T‐DNA insert rescued from the pale mutant were used as probes for the isolation of genomic and full‐length cDNA clones of the wild‐type cs gene. Transformation of the pale mutant with T‐DNA vectors carrying these clones resulted in a normal green phenotype, thus demonstrating positive complementation of the T‐DNA induced mutation. DNA sequence comparison of the cs mutant and its wild‐type allele revealed that the T‐DNA insertion occurred 11 bp upstream of the stop codon. A fusion protein, seven amino acids longer than its wild‐type counterpart of Mr 46,251, is therefore synthesized in the pale mutant. Transcript analysis during dark‐light transition, in vitro protein transport assay, and the absence of DNA sequence homology between cs and known genes indicates that the light regulated expression of the cs gene results in the synthesis of a novel chloroplast protein.

In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plants accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-micron granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB that accumulated. We conclude that there does not appear to be any biological barrier to high-level production of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand.

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. Formation of precise junctions at the right T‐DNA border, and DNA sequence homology between the left termini of T‐DNA segments and break‐points of target deletions were observed in those cases where full‐length canonical T‐DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T‐DNA segments showed no homology to break‐points of target sequence deletions. Homology between short segments within target sites and T‐DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T‐DNA integration results from illegitimate recombination. The data suggest that while the left T‐DNA terminus and both target termini participate in partial pairing and DNA repair, the right T‐DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

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. Plant boundaries of the T‐DNA insert rescued from the pale mutant were used as probes for the isolation of genomic and full‐length cDNA clones of the wild‐type cs gene. Transformation of the pale mutant with T‐DNA vectors carrying these clones resulted in a normal green phenotype, thus demonstrating positive complementation of the T‐DNA induced mutation. DNA sequence comparison of the cs mutant and its wild‐type allele revealed that the T‐DNA insertion occurred 11 bp upstream of the stop codon. A fusion protein, seven amino acids longer than its wild‐type counterpart of Mr 46,251, is therefore synthesized in the pale mutant. Transcript analysis during dark‐light transition, in vitro protein transport assay, and the absence of DNA sequence homology between cs and known genes indicates that the light regulated expression of the cs gene results in the synthesis of a novel chloroplast protein.